Abstracts for the IFPA Meeting 2011

Placenta 32 (2011) A1–A149

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Abstracts for the IFPA Meeting 2011

Abstract Outline - IFPA 2011 Plenary Session 1

(PL1.1 – PL1.2)

Plenary Session 2

(PL2.1 – PL2.2)

Plenary Session 3 - Trophoblast Research Award and New Investigator Oral Session 1

(PL3.TR1 - PL3.NI1 – PL3.NI6)

Plenary Session 4 - New Investigator Oral Session 2

(PL4.NI1 – PL4.NI6)

Plenary Session 5

(PL5.1 – PL5.4)

Plenary Session 6

(PL6.1 – PL6.2)

Plenary Session 7

(PL7.1 – PL7.2)

Poster Session 1

(P1.1 – P1.140)

Poster Session 2

(P2.1 – P2.145)

doi:10.1016/j.placenta.2011.07.004

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Abstracts / Placenta 32 (2011) A1–A149

Plenary Session 1 (PL1.1 – PL1.2) PL1.1 EVOLUTION, DEVELOPMENT AND LIFELONG HEALTH: THE WOMB, THE PLACENTA AND LATER Mark Hanson University of Southampton, Southampton Non-communicable diseases (NCDs) present enormous humanitarian and economic threats to developed, and increasingly to developing, societies. NCDs are often thought to be direct consequences of sedentary lifestyle and unbalanced diets in adulthood. Yet this does not explain why some individuals or population groups are at greater risk in the same environment: nor why interventions targeted at improving adult lifestyle are sometimes ineffective. We are focussing on the proximate causes of NCD rather than asking about the ultimate causes especially those influencing responses to environmental challenges. Developmental processes provide better answers, especially those we believe to have evolved to provide a fitness advantage – viz. survival to successful reproduction. Since evolution drives fitness, rather that health, in a predicted environment and incorporates a degree of variation into phenotypes developed, it can explain the ranges of responses to a mismatched contemporary environment, and the associated risks of NCD. In many species, maternal and paternal effects induce offspring phenotype depending on environmental conditions (e.g. nutrition, stress levels) and aspects of parental phenotype (body composition, age, parity etc). Such processes operate within the normal range and, it is incorrect to view them as disrupting development or as early signs of pathophysiology. We are now learning how epigenetic mechanisms can affect “heritable” risk without changes in the genome. They can affect embryo, fetus, infant and placental development. Epigenetic differences may provide valuable perinatal biomarkers of an individual’s responses to their developmental environment and predictors of their later NCD risk. They may help customise interventions appropriately. Consideration of more ultimate “why” questions about the origins of NCD risk stresses the importance of education and other initiatives to promote the lifestyle and body condition of parents-to-be, initiatives which will repay investment substantially even in the short-term by assisting the next generation to have a healthier start to life. MAH is supported by the British Heart Foundation

PL1.2 EVOLUTION, DEVELOPMENT AND LIFELONG HEALTH; THE PLACENTAL CONTRIBUTION G. Burton University of Cambridge, Cambridge, UK Dysfunction of any one of the myriad of activities of the placenta may impact adversely on fetal development, resulting in programming and predisposition to adult disease. Of particular relevance are the capacity for active and passive transfer of nutrients and oxygen, and protection against transfer of potentially harmful maternal hormones, such as cortisol. These functions may, in turn, be influenced by a diverse range of factors, including the maternal body composition and diet, the intrauterine hormonal environment, placental perfusion and maternal-fetal immune interactions. Acting through alterations in the pattern of placental cell proliferation and differentiation, and epigenetic changes in gene expression, these factors can result in structural differences, and changes in placental metabolism and transport capacity. The placenta is not just a passive target, however, and data showing that mice can adapt to genetically induced reductions in growth by increased expression of imprinted genes encoding transporter proteins will be reviewed. In more pathological situations, the ability of the placenta to adapt may be compromised. Vascular or hypoxic insults to the human and murine placenta cause both oxidative and endoplasmic reticulum (ER) stress. One of the consequences of the latter is suppression of non-essential protein synthesis in order to preserve energy and nutrient resources. Data will be presented demonstrating that ER stress is a sufficient cause of placental and fetal growth restriction in the mouse. Induction of ER stress results in the loss of bioactivity of secreted growth factors from embryonic fibroblast cells. Consequently, trophoblast stem cell differentiation is perturbed through loss of paracrine signalling, leading to structural alterations and a compromise in placental function. ER stress can be induced through a variety of causes, including hypoxia, energy or nutrient deficiency, and pro-inflammatory cytokines. Hence, it may provide a common link in the pathophysiology of placental insufficiency of diverse aetiologies. Supported by the Wellcome Trust. Plenary Session 2 (PL2.1 – PL2.2) PL2.1 SPATIOTEMPORAL DYNAMICS OF HCG/CAMP SIGNALLING AND REGULATION OF PLACENTAL FUNCTION Kjetil Taskén University of Oslo, Oslo, Norway Human chorionic gonadotrophin acts on G-protein coupled LH receptors expressed on trophoblasts in the placenta to regulate differentiation and function. LH receptor signal transduction is mediated by the second messenger cyclic AMP (cAMP), acting through protein kinase A (PKA). The cAMP - PKA pathway is one of the most common and versatile signal pathways in eukaryotic cells. A kinase anchoring proteins (AKAPs) target PKA to specific substrates and distinct subcellular compartments, providing spatial and temporal specificity for mediation of biological effects channelled through the cAMP-PKA pathway. We have recently identified new AKAP-coordinated signalling complexes in cytotrophoblast cells and in syncytiotrophoblast through a chemical proteomics approach, and are mapping their functional effects through a variety of molecular interaction techniques, imaging and functional assays.

Abstracts / Placenta 32 (2011) A1–A149

PL2.2 EVOLUTION OF HUMAN KILLER-CELL IMMUNOGLOBULIN-LIKE RECEPTORS FOR BATTLING BUGS AND BUILDING BIGGER-BRAINED BABIES Peter Parham Stanford University, California Natural killer (NK) cells make vital contributions to human immunity and reproduction. Diversifying these functions are variable killer cell immunoglobulin-like receptors (KIR) that recognize HLA class I. Interactions between maternal NK-cell KIR and HLA-C on fetal extra-villous trophoblast influence embryo implantation and reproductive success. Notably, the combination of maternal KIR2DL1 with fetal HLA-C having the C2 epitope correlates with pregnancy disorders. Natural variation in KIR2DL1 affects the avidity and specificity of the interaction with HLA-C, as well as signal transduction and the likelihood of disordered pregnancy. Counterparts to the human KIR are present only in simian primates and they exhibit striking species-specific divergence. HLA-C is restricted to great apes and humans, species where there is corresponding expansion of cognate lineage III KIR. Absence of HLA-C and –G counterparts in gibbons is accompanied by a degraded KIR locus, having KIR3DL3 as the only functional gene. Uniquely distinguishing the human KIR locus are its two KIR haplotype groups, A and B, which appear specialized towards immune and reproductive functions, respectively. Human populations all have both haplotype groups, including small Amerindian tribes that survived successive episodes of epidemic infection and population bottleneck. The distinctive properties of the A and B haplotypes point to a selective episode during human evolution that increased the placental blood supply, with consequential heightened likelihood for pregnancy disorder. This in turn led to the emergence of the B KIR haplotype, providing protection against pregnancy disorder, but at the cost of immune defence. A candidate cause of the selective episode was increasing fitness of individual humans with bigger brains, and whose development in utero demanded extraordinary supply of maternal blood to the placenta. Because chimpanzees were less subject to such clever compromise, their NK cells are predicted to be more reliable at both eliminating infection and implanting embryos than human NK cells.

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Plenary Session 3 - Trophoblast Research Award and New Investigator Oral Session 1 (PL3.TR1 - PL3.NI1 – PL3.NI6) PL3.TR1 NEW INSIGHTS INTO ESTROGEN EFFECTS IN TROPHOBLASTIC CELLS. FOCUS ON LEPTIN EXPRESSION Yésica Paola Gambino1, Antonio Pérez Pérez2, José Luis Dueñas3, Juan Carlos Calvo1,4, Víctor Sánchez-Margalet2, Cecilia Laura Varone1 1 Dpto. de Química Biológica, FCEN, UBA, Buenos Aires, Argentina, 2Dpto. de Bioquímica Médica y Biología Molecular, Universidad de Sevilla, Sevilla, Spain, 3Servicio de Ginecología y Obstetricia, Hospital Universitario Virgen Macarena, Sevilla, Spain, 4IByME, Buenos Aires, Argentina Estradiol influences various aspects of placental function and fetal development as the regulation of implantation, placental development, fetal growth, onset of parturition, release of neuropeptides and glycoproteins, and leptin secretion. Leptin is a key hormone in trophoblast proliferation and survival. It regulates cell proliferation, inhibits apoptosis, stimulates protein synthesis, and regulates fetal growth and development. The mechanisms involved in regulation of placental leptin expression are still poorly understood. In the present study, we analyzed the effect of 17beta-estradiol (E2) on leptin expression in human placental cells. We have found a stimulatory effect of E2 on endogenous leptin expression in both BeWo choriocarcinoma cell line and human placental explants. E2 treatment enhanced leptin promoter activity, as evaluated by reporter plasmids. Deletion analysis showed that a minimal promoter region between -1951 and -1847 bp is both necessary and sufficient to achieve E2 effects. These effects involved estrogen receptors (ER), as the antagonist ICI 182,780 inhibited E2-induced leptin expression. Moreover, the overexpression of ERalpha, but not ERbeta, increased E2 effect on leptin promoter activity. E2 action probably involves membrane receptors too, as treatment with a membrane-constrained E2 conjugate, E-BSA, enhanced leptin expression. Furthermore, E2 and E-BSA rapidly activated different MAPKs and AKT signaling pathways. Inhibition of these pathways with dominant negative mutant kinases or pharmacologic inhibitors prevents E2 effect on leptin expression. On the other hand we demonstrated the presence of ERalpha associated to the plasma membrane of BeWo cells. We also showed that the downregulation of ERalpha level through a specific siRNA, decreased E-BSA effects on leptin expression, suggesting that E2 genomic and nongenomic actions could be mediated by this receptor. Taken together, our results provide new evidence on the mechanisms whereby E2 regulates placental leptin expression and support the importance of leptin in placental physiology.

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Abstracts / Placenta 32 (2011) A1–A149

PL3.NI1 CYTOLYTIC CAPACITY OF DECIDUAL NATURAL KILLER (DNK) CELLS AT THE MATERNAL-FETAL INTERFACE

PL3.NI2 CHARACTERISING THE PATHOLOGICAL INTERACTIONS BETWEEN ANTIPHOSPHOLIPID ANTIBODIES AND TROPHOBLASTS

Ângela Crespo1,2, Tamara Tilburgs1, Jack Strominger1

Chez Viall1, Qi Chen1,2, Peter Stone1, Larry Chamley1 1 The Department of Obstetrics and Gynaecology, The University of Auckland, Auckland, New Zealand, 2Hospital of Obstetrics and Gynaecology, Fudan University, Fudan, China

Harvard University, Cambridge, MA, United States, 2PhD Programme in Experimental Biology and Biomedicine - Center for Neuroscience and Cell Biology - University of Coimbra, Coimbra, Portugal

1

Objectives: dNK cells contain high levels of cytolytic molecules like perforin, granzyme B and granulysin. However, dNK cells are believed to be less cytotoxic than their peripheral blood counterparts. Cytolytic capacity of dNK cells may highly depend on the initial NK cell activation, killing assay used and, most importantly, NK cell subtype studied. Methods: dNK cells from first trimester pregnancies and pNK cells from healthy blood donors were characterized by flow cytometry and quantitative PCR for expression of the MHC class-I receptors KIR2DL1/S1/L2/L3/ L4, ILT-2, ILT-4, CD94/NKG2A, CD94/NKG2C and the cytolytic molecules perforin, granzyme B and granulysin. Cytotoxicity of dNK cells and pNK cells was tested against the MHC Class I negative K562 target cells, and K562 cells transfected with different MHC Class I molecules, using a flow cytometry based method. Results: The percentage of dNK cells expressing MHC Class I receptors is significantly higher than pNK cells, and both exhibit great variety of subsets with different receptor combinations. Expression of perforin and granzyme B is not significantly different between dNK and pNK cells. Granulysin is highly expressed in dNK cells, being virtually absent in pNK cells. However, freshly isolated dNK cells are less cytotoxic against K562 cells than freshly isolated pNK cells. After overnight stimulation with IL-15, dNK cell cytotoxicity increases and becomes comparable to pNK cells (Kopcow et al, 2005). In contrast to previous reports (Rouas-Freiss et al, 1997), our data do not demonstrate a consistent reduction in cytotoxicity against HLA-Gþ cell lines by either dNK or pNK cells, due to high variations between donors. Conclusion: NK subset distribution is highly variable among individuals. Analysis of cytotoxicity of the total NK population may therefore not give a clear picture of NK cell function. Evaluation of individual NK cell subtypes may provide better insights into NK cell function during pregnancy.

Antiphospholipid Antibodies (aPL) are auto-antibodies present in 1-3% of pregnant women. These antibodies increase the risk of a woman developing preeclampsia, recurrent miscarriage, stillbirth and intrauterine growth restriction. Antiphospholipid antibodies may predispose to these pathologies by causing trophoblast dysfunction, and there is evidence that both villous and extravillous trophoblasts are affected by aPL in vitro. How aPL have this affect on trophoblasts is unknown. Objective: To understand how aPL cause trophoblast dysfunction by characterising the interactions between aPL and trophoblasts of the villous and extravillous lineages. Methods: To study villous trophoblasts, first trimester human placental explants were incubated with the murine monoclonal aPL, IIC5 or ID2 or a control antibody for 2 hours before being frozen in liquid nitrogen. Cryosections were prepared and the antibodies probed for by immunohistochemistry. To study extravillous trophoblasts, first trimester placental explants were cultured on MatrigelTM for 48 hours. Explants that had produced extravillous trophoblast outgrowths were incubated with fluorescently-labelled murine monoclonal aPL IIC5, ID2 or JAB1, or a control antibody for 2 hours. The outgrowths were visualised live on Nikon Ti and Zeiss LSM_710 microscopes. Lysates of first trimester placenta were probed using the monoclonal aPL IIC5, ID2 and JAB1 on western blots. Experiments were conducted in triplicate on separate placentae. Results: The syncytiotrophoblast rapidly internalised aPL but not control antibodies. The aPL appeared to be excluded from the villous cytotrophoblast. Western blotting suggested that in the villous placenta, aPL interact with proteins of 250kDa, 170kDa and 130kDa. Fluorescent aPL bound to but were not internalised by extravillous trophoblasts. Conclusion: These data show that aPL interact differently with different trophoblast populations. Further characterisation of these interactions and the receptors involved will aid our understanding of aPL-associated pregnancy morbidity and may provide potential therapeutic strategies to block the pathological interaction between aPL and the placenta.

Abstracts / Placenta 32 (2011) A1–A149

PL3.NI3 CHARACTERIZATION OF ACTIVATOR PROTEIN-1 (AP-1) FAMILY GENE EXPRESSION IN PLACENTAL MESENCHYMAL STEM CELLS DERIVED FROM NORMAL AND PREECLAMPTIC PREGNANCIES Anna Maria Nuzzo1, Simona Cardaropoli1, Ettore Piccoli1, Annalisa Piazzese2, Tullia Todros1, Alessandro Rolfo1 1 Department of Obstetrics and Gynaecology, University of Turin, Turin, Italy, 2O.I.R.M. S. Anna Hospital, Turin, Italy Objective: Mesenchymal Stromal Cells (MSCs) are adult stem cells able to control proliferation and apoptosis in neighbouring cells. Less is known about placenta-derived MSCs (PDMSCs) and their contribution to proper trophoblast development. Herein, we characterized the expression of AP-1 related genes, key regulators of cellular proliferation and death, in both PDMSCs and placental chorionic tissue from normal and preeclamptic (PE) pregnancies characterized by immature proliferative trophoblast and excessive trophoblast apoptosis. Methods: PE (n¼20) and control (n¼14) placentae were collected. PDMSCs were isolated and characterized for HLA-I, HLA-II, CD133, CD166, CD45, CD14, CD20, CD34, CD73, CD90 and CD105 expression by FACS analysis. mRNA expression of AP-1 genes JunD, JunB, FosB, c-fos, c-jun, Fra-1 and Fra-2 was assessed by quantitative RT-PCR. Specific primers were designed to detect c-fos-2 and delta-FosB splicing variants. Results: PDMSCs presented proper MSC phenotype by FACS analysis. PE and control PDMSCs expressed all AP-1 molecules and c-fos-2 variant was predominant relative to c-fos. Moreover, RT-PCR showed increased JunB mRNA expression (p<0.05) in PE-PDMSCs vs controls. We found decreased JunD (p¼0.047) and increased Fra1 (p¼0.018) mRNA levels in PE vs control placentae. No differences were found for the other AP-1 genes between groups. c-fos 2 variant was expressed only in PE placentae. Conclusion: Herein, we reported that PDMSCs and chorionic tissue differentially expressed AP-1 genes. JunB inhibits embryonic cell proliferation and promotes cellular senescence. Its increased expression in PE-PDMSCs suggests that these cells may contribute to the abnormal development of the surrounding trophoblast. In PE chorionic tissue, decreased expression of anti-proliferative JunD may account for the immature proliferative trophoblast phenotype. Increased Fra1 mRNA, transcription factor able to promote cytokines expression, may contribute to the exacerbated inflammation typical of PE placentae. The biological meaning of c-fos-2 expression in PDMSCs and PE chorionic tissue remains to be investigated.

PL3.NI4 SECOND TRIMESTER ESTIMATED PLACENTAL VOLUME SCREENING IS PREDICTIVE OF SGA AND LGA AT BIRTH

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(EPV)

Anne Cathrine Staff1,4, Katariina Laine1,4, Gulim Lahmami1, Jaana Gustafsson3, Hanne Surbehan4, Mark Lewis Barbero1, Harvey J. Kliman2 1 Oslo University Hospital, Oslo, Norway, 2Yale University School of Medicine, New Haven, United States, 3Akershus University Hospital, Lørenskog, Norway, 4University of Oslo, Oslo, Norway Objectives: We hypothesized that estimated placental volume (EPV) measurement based on 2-dimensional ultrasound (Azpurua et al) at gestational weeks 17þ0 to 21þ6 could predict small for gestational age (SGA) and large for gestational age (LGA) babies at birth. Methods: An unselected population of 1,000 pregnant women attending routine ultrasound screening by midwives at Oslo University Hospital at 17þ0 to 21þ6 weeks were recruited to the study between September 2009 and February 2011. This national screening is performed on 99% of pregnant women in Norway. Placentas were imaged at maximal width so that width, height and thickness measurements could be recorded. Image quality and measurement accuracy were routinely confirmed by two study investigators (KL, GL), while problematic images were evaluated by the senior investigators (ACS, HJK). After delivery, all medical charts were retrospectively reviewed and EPV was calculated. Birth weight percentiles were compared to the EPV. Results: By March 2011, 495 of the 1000 registered pregnancies delivered and were retrospectively evaluated. Exams were performed at a mean of 18.840.78 weeks with a resultant mean EPV of 23086 cc. Women with the placentas in the lowest 10th percentile of EPV had a 2.5 increased odds of producing an SGA infant compared to those above the 10th percentile (10/49 vs 42/446, p ¼ 0.0257; sens 0.19, spec 0.91, OR 2.47 (95% CI: 1.155.29)), while women above the 90th percentile of EPV had a 2.7 increased odds of producing an LGA infant compared to those  90th percentile (9/49 vs 34/446, p ¼ 0.018; sens 0.21, spec 0.91, OR 2.73 (95% CI: 1.22-6.09)). Conclusion: This simple and rapid placenta volume evaluation is feasible in the daily routine of ultrasound screening by midwives even in low-risk pregnancies. Identifying a small or large EPV before mid-pregnancy will assist in selecting at-risk pregnancies for closer surveillance.

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Abstracts / Placenta 32 (2011) A1–A149

PL3.NI5 ALTERED ENDOTHELIAL CELL DECORIN EXPRESSION LEADS TO INCREASED THROMBIN GENERATION: A POTENTIAL MECHANISM IN THE PATHOGENESIS OF HUMAN FETAL GROWTH RESTRICTION

PL3.NI6 A2A ADENOSINE RECEPTOR INDUCES FETAL ENDOTHELIUM PROLIFERATION THROUGH A NITRIC OXIDEINDEPENDENT INTRACELLULAR PATHWAY IN GESTATIONAL DIABETES.

Amy Chui1, Padma Murthi1, Shaun Brennecke1, Vera Ignjatovic2, Paul Monagle2, Joanne Said1 1 Department of Perinatal Medicine, Pregnancy Research Centre, The Royal Women’s Hospital and Department of Obstetrics and Gynaecology, The University of Melbourne, Parkville, Victoria, Australia, 2Murdoch Children’s Research Institute and 4Department of Clinical Haematology and Department of Paediatrics, The Royal Children’s Hospital and The University of Melbourne, Parkville, Victoria, Australia

Andrea Escudero1, Jesenia Acurio1, Claudia Triviño1, Patricio Bertoglia2, Carlos Escudero1 1 Vascular Physiology Laboratory, Basic Sciences Department, Universidad del Bio Bio, Chillan, Region del Bio Bio, Chile, 2Obstetric and Gynecology Department, Hospital Clinico Herminda Martin, Chillan, Region del Bio Bio, Chile

Objectives: Fetal growth restriction (FGR) is a leading cause of perinatal morbidity and mortality. FGR pregnancies are associated with abnormal umbilical artery Doppler velocimetry and histological evidence of placental thrombosis. Proteoglycans (PGs) are macromolecules with anticoagulant roles in vascular endothelial environments. Decorin, a small leucine-rich PG localised to placental fetal endothelial cells, may act as an important anticoagulant in the placenta by activating plasma thrombin inhibitor heparin cofactor II. We have previously shown that decorin expression is decreased in FGR. However, the possible effect of this decrease on haemostatic function is not known. Therefore, our hypothesis was that reduced decorin expression in an endothelial cell line will result in decreased anticoagulation. Methods: The aim of this study was to investigate the functional anticoagulant activity of decorin in a vascular endothelial environment. To model decreased expression of decorin, the human microvascular endothelial cell line (HMVEC) was used. Downregulation of decorin was achieved by short interference RNA (siRNA) transfection. Human plasma was added to the HMVEC surface and the Calibrated Automated Thrombogram (CAT) system (Stago, Australia) was used to measure the total endogenous thrombin generation potential (ETP) of the cells. Results: A timecourse experiment demonstrated maximal downregulation of decorin after 48h (Control: 1.00.017 vs. siRNA: 0.150.035, p<0.005, n¼3, t-test). The total ETP measured by the CAT system following downregulation of decorin in HMVECs was increased compared to the mock or non-specific siRNA control (Control: 2457.830.1 vs. siRNA: 2580.46.9, p<0.005, n¼3, t-test), indicating an increase in thrombin generation. Conclusion: These data demonstrate that downregulation of decorin in an endothelial cell line model resulted in reduced anticoagulant function and thus, an increase in the amount of thrombin generated by the HMVECs. Therefore, we speculate that it is biologically plausible that reduced decorin expression may contribute to the placental thrombotic lesions observed in human FGR.

Human umbilical vein endothelial cells (HUVEC) isolated from gestational diabetes exhibit high adenosine extracellular level and nitric oxide (NO) synthesis. Stimulation of adenosine receptor A2A and A2B promote endothelial cell proliferation in HUVEC isolated from normal pregnancies. Moreover, several reports have showed that A2A stimulation trigger an intracellular pathway dependent of NO synthesis in several cell types including HUVEC. Aim: Investigate whether NO signalling pathway is involved in fetal endothelium proliferation induced by adenosine A2A adenosine receptor stimulation. Methods. Human umbilical vein endothelial cells (HUVEC) were isolated by collagenase (0.25 mg/ml) from normal (n¼15) or gestational diabetes (n¼12) and cultured under standard condition (37oC, 5% CO2) up to passage 2. The experiments were performed after overnight serumdeprivation and cells incubation with or without nonselective adenosine receptor agonist (NECA 10mM), adenosine A2A receptor selective agonist (CGS-21680, 30nM), and/or the antagonists ZM-241385 (10nM) for A2A receptors during additional 48 hours. In parallel experiments a dose response curve of ZM-241385 (0 to 100nM) was performed. Additionally, NOS inhibitor (L-NAME, 100 mM) was used in co-incubation by either adenosine receptor agonist or antagonists. Cell proliferation was performed by MTT-assay. Results: Gestational diabetes was associated with high A2A and A2B adenosine receptors expression in w6 and w2-fold, respectively. CGS21680 and NECA increase (w2 and 2.5- fold) cell proliferation in both gestational diabetes and normal pregnancies; and effect that was blocked by ZM-241385 (10nM) only in normal pregnancies. A shift in the curve of ZM-241385 for inhibiting the stimulatory effect of CGS-21680 was observed in diabetic pregnancies compared with normal pregnancies (calculated Ki 1.3  0.2 and 12.8  3.4nM, respectively). L-NAME coincubation did not block CGS-21680 stimulatory effect in both normal and pathological pregnancies. In fact, L-NAME alone increases (w1.5 fold) cell proliferation in normal and diabetic derived cells. Conclusions: A2A adenosine receptor stimulation increases cell proliferation in gestational diabetes through an intracellular pathway independent of NO synthesis. A posttranslational modification in A2A receptor could be involved in the reduced affinity

Abstracts / Placenta 32 (2011) A1–A149

Plenary Session 4 - New Investigator Oral Session 2 (PL4.NI1 – PL4.NI6) PL4.NI1 VASCULAR ENDOTHELIAL GROWTH FACTOR EXPRESSION IN NONPREGNANT HUMAN ENDOMETRIUM AND ITS GENETIC POLYMORPHISMS: CONTRIBUTION TO RECURRENT PREGNANCY LOSS. Olga N. Sadekova1, Liudmila A. Nikitina1, Timur N. Rashidov2, Elena M. Demidova2, Larisa M. Samokhodskaya1 1 Faculty of fundamental medicine, Moscow State University named after M.V. Lomonosov, Moscow, Russian Federation, 2Department of obstetrics and gynecology, PFUR, Moscow, Russian Federatio Objectives: Vascular endothelial growth factor (VEGF) is one of the key molecules coordinating angiogenesis in reproductive organs. Adequate angiogenesis during implantation and placentation contributes to successful pregnancy outcome. Altered VEGF expression and placental vessels development are observed in placentas from recurrent miscarriages (RM). However, changes of local VEGF expression in non-pregnant endometrium of RM patients remain unclear. We aim to study association between RM and VEGF expression in the endometrium during implantation window. Methods: Endometrial biopsies were collected at Days 21-23 of menstrual cycle from 24 women with history of RM and 15 fertile age-matched controls. VEGF mRNA transcription level was measured by quantitative PCR, VEGF protein expression was evaluated using semi-quantitative immunohistochemical method. Venous blood was collected from 150 women with RM and 120 female controls. Genotyping has been performed to evaluate allelic frequency of VEGF single nucleotide polymorphisms (G1154A, C2578A, C936T, G(-634)C) presumably associated with impaired gene expression. Results: VEGF mRNA expression was significantly lower in endometrium of women with RM compared with control group (P ¼ 0.03). The immunostaining of endometrial vessels and stroma in endometrium from RM women was less intensive than in control group, although the difference was not significant (p ¼ 0.11 and 0.08 respectively). Frequency of 1154A allele in RM group was higher compared to control group (p¼0.06). We observed no difference in mutant allele frequency of 2578A, 936T and 634C alleles in women with RM and control group (P¼0.64, 0.78 and 0.28 respectively). Conclusion: Altered expression of VEGF in the non-pregnant endometrium of women with RM suggests that inadequate endometrial angiogenesis may be one of the prerequisites of pregnancy loss.

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PL4.NI2 ENDOTHELIN-1 INDUCES ENDOPLASMIC RETICULUM STRESS BY ACTIVATING THE PLC/IP3 PATHWAY; IMPLICATIONS FOR PLACENTAL AUTOCRINE SIGNALLING IN THE PATHOPHYSIOLOGY OF PRE-ECLAMPSIA AND INTRAUTERINE GROWTH RESTRICTION Arjun Jain, Hong-wa Yung, Graham Burton University of Cambridge, Cambridge, United Kingdom Objectives: Recent evidence implicates placental endoplasmic reticulum (ER) stress in the pathophysiology of pre-eclampsia and intrauterine growth restriction (IUGR). The ER is involved in synthesis and packaging of membrane and secreted proteins, and serves as a reservoir of Ca2þ. Loss of ER Ca2þ homeostasis suppresses post-translational processing, triggering ER stress pathways. Endothelin (ET)-1 can induce Ca2þ release from the ER, and is increased in both IUGR and pre-eclampsia. This study therefore investigated whether ET-1 induces ER stress. Methods: Trophoblast-like JEG-3 choriocarcinoma cells were treated with different doses of ET-1 for 1-hour with or without inhibitors. Activation of signalling pathways was assessed by immunoprecipitation and Western blots. Live-cell imaging was used to monitor Ca2þ release by ET-1. Conditioned medium was prepared from term placental explants, collected with ethical approval, exposed to hypoxia/reoxygenation (H/R) or 10%O2 over a 16-hour time-course. Results: JEG-3 choriocarcinoma cells treated with ET-1 displayed a dosedependent increase in ER stress markers. Immunoprecipitation of cell lysates showed ET-1 induces phospho-activation of the ETB receptor. Treatment with BQ788, an ETB receptor antagonist, and siRNA knockdown of the receptor, both inhibited the induction of ER stress. Increased intracellular Ca2þ was observed upon ET-1 treatment of JEG-3 cells. ET-1 also stimulated p-PLC levels, which could be inhibited by U73122. Xestospongin-C, which inhibits the IP3 receptor, also abolished induction of ER stress by ET-1. Furthermore, levels of ET-1 increased in the syncytiotrophoblast of placental explants and conditioned medium following H/R, compared to treatment at 10%O2. Conditioned medium from explants exposed to H/R induced ER stress in JEG-3 cells, which could be abrogated by ET-1 blocking antibodies. Conclusion: ET-1 induces ER stress via the ETB receptor by initiating Ca2þ release and signalling through the PLC/IP3 pathway. Increased ET-1 in the syncytiotrophoblast following H/R suggests a possible adverse autocrine action of ET-1 in pathological pregnancies.

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Abstracts / Placenta 32 (2011) A1–A149

PL4.NI3 INTERFERON-GAMMA-INDUCED-PROTEIN 10 IN SPIRAL ARTERY REMODELLING: TROPHOBLAST – VASCULAR INTERACTIONS REVEALED USING 3-D CO-CULTURE Alison Wallace, Judith Cartwright, Baskaran Thilaganathan, Guy Whitley St George’s University of London, London, United Kingdom Objectives: During placentation, fetal extravillous trophoblast (EVT) enter maternal spiral arteries and transform them into large, non-vasoactive vessels capable of transporting nutritional and oxygen requirements to the fetus. 2-dimensional in vitro models have been developed to combat the difficulties of studying first trimester human pregnancy. However coculturing endothelial cells (EC) with vascular smooth muscle cells (VSMC) in 3-D spheroids may better represent vascular cell interactions occurring in vivo, as EC and VSMC orientate to mimic a vessel lumen. This study investigated changes in gene expression in vascular spheroids induced by EVT, and examines the role of interferon-gamma-induced-protein-10 (IP10) in spiral artery remodelling using spheroids and ex vivo dissected spiral arteries. Methods: EC and VSMC cell lines were co-cultured in hanging droplets to form spheroids. Control media (C) or conditioned media (CM) incubated with the EVT cell line, SGHPL-4, was added to spheroids for 24 hours. Spheroid RNA was isolated and analysed by Illumina Sentrix BeadChip gene array. IP-10 expression by spheroids was examined by quantitative PCR and western blot, and in spiral arteries incubated with C or CM (dissected from non-placental bed term biopsies) by immunohistochemistry. Timelapse microscopy was used to analyse VSMC motility and apoptosis in response to IP-10. Results: EC migrated to form a single cell layer around a core of VSMC, representing an inverted vessel lumen. Spheroids incubated with EVT CM showed significant up or downregulation of 101 genes (>1.5 fold, p<0.05), including an upregulation of IP-10 (2.24 fold, p<0.01). IP-10 expression was confirmed by RT-PCR and western blotting. EVT CM increased expression of IP-10 in dissected spiral arteries. IP-10 decreased apoptosis, and increased migration of VSMC, indicating de-differentiation of VSMC. Conclusion: 3-D spheroids present a good model to study EVT-spiral artery interactions in vitro. EVT induced IP-10 expression may contribute to spiral artery remodelling during pregnancy.

PL4.NI4 INSULIN RECEPTOR A IS INVOLVED IN GESTATIONAL DIABETESREDUCED ADENOSINE TRANSPORT VIA HUMAN EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 1 IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS. Francisco Westermeier, Carlos Salomón, Enrique Guzmán-Gutierrez, Carlos Puebla, Andrea Leiva, Paola Casanello, Luis Sobrevia Pontificia Universidad Católica de Chile, Santiago, Chile Objectives: To identify whether insulin reversal of gestational diabetes (GD)-reduced expression and activity of human equilibrative nucleoside transporters 1 (hENT1) in human umbilical vein endothelial cells (HUVEC) involves insulin receptor isoforms A (IR-A) and/or B (IR-B). Methods: Primary cultured HUVECs from full-term normal and diettreated GD pregnancies (n¼44) were used. Insulin (1 nM, 8 hours) was assayed on hENT1 expression [SLC29A1 promoter activity (firefly/renilla luciferase for pGL3-hENT1-3198 and pGL3-hENT1-1670), mRNA expression (quantitative real time PCR), protein abundance (Western blot)] and adenosine transport (4 mCi/ml, 20 seconds, 22oC). Total and phosphorylated endothelial nitric oxide synthase (eNOS), Akt and p42/44mapk phosphorylation (Western blot), and IR-A and IR-B expression (qRT-PCR) were determined. Adenoviral-based siRNA delivering system was used to knockdown IR-A (Ad-IR-A) and IR-B (Ad-IR-B). Assays were done in absence or presence of NG-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), PD-98059 (p42/44mapk inhibitor) or Akt inhibitor IV. Results: hENT1-adenosine transport (w75%), mRNA expression (w47%) and protein abundance (w80%) were reduced (Student’s unpaired t test and ANOVA, P<0.05) in a NO-dependent manner in GD compared with normal pregnancies, effects that were blocked by insulin. Insulin increased phosphorylated/total eNOS protein ratio (2.7-fold) in cells from normal pregnancies, but decreased this ratio in GD. GD increased only IR-A mRNA expression (1.5-fold) and p42/44mapk phosphorylation (3.5-fold), effects blocked by insulin. Similarly, GD effect on p42/44mapk phosphorylation was blocked in cells Ad-IR-A. Conclusion: GD reduces insulin response due to overexpression of IR-A isoform in human umbilical vein endothelium. These receptors could play crucial roles in the adaptive response to insulin in fetal endothelium from GD pregnancies acting as protecting factor for endothelial dysfunction characteristic of this syndrome. Support: CONICYT (ACT-73 PIA, AT-24100210), FONDECYT (1110977, 1080534), Faculty of Medicine, Pontificia Universidad Católica de Chile (FM-PUC) (PMD 03/10). CS, FW and EG-G hold CONICYT-PhD fellowships. CS holds a FM-PUC PhD fellowship.

Abstracts / Placenta 32 (2011) A1–A149

PL4.NI5 CHARACTERIZATION OF SYNCYTIN-2 PROMOTER: A CRE/AP-1 MOTIF IS ESSENTIAL FOR THE REGULATION OF TRANSCRIPTION OF THIS GENE. Chirine Toufaily, Amandine Vargas, Éric Rassart, Benoit Barbeau Université de Québec à Montréal, Montreal, Quebec, Canada Objectives: We hypothesise that the CREB protein, as well as forskolin, are implicated in the expression of syncytine-2, trophoblast fusion and its promoter activity. Thus we investigated the transcription factors essentials for the induction of the syncytin-2 promoter upon stimulation with forskolin. Methods: Reporter vectors containing the promoter of syncytin-2 and its deletion mutants were tested in BeWo cells for their promoter activities induced by forskolin. A region from -300 to -150 was cloned upstream a TATA box in a reporter vector and tested for its luciferase activity following stimulation with forskolin. Identification of the CRE/AP-1 motif in this region was done by using the EMSA assay after testing 6 different probes covering the 150 nucleotides with nuclear extracts from BeWo cells stimulated or not with forskolin. CREB was also tested for syncytin-2 mRNA level increase and cell-cell fusion in BeWo cells. Results: Following induction with forskolin, we identified that the promoter region of syncytin-2 is situated in its LTR. A region from -300 o -150 seems to contain a forskolin responsive element. Our EMSA assays showed that two proteins, from nuclear extract stimulated with forskolin, interacted with 2 motifs on the probe called 5-2 covering the -215 to -171 nt. One protein is specific to a CRE/AP-1 like motif (CCTGACT) while the other protein is specific to the probe 5-2 but different from CRE/AP-1 motif. CREB was also shown to increase cell-cell fusion and the expression of syncytin-2 transcript. Conclusion: Ours results showed that the promoter region of syncytin-2 is located in the LTR of HERV-FRD. CREB, as well as forskolin were able to induce the promoter activity of syncytin-2. A potentiel CRE/AP-1 binding site located in -215 to -171 seems to be critical for the transcription of this gene.

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PL4.NI6 DOES CIRCULATING BIOMARKERS ASSOCIATED WITH PLACENTAL DYSFUNCTION OR ENDOTHELIAL FUNCTION DIFFER IN MOTHERCHILD PAIRS SEVERAL YEARS AFTER PREECLAMPTIC PREGNANCIES COMPARED TO CONTROLS? Anne Stine Kvehaugen1,6, Ralf Dechend2, Heidi Ramstad3, Rebecca Troisi4, Drude Fugelseth5,6, Anne Cathrine Staff1,6 1 Department of Obstetrics and Department of Gynecology, Oslo University Hospital, Ulleval, Oslo, Norway, 2Experimental and Clinical Research Center, Max-Delbrück Center and Charité Medical Faculty, Berlin, Germany, 3Department of Pediatrics, Oslo University Hospital, Ulleval, Oslo, Norway, 4National Cancer Institute, National Institutes of Health, U.S. Department of Health and Human Services, Bethesda, MD, United States, 5 Department of Neonatal Intensive Care, Oslo University Hospital, Ulleval, Oslo, Norway, 6Faculty of Medicine, University of Oslo, Oslo, Norway Objectives: Women with a history of preeclampsia and offspring of preeclamptic pregnancies exhibit an increased risk of cardiovascular diseases later in life. Endothelial dysfunction may represent the mechanistic link between preeclampsia and subsequent cardiovascular disease risk in women. Complications which often accompany preeclampsia, such as premature delivery and restricted fetal growth, may adversely affect short and long term health in the offspring. Circulating inflammatory and angiogenic proteins, the latter mainly placenta derived in pregnancy, are typically dysregulated in preeclampsia. We wanted to investigate whether biomarkers reflecting inflammation and angiogenesis, as well as a noninvasive measure of endothelial function, differed between mother-child pairs after preeclamptic pregnancies compared to mother-child pairs after uncomplicated pregnancies. Additionally, we wanted to address the effect of premature delivery and of small for gestational age (SGA) in preeclampsia. Methods: Twenty-six mother-child pairs after pregnancies complicated by preeclampsia (prematurely delivered < 34 weeks: n¼17, SGA: n¼12) and 17 mother-child pairs after uncomplicated pregnancies were studied 5-8 years after delivery. Blood samples were analyzed for inflammatory markers (high-sensitivity C-reactive protein (hs-CRP) and calprotectin) and angiogenic factors (soluble fms-like-tyrosine kinase-1 (sFlt1), soluble endoglin, vascular endothelial growth factor and placental growth factor). Endothelial function was measured using finger pulse wave amplitude testing (EndoPAT). Results: Maternal hs-CRP (p¼0.02) and sFlt1 (p¼0.05) were elevated in the preeclampsia group compared to controls. These results remained unaltered after adjustment for age and BMI. Endothelial function was impaired in both women and children following SGA preeclamptic pregnancies, compared to non-SGA pregnancies (mothers: p<0.001, children: p<0.05) and uncomplicated pregnancies (mothers only: p¼0.005). Conclusion: Our study identified parallel findings of reduced endothelial function in mother and child pairs 5-8 years after SGA preeclamptic pregnancies, while women with previous preeclampsia, independent of early or late onset disease, had increased hs-CRP and sFlt1 compared to controls.

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Abstracts / Placenta 32 (2011) A1–A149

Plenary Session 5 (PL5.1 – PL5.4) PL5.1 IMPRINTING AND EARLY PLACENTAL DEVELOPMENT J. Richard Chaillet1,2 1 University of Pittsburgh, Pittsburgh, Pennsylvania, USA, 2Magee-Womens Research Institute, Pittsburgh, Pennsylvania, USA Genomic imprinting is a form of transcriptional gene regulation in which parental alleles are epigenetically distinguished by their parental origin such that one allele is transcribed and the opposite allele is silent. Of the approximately 80 known imprinted genes, most are organized into 16 clusters, with the monoallelic, imprinted expression of genes within a cluster regulated by a differentially methylated domain (DMD). Each DMD is w3 kilobases in size. Thus, the molecular process that establishes and perpetuates imprinted gene expression depends on a fraction of a mammal’s genome. A DMD is established in two steps. One parental allele of a DMD acquires methylation de novo through the action of the DNMT3a cytosine methyltransferase. The opposite parental allele remains unmethylated. DMD methylation is then perpetuated after fertilization by the action of the DNA cytosine methyltransferase 1 (DNMT1) enzyme. There are oocyte- and embryo-derived forms of DNMT1, which work together during preimplantation to perpetuate imprinted DMD methylation. At the 8-cell stage, the oocyte-derived DNMT1o variant maintains the heritable methylation marks associated with DMDs. Mouse embryos and placentas derived from mice that are deficient in DNMT1o have, on average, 50% of the normal level of imprinted methylation. This loss of methylation leads to altered expression of imprinted genes, manifested as biallelic expression of some genes and loss of expression in most genes. Consistent with the central role imprinted genes play in fetal growth, many imprinted genes are uniquely expressed in the placenta. DNMT1odeficient placentas exhibit a wide range of morphologic and molecular abnormalities, including defects in the maternal-fetal interface and the distribution of glycogen trophoblast cells. Consequently, DNMT1o deficiency in mice is a powerful model to study the contributions of imprinted genes to placental development.

PL5.2 THE PLACENTA OF CLONES: LESSONS FROM THE BOVINE Pascale Chavatte-Palmer1,2, Rita Lee3, Sylvaine Camous1, Hélène Jammes1, Michel Guillomot1 1 INRA, Jouy en Josas, France, 2PremUp Foundation, Paris, France, 3AgResearch, Hamilton, New Zealand Since the first success in sheep, the production of viable cloned offspring by somatic cell nuclear transfer (SCNT) in mammals has increased significantly, with currently most domestic mammals having been cloned. The incidence of pregnancy failure and fetal death, however, is still very high, even in the bovine species where the success rates are highest compared to other species. In cattle, 30 to 70% of initiated pregnancies are lost during the early postimplantation period (D30 to D70), due to abnormal implantation and poor placental development. All SCNT fetuses appear to suffer from slight growth restriction in these early stages. Thereafter, losses occur in the last third of gestation, affecting 0-100% of the remaining pregnancies, depending on the genotype. These pregnancies are associated with fetal overgrowth, a phenomenon commonly referred to as Large Offspring Syndrome. The placenta appears to be central to the onset of the pathology, with placentomegaly and hydrallantois being the most common features. Clinically, transabdominal ultrasound monitoring of fetoplacental development, as well as the measurement of maternal plasma concentrations of pregnancy associated glycoproteins, are used to monitor the pregnancies. Pregnancy termination by Caesarian section or slaughtering is necessary to avoid unnecessary suffering of affected animals. The etiology appears multifactorial. Abnormal placental growth, vascularization and endocrine function, modified transplacental exchanges of glucose, modified responses to oxidative stress, disturbed expression of placental genes, especially imprinted genes, and epigenetic modifications have been shown to play a role in the placental phenotype of animals affected by the Large Offspring Syndrome. Some of these observations were also made, to a lesser extent, in placentas from animals that were alive at birth, suggesting a continuum in the placental pathology in SCNT.

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PL5.3 NEW BIOMARKERS TO PREDICT PREECLAMPSIA Stefan Hansson, Ulrik Dolberg-Anderson, Karl Kristensen, Bo Åkerström 1 Dep. Obstetrics and Gynecology, IKVL, Lund University, Lund, Sweden, 2 Dep. for Experimental Medical Science, Lund University, Lund, Sweden Background: The worldwide prevalence of preeclampsia (PE) ranges from 3 to 8% of pregnancies. Yearly, 8 500 000 cases are reported worldwide, although this is probably an underestimate due to the lack of proper diagnosis. PE is today the most common cause of death for both children and mothers during pregnancy, and yet no specific treatment is available. A reliable biomarker for prediction and diagnosis of PE would have a great impact on maternal health. A perfect biomarker should:

     

be part of the pathogenesis appear early or before clinical manifestations should correlate with the severity of the condition not be measurable in the normal condition be easy to measure technically in plasma and/ or urine show a high sensitivity and specificity.

Aim and Methods: This review describes PE biomarkers in general, and first trimester PE biomarkers specifically. It is based on a literature search through PubMed from 2006 to the present, with the search terms “preeclampsia, prediction, diagnosis, biomarker, screening” individually or in combination. Results: 40 publications were retrieved. The main categories described are angiogenesis factors, placental proteins, free hemoglobin (HbF), kidney markers, ultrasound and maternal risk factors. The specific biomarkers discussed are: PAPP-A, s-Flt-1/PLGF, s-Endoglin, pp13, cystatin-C, alpha-1microglobulin (A1M) and HbF. Conclusions: Both PAPP-A and HbF show potential as predictive biomarkers in the first trimester, with 70% sensitivity at 95% specificity. However, PAPP-A is not PE -specific and needs to be combined with Doppler ultrasound to achieve the same sensitivity as HbF. Soluble Flt -1/PLGF are both promising markers that show high sensitivity from the mid-second trimester. Implementations: Screening pregnant women with new and more specific biomarkers for PE can avoid unnecessary suffering and major health care costs by:

 enabling early detection of mothers at increased risk for PE.  avoiding unnecessary hospitalization of mothers with suspected or mild PE.  enabling monitoring of the progression of the disease and thereby the optimizing time for delivery and reducing the number of premature births.

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PL5.4 HUMAN TROPHOBLAST DIFFERENTIATION; FROM IN VITRO MODELS TO PREGNANCY PATHOLOGIES Danièle Evain-Brion1,2,3 Inserm, Paris 75006, France, 2University Paris Descartes, Paris 75006, France, 3PremUP foundation, Paris 75006, France 1

Different in vitro models have been established to study human trophoblast differentiation along the extravillous and the villous pathway. Purified villous cytotrophoblasts, cultured on plastic dishes, aggregate and fuse, forming the multinucleated syncytiotrophoblast with pregnancy specific hormonal production (ie hCG, Progesterone). This physiological model allowed us to perform accurate biochemical studies, and to decipher the dynamics of trophoblast membrane fusion, a limiting step in human trophoblast differentiation. As we have previously demonstrated, cultured cytotrophoblasts isolated from Trisomy 21 (T21)-affected placentas aggregate, but fuse poorly or after a delay. In this talk, we will show that the abnormal syncytiotrophoblast formation observed in T21 is reversible in vitro by stimulation of the cAMP-dependent pathway, or TGF beta signaling. These data allowed us to unravel the role of, and the interactions between, the major membrane proteins identified as being involved in the dynamics of trophoblast fusion, such as syncytins, connexin 43, ZO-1 and ezrin. In addition, we will discuss the stimulatory role of trophoblastic autocrine factors, but also paracrine factors, including activin A arising from the mesenchymal core of the villi, on human trophoblast differentiation. As illustrated in T21, syncytiotrophoblast formation appears to be dependent on the glycosylation status of major actors involved in trophoblast fusion and differentiation, and to the paracrine cross talk between the trophoblast and the mesenchymal core within the placenta. Plenary Session 6 (PL6.1 – PL6.2) PL6.1 THE GREAT DEBATE: WHO IS CALLING THE SHOTS ON PLACENTAL FUNCTION? THE MOTHER OR THE FETUS? Vicki Clifton1, Tom Moore2 Adelaide, Australia, 2 Cork, Ireland

1

The placenta has an ability to adapt and change its structure and function in response to its environment to ensure the fetus can grow normally and survive. However the question is who controls placental adapatation? Is it the fetus or the mother that ultimately determines placental function? There is a great deal of evidence to suggest the mother is central to determining placental function since a placenta cannot have a “one size fits all” approach to a heterogeneous range of maternal environmental factors. The placenta must develop in a maternal environment that may be exposed to, for example, cigarettes, drugs, disease, ranges of maternal age, variations in maternal nutrition or altered oxygen concentrations. If the placenta was incapable of recognising and adapting to maternal conditions then very few pregnancies would be viable. For example, the presence of maternal stress results in a rise in maternal cortisol, an alteration in placental glucocorticoid regulated pathways and a subsequent adjustment in the fetal growth trajectory. This evidence would suggest it is the mother who controls placental adaptation and function. An alternative view, derived from evolutionary arguments, suggests that because the fetus and mother are genetically distinct individuals, there is a ‘conflict’ between maternal and fetal interests. Notwithstanding the fact that fetal and maternal genes may cooperate in maintaining general homeostasis, kin selection theory predicts that the fetal trophoblast will promote the ‘interests’ of the fetus over those of the mother, and the maternal decidual component of the anatomically defined placenta will promote maternal interests. Therefore, the ‘control’ of placental function may be a contested, rather than a cooperative, exercise. Fetal genes can modulate placental function by promoting or inhibiting trophoblast differentiation, growth and invasion, and by producing placental hormones with wide-ranging metabolic, immune and angiogenic effects. The fact that preeclampsia may originate from abnormal development of the fetal component of the placenta indicates that considerable ‘power’ resides with the fetus.

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PL6.2 THE EVOLVING PLACENTA: DIFFERENT STARTING POINTS BUT REACHING THE SAME GOAL Allen Enders University of California, Davis CA, USA It is widely recognized that there are many different definitive arrangements of hemochorial placentas with regard to the number and nature of the trophoblast layers as well as their pattern of organization, but the way in which these placentas are established developmentally receives less attention. Although it might be considered logical that developing placentas would pass through an endotheliochorial stage to become hemochorial, such a developmental pattern is seen only as a brief transient stage in several species of bats and sciuromorph rodents. Instead, maternal blood is tapped well before tertiary villi are formed. Commonly, a mass of trophoblast at the junction with the endometrium serves as a meshwork through which maternal blood passes, with subsequent organization of a labyrinth when fetal connective tissue and vessels intrude. The initial trophoblast meshwork may be cellular or syncytial, often leading to a similar relationship in the spongy zone and labyrinth. Old World monkeys, apes and humans pass through a lacunar stage before establishing a villous hemochorial condition. New World monkeys lack a true lacunar stage, retaining portions of maternal vessels for some time and initially form a trabecular arrangement similar to that in the tarsier. In armadillos, preexisting maternal venous sinuses are converted into an intervillous blood space by intruding fetal villi. In shrew tenrecs, a blood space between the chorion and a trophoblast pad has villi before establishment of a labyrinth within the trophoblastic pad. It is concluded that early tapping of maternal blood provides nourishment while restricting blood flow. It is only subsequently that both fetal vascular development and modification of maternal vessels result in more efficient exchanges. Plenary Session 7 (PL7.1 – PL7.2) PL7.1 DOES SIZE REALLY MATTER? – PLACENTAL DEBRIS, DANGER MOLECULES AND PRE-ECLAMPSIA Ian Sargent, Chris Redman Oxford University, Oxford, UK The maternal syndrome of pre-eclampsia is characterised by an excessive inflammatory response associated with endothelial dysfunction, caused by the release of multiple factors from the placenta into the maternal circulation. Candidate factors include anti-angiogenic molecules (e.g. sFlt-1 and sEndoglin), proinflammatory “danger” molecules (e.g. cytokines, HMGB-1, HSP-70, S100B) and miRNA’s. While some of these factors are released as soluble molecules, increasing evidence suggests that many are associated with cellular debris shed from syncytiotrophoblast. This debris ranges in size from large syncytial sprouts (5-20mm) to much smaller microvesicles (100nm-1mm) and exosomes (50-100nm). Increased levels are found in the circulation of women with pre-eclampsia. Both exosomes and microvesicles are involved in cell–cell signalling. Their cargo includes membrane and cytosolic proteins and miRNAs, which can affect the physiology of target cells, Syncytiotrophoblast microvesicles and exosomes (STBM) are variously proinflammatory, cause endothelial dysfunction and, in addition, inhibit T and NK cell responses. We propose that the different effects of STBM result from different types of vesicles within the preparation, the smaller exosomes being immunoregulatory and the larger microvesicles being proinflammatory, with a shift to the latter in pre-eclampsia. Furthermore, the range and types of factors they carry (and hence their functions) may differ in pre-eclampsia, where the syncytiotrophoblast is subjected to oxidative and inflammatory stress. To explore this hypothesis we are employing a range of tools to size, count and phenotype STBM, including proteomics, miRNA analysis, multi-colour flow cytometry and a new technique, Nanoparticle Tracking Analysis, which allows us for the first time to directly measure nano-sized vesicles in these preparations. Together these techniques will enable us to answer the question – does size really matter?

PL7.2 MOLECULAR REGULATION OF TROPHOBLAST INVASION Martin Knöfler Medical University of Vienna, Department of Obstetrics and Fetal-Maternal Medicine, Reproductive Biology Unit, Vienna, Austria Correct development of the human placenta and its epithelial trophoblast layers is critical for fetal wellbeing and successful pregnancy outcome. In particular, placental anchoring villi give rise to different invasive trophoblast cell types, such as interstitial trophoblasts invading the uterine stroma and endovascular trophoblasts migrating into the maternal spiral arteries. Interstitial trophoblasts produce a variety of hormones, angiogenic factors and cytokines controlling trophoblast invasion, uterine NK cell function, decidualization as well as angiogenesis within the placental bed. Endovascular trophoblasts, on the other hand, are required to complete the remodelling process of spiral arteries characterized by elastolysis, disruption and degradation of the vascular wall. Transformation of the vessels into dilated conduits ensures appropriate transfer of nutrients and oxygen to the developing foetus. Failures in this remodelling process are thought to be associated with gestational diseases, such as preeclampsia and severe forms of intrauterine growth restriction. However, the underlying causes remain largely obscure, warranting studies on the molecular regulation of trophoblast invasion and differentiation. Indeed, numerous growth factors of the fetal-maternal interface acting through signalling pathways such as MAPK or PI3K/AKT were shown to control trophoblast proliferation and invasion in an autocrine or paracrine manner. However, the cross-talk between signalling cascades, as well as key regulatory factors committing, and differentiating, invasive trophoblast cells remain largely elusive. To gain more insights into these processes, we recently performed comparative gene chip analyses of invasive and non-invasive first trimester trophoblasts. Gene set enrichment analyses led to the identification of extravillous trophoblast-enriched protein families, transcription factors and signalling pathways potentially controlling trophoblast motility and/or differentiation. Based on these data, descriptive as well as functional analyses of ADAM family members, Wingless (Wnt)-dependent T-cell factors (TCFs) as well Notch signalling components were performed. Whereas ADAM12 and TCF-4 promote trophoblast invasion, Notch signalling negatively affects trophoblast migration, cell column and villous trophoblast proliferation. In summary, developmental pathways such as Notch and Wnt signalling could play crucial roles in cell growth and differentiation of the placental anchoring villus. Research in the laboratory of M. Knöfler is supported by grant P-22687-B13 of the Austrian Science Funds, by grant APP00323OFF of the Herzfelder’sche Familienstiftung as well as by a grant from the Austrian National Bank (grant Nr. 12487).

Abstracts / Placenta 32 (2011) A1–A149

Poster session 1 (P1.1 – P1.139) P1.1 PERINATAL OUTCOMES ACCORDING TO PLACENTAL POSITION IN PLACENTAL PREVIA Dong gyu Jang, Gui Se Ra Lee St.Vincent’s Hospital, Suwon, Kyeonggi, Republic of Korea Objectives: The purpose of this retrospective cohort study was to elucidate whether the location of placenta below uterine incision in cesarean section is more important in the development of maternal as well as neonatal complications than the level of placental coverage over internal os of cervix in placenta previa patients. Methods: The study was conducted on 487 patients 492 parturition at 3 Catholic University Hospitals from May 1999 to December 2009. The subjects were divided to two groups: the group whose placenta was located in the anterior body or central portion of internal os of cervix (anterior group) and the group whose placenta was located in the posterior or lateral portion (posterior/lateral group). And then they are compared. Logistic regression was used to control for confounding factors including complete previa. Results: In the anterior group, regardless of maternal age, parity, previous abortion, previous cesarean section, or complete previa, the incidence of placental accrete (OR 2.66; 95% CI: 1.52-4.66), excessive blood loss (OR 3.32; 95% CI: 1.94-5.66), massive transfusion (OR 3.61; 95% CI: 1.58-8.27), and hysterectomy (OR 3.35 95% CI 1.50-7.49) was significantly high. Regardless of maternal age, gestation age, or complete previa, neonatal anemia was increased (OR 2.21; 95% CI: 1.05-4.62), and their influence on maternal or neonatal complications was greater than that of complete previa. Conclusion: To determine maternal or neonatal prognosis in placenta previa patients, sonographic evaluation of placental location, whether it located below the uterine incision, is most important, even more important than complete previa, and for such cases, close attention is required for massive hemorrhage and neonatal anemia. P1.2 PATHOLOGIC DIFFERENCES BETWEEN PLACENTAS FROM INTRAUTERINE GROWTH RESTRICTION PREGNANCIES WITH AND WITHOUT ABSENT OR REVERSED END DIASTOLIC VELOCITY OF UMBILICAL ARTERIES Dong Gyu Jang1, Gui Se Ra Lee1, Sun Jung Hwang2 1 St. Vincent’s Hospital, Suwon, Republic of Korea, 2Daejeon St. Mary’s Hospital, Daejeon, Republic of Korea Objectives: The aim of this study was to determine the pathologic differences in the placentas from IUGR pregnancies with and without the absent or reversed end diastolic velocity (AREDV). Methods: Among the cases that had undergone prenatal follow-up in our institute, a retrospective slide review was conducted for 18 cases of IUGR with AREDV and 17 cases with IUGR that had normal end-diastolic flow of the umbilical artery. Results: The birth weight and the other clinical parameters were not different among the two groups. Grossly, the placental weight percentiles were significantly smaller in AREDV group when they were adjusted according to gestational age. Histologically, chronic deciduitis, mural hypertrophy of the decidual arteries, an intimal fibrin cushion of the large fetal vessels, increased syncytial knots, villous agglutinations, avascular villi, villous stromal-vascular karyorrhexis, and acute atherosis were more frequently found in the AREDV group and their presence showed statistical significance. Conclusions: These findings suggest that pathologic abnormalities due to fetal and maternal vasculopathies in the placenta may be the cornerstone for inducing AREDV in the umbilical artery.

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P1.3 INDOMETHACIN FOR THE PREVENTION OF PRE-TERM LABOR: IS THERE AN ASSOCIATION WITH PLACENTAL INFLAMMATION? Karen Florio, Carolyn Salafia, Sanford Lederman, John Nguyen, Rocco Florio, Kathryn Keaty, Michael Klug, Ashley Bartalot, Allison Crockett, Lindsey Haga, Samantha VanHorn New York Methodist Hospital, Brooklyn, NY Objective: To evaluate the severity of placental inflammation in pregnant women who received indomethacin during the second trimester. Methods: 33 women who received indomethacin for the prevention of preterm labor (between the gestational ages of 180/7 and 320/7 weeks) with singleton pregnancies delivering at NYMH and Good Samaritan Hospital between 1/2009 – 12/2010 were included as well as 48 age-, race- and gestational age-matched controls. Medical records and placental pathology were reviewed and the following parameters were evaluated: maternal age, BMI, race, parity, gestational age at delivery, birth weight, maternal medical history, use of Lovenox, procardia, and progesterone, placental weight, and placental inflammation including chronic villitis, maternal and fetal thrombotic lesions, acute inflammation and uteroplacental insufficiency. The Pearson Chi-square test was used to analyze the differences in placentas exposed to indomethacin against those that were not exposed. Results: There was no significant difference between cases and controls in the following pathology categories: fetal inflammatory response (p< ), intervillous thrombi (p¼0.126), chronic non-specific villitis (p¼0.46), and non-marginal placental infarcts (p¼0.235). However, there was a statistically significant difference in maternal vascular lesions (p<0.0001). Conclusion: Our data supports the hypothesis that indomethacin has an anti-inflammatory response on the placenta when given to women for the prevention of preterm labor. Prolonged labor could be a result of this antiinflammatory response leading to longer latency periods and superior pregnancy outcomes.

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Abstracts / Placenta 32 (2011) A1–A149

P1.4 A CASE OF COMPLETE MOLE WITH CO-EXISTENT TWIN FETUS WITH SUBSEQUENT DEVELOPMENT OF PERSISTENT GESTATIONAL TROPHOBLASTIC NEOPLASM Karen Florio, Erin Stevens, Carolyn Salafia, Constantine Gorelick, Gary Fiasconaro New York Methodist Hospital, Brooklyn, NY Background: Hydatiform mole with a coexistent fetus is a rare occurrence. The incidence reported varies from 1:22,000 to 1:100,000 pregnancies. We present a case of complete hydatiform mole with co-existent fetus in a patient who later developed persistent gestational trophoblastic neoplasm. Case: The patient is a 33 y/o G1P0 who conceived by IVF after transfer of four embryos. She had routine obstetrical care with a private physician. She presented to the ER with vaginal bleeding and elevated BP (158/101) at 23 0/7 weeks and underwent an ultrasound, which revealed a single intrauterine gestation with measurements compatible for gestational age and a molar pregnancy at the level of the anterior uterine wall likely representing a twin complete mole. Her beta HCG was 256,931 mIU/mL. She was observed in the hospital until she had no further bleeding and it was felt she could be managed as an outpatient. At 36 5/7 weeks, patient presented again with vaginal bleeding and contractions. Upon admission, patient was again noted to have elevated blood pressure (158/81) with trace proteinuria. Decision at that time was made to deliver via primary cesarean section. A live male fetus was delivered weighing 3020 grams with APGARS 9/9. The placenta and coexisting complete molar gestation were delivered manually and sent to pathology. Pathology specimen revealed a placenta weighing 461grams measuring 20x18x2.2 cm. There was a 22x16.5x2.8 cm aggregate cluster of translucent grape-like cysts ranging in size from 0.4 to 4 cm adjacent to the normal placenta. Histology revealed large villi with typical central cisterns with the majority of the tissue demonstrating significant necrosis. Viable cells remained readily identifiable with a phenotype of complete mole. After delivery, her beta HCG was followed weekly. The nadir was reached two weeks postpartum and was measured at 4265 mIU/mL. Subsequently, her HCG levels began to rise; two weeks after the nadir it was measured at 8067 mIU/mL. She then underwent a chest XR and CT of the abdomen and pelvis revealing no metastatic disease. Her FIGO-WHO score was 3. She was treated with weekly methotrexate 30 mg/m2 for 14 weeks until her levels began to rise from a nadir of 4 mIU/mL to 16.6 mIU/mL over 4 weeks. Repeat imaging via CT of the chest, abdomen and pelvis revealed no evidence of metastatic disease. Her FIGO-WHO score was recalculated at 5. The decision was made to change her chemotherapy regimen to actinomycin D 1.25 mg/m2 every other week, which she is currently receiving. Conclusion: More than 50% of complete hydatiform mole with a coexistent fetus later develop persistent gestational trophoblastic neoplasm and 22% develop metastatic tumors. Patients who fail primary chemotherapy should have the FIGO-WHO score recalculated to determine risk status and subsequent chemotherapy regimens.

P1.5 DETERMINATION OF TRACE ELEMENTS AND THEIR DISTRIBUTION MAPS IN BOVINE PLACENTA Sonia Elisabete Will, Rose Eli Grassi Rici, Maria Angelica Miglino, Andrea Antunes Faculdade de Medicina Veterinaria e Zootecnia de São Paulo, Brazil Objectives: The aim of this study was to determine the concentration of trace elements for non-manipulative animals (GCO) and cloned animals (GCL) for gestational periods and to obtain maps of qualitative distribution for the placenta of bovines GCO and GCL. Methods: We used bovine receptor 5 placentas of cloned embryos and placenta 15 unhandled beef (bulls) were collected at ages 90, 135, 225 days of gestation. After lyophilization of the material analysis was performed at the Institute for Energy and Nuclear Research (IPEN-USP) using fluorescence X-ray energy dispersive (EDXRF) for the quantification of Fe, Cu and Zn, dry weight. Results: The results indicated significant variations for the elements Na, Mg, P, Cl, K e Ca. The maps of elementary distribution confirmed that the highest elementary concentration occurred in the region of inter-digitation of carunculate vaults of the uterine epithelium and cotiledonary vilosities with trophoblast cells, in the regions of the placentons and endometrial glands of the interplacentomal region, confirming that, in these regions, nutrient exchanges intensify between mother and fetus. For the results obtained in evaluation of the elementary interrelation in relation to the samples of cloned bovine placentons we observed that the maps demonstrated several interrelations between Ca-Fe, Cu-Fe, Zn-Ca, Fe-Zn, Ca-Cu, and Cu-Zn. Conclusion: The chemical analyses were adequate for the determination of the elementary concentration of constituent elements and trace and also that the qualitative analysis of the bi-dimensional maps and correlated maps obtained through experiments with Synchrotron radiation were essential for the discussion on alterations on selected regions and on evaluated periods of gestations.

Abstracts / Placenta 32 (2011) A1–A149

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P1.6 GESTATIONAL AGE CORRELATION OF CLINICAL CONDITIONS AND PLACENTAL LESIONS

P1.7 PLACENTA ACCRETA MIMICKING PLACENTAL SITE TROPHOBLASTIC TUMOR: A CASE REPORT

Jerzy Stanek1, Jacek Biesiada2 Division of Pathology and Laboratory Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States, 2Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States

Sawako Fujiwara, Kaoru Niimi, Kanako Shinjyo, Eiko Yamamoto, Yukio Mano, Seiji Sumigama, Tomomi Kotani, Fumitaka Kikkawa Nagoya University, Nagoya/Aichi, Japan

1

Objectives: Gestational age (GA) dependence of several clinical conditions and outcomes and placental lesions is well known. However, we are not aware of any all-inclusive comprehensive analysis of the GA correlation of routinely documented placental and clinical findings. Methods: This is a retrospective statistical analysis of 29 clinical (maternal and fetal) and 44 gross and microscopic placental features from 3081 from 20 weeks pregnancies consecutively signed by one of us (JS) in years 2001-2010. Pearson or Spearman correlation coefficients were used to correlate those parameters with GA, where appropriate. Results: Strong positive correlation with GA (R0.7,p<0.0003) was found in: abnormal cardiotocography, thick meconium, cesarean section rate, placental weight, deep meconium, chronic villitis, chorangiosis, massive perivillous fibrin deposition, clusters of avascular chorionic villi, perivascular stem edema, microscopic chorionic pseudocysts, and membrane laminar necrosis. Strong negative correlation with GA (R-0.7,p<0.0003) was found in: macerated stillbirths, neonatal deaths, terminations of pregnancy, congenital anomalies, premature rupture of membranes, retroplacental hematoma, placental edema, amnion nodosum, intravillous hemorrhage, villous hemosiderosis, and fetal chorioamnionitis. Moderate positive correlation (0.7>R0.4, p<0.05) was found in: pregnancyinduced hypertension, mild preeclampsia, diabetes mellitus, polyhydramnios, thick meconium, fetal growth restriction, gross chorionic cysts, succenturiate lobe, intimal cushions of fetal veins, intervillous thrombi, erythroblasts in fetal blood, hypercoiled umbilical cord, and uterine pattern of hypoxic placental injury. Moderate negative (-0.7
Placental site trophoblastic tumors (PSTT) are rare and account for 1-2% of gestational trophoblastic disease (GTD). PSTT produce less human chorionic gonadotropin (hCG) than choriocarcinoma and the expression of human placental lactogen (hPL) is the hallmark of PSTT. However, it is difficult to diagnose PSTT clinically and histological analysis after hysterectomy is necessary. Case Report: A 31-year-old woman (gravida 4, para 3) had an amenorrhea for 3 months and abnormal irregular vaginal bleeding. Urine beta-hCG was slightly raised and pelvic ultrasound showed a heterogeneous lesion in the uterine corpus. GTD was suspected at a clinic and she was referred to our hospital. Computed tomography (CT) scan showed a tumor of the uterine corpus which had contrast enhancement with an irregular surface and ultrasound demonstrated hypervasucularity around the tumor in color Doppler mode. Serum hCG level was 96 mIU/ml and hPL level was within the normal range. A hysterectomy was performed under a preoperative diagnosis of PSTT. We found a 4cm tumor with hematomas on the endometrium infiltrating into the myometrium macroscopically. Histologic examination of the tumor demonstrated chorionic villi with severe degeneration but not trophoblastic disease. At the site of placental implantation, mononuclear intermediate trophoblasts were infiltrating into the decidua and myometrium but we found a part of villi invaded into myometrium which meant placenta acrreta. The morphology of chorionic villi indicated that the pathological diagnosis was retained placenta after an abortion within 15 gestational weeks. Discussion: PSTT is difficult to be diagnosed clinically with radiologic studies and the measurement of hCG level. The differential diagnosis for PSTT includes epithelioid trophoblastic tumor (ETT), exaggerated placental site, placental site nodule, choriocarcinoma, retained portions of placenta, and epithelioid type of leiomyosarcoma. In these cases, a hysterectomy is necessary for making a pathological diagnosis and a right decision for treatment.

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Abstracts / Placenta 32 (2011) A1–A149

P1.8 QUANTITATIVE ESTIMATION OF PROLIFERATIVE ACTIVITY IN TERMINAL VILLI OF NORMAL AND DIABETIC TERM PLACENTA.

P1.9 APOPTOSIS IN PLACENTAL VASCULATURE IN PREGNANCIES COMPLICATED BY DIABETES MELLITUS TYPE I

Marie Jirkovská, Tomá
s Kucera, Martin Jadrnícek, Veronika Niedobová,

Milena Moravcová, Vratislav Krejcí, Zdenek Zi zka Charles University in Prague, First Faculty of Medicine, Prague, Czech Republic

Tomas Kucera, Martin Jadrnicek, Veronika Niedobova, Zdenek Zizka, Vratislav Krejci, Milena Moravcova, Marie Jirkovska Charles University in Prague, First Faculty of Medicine, Prague, Czech Republic

Objectives: The growth and development of placenta is conditioned by continual cell proliferation during pregnancy. In the third trimester it manifests itself above all by massive growth of terminal villi and their capillary bed. In our previous study we have found higher degree of capillary branching and signs of active angiogenesis in terminal villi of diabetic placentas at term. In this study we tested the hypothesis that this finding is connected with higher proliferation of cells constituting villous capillaries. Methods: Specimens collected by systematic random sampling from eight normal and twelve diabetic placentas (type I diabetes) were fixed in formaldehyde and embedded in paraffin. Histological sections were cut from five randomly chosen blocks per placenta and immunohistochemical detection of Ki-67 antigen as a marker of proliferation was performed. The area of terminal villi was measured and the number of Ki-67 positive nuclei was counted in trophoblast, stroma and capillary wall in 20 fields of view per section using Leica IM 500 program. Mean numbers of labeled nuclei per squared millimeter of section were calculated. Results: In both groups of placenta the Ki-67 positive nuclei occurred in cytotrophoblast, stromal cells as well as in endothelium and pericytes but in diabetic placentas we observed conspicuously lower frequency of those nuclei in pathological forms of villi. The mean number of labeled nuclei of cytotrophoblast just like the mean number of labeled nuclei of capillary wall cells expressed per squared milimeter of section were significantly lower in diabetic placentas (32.22  14.69 vs. 15.13  10.76 and 10.11  4.65 vs. 4.6  5.64 respectively). Conclusion: These data suggest that maternal diabetes mellitus decreases proliferative potential of cytotrophoblast and capillary wall cells in term placenta. Lower proliferative activity may influence fetal well-being in terminal phase of pregnancy. Supported by GACR, Project number 304/09/0733.

Objectives: There is an increased level of angiogenesis in placentas from pregnancies complicated by diabetes mellitus type I (DM type I). During angiogenesis and vascular remodeling, certain parts of microvasculature might be eliminated via apoptosis. We decided to quantify the number of apoptotic cells in placental vessels from normal pregnancies and pregnancies complicated by DM type I. Methods: The placentas from normal pregnancies (n¼8) and placentas from mothers with DM type I (n¼18) were obtained at the time of delivery. The specimens were collected using unbiased systematic random sampling, fixed with 4% formaldehyde and embedded in paraffin. The number of active caspase-3 immunoreactive cells (apoptotic cells) was quantified and normalized to the cross-sectional surface of villi. The double immunohistochemical study was performed with anti-active caspase-3 rabbit polyclonal antibody and anti-CD34 mouse monoclonal antibody to visualize apoptotic endothelial cells. Results: Apoptotic cells including endothelial cells were detected in both groups. In the diabetic group, the average number of apoptotic cells in vessels was 18.118.7/mm2. In placentas from normal pregnancies, this value was 8.35.7/mm2. We also compared the number of apoptotic cells in vessels form pregnancies of mothers with good and poor metabolic compensation. Those with good compensation had 17.015.2 apoptotic cells in vessels per mm2 of villous cross-sectional surface, while in those with poor compensation this value was 18.822.1/mm2. The differences between the groups were not statistically significant. Conclusion: We found that the number of apoptotic cells in placental vessels from DM type I pregnancies as well as in normal pregnancies is rather variable. Regarding vascular changes previously observed in DM type I placentas, it could be concluded that these arise without an increased apoptosis of vascular cells. The work was supported by the Research Project MSM 0021620807 and Grant GACR No.304/09/0733.

Abstracts / Placenta 32 (2011) A1–A149

P1.10 CHRONIC UTEROPLACENTAL INSUFFICIENCY CAN BE ASSOCIATED WITH NORMAL BIRTH WEIGHT IN BOTH TWIN AND SINGLETON PLACENTAS: A STEREOLOGICAL STUDY. Aiveen O’Malley1, Angel Mthunzi1, Etaoin Kent2, Jennifer Donnelly1, Sharon Cooley3, Fionnuala Breathnach2, Michael Geary1, Fergal Malone2, John Gillan1 1 Rotunda Hospital, Dublin, Ireland, 2Royal College of Surgeons, Dublin, Ireland, 3National Maternity Hospital, Dublin, Ireland Objective: Accelerated Villus Maturation is classically associated with chronic uteroplacental insufficiency (CUPI) of placental chorionic villi e.g. pre-eclampsia with IUGR (PET-IUGR), thrombophilia and smoking. These small sized villi represent a reduced fetomaternal interface for both nutrient absorption and gas exchange. Our group have studied accelerated villus maturation associated with normal birth weight in both singleton and twin placentas. Study Design: Case selection: Histopathological examination of placentas from 800 low risk singleton pregnancies and 154 dichorionic twin gestations allowed selection of the study cohorts. 4 cohorts of 10 placentas were examined, these comprised :- i) singleton accelerated villus maturation associated with normal birth weight; ii) twin gestation with accelerated villus maturation in one placenta (and normal co-twin); iii) PET-IUGR and iv) normal term singleton pregnancies. Stereological examination of terminal villous surface area and volume and that of the fetal vasculature was carried out. Results: 53 (6.7%) of singleton placentas and 10 of 154 (6.5%) dichorionic twins had accelerated villus maturation associated with normal birth weight (in one twin placenta only). When examined by Stereology, the CUPI normal birth weight placenta from both singleton and twin placentas had a reduced capillary volume fraction (p¼0.006; p¼0.005 respectively) and surface area (p¼0.0008; p¼0.005 respectively) when compared to their respective controls, but had a normal villous surface area. The PETIUGR capillary surface area was significantly reduced compared to normal controls (p¼0.0003). Conclusion: The normal villus surface area in cases of accelerated villus maturation means increased numbers of small villi which is commensurate with normal birth weight. The reduced vascular volume similar to PET-IUGR provides a possible explanation of previously unexplained intrapartum hypoxia.

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P1.11 IUGR SECONDARY TO CHRONIC UTEROPLACENTAL INSUFFICIENCY (CUPI) AFFECTING ONLY ONE TWIN IN DICHORIONIC PREGNANCIES STEREOLOGICAL EVIDENCE FOR CUPI LEADING TO DISCORDANT GROWTH Angel Mthunzi1, Aiveen O’Malley1, Etaoin Kent2, Fionnuala Breathnach2, Fionnuala McAuliffe3, Michael Geary1, Sean Daly4, John Higgans5, James Dornan6, John Morrison7, Gerard Burke8, Shane Higgans9, Stephen Carroll10, Patrick Dicker2, Fiona Manning2, Fergal Malone2, John Gillan1 1 Rotunda Hospital, Dublin, Ireland, 2Royal College of Surgeons, Dublin, Ireland, 3UCD School of Medicine and Medical Science, Dublin, Ireland, 4 Coombe Womans and Infants University Hospital, Dublin, Ireland, 5Cork University Maternity Hospital, Cork, Ireland, 6Royal Victoria Maternity Hospital, Belfast, Ireland, 7Nation University of Ireland, Galway, Ireland, 8 Graduate Entry Medical School, Limerick, Ireland, 9University of Limerick , Department of Obstetrics and Gynaecology, Limerick, Ireland, 10Our Lady of Lourdes, Drogheda, Israel, 11National Maternity Hospital, Dublin, Ireland Objective: Stereological evaluation of accelerated villous maturation associated with birth weight (BW) discordance in dichorionic twins. Study Design: Twin placentas were examined to establish chorionicity and the incidence of accelerated villous maturation in one or both twins. 42/ 154 (27%) dichorionic twins demonstrated accelerated villous maturation. This was associated with a 20% BW discordance profile in 27/42 (43%) cases. Stereology was performed on coded dichorionic twin placentas with 20% BW discordance and accelerated villous maturation in one twin only; 3 cohorts of controls comprising: (i) 10 twins with 10% BW discordance and normal terminal villi in both twins; (ii) 10 singleton placentas from gestational-age matched normal pregnancies and (iii) 10 preeclampsia-associated IUGR singleton placentas as positive controls for CUPI IUGR. Stereology was used to quantify the terminal villous interface and internal vasculature by measurement of surface area and volume fraction. Results: Stereological evaluation of the Surface Area in terminal villi and capillaries were significantly decreased when compared with their cotwins (p¼0.04; p¼0.049 respectively). Volume Fraction of terminal villi was significantly reduced when compared to their co-twins (p¼0.02), although capillary volume fraction was reduced when compared to cotwins this was not quite significant (p¼0.06). This profile was similar to the contrast observed between the preeclampsia-associated IUGR and normal control cohorts. Conclusion: Our results demonstrate that chronic uteroplacental insufficiency affects a significant number of discordant dichorionic twin pregnancies. Stereology has confirmed the histological finding of chorionic villi demonstrating accelerated villous maturation. In contrast to maternal conditions, which necessarily affect both twins, accelerated villous maturation affecting one twin suggests a primary disorder of the nonvillous trophoblast causing impaired physiological adaptation of maternal vessels in these pregnancies.

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Abstracts / Placenta 32 (2011) A1–A149

P1.12 ABRUPTIO PLACENTAE IN THREE NON-HUMAN PRIMATE SPECIES: MACACA FASCICULARIS, PAPIO SPP. AND CALLITHRIX JACCHUS.

P1.13 FETAL VESSELS DILATATION CONTRIBUTE UNIPARENTAL DISOMY CHROMOSOME 14

Natalia Schlabritz-Loutsevitch1, Mauro Schenone1, Jacques Samson1, Jie Zhang1, Giancarlo Mari1, Gene Hubbard2,3, Edward Dick2 1 University of Tennessee Health Science Center, Memphis, TN, United States, 2Texas Biomedical Research Institute, San Antonio, TX, United States, 3University of Texas Health Science Center, San Antonio, TX, United States

Kentaro Matsuoka1, Masayo Kagami2, Tsutomu Ogata2 1 National Medical Center for Children and Mothers, Tokyo, Japan, 2 National Research Institute for Child Health and Development, Tokyo, Japan

Introduction: Abruptio placentae (AP) is a serious clinical condition associated with a defective deep trophoblast invasion. Another theory connects the underdevelopment of the subperitoneal uterine vascular layer to the AP. Old World Primates : Macaca fascicularis (MAF) and Papio spp (PS) and have shallow trophoblast invasion, New World primates: Callithrix jacchus (CTJ) have a trabecular placenta. The information regarding naturally occurring placental abruption in these species is sparse. The goal of this study: To evaluate the pathological changes in naturally occurring AP in three non-human primate (NHP) species: MAF, PS and CTJ. Material and methods: Placentas were collected as part of the routine pathological examination between 2002-2008 at the Texas Biomedical Research Institute. After fixation in formalin, 5 mm slides were cut and stained with H&E, the slides were evaluated by two independent veterinary pathologists and the microscopic pictures were additionally examined by a certified MD pathologist. Results: AP was diagnosed in 14 PS, 9 MAF and one CTJ. The typical features of AP at the end of gestation were intra- and retro-placental hemorrhage with or without hematoma formation. Neutrophil infiltration and decidual necrosis were present in some cases. Increased neutrophil infiltration was noticed in PS at earlier gestational ages: at the end of the first to beginning of the second trimester. Conclusion: Despite the differences in the early placentation of NHP (shallow trophoblast invasion) and humans (deep trophoblast invasion), the clinical and histological pictures of Abruptio placentae in the three NHP species were very similar to the conditions in humans. Our findings challenge the current theory of shallow placentation as an etiologic factor in AP.

PLACENTOMEGALY OF

Objectives: Placentomegaly is observed in various conditions such as hydrops fetalis, Beckwith-Wiedemann syndrome, mesenchymal dysplasia and poorly controlled diabetes mellitus. Placenta of patient with paternal uniparental disomy chromosome 14 (upd(14)pat) also displays placentomegaly. Human chromosome 14q32.2 imprinted region carries a cluster of imprinted region including protein coding paternal expressed genes (PEGs) such as DLK1, RTL1 and DIO3, non-coding maternal expressed genes (MEGs) such as MEG3, RTL1as, MEG8, microRNAs and snoRNAs. Virtually, all the imprinted gens are expressed in the placenta. The roles of each gene of human 14q32.2 imprinted region in placenta are still unknown. In this study, we performed histological study using placental samples of cases with upd(14)pat phenotypes. Methods: We studied three placental fresh samples of upd(14)pat and a single paraffin enveloped placental sample of case with maternal microdeletion involving DLK1, IG-DMR, MEG3-DMR and MEG3. Results: According to the expression study, the levels of DLK1 and RTL1 using fresh placental sample of upd(14)pat detected an approximately 3.0to 4.5- fold and 10- to 34- fold, respectively. Placentomegaly was observed in all placental samples. Histologically, fetal vessels of intermediate villi are dilated with prominent endothelial cells. Electron micrographs of each sample demonstrated swelling of the endothelial cells. Immunohistochemical study was performed with antibody to DLK1 and RTL1. DLK1 and RTL1 were detected in the endothelial cells of fetal vessels of normal placenta. Interestingly, in placental samples of upd(14)pat, DLK1 and RTL1 were stained stronger than normal placentas. In placental sample of case with maternal microdeletion involving DLK1, DMRs and MEG3, only RTL1 was stained stronger than normal control. Conclusion: These results indicated that dilation of fetal villous vessels contributes to placentomegaly in upd(14)pat and RTL1 plays a key role in placental development.

Abstracts / Placenta 32 (2011) A1–A149

P1.14 CLUSTERING OF MATERNAL/FETAL OUTCOMES AND PLACENTAL LESIONS

CLINICAL

CONDITIONS

AND

Jerzy Stanek1, Jacek Biesiada2 1 Division of Pathology and Laboratory Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States, 2Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States Objectives: To present a comprehensive retrospective analysis of mutual clustering of complications of pregnancy, perinatal outcomes and placental lesions performed on a large unselected placental material. Methods: Twenty nine clinical (maternal and fetal) and 44 gross and microscopic placental features from 3081 20 weeks placentae consecutively signed by one of us (JS) in years 2001-2010 were analyzed using the Ward agglomerative hierarchical clustering method and results were visualized with multidimensional scaling. Results: The following most informative clinical pathological clusters comprising 32 parameters were identified (arranged according to the rising coefficient of dissimilarity): (1) Fetal thrombotic vasculopathy: clusters of avascular terminal villi and haemorrhagic endovasculitis; (2) Macerated stillbirths: luminal abnormalities of stem vessels, villous fibrosis; (3) Uteroplacental malperfusion: severe preeclampsia, hypertrophic decidual arteriolopathy, acute atherosis of decidual spiral arterioles, uterine pattern of chronic hypoxic placental injury, villous infarction, excessive extravillous trophoblasts, microscopic placental disc chorionic pseudocysts, decidual clusters of multinucleate trophoblastic giant cells; (4) Fetus-derived placental pathology: terminations of pregnancy, neonatal mortality, congenital anomalies, oligohydramnios, polyhydramnios, dilatation of chorionic and stem veins, villous edema and haemosiderosis, amnion nodosum; (5) Fetal stress/distress: term pregnancies, clinical thick meconium, histological deep meconium penetration; (6) Antepartum hemorrhage, retroplacental hematoma and intravillous hemorrhage, (7) Primary placental insufficiency: abnormal Dopplers, fetal growth restriction, low placental weight. Conclusion: The knowledge of mutual clustering of clinical conditions and placental lesions is not only practically useful for a pathologist but could be a starting point for further research on ethiopathogenesis of clinical conditions and outcomes based on placental pathology. In particular, the conditions and lesions of Cluster 4 link the placental edema and oligohydramnios-related fetoplacental pathophysiology to fetal congenital malformations, particularly those associated with fetal heart failure and/or those producing a mass effect with impaired blood return from the placenta to the fetus.

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P1.15 CLINICAL AND PATHOLOGICAL CORRELATION IN PLACENTA ACCRETA Arun Singavi2, Stacy Zamudio1, Manuel Alvarez1, Abdulla Al-Khan1 1 Center for Abnormal Placentation, Hackensack University Medical Center, Hackensack, NJ, 2St. Georges University Medical School, Grenada, Grenada Objectives: Placenta accreta results when the trophoblastic tissue invades beyond Nitabuch’s layer. Post partum hemorrhage (PPH) is a common, severe and life threatening outcome. PPH is due not only to traumatic separation of the placenta, but also to excess vascularity in the pelvic region. We asked whether excess vascularity correlates with the severity of accreta (A ¼ accreta – minimal invasion, I ¼ increta – w50% of myometrium, P ¼ percreta – bordering on or through uterine serosa). Methods: Patients included 19 pathologically confirmed accreta cases operated on by a single surgical team between 2006-2010 (A, n¼4; I, n¼6; P, n¼9). Clinical assessment of excess vascularity occurred intraoperatively and was recorded and photographed. Patient variables included units of pRBCs transfused, hospital length of stay (LOS), and maternal and neonatal complications. Data were analyzed using chi square, ANOVA and correlation, with accreta serving as the comparison group. Results: Vascularity increased with degree of invasion (100% P > 83% I > 0% A; p¼<0.001). pRBC units transfused was highest for I (7  4; p<0.01, 100% of patients); followed by P (5  4, 88%); and A (2  1, 75%). Adjuvant vascular intervention (embolization or hypogastric artery ligation) was required in 25% (A), 33% (I), and 78% (P). LOS (days) increased with disease severity (accreta 8  5; increta 8  4; percreta 13  9). There were no adverse neonatal outcomes. NICU LOS correlated with gestational age (r2 ¼ 0.69, p<0.001). Unlike prior reports suggesting growth restricted babies, we found them to be normally grown relative to gestational age (61st  18th percentile). Maternal clinical interventions correlated with severity: 0% (A), 17% (I) and 50% (P) required staged surgeries (>1 abdominal surgery). Conclusion: Excess pelvic vascularity, increasing risk of hemorrhage, is correlated with severity of accreta. While this seems intuitively obvious, it has not before been reported. Our present research is directed at antenatal evaluation of severity of vascularity, in order to anticipate degree of surgical complexity; we hope to present these results as well.

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Abstracts / Placenta 32 (2011) A1–A149

P1.16 EXPLORATION OF PLACENTAL DEVELOPMENT BY IN SILICO MATHEMATICAL MODELLING DEMONSTRATES THAT PLACENTA SHAPE AND CORD INSERTION RESULT FROM EVENTS EARLY IN PREGNANCY Alexander Heazell1, Simon Cotter2, Vaclav Klika2, Laura Gallimore2, Adriana Setchi3, Katharine Preedy4, Jennifer Siggers3, Ida Pu5, Robert Whittaker6 1 Maternal and Fetal Health Research Centre, University of Manchester, Manchester, United Kingdom, 2Mathematical Institute, University of Oxford, Oxford, United Kingdom, 3Department of Bioengineering, Imperial College, London, United Kingdom, 4University of St Andrews, St Andrews, United Kingdom, 5Goldsmiths College, University of London, London, United Kingdom, 6University of East Anglia, Norwich, United Kingdom Objective: Placental shape and the site of umbilical cord insertion are distorted in intra-uterine growth restriction (IUGR). The precise origins of placental shape and the site of cord insertion are difficult to study in vivo. We aimed to develop a mathematical model that simulates early placental development, and incorporates several growth mechanisms, with the aim of assessing their effect on placental shape and cord insertion. Methods: This model is based on the assumptions that the sources of nutrition from the occluded spiral arteries diffuse to create a gradient of

Ă

growth-promoting factors. This has a regular pattern, and the primary villi react to this trophic signal. The model makes use of the Langevin equation for a Brownian particle, and it is implemented numerically using the EulerMarayama discretization. Results: By representing the distribution of nutrient sources on a grid, shown by green circles (Figure 1A), the placenta develops evenly with a branching system of arteries (Figure 1B). By blocking a single nutrient source (Figure 1C), changes in the placental shape, and vascularisation can be seen, giving a marginal cord insertion (Figure 1D). By larger disruption near to the centre of placental mass (Figure 1E), a bipartite placental shape can be reproduced (Figure 1F). Discussion: Using this model, we can simulate and alter placental development. Small disruptions in nutrient supply can alter placental shape and the site of cord insertion. Therefore, abnormalities of placental shape may be an effect of the suboptimal early pregnancy environment. Figure 1 – A) Arrangement of nutrient sources; centre of placental mass in black. B) Normal placental shape and arterial network determined by the mathematical model. C) Single nutrient source blocked (red) D) Placental shape and cord insertion generated by the model E) Larger number of nutrient sources blocked (red). F) Resulting bipartite placental shape.

Abstracts / Placenta 32 (2011) A1–A149

P1.17 REVISITING MATERNAL SPIRAL ARTERY REMODELLING IN NORMAL PREGNANCIES. Ruta Deshpande1,2, George Bugg2, Lopa Leach1 1 School of Biomedical Sciences, University of Nottingham, Nottingham, 2 Department of Obstetrics and Gynaecology, Nottingham University Hospitals NHS Trust, Nottingham Objectives: Remodelling of the maternal spiral arteries (SA) by extravillous trophoblast to create high flow, low resistant SA is accepted as a key mechanism by which the fetus ensures adequate blood supply and overrides maternal regulation of blood flow in SA. Antibodies against cytokeratin 7 (C7) are used as reliable markers for the trophoblast in SA, although there is some evidence that it may localise to endothelial cells. We wanted to first ascertain the specificity of C7 as a trophoblast marker before testing the hypothesis that all the decidual SA identified at central cotyledons are remodelled in normal pregnancies. Methods: Term placenta, umbilical cord and omental biopsies were obtained from women with uncomplicated pregnancies leading to normal fetal outcomes. Chorionic villi and decidual biopsies were collected from three central cotyledons. Immunohistochemistry was performed on all tissue-types, using double labelling with C7 and the endothelial markers, von willebrand factor (vWF) and vascular endothelial (VE) cadherin. SA profiles were classified as fully remodelled (C7 immunolabelling alone), partially remodelled (C7 þ VE-cadherin/vWF) and no remodelling (VEcadherin/vWF only). Results: C7 was localised to the syncytiotrophoblast. In the same subjects, the endothelial vascular lining in the omentum, umbilical cord and chorionic villi demonstrated positive immunolabelling for vWF and VEcadherin but was negative for C7. In the 7 placenta studied, remodelling in SA was present in 57 of the total 73 vascular profiles identified (78%) whilst 16 (22%) showed no remodelling. Conclusion: Cytokeratin 7 is a useful trophoblast specific marker for studies on term placental SA when used in conjunction with endothelial markers. Remodelling is not an all or nothing phenomenon, a proportion of non remodelled decidual spiral arteries exist in normal pregnancies. We suggest the possibility of a physiological advantage to the mother and fetus by having a proportion of vessels under maternal regulatory control.

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P1.18 DECREASED VILLOUS CAPILLARIZATION IS ASSOCIATED WITH THE INCREASED PLACENTAL FAT CONTENT, BUT NOT WITH OBESITY PHENOTYPE IN A BABOON MODEL OF OBESITY Natalia Schlabritz-Loutsevitch1, Jacques Samson1, Mauro Schenone1, Gene Hubbard2, Edward Dick3, Giancarlo Mari1 1 University of Tennessee Health Science Center, Memphis, TN, -, 2University of Texas Health Science Center at San Antonio, San Antonio, 3Texas Biomedical Research Institute, San Antonio, TX Introduction: Maternal obesity has been associated with stillbirths, preeclampsia and preterm birth. The cause of this effect remains unknown. Due to striking similarity in placental physiology, baboons (Papio spp.) have been a great model to study pregnancy related problems. Maternal obesity in baboons has been associated with increased incidence of stillbirths (J Med Primatol. 37(6):337-45) and placental inflammatory changes (Placenta. 30(9):752-60). Recently placental vascular changes have been described in another Old World non-human primate - Rhesus monkeys fed high fat diet (Endocrinology. 2011). The Goal of this study was to evaluate placental vascular structure and placental fat content in a baboon (Papio sp.) model of obesity. Material and methods. 4 non-obese and 4 obese animals underwent caesarean section at 09.G. The fetus and placenta were weighed; placenta samples were randomly selected and fetal capillary volume (Vc) and villous tree volume (Vv) were estimated using Computerized Stereology approach as previously described (Placenta. 30(9):752-60). Villous capillarization was calculated as a ratio Vc/Vv. The total fat content was estimated using petroleum extraction methodology (CV¼3.6%). Results. Fetal and placental weight, fat content and capillarization did not differ between obese and non-obese groups. Placental fat content correlated negatively with the placental villous capillarization (r¼-0.82, p¼0.012). Conclusion. Our data suggest that increased placental fat deposition, but not maternal obesity itself, might trigger the adverse effects of maternal obesity on pregnancy outcome.

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P1.19 AUTOMATED TRACKING OF FETAL STEMS FROM INACCURATE INITIAL GUESS Prashant Athavale1, Luminita Vese1, Carolyn Salafia2 1 University of California, Los Angeles, 2Placental Analytics, New York The fetal stem vasculature in a placenta could be used in the diagnosis of some diseases like autism. To study the fetal stem structure effectively, we need to automatically and accurately track fetal stems through a sequence of digitized hematoxylin and eosin (H&E) stained histology slides. We start with an initial manual tracing in one of the images (slide # 1) in the sequence as shown in figures 1 and 2 below. We need to achieve automatic tracing of the same fetal stem in other images in the sequence based on this tracing. There are many problems in successfully achieving this goal. A few of the problem are: large size of the images (of the order of 500 MB), misalignment of the consecutive H&E slides, unpredictable inaccuracies of manual tracing, just to name a few.

We first use global rigid image registration of all the images based on displacement, scaling and rotation. This gives us approximate location of the corresponding fetal stem in the image that needs to be traced. Figure 3 shows details of a slide # 4 ‘near the approximate location’ that needs to be traced. We then use the rigid registration algorithm ‘locally’ near this location. At this point, we use a fast non-rigid registration based on L2similarity measure and diffusion regularization to get a better location of the fetal stem. Finally, we have to take into account inaccuracies in the initial tracing. This is done by using a novel version of Chan-Vese segmentation where the coefficients of the fitting terms are computed iteratively to ensure that we get a unique stem in the segmentation, as shown in figure 4. It is noteworthy that our algorithm gives accurate tracing in slide # 4 even though the initial manual tracing in slide # 1 is only an approximate one, with irregular boundary thickness. This tracing then can be used as an initial guess to obtain tracing in the rest of the images in the sequence. This constitutes an important step in the extraction of the vasculature.

Fig. 1. Details of the untraced fetal stem in slide # 1 in the sequence.Ă

Fig. 2. Manually traced fetal stem in slide # 1 in the sequence.Ă

Fig. 3. Manually traced fetal stem in slide # 1 in the sequence.Ă

Fig. 4. Details of “automatically traced”slide # 4 in the sequence.Ă

Abstracts / Placenta 32 (2011) A1–A149

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P1.20 INCREASED RESISTANCE INDEX AND DIMINISHED DIAMETER OF THE UTERINE ARTERY IS ASSOCIATED TO REDUCED VOLUME EXPANSION DURING GESTATION IN THE RAT.

P1.21 PLACENTAL MORPHOLOGY NEAR TERM AFTER TRANSFER OF CLONED AND TRANSGENIC EMBYOES: MISCARRIAGE SHOWED ENDOMETRIAL OEDEMA AND BLOOD EXTRAVASATION.

J. St-Louis, B. Sicotte, M Brochu Research centre, CHU Sainte-Justine, and Departments of physiology and of Obstetrics & Gynecology Faculty of Medicine, Université de Montréal, Montréal, Canada

V. Dantzer1, M. Schmidt2, H. Callesen3, K.D. Winter5, P.M. Kragh3, J. Li3, Y. Du3,4, L. Lin3,4, Y Liu3, G. Vajta3, J.S. Agerholm2 1 Section for Anatomy and Cell Biology, University of Copenhagen, Frederiksberg, Denmark, 2Reproduction and Obstetrics, University of Copenhagen, Frederiksberg, Denmark, 3Genetics and Biotechnology, Aarhus University, Tjele, Denmark, 4BGI/HuaDa Shenzhen, ., China, 5Landbrug & Fødevarer, Kjellerup, Denmark

Introduction: Optimal delivery of oxygen and nutrients by the uteroplacental circulation is essential for fœtal growth and successful pregnancy. To do so, uterine circulation undergoes important structural changes. This study was undertaken to test the hypothesis that intrauterine growth restriction induced by the absence of volume expansion during pregnancy is consequent of reduced utero-placental perfusion and/ or altered uterine vascular remodelling. Methods: The experimental condition was induced in the rat while giving a low sodium diet during the last 7 days of gestation (over 22). Pregnant Sprague-Dawley rats were randomly assigned to a control group or to a group fed the low-sodium diet. Blood velocity wave forms in the main uterine artery were obtained by Doppler sonography under isoflurane anaesthesia on days 14, 18 and 21 of gestation. Results: Peak systolic and end diastolic velocity increased between on day 18 in both groups but on day 22, this was reduced in pregnant rats on low sodium diet leading increased resistance index compared to controls. Rats were sacrificed on day 22 to evaluate the diameter and myogenic response of uterine radial artery supplying a placenta in a pressurized myograph (LSI, Burlington VT). Radial artery diameter was significantly diminished by low salt diet while myogenic tone was augmented by this treatment. Discussion: Together, these alterations could be responsible for the increased resistance index of main uterine artery. The present results are indicative of reduced utero-placental blood flow that could explain the intra uterine growth restriction observed in the offspring of these pregnant rats. Work supported by Canadian Institute for Health Research (grant MOP 84450).

Introduction and aims: The development of placenta, growth and angiogenesis is a prerequest for successful gestation. The perinatal mortality of cloned animals is a well-known problem and might be due to compromised placental function and sub-optimal fetal-maternal communication. The aim of this study was to investigate the diffuse, folded, epitheliochorial porcine placenta at term in order to elucidate possible changes that might lead to the increased mortality in piglets seen after transfer of cloned and/ or transgenic embryos. In a large retrospective study of mortality after transfer of cloned and or transgenic minipig blastocysts to Large White (LW) sows was compared to standard AI. Methods and results: Placental/endometrial tissue was sampled from 14 sows during caesarean section and endometrium from five empty sows and prepared for histology. We found changes in placenta from cloned embryos when compared with AI. In the cloned placentas an extensive diffuse oedema of the deep endometrium was a consistent finding. The presence and severity of other lesions varied among the individual placentas without any obvious patterns: loss of feto-maternal mutual complementary foldings, endometrial hyperplasia with polyps covered by a hyperplastic epithelium, multiple focal to widespread acute hemorrhages in the superficial endometrial connective tissue.The endometrium from empty sows showed severely endometrial oedema with scarce vascularisation and local areas with subepithelial haemorrhage. In the fetal placenta a generally sparse edema was seen and in several cases ballooning degeneration of the trophoblasts. In some cases thick walled large vessels were seen. Conclusion: Despite a rather high success rate for cloning, the changes in the endometrium are new observations indicating a feto/maternal disharmonie with long lasting effect.

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P1.22 CYTOTROPHOBLAST ALTERATIONS IN PLACENTAS FROM DISORDERS ASSOCIATED WITH ANTI-PHOSPHOLIPID ANTIBODIES AND DYSREGULATED CLOTTING FACTORS Rie Kawaguchi1,2, Monika Siwetz1, Monika Sundl1, Yuki Nakada2, Michiko Suzuki2, Eri Takahashi2, Taizan Kamide2, Nagayoshi Umehara2, Tadao Tanaka2, Berthold Huppertz1 1 Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Graz, Austria, 2Department of Obstetrics and Gynecology, The Jikei University School of Medicine, Tokyo, Japan Objectives: Pregnancy pathologies such as preeclampsia, fetal growth restriction and abruption are well known to be affected by an impaired placental development. Antiphospholipid antibodies (APL) are associated with placental insufficiency by activation of the complement cascade. However, also dysregulated clotting factors (CF) may directly lead to injuries of placental tissues. Here we investigated the effect of APLs and dysregulated clotting factors on the biology of villous trophoblast. Methods: Placentas were collected from a normal control group (NC; n¼8, 27-38 gestational weeks), an APL group (n¼10, 25-36 weeks), and a CF group (n¼10, 27-35 weeks), where patients had no APL, but disorders of serum clotting factors. Placentas were stained for anti Ki-67 and anticytokeratin 7 to selectively identify proliferating villous cytotrophoblasts. Images were systematically randomly selected and cytotrophoblasts counted. One way ANOVA and post hoc analysis were applied as statistical analysis. Results: The ratio of Ki-67 positive cytotrophoblasts in NC gradually decreased in accordance with gestational age, while this was not apparent in APS and CF. The mean of Ki-67 positive cells was significant reduced in APL (12.1  7.1%) and CF (13.3  5.8%) compared to NC (22.6  6.4%). The total number of cytotrophoblasts was reduced in APS (578  209) compared to CF (822  232) and NC (780  149). Conclusion: Throughout the third trimester of pregnancy there are significant differences in cytotrophoblast features such as total number and rate of proliferating cells in patients with APL and a dysregulated clotting system. Although cytotrophoblast proliferation was reduced in all compromised placentas, the total number of cytotrophoblasts only decreased in APS. We suggest that APL promote inhibition of cytotrophoblast proliferation throughout pregnancy, while in the CF group, an increasing amount of fibrin depositions finally leads to a decrease in trophoblast proliferation but does not affect total cell count.

P1.23 LYSOPHOSPHATIDYLINOSITOL IS A PUTATIVE INDUCER OF VILLOUS TROPHOBLAST DIFFERENTIATION Martin Gauster, Julia Kremshofer, Veronika M. Berghold, Gerit Moser, Berthold Huppertz Medical University of Graz, Graz, Austria Objectives: The endogenous lysophospholipid lysophosphatidylinositol (LPI) regulates many physiological processes including angiogenesis, apoptosis, cell proliferation, inflammation and reproduction. Here the hypothesis was tested whether or not LPI is able to induce villous trophoblast differentiation. Methods: The trophoblast derived cell lines ACH-3P and BeWo were incubated with various LPI concentrations for 48h. After incubation Ecadherin expression was analysed by immunoblotting and subsequent band densitometry. Secretion of human chorionic gonadotropin, beta subunit (beta-hCG) was determined in culture supernatants by routine immunoassay. Mitogenic activity was determined by staining cells for phosphorylated histone H3 (phospho-histone H3), which is tightly correlated with chromosome condensation during mitosis. Results: LPI treatment induced a concentration dependent downregulation of E-cadherin after 48h. In presence of 10mM LPI E-Cadherin was downregulated to 77.2% and 84.6% in ACH-3P and BeWo, respectively. At the same time, LPI increased secretion of beta-hCG in ACH-3P and BeWo by 28.5% and 17.1%. In presence of 5mM LPI the relative number of phosphohistone H3 positive nuclei in ACH-3P and BeWo decreased to 83.1% and 82.3% when compared to control after 48h. Conclusion: Downregulation of E-cadherin together with increased hCG secretion and reduced mitogenic activity are characteristic for villous trophoblast differentiation. Our study suggests that the endogenous lysophospholipid LPI is able to trigger these processes and thus represents a novel player in regulation of villous trophoblast differentiation. P1.24 PHOSPHOLIPID SCRAMBLASE 1 IN THE HUMAN PLACENTA Veronika M. Berghold, Martin Gauster, Gerit Moser, Monika Siwetz, Monika Sundl, Berthold Huppertz Medical University of Graz, Graz, Austria Objectives: Maintenance of regular cell membrane asymmetry is accomplished by scramblases, translocases and floppases. During villous trophoblast fusion this asymmetry is transiently abolished through externalization of phosphatidylserine to the outer membrane leaflet. One of the enzymes involved in rearrangement of membrane architecture is phospholipid scramblase 1 (PLSCR1). Expression of PLSCR1 was analyzed in isolated primary trophoblasts, the trophoblast-derived cell line BeWo and placental tissues. Methods: Microarray was performed in primary trophoblasts from human first trimester and term placenta as well as in BeWo cells. BeWo cells were stimulated with forskolin to syncytialize while DMSO treated cells served as controls. Placental tissues and cells were immunostained for PLSCR1 and b-hCG. Expression was analyzed by Western Blot and quantitative real time PCR. Results: Microarray analysis revealed that PLSCR1 showed the strongest expression among the three transporter families. While PLSCR1 protein was abundantly expressed in syncytiotrophoblast, macrophages and endothelial cells, cytotrophoblasts showed only weak staining in first trimester and term placenta. In contrast, in BeWo cells PLSCR1 was not colocalized with b-hCG positive syncytia. No significant changes in PLSCR1 mRNA and protein expression were observed between forskolin treated and control cells. Time course experiments with BeWo cells treated with or without forskolin showed no significant changes in PLSCR1 mRNA expression over 48 hours. Conclusion: Localization of PLSCR1 in the villous trophoblast compartment suggests a putative role in regulation of trophoblast membrane architecture. However, so far no obvious changes in protein expression during BeWo differentiation process could be detected. Work is in progress to identify the level of involvement of PLSCR1 in trophoblast fusion.

Abstracts / Placenta 32 (2011) A1–A149

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P1.25 ANALYSIS OF HCG GLYCOFORMS SECRETED BY THE NORMAL TROPHOBLAST AND THE TRISOMIC TROPHOBLAST.

P1.26 ESTABLISHMENT OF IMMORTALIZED HUMAN COMPLETE HYDATIDIFORM MOLE CELL LINES

marie clemence leguy1,2, arnaud bruneel4, agnes choiset1,3, fanny lewin1, daniele evain-brion2,5, nathalie seta3,4, thierry fournier2,3, jean guibourdenche1,5 1 AP-HP CHU Cochin, paris, France, 2INSERM U 767, paris, France, 3Université ParisDescartes, paris, France, 4AP-HP CHU Bichat, paris, France, 5 PremUp foundation, paris, France

Eiko Yamamoto, Kaoru Niimi, Kanako Shinjo, Sawako Fujiwara, Yukio Mano, Seiji Sumigama, Tomomi Kotani, Fumitaka Kikkawa Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan

The human chorionic gonadotropin hormone (hCG) is a maternal serum marker used for the screening of fetal Trisomy 21 (T21). It is well established that its concentration increases in T21 and decreases in Trisomy 18 (T18). We previously showed that, in T21 in vitro, there is a defective differentiation of the villous cytotrophoblasts involving hCG which is different in its glycosylation and its bioactivity (Frendo, JCEM, 2004). Objectives: To analyse the hCG glycoforms secreted in vitro by the different cytotrophoblast subtypes (villous, VCT ; extravillous, EVCT) and by the syncytiotrophoblast (ST). To compare those hCG glycoforms to those circulating in maternal blood, in normal and pathological pregnancies. Methods: We studied the hCG glycoforms profile by 2D electrophoresis. The first dimension allowed a separation based on the isoelectric point, completed by a western blot with glycoforms detection performed by an anti-hCG ß subunit polyclonal antibody. We analysed four cultures supernatants from each trophoblast subtypes, isolated from first trimester normal placentas, and 12 maternal sera (3 controls, 3 T21, 3 T18, 3 T13). Results: EVCT secretes acidic hCG glycoforms (3.4
Objectives: Choriocarcinoma is derived from trophoblasts in all kinds of pregnancies but a hydatidiform mole seems to have a higher potential to develop into choriocarcinoma than a normal pregnancy. However, the mechanism for trophoblastic carcinogenesis remains unclear. We aimed to produce human complete hydatidiform mole (CHM) cell lines with a long lifespan and to provide an ideal in vitro cell model. Methods: CHM tissue was obtained from a patient who underwent an evacuation and was diagnosed as CHM by pathological examination. Informed consent was obtained from the patient and her husband for the use of their tissue samples. The CHM tissue was cultured on collagen coated dishes and outgrowth cells were confirmed to be trophoblastic cells by immunocytochemistry. The cells were immortalized by infection of human telomerase reverse transcriptase (hTERT), Cdk4, cyclinD1, p53 with retroviral expression vectors. We performed immunocytochemistry of the immortalized cells using CK8/ 18, hCG, hPL and vimentin Abs. Chromosomal number and genetic origin were examined by karyotype analysis and DNA polymorphism analysis, respectively. Cell proliferation and hCG secretion were examined by MTS assay and ELISA. Results: Primary culture cells from CHM were confirmed to be positive for CK7, hPL and hCG by immunocytochemistry. HMol1-8 (hTERT/Cdk4), HMol1-3B (hTERT/cyclinD1/Cdk4), and HMol1-2C (hTERT/Cdk4/cyclinD1/ p53) were established. Immunocytochemistry revealed that all the three cell lines expressed CK8/18, hCG, hPL, but not vimentin. Karyotype analysis showed that the chromosomal number of HMol1-8 was almost normal but those of HMol1-2C and HMol1-3B were about 96. HMol1-2C and Hmol13B had the same alleles as CHM tissue which had only the paternal gene but the result of HMol1-8 was completely the same as that of maternal tissue. Conclusion: HMol1-2C and Hmol1-3B were showed to be cell lines derived from CHM and these newly immortalized cell lines are useful models for the study of trophoblastic disease.

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P1.27 THE NOVEL IL-1 FAMILY MEMBER IL-33 IS PRODUCED DURING HUMAN PREGNANCY AND CONTROLS FUNCTION OF PRIMARY TROPHOBLASTS IN VITRO. Valerie Fock, Harald Zeisler, Martin Knöfler, Jürgen Pollheimer Medical University of Vienna, Vienna, Austria Objectives: In this study we describe expression and function of the novel IL-1 superfamily member IL-33 during human placentation. In a first step we studied expression pattern of both IL-33 and its receptor ST2L in the placenta and at the fetal-maternal interface. Secondly, we investigated the effects of human recombinant (rhu) IL-33 in primary trophoblast model systems. Methods: Western Blot analyses, immunohistochemistry and RT-PCR were performed to analyze short and long isoforms of ST2 (sST2/ST2L) and IL-33 expression in human reproductive tissues, various trophoblast cell lines, isolated primary decidual cells and first trimester trophoblasts. In addition, we studied ST2L expression in freshly isolated CTBs (EGFRþ) and EVTs (HLA-G1þ) by FACS analyses. Moreover, primary isolated trophoblasts were stimulated with rhuIL-33 and analyzed by Western Blot. Migration was evaluated by studying differentiating villous explant cultures on collagen-I. Proliferation of primary trophoblasts was assessed by evaluating BrdU incorporation in the presence or absence of rhuIL-33. Results: Various descriptive analyses revealed expression of sST2/ST2L in first trimester CTBs and EVTs. ST2, however, was absent from villous/ decidual fibroblasts (CD90þ/Vimþ, CD45-), endothelial cells (CD31þ) and leukocytes (CD45þ). Interestingly, we detected cytoplasmic IL-33 expression in Hofbauer cells (CD68þ), decidual cells and not yet fully characterized leukocytes of the fetal-maternal interface. In addition, primary isolated decidual cells express IL-33 in vitro. Finally, rhuIL-33 enhanced BrdU incorporation in primary isolated trophoblasts and induced outgrowth in first trimester villous explant cultures. Conclusion: Cells of the placental villus and maternal decidua, including Hofbauer cells and decidual fibroblasts represent putative sources for extracellular IL-33 to activate ST2L-expressing CTBs and EVTs. Functional in vitro assays further suggest that rhuIL-33 influences trophoblast invasion and proliferation. Taken together, our results indicate that ST2/IL-33 signalling might play an important role in trophoblast function.

P1.28 ROLE OF CDKN1C IN HUMAN HLA-GD EXTRAVILLOUS TROPHOBLASTS (EVT) Tamara Tilburgs, Basya Rybalov, Jack Strominger Harvard University, Cambridge, MA, United States Objectives: Invading HLA-Gþ EVT play a key role in the process of placental anchoring, opening of the uterine spiral arteries and prevention of a maternal immune attack to the foreign fetal tissue. However, HLA-Gþ EVT are extremely difficult to study due to their low frequency and lack of methods to culture or expand them. Cyclin Depended Kinase Inhibitor 1C (CDKN1C or KIP2 or p57) is a potent inhibitor of cell cycle progression and is highly expressed in trophoblast cells. In mouse models CDKN1C together with Fibroblast Growth Factor-4 (FGF-4) have been shown to regulate trophoblast giant cell formation and trophoblast proliferation (Ullah et al. 2009). The aim of this study was to investigate the role of CDKN1C in human trophoblast cells. Methods: A previously developed method (Apps et al. 2009) was used to isolate HLA-Gþ EVT from human 1st trimester tissue. Purification using FACS sort provided 1.1-7.4 x105 HLA-Gþ EVT and 8.9-33 x 105 HLA-Gvillous trophoblasts (VT), both with >95% purity. Microarray analysis confirmed high expression of CDKN1C in human EVT and VT cells, whereas no expression of p57 was found in proliferating JEG-3 cells. Lentiviral vectors with shRNA for CDKN1C and scrambled shRNA controls were generated and used to infect human trophoblast cell cultures. Western blots confirmed knockdown of p57 compared to control vectors. Results: Knockdown of CDKN1C did not induce VT or EVT to proliferate, demonstrated by CFSE analysis and Ki-67 staining. Knockdown of CDKN1C did increase the life span of HLA-Gþ EVTs from 3-5 days up to 7-9 days. Additional stimulation with FGF-4 or EGF did not induce differentiation or proliferation of HLA-Gþ EVT. Conclusions: This study shows again the complexity of trophoblast biology and the little knowledge that is available on factors that may induce human HLA-Gþ EVT or HLA-G- VT to proliferate or differentiate.

Abstracts / Placenta 32 (2011) A1–A149

Sandra Haider, Peter Haslinger, Jürgen Pollheimer, Martin Knöfler Medical University of Vienna, Department of Obstetrics and Fetal-Maternal Medicine, Reproductive Biology Unit, Vienna, Austria Objective: Notch signalling is a highly conserved pathway controlling diverse developmental processes. Here, we investigated for the first time localization of all Notch receptors (Notch1-4), their ligands (Jagged1 and 2, Dll1, 3,4) and the nuclear downstream effector RBPJk in early human placentae. Moreover, the role of Notch signalling in cell column proliferation, trophoblast invasion and decidual cross-talk was analyzed. Methods: Expression patterns of proteins were analyzed in tissue sections of first trimester placentae using immunofluorescence. Notch activity was evaluated in the trophoblast cell line SGHPL-5 using a luciferase reporter containing wildtype or mutant RBPJk binding sites. Invasion and migration were studied in transwell assays of SGHPL-5 cells and villous explant cultures on collagen-I, respectively. Proliferation was measured by BrdU labelling of floating explant cultures. Influence of the Notch pathway on Wingless (Wnt) signalling was analyzed by using a canonical Wnt reporter. Experiments were performed in the absence or presence of the Notch inhibitor DAPT. Moreover, constitutive activation of the pathway was achieved by overexpression of the Notch1 intracellular domain (NICD). Results: Immunofluorescence indicated predominant expression of Notch 1-3 in cell columns, whereas non-proliferating, extravillous trophoblasts specifically expressed Dll3. RBPJk reporter assays revealed basal Notch activity in SGHPL-5 cells which was enhanced by overexpression of NICD and upon co-cultivation with decidual stromal cells. Activation of the pathway through NICD also suppressed Wnt signalling. Inhibition of Notch signalling increased invasion and migration in the trophoblast model systems, but also elevated proliferation in cell columns. Conclusion: Notch signalling could play a major role in controlling cell column proliferation and trophoblast differentiation, partly by regulating Wnt signalling. Moreover, decidual cells may regulate the pathway in invasive trophoblasts. However, complex expression patterns of Notch and ligands in the placenta suggest trophoblast subtype-specific roles of the pathway warranting further functional studies on individual Notch receptors and ligands. This work was supported by a grant from the Austrian Science Funds (P22687-B13).

P1.30 CAN THE FORMATION OF SYNCYTIAL NUCLEAR AGGREGATES BE STUDIED IN THIRD TRIMESTER PLACENTAL EXPLANTS? Sarah Coleman, Carolyn Jones, Colin Sibley, John Aplin, Alexander Heazell Maternal and Fetal Health Research Centre, University of Manchester Objectives: The mechanism of syncytial nuclear aggregate (SNA) formation is unknown, but it is estimated to take 14-21 days. We hypothesise that the cytoskeleton is involved in the lateral movement of nuclei through the syncytiotrophoblast into SNAs. Methods: Term villous explants (n¼6) were cultured in supplemented CMRL in either 6% or 20% O2 and samples fixed on days 0, 4, 8, 12 and 16. The number of SNAs per mm2 of tissue and size of SNAs were quantified. Immunofluoresence was performed to identify cytoskeletal components including; actin and tubulin. Results: The absolute number of SNAs per mm2 of tissue did not alter in different O2 tension or over time (median 131 SNAs/mm2 villous area) (Figure 1). Furthermore, there was no change in SNA size (median 179mm2). Immunofluorescence showed that tubulin and g-actin localised to SNA structures (Figures 2 and 3). Conclusion: No change in SNA number may be due to a lack of de novo production in vitro or because the rate of synthesis matched the rate of loss. The former seems more likely as explants do not generate a large number of new nuclei. This would suggest that SNAs in term placental explants are stable: further work investigating material shed into the culture medium will clarify this. Finally cytoskeletal proteins including g-actin and tubulin may be involved in nuclear movement into SNAs.

Average number of SNAs /1mm2 villous area

P1.29 NOTCH SIGNALING PLAYS A ROLE IN HUMAN PLACENTAL DEVELOPMENT: REGULATION OF CELL COLUMN PROLIFERATION AND TROPHOBLAST INVASION

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250

SNA number in explants at 6% O 2

200

SNA number in explants at 20% O 2

150 100 50 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Day Fig. 1. Density of SNAs on explants cultured for 16 days.Ă

Fig. 2. Immunofluorescence of an explant (day 8). a-tubulin (green) is seen in cytotrophoblasts and the regenerating syncytiotrophoblast layer.

Fig. 3. Immunofluoresence (day 16). g-actin (green) is highly stained within a SNA region of the newly formed syncytiotrophoblast. Both images counterstained with PI (some background in figure 3).

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Abstracts / Placenta 32 (2011) A1–A149

P1.31 RHOE REGULATES FUSION OF BEWO CHORIOCARCINOMA CELLS Gavin Collett, Xue Fang Goh, Elizabeth Linton, Christopher Redman, Ian Sargent University of Oxford, Oxford, United Kingdom Objectives: Differentiation and fusion of villous cytotrophoblasts are essential for the normal formation, growth and maintenance of the syncytiotrophoblast layer of the human placenta. Rho GTPases are involved in numerous cellular processes, including proliferation, migration and differentiation. Several of these proteins, including RhoA, Rac1 and RhoE, have been shown to play a role in myoblast fusion. In this study we examined the role of RhoE in trophoblast fusion using the fusogenic BeWo human choriocarcinoma cell line. Methods: Confluent BeWo cells were cultured with 1mM dibutryl cyclic AMP (dbcAMP) to induce fusion. At 24h and 48h after the start of treatment cells were either fixed in methanol for the assessment of fusion by desmosomal protein staining, or lysed for western blotting. The effect of RhoE on fusion was assessed by transfecting cells with RhoE siRNA or a non-silencing control for 48h prior to treatment with dbcAMP. For hypoxia studies, experiments were carried out at 1% O2. Results: Treatment with dbcAMP resulted in a strong upregulation of RhoE at 24h, coinciding with the onset of fusion. In RhoE siRNA-transfected cells there was a significant decrease in fusion compared with cells transfected with non-silencing control (7.0% vs 11.9% and 16.6% vs 25.2% at 24h and 48h respectively). However, expression of differentiation markers human chorionic gonadotrophin and placental alkaline phosphatase was unaffected by RhoE silencing. Finally, RhoE upregulation by dbcAMP was significantly reduced under hypoxic conditions in which cell fusion is impaired. Conclusions: Our results show that induction of RhoE by dbcAMP promotes BeWo cell fusion but has no effect on functional differentiation, supporting evidence that these two processes may be controlled by separate or diverging pathways. Furthermore, we show that a reduction in RhoE expression may contribute to the inhibition of fusion under hypoxia.

P1.32 DESCRIPTIVE MORPHOLOGICAL CHARACTERISATION OF SYNCYTIAL KNOTS SHED IN VITRO FROM FIRST TRIMESTER PLACENTA Olivia Holland, Maria McDowell-Hook, Peter Stone, Larry Chamley University of Auckland, Auckland, New Zealand Objectives: The syncytiotrophoblast is a single multinucleated cell that covers the surface of the human placenta. Large multinucleated portions of the syncytiotrophoblast, syncytial knots/sprouts, are shed from the placenta into the maternal blood. Although syncytial knots were first described over a century ago some of the most basic features of these structures are unknown. We have begun to characterise some of the key features of syncytial knots. Methods Individual syncytial knots shed from placental explants which had been stained with CMFDA were collected, exposed to proprodium iodide, and visualised utilising the Zeiss LSM 710 inverted confocal microscope. Z-series images were captured and 3D models reconstructed for 47 syncytial knots collected from 12 placentae (8-12 weeks gestation). Values of volume, height, width and depth were measured, and nuclei were counted manually. Results None of the syncytial knots were able to exclude proprodium iodide. Three broad morphological categories were identified; teardrop, spherical and asymmetrical. Syncytial knots had a mean volume of 48,321 mm3, and the mean number of nuclei was 61. Nuclei were either heterochromatic (55%) or euchromatic (45%). Blebbing of the plasma membrane was seen in 23% of the syncytial knots and was usually associated with heterochromatic nuclei (82%). Conclusion Syncytial knots had highly variable morphology and may represent a mixture of syncytial knots (aged nuclei) and syncytial sprouts (prominent nucleoli). Alternatively, since all of these structures took up proprodium iodide indicating they were not viable, it may be that they all have a single origin but have been visualised at different stages of the death process.

Examples of syncytial knots shed in vitro with spherical (A) and teardrop (B) morphology are shown. Note heterochromatic (A) and euchromatic (B) nuclei, nucleoli (B) and blebbing of plasma membrane (A; arrows). Cytoplasm ¼ green; nuclei ¼ red. Scale bars 20 mm.

Abstracts / Placenta 32 (2011) A1–A149

P1.33 PLACENTA PERFUSION - A SUITABLE EX VIVO MODEL TO CHARACTERISE INTERACTION OF NANO-PARTICLES WITH HUMAN TISSUE? Lydia Seyfarth1, Uta Enke1, Rolf Bräuer2, Nadine Brendel1, Markus Büttner3, Andrea Csaki5, Florian Schlenk4, Christian Bergemann6, Dagmar Fischer4, Wolfgang Fritzsche5, Paul Seidel3, Ekkehard Schleussner1 1 Department of Obstretrics and Gynecology, Placentalabor Jena University Hospital, Friedrich-Schiller-University Jena, Jena, Germany, 2Institute of Pathology, Jena University Hospital, Friedrich-Schiller-University Jena, Jena, Germany, 3Institute of Solid State Physics, Friedrich-SchillerUniversity Jena, Jena, Germany, 4Department of Pharmaceutical Technology, Institute of Pharmacy, Friedrich-Schiller-University Jena, Jena, Germany, 5Institute of photonic technology (IPHT), Jena, Germany, 6 Chemicell GmbH, Berlin, Germany Objectives: Nanotechnology is a rapidly expanding field of industry in which nanoparticles (NP) are developed for a wide range of purposes, including medical applications. Nevertheless, the current lack of knowledge about their potential toxicity requires the establishment of new test procedures to estimate potential human health risks. Placentae can play a central role in the establishment of human tissue models as they do not constitute an ethical problem. To date, no standardised methods are available for quantification and toxicological characterisation of NP in human tissue. Methods: The cooperation project NanoMed focuses on iron oxide and gold NP for later use in diagnostic treatments for which especially the quantification of these NP in human tissue is essential. By optimising the magnetorelaxometry (MRX) technique, we developed an appropriate tool for a standardised quantification of NP with magnetic properties in human tissue. Dynamic light scattering (DLS) was used to characterize NP size and stability in perfusion media. For determination of NP surface charge zeta potential measurements were performed. To analyse the reaction of placental tissue to iron oxide or gold NP suspended in perfusion media, we infused these NP into placental tissue for up to 6 h. In (circulating) perfusate media, different metabolic control parameters and the release of cytokines from the perfused cotyledons were analysed. Histological analysis was performed in paraffin embedded tissue. Results: Distribution of NP between tissue and perfusion media depends on NP characteristics (e.g. size, surface). Similarly, metabolic parameters (secretion of b-HCG, synthesis of lactate) as well as cytokine levels (e.g. IL6, IL-8, TNF-a) detectable in perfusion media and their release profile, are dependant upon NP characteristics. Conclusion: Placenta perfusion constitutes is an adequate method to characterise specific effects of NP on placental tissue.

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P1.34 DREAM REPRESSES GLIAL CELL MISSING 1 (GCM1) TO IMPAIR DEVELOPMENT OF THE MURINE LABYRINTH: INSIGHTS INTO THE PATHOGENESIS OF PREECLAMPSIA. Dora Baczyk1, Marcos Rivas2, Sascha Drewlo1, John CP Kingdom3, Jose R. Naranjo2 1 Samuel Lunenfeld Research Inst, Toronto, ON, Canada, 2Departamento Biologia Molecular y Celular, Madrid, Spain, 3Department of Ob/Gyn at Mt. Sinai Hosp, Toronto, ON, Canada Objective: Determine the role of DREAM in Gcm1-mediated development of the placental labyrinth in mice. Methods: Transgenic mice expressing dominant active DREAM mutant insensitive to calcium (Jn1, n¼5) and wild type (wt) controls (n¼5) were sacrificed at E13.5 and E18.5 for placental and embryo weights. Their placentas were dissected and fixed for paraffin and semi-thin histologic examination, and Ki67, CD34 and sFLT immuno-histochemistry. Their kidneys were fixed for histologic examination of glomeruli and stained with MSB to assess fibrin and collagen deposition (n¼5). Placental RNA extracts were subjected to qRT-PCR analysis of steady-state DREAM and Gcm1 transcription. Results: Compared to wt, Jn1 mice exhibited a 30-fold increase in DREAM mRNA levels while Gcm1 mRNA was down-regulated by 40%. These findings coincided with reduced placental size at E13.5 as well as reduced pup size at both time points. Histologic assessment of placentas from Jn1 animals revealed alterations in the labyrinth including; reduced blood spaces, pockets of undifferentiated Ki67-positive cytotrophoblast progenitors, disorganized syncytial layers and more intense sFLT staining. At E13.5, Jn1 placentas show increased rates of proliferation in the labyrinth layer as compared to age-matched wt controls. By term, placentas from Jn1 animals lost the excess proliferation capability, and were similar to wt mice. Kidneys of Jn1 mice showed excess fibrin deposition and collagen formation in glomeruli (positive MSB staining) indicative of early glomerular injury (thrombotic microangiopathy). Conclusion: In mice DREAM functions as an upstream negative regulator of Gcm1-directed labyrinthine trophoblast development analogous to observations in human placental villi. The resultant syncytiotrophoblast abnormalities including increased sFLT1 expression and glomerular injury in Jn1 mice represents elements of the preeclamptic disease phenotype.

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Abstracts / Placenta 32 (2011) A1–A149

P1.35 DRAMATIC MITOCHONDRIAL MODIFICATIONS ACCOMPANY FUSION OF BEWO CELLS

P1.36 DRAMATIC MODULATION OF THE GOLGI AND TRANS-GOLGI NETWORK IN FUSED BEWO CELLS

William Ackerman, Dale Vandre, John Robinson The Ohio State University, Columbus, OH, United States

Gen Ishikawa1,2, Toshiyuki Takeshita2, William Ackerman1, Dale Vandre1, John Robinson1 1 The Ohio State University, Columbus, OH, United States, 2Nippon Medical School, Tokyo, Japan

Objectives: Trophoblast fusion leads to a number of changes associated with syncytium formation. We hypothesize that such changes are reflected at the organelle level. Herein, we focus on alterations to mitochondria associated with BeWo cell fusion. Methods: Cultured BeWo cells were used as a surrogate for cytotrophoblasts and were induced to fuse by treatment with forskolin (FK). BeWo cells treated with FK for 24, 48, and 72 hours were compared to untreated control cells and assessed using immunoblotting, immunofluorescence, and electron microscopy methods. In some experiments, JAR cells in the presence or absence of FK treatment were also used. Results: Control cells exhibited a heterogeneous population of mitochondria the morphologies of which ranged from elongated tubular- to shorter rod-shaped structures as determined by immunofluorescence microscopy. Following cell fusion, however, the tubular mitochondria largely disappeared and were replaced by those with smaller rod-shaped and spherical appearances, suggesting mitochondrial fission. Despite these dramatic alterations in morphology, immunoblotting data using anti-complex II subunit 70kD, a mitochondrial marker protein, indicated that the total amount of mitochondria remain unchanged following cell fusion. Concomitant with mitochondrial fission there were changes in their ultrastructural appearance. Mitochondria in mononuclear cells exhibited primarily plate-like cristae while those of fused cells had increased numbers of circular cristae. These morphological changes correlated with induction of mitochondrial P450scc (CYP11A1). Conclusion: We report dramatic alterations in mitochondrial appearance in FK-treated BeWo cells associated with both mitochondrial fission and modification of the pattern of cristae structure. However, it appears that the complement of mitochondria in non-fused and fused cells remains unchanged despite the dramatic morphological alterations in fused cells. Thus following cell fusion, BeWo mitochondria are transformed to have an appearance more like those of mitochondria in steroidogenic cells.

Objectives: Formation of syncytia from mononuclear cells leads to a number of changes in cellular physiology and function. We hypothesized that such changes will be reflected at the organelle level. Herein, we focus on alterations to the Golgi complex (GC) and trans-Golgi network (TGN) associated with BeWo cell fusion. Methods: Cultured BeWo cells were used as surrogates for cytotrophoblasts and were induced to fuse by treatment with forskolin (FK). BeWo cells treated with FK for 24, 48, and 72 hours were compared to untreated control cells using immunofluorescence, immunoblotting, and electron microscopy methods. Antibodies to the GC marker proteins GM130, giantin, and beta-COP were used along with TGN 46 and p230 for the TGN. In some experiments, JAR cells in the presence or absence of FK treatment were also used. Results: In control cells (non-fused), the GC and TGN had a perinuclear distribution typical of cultured cells as determined by immunofluorescence microscopy. In contrast, fused cells had dramatically altered GC and TGN architecture, which were typically very enlarged and often formed giant GC and TGN structures. There was a strong association between the GC and TGN with nuclei in syncytial structures. The immunofluorescence staining patterns suggested that the GC and TGN had increased in amount when compared to control cells. This was further investigated using immunoblotting methods; a time-dependent increase in GC and TGN markers proteins was found in FK-treated fused cells. Conclusions: We show that there is a dramatic alteration in the architecture of GC and TGN along with an increase in GC and TGN marker proteins in fused BeWo cells. We propose that this is due to increases in protein synthesis and subsequent post-translational processing occurring in syncytial structures. These results may have implications for increased protein trafficking and export necessary for syncytiotrophoblast function.

Abstracts / Placenta 32 (2011) A1–A149

P1.37 DRAMATIC MODULATION OF ADHESION COMPLEXES ACCOMPANY CELL FUSION Atsuko Ishikawa1,2, Toshiyuki Takeshita2, William Ackerman1, Dale Vandre1, John Robinson1 1 The Ohio State University, Columbus, OH/, United States, 2Nippon Medical School, Tokyo, Japan Objectives: Cell-cell fusion leading to syncytium formation is a hallmark of placental biology. The interaction of cells with the extracellular matrix occurs at specialized structures; several types of which have been identified in various cell types (focal complexes, focal adhesions, fibrillar adhesions, podosomes, and invadopodia). We hypothesized that cell-cell fusion alters the adhesive properties of cells and compared adhesion sites in mononuclear BeWo with those in fused cells. Methods: Cultured BeWo cells were used as surrogates for cytotrophoblasts and were induced to fuse by treatment with forskolin (FK). BeWo cells treated with FK for 24, 48, and 72 hours were compared to untreated control cells using immunofluorescence, immunoblotting, and fluorescence microscopy. Antibodies to proteins associated with adhesion were used to map the distribution and types of adhesion sites present in fused and non-fused cells. Fluorophore-labeled phalloidin was used to localize filamentous actin. Immunoblotting was used to determine the relative abundance of adhesion-related proteins. Results: Mononuclear BeWo cells had adhesion sites primarily of the focal adhesion type. Following cell-cell fusion, focal adhesions largely disappear and were replaced by smaller structures including podosome-like adhesion sites. The alterations in adhesion site morphology observed in fused cells were accompanied by dramatic alterations in the organization of intracellular actin. Control BeWo cells displayed prominent stress fibers and cortical actin as detected by phalloidin staining. The stress fibers terminated in vinculin-positive structures as in classical focal adhesions. Stress fibers largely disappeared from fused cells but phalloidin-labled actin was associated with the smaller adhesion sites. Conclusions: There was a dramatic remodeling of adhesion sites in BeWo cells following fusion that was accompanied by dramatic changes in the distribution of filamentous actin. We propose that these changes reflect the differing mechanical properties of mononuclear and fused BeWo cells.

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P1.38 EXPRESSION AND LOCALIZATION OF DLX3 AND PPARG IN BOVINE EXTRA-EMBRYONIC TISSUES DURING BINUCLEATED CELL DIFFERENTIATION Severine Degrelle1,3, Padma Murthi4,5, Daniele Evain-Brion2,3, Thierry Fournier2,3, Isabelle Hue1 1 INRA,UMR1198 Biologie du Développement et Reproduction, F-78352 Jouy-en-Josas, France, 2INSERM, U767, Université Paris Descartes, F-75006 Paris, France, 3PremUP Foundation, Faculté des Sciences Pharmaceutiques et Biologiques, F-75006 Paris, France, 4Department of Perinatal Medicine, Pregnancy Research Centre, The Royal Women’s Hospital, Parkville, Victoria 3052, Australia, 5Department of Obstetrics and Gynaecology, The University of Melbourne, Parkville, Victoria 3052, Australia Objectives: In ruminants, the differentiation of the trophoblast is marked by the formation of binucleated cells (BNC). Recent studies in human studies showed that the nuclear transcription factors DLX3 and PPARg play an essential role on trophoblast differentiation [1, 2]. However, their expression in bovine extra-embryonic tissues are largely unknown. Therefore, in this study we determined expression patterns of DLX3 and PPARg and their potential partner SP1 on proliferating and differentiating bovine extra-embryonic tissues. Methods: Bovine conceptuses were collected at Day-15 (D15), Day-18 (D18) and D-25 (D25) after artificial insemination (n¼5 in each group). After embryonic disc dissection, the extra-embryonic tissues were snapfrozen or fixed with 4% PFA. The expression of DLX3, PPARg and SP1 mRNA were quantified by real-time PCR and protein localization by confocal microscopy imaging following immunofluorescence labelling. Results: A progressive increase in the mRNA expression of DLX3 was observed from D18. However, PPARg and SP1 mRNA expression was evident only after D25. The protein expression by immunofluorescence on in toto extra-embryonic tissues detected nuclear localization of DLX3 and SP1 at D18 and co-localisation of DLX3, SP1 and PPARg in the nuclei of BNC was evident only in D25. Conclusion: Co-expressions of DLX3, PPARg and SP1 mRNA following implantation and the co-localization of the corresponding proteins in the nuclei of BNC suggest a potential role for these transcription factors in bovine BNC differentiation. [1] Chui et al., Placenta. 2010 Aug;31(8):691-7. [2] Tarrade et al., J Clin Endocrinol Metab. 2001 Oct;86(10):5017-24.

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Abstracts / Placenta 32 (2011) A1–A149

P1.39 IS STROMAL CELL DERIVED FACTOR-2 INVOLVED IN TROPHOBLAST DIFFERENTIATION?

P1.40 TROPHOBLAST DEVELOPMENT IS COMPROMISED BY THE MITOCHONDRIAL DYSFUNCTION.

Aline Lorenzon1,2, Shaker Farah3, Susan Fisher2, Estela Bevilacqua1 Cell and Developmental Biology Department - University of Sao Paulo, Sao Paulo, SP, Brazil, 2Department of Obstetrics, Gynecology, and Reproductive Sciences - University of California San Francisco, San Francisco, CA, United States, 3Biochemistry Department, Chemistry Institute, - University of Sao Paulo, Sao Paulo, SP, Brazil

Mateusz Kolanczyk1,6, Markus Pech1, Tomasz Zemojtel1, Hiroshi Yamamoto1, Ivan Mikula2, Maria-Antonietta Calvaruso3, Ricarda Richter4, Björn Fischer6, Anita Ritz1, Nadine Kossler1, Pavel Martasek2, Ralf Spörle1, Jan Smeitink3, Uwe Kornak1,6, Martin Vingron1, Leo Nijtmans3, Knud Nierhaus1, Robert Lightowlers4, Markus Schuelke5, Stefan Mundlos1,6 1 Max Planck Institute for Molecular Genetics, Berlin, Germany, 2Department of Pediatrics and Center for Applied Genomics, Ist Faculty of Medicine, Charles University, Prague, Czech Republic, 3Centre for Mitochondrial Disorders at the Department of Pediatrics, Radboud University Nijmegen Medical Centre, NijCentre for Mitochondrial Disorders at the Department of Pediatrics, Radboud University Nijmegen Medimegen, -, 4Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, -, 5Department of Neuropediatrics and NeuroCure Clinical Research Center, Charité University Medical Center, Berlin, Germany, 6for Medical Genetics, Charitè, Universitätsmedizin, Berlin, Germany

1

Background: We have previously shown that SDF-2 is widely expressed in human placenta, although its function is still unknown in mammals. Objectives: Herein we aim to elucidate whether SDF-2 is associated with human trophoblast differentiation. Methods: Primary cytotrophoblast cells were obtained from second trimester placentas by enzyme digestion and Percoll gradients. Cells were plated in matrigel and maintained in culture, with 5% CO2 and 21% O2, for 30 min, 24 h and 48 h for differentiation assays. An additional group was maintained for 24 h in 21% O2 and then submitted overnight to 2 % O2 for 1 h. Protein lysates were loading for Western Blotting analysis for SDF-2, and cells in coverslips were stained for SDF-2 and cytokeratin. Beta-actin was used as loading control and HIF and hCG were used as positive controls for hypoxia condition and trophoblast differentiation, respectively. Results: Protein concentration of SDF-2 was upregulated in normoxia after 24 h and down-regulated in hypoxia after 1 h. HIF and hCG confirmed hypoxia and differentiation conditions. Morphological analysis confirmed the differentiation process, as 24 h-cultured cytotrophoblast cells formed columns. Conclusions: Our preliminary data suggest that SDF-2 is somehow involved in the differentiation of human trophoblast. We are now focusing in understanding the exact role played by this protein in the trophoblast differentiation process and its biological meaning at the maternal-fetal interface. Supported by FAPESP.

Objectives: We have recently cloned and functionally analyzed a new gene which is of major importance for mitochondrial function. NOA1 is en evolutionarily conserved GTPase localizing to mitochondria in mammalian cell. We bioinformatically predicted and subsequently experimentally shown that NOA1 is required for mitoribosomal biogenesis. Methods: We analyze NOA1 function through generation of knock-out mice and analysis of the purified to homogeneity protein. Results: These studies revealed NOA1 is an essential mitochondrial protein, indispensable for normal development. Specifically, in-situ gene expression analysis revealed that NOA1 is required for development of trophoblast. Noa1-/- mice are severely growth retarded and die at gestation stage wE10.5. We could show that Noa1 inactivation ablates mitochondrial protein synthesis and causes global defect of phosphorylative oxidation. Consequently, NOA1 deficient cells exhibit diminished viability and reduced ATP level upon nutrient starvation. Additionally, Noa1-/- cells are impaired in caspase dependent apoptosis. The mito-protein synthesis defect was traced back to the defective assembly of the large mitoribosomal subunit. Conclusions: Thus NOA1 is a critical factor required for the mitochondrial function. The dependence of trophoblast development on NOA1 function exposes a crucial role of mitochondria in the process of maternal-fetal interface development.

Abstracts / Placenta 32 (2011) A1–A149

P1.41 ACETALDEHYDE EFFECTS ON APOPTOSIS AND MIGRATION

PLACENTAL

CELL

PROLIFERATION,

Sylvia Lui, Rebecca L. Jones, John D. Aplin, Clare L. Tower Maternal and Fetal Health Research Centre, University of Manchester, Manchester, United Kingdom Objective: Acetaldehyde is the major metabolite of ethanol following oral ingestion and some studies have suggested it to be the more harmful compound. In pregnant animal models, acetaldehyde is associated with smaller offspring and reduced litter size. Aberrations in placental development are known contributors to fetal growth restriction. We have previously shown that ethanol has an adverse affect on placental development and hypothesise that acetaldehyde also has a deleterious effect on extravillous trophoblast migration, trophoblast proliferation and apoptosis. Methods: Immunohistochemistry was used to analyse proliferation (Ki67) and apoptosis (cytokeratin M30) in a BeWo cell model (N¼5) and first trimester placenta (6-9wks; N¼5). Colorimetric readings of MTT assay were used to measure mitochondrial dehydrogenase activity of BeWo cells (N¼8). Treatment was over 48h with 0, 10, 20 and 40mM acetaldehyde. Invasion was measured by counting migration of the extravillous cytotrophoblast-derived cell line SGHPL-4 through a matrigel layer (N¼5) after 24h treatment. Statistical analysis was performed using Wilcoxon signed rank for fold change. Results: BeWo cells showed a trend towards decreased in proliferation at 10mM and 40mM acetaldehyde (p¼0.06). First trimester placental explants showed a similar trend for decreased proliferation at 40mM (p¼0.06). There was a significant decrease in mitochondrial activity at 10mM (p¼0.01) and 20mM (p¼0.02). SGHPL-4 invasion was reduced by acetaldehyde treatment at 20mM (p¼0.05) and at 40mM (p¼0.06). There was no effect on apoptosis in either BeWo cells or first trimester cytotrophoblasts. Conclusion: Acetaldehyde has a negative effect on cytotrophoblast mitochondrial activity, proliferation and invasive ability. This suggests it may contribute to the fetal growth restriction seen in fetal alcohol syndrome by adversely affecting placental development. Moreover the effects shown in this study are from concentration levels that reflect moderate levels of social drinking.

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P1.42 THE INFLUENCE OF BISPHENOL A ON OXIDATIVE STRESS AND SYNCYTIALISATION IN PLACENTAL TROPHOBLASTS Muralitharan Ponniah, Ellen Billett, Luigi De Girolamo Nottingham Trent University, Nottingham, United Kingdom Trophoblasts differentiate to form the outermost layer of the placenta (syncytiotrophoblasts), separating the foetus from the maternal circulation. These cells are subject to adverse environmental compounds, which may arise during pregnancy, from the maternal circulation and potentially harm placental development. The environmentally prevalent, endocrine disrupting compound, Bisphenol A (BPA) is used in various products including food packaging. This compound is known to leach into food and drinks and has been reported to be present in maternal serum and placental tissue. In this poster we report the effect of BPA on trophoblasts and syncytialisation using a human trophoblast cell model. We have established that physiologically ( 200 ng/ml) and environmentally ( 1000 ng/ml) relevant concentrations of BPA can increase the viability of trophoblasts that are subject to conditions of oxidative stress. Indeed, when glutathione levels are depleted for period of 72 hours leading to an increase in reactive oxygen species, a reduction in ATP levels and subsequently cell viability, BPA can reverse these effects and ameliorate cell death. In parallel studies we have also investigated the effect of BPA on syncytialisation. Firstly, we characterised forskolin-induced differentiation of human choriocarcinoma BeWo cells. Differentiation for 72 hours results in a decrease and re-distribution in the levels of e-cadherin and zonaoccludens-1 with a concomitant increase in cell fusion and human chorionic gonadotrophin secretion; all indicative markers of syncytialisation. In the presence of physiologically relevant concentrations of BPA these markers are altered corresponding to an impairment of syncytialisation. Furthermore, we have begun to characterise the interaction of BPA with cellular proteins using drug affinity responsive target stability (DARTS). This approach has identified selected targets which have relevance to syncytialisation. Taken together, these results suggest that BPA may interfere with normal placental development and function and requires further study.

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Abstracts / Placenta 32 (2011) A1–A149

P1.43 TRANSCRIPTIONAL ACTIVITY IN THE HUMAN SYNCYTIOTROPHOBLAST; UNRAVELLING DIFFERENCES BETWEEN SPROUTS AND KNOTS

P1.44 ESTABLISHMENT OF 3D SPHEROID CULTURE MODELS TO STUDY BOVINE TROPHOBLAST CELL DIFFERENTIATION IN VITRO

Norah M.E Fogarty1,2, Anne C. Ferguson-Smith1,2, Graham J. Burton1,2 1 Centre for Trophoblast Research, University of Cambridge, Cambridge, United Kingdom, 2Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom

Jan-Dirk Haeger, Nina Hambruch, Christiane Pfarrer University of Veterinary Medicine Hannover, Hannover, Germany

Background: Villous trophoblast consists of two compartments; the proliferating cytotrophoblast cells (CTBs) and the terminally differentiated syncytiotrophoblast (STB). CTB nuclei contain diffuse chromatin whereas STB nuclei range from being euchromatic to heavily heterochromatic. The most heavily condensed are seen in aggregates called syncytial knots. It was thought that the CTB-to-STB differentiation process involved induction of apoptosis and transcriptional shut-down, accompanied by increased nuclear condensation. It is now known that a proportion of STB nuclei are transcriptionally active. Immunostaining for RNA Polymerase II (RPol II) and Upstream Binding Protein (pUBF) show a range of transcriptional statuses in the syncytium. Stretches of active nuclei are seen to be punctuated by presence of an inactive nucleus, suggesting that transcriptional activity is regulated at the level of individual nuclei. We focus on the transcriptional status of recently incorporated STB nuclei, sprouts and knots. Transcriptional status in STB nuclei is currently being correlated with dynamic changes in epigenetic modifications. Method: Immunostaining for RPol II, pUBF and repressive histone modifications was performed on serial sections (11-39 weeks). As there are no known markers of knots and sprouts, suspected knots and sprouts were tracked through serial sections to confirm “true” status and to eliminate tangential sections. Immunofluorescence for Proliferating Cell Nuclear Antigen (PCNA) identified recently incorporated nuclei. Results: PCNA co-localised with RPol II. Sprouts contained RPol II and UBFpositive nuclei. Any “knots” which appeared to be transcriptionally active were revealed to be false knots upon tracking. Conclusion: Transcriptional activity in STB nuclei may be a feature of recent incorporation. Syncytial sprouts, which are associated with regions of proliferation, can be seen to contain transcriptionally nuclei. Syncytial knots are transcriptionally inactive and may be aggregations of aged effete nuclei. We hypothesise that transcriptionally inactive nuclei contain greater amounts of repressive histone modifications. NMEF is supported by the Anatomical Society.

Objectives: Invasive, multinucleated bovine trophoblast giant cells (TGC) are a hallmark of the bovine placenta but their differentiation in vivo is barely understood. In traditional culture system these cells are lost and cannot be studied. We hypothesized that confrontation of bovine trophoblast/fibroblast 3D co-culture spheroids (CCS) with uterine epithelial cell spheroids (BCECsp) could be particularly suitable to investigate feto-maternal communication and TGC differentiation in vitro. Methods: Fibroblasts were grown to confluence and trophoblasts were seeded on the latter. Resulting cells sheets were transferred to coated wells and cultured until CCS formation. BCECsp were generated in parallel. CCS were characterized by light microscopy, electron microscopy (EM) and immunofluorescence. BCECsp were analyzed by EM and immunohistochemistry. Proliferating cells in CCS were detected by Ki-67 antibody and apoptotic cells by active caspase-3 antibody. Furthermore CCS were either co-cultured with BCECsp or embedded in gels. Results: CCS and BCECsp were compact and round with outer transparent layers consisting of epithelial cells. CCS had three compartments (outside to inside): trophoblast cells, fibroblasts and spheroid core, while BCECsp consisted of two compartments: uterine epithelial cells and core. The outer epithelial cells of both spheroids were intact, cytokeratin positive and polarized. Ezrin was always located at the apical cell poles. In CCS the fibroblasts underneath the trophoblasts were morphologically intact and vimentin positive. The trophoblasts and fibroblasts proliferated (Ki-67 positive), while caspase-3 staining was only located in the spheroid core which consisted of debris and dying cells. In preliminary studies CCS and BCECsp interacted forming firm spheroid pairs. Additionally, CCS cells displayed invasion into collagen-I gels. Conclusion: We present CCS and BCECsp and their experimental utilization to study bovine trophoblast cell differentiation and feto-maternal interaction. The established techniques may also be valuable tools in regard to the differentiation of mouse and human trophoblast cells.

Abstracts / Placenta 32 (2011) A1–A149

P1.45 LOCALIZATION OF MELATONIN RECEPTORS IN VILLOUS AND EXTRAVILLOUS TROPHOBLAST CELLS FROM FIRST TRIMESTER PREGNANCIES Dave Lanoix1, Mélanie Cocquebert2, Thierry Fournier2, Cathy Vaillancourt1 INRS-Institut Armand Frappier, Université du Québec, Laval, Québec, Canada, 2INSERM, Unité 767, Université René Descartes, Paris, France 1

Melatonin-synthesizing enzymes and melatonin receptors (MT1, MT2 and RORa) are expressed and functional in human term villous trophoblast. Melatonin protects term villous trophoblast by inhibiting hypoxia/ reoxygenation-induced apoptosis. However the expression of melatonin receptors in human first trimester trophoblast has never been studied. Objective: The objective of this study was to determine the expression and intracellular localization of MT1 and MT2 G protein-coupled receptors and RORa nuclear melatonin receptors in first trimester trophoblast. Methods: The expression of MT1, MT2 and RORa melatonin receptors was analysed by immunohistochemistry in tissue and by real time RT-PCR in primary extravillous cytotrophoblast, villous cytotrophoblast and syncytiotrophoblast from first trimester placenta. Intracellular localization of melatonin receptors was determined by fluorescent immunocytochemistry in first trimester primary extravillous cytotrophoblast, villous cytotrophoblast and syncytiotrophoblast. Results: Melatonin receptors are expressed in first trimester villous and extravillous trophoblast. In first trimester placental tissue, melatonin receptor expression is stronger in villous cytotrophoblast than in syncytiotrophoblast or extravillous cytotrophoblast. In extravillous cytotrophoblast and villous cytotrophoblast, MT1 and MT2 receptors are localized on the membrane, cytoplasm and nucleus while RORa is only expressed in the nucleus. In the syncytiotrophoblast, the three receptors are only localized in the nucleus. Conclusion: Differential expression of melatonin receptors between the types of trophoblast cells suggests that melatonin may act through different signalling pathways in these cells. Taken together, these results show that melatonin could have a protective role in first trimester villous and extravillous trophoblast.

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P1.46 INFLAMMATION INDUCED BY LPS AFFECTS MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) AND RECEPTOR EXPRESSION AT MATERNAL-FETAL INTERFACE Miriam Faria1, Karollina Nascimento1, Mariane Martucci1, Adriana Costa1, Simone Correa Silva1, Luana Paulesu2, Estela Bevilacqua1 1 Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil, 2University of Siena, Siena, Italy Background: As a proinflammatory molecule MIF serum profile has been positively correlated with the severity of bacterial infection, whereas deletion of the MIF gene in mice seems to confer protection against lethal endotoxemia. MIF is expressed during pregnancy, particularly by trophoblast cells, in different species. Binding of MIF to the membrane receptor CD74 and co-receptor CD44 triggers specific intracellular signaling pathways and gene expressions associated with cellular death, cell proliferation or cell survival, among many others. Objectives: In this study, we sought to analyze in mice the expression and distribution of MIF and MIF complex receptor at maternal-placental interface in healthy and LPS-treated pregnant females. Injection of lipopolysaccharide into pregnant mice was used to induce an inflammatory condition on early gestation (gd7.5). Methods: Gene expression by RT-PCR and immunolocalization of MIF, CD74 and CD44 were performed in parallel with an enzyme-linked immunosorbent assay (ELISA) to measure the level of TNF-a on gd7.5 implantation sites in pregnant females that received 0.1 mg/g of body weight of LPS or saline (control). Results: We found that in non-treated pregnant mice gene expression and immunoreactivity for MIF was prevalent at the trophoblastic cells whereas for CD74 and CD44 gene expression and reactivity was observed mainly in decidual cells and leukocytes of the maternal-fetal interface. In LPS-treated animals there was a relevant decrease in MIF-MIF receptors expression during the following 6h after treatment. This finding was not correlated with TNF-a levels in the maternal-fetal interface; TNF-a was higher in LPStreated animals. Conclusion: Our results indicate that the LPS-induced inflammatory condition includes the increase of TNF-a levels at the maternal fetal interface, but not of MIF and receptors. This data suggests that MIF expression might be associated with a possible compensatory control mechanism in maintaining the local homeostasis by reducing the inflammatory reaction and allowing the pregnancy progress. Financial support: FAPESP, CNPq and CAPES.

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Abstracts / Placenta 32 (2011) A1–A149

P1.47 EFFECTS OF STAT1 SUPPRESSION ON ERK1/2 IN TROPHOBLASTIC CELLS

P1.48 CYTOGENETIC AND STAT3 EXPRESSION ANALYSIS OF HTR8/SVNEO

Francisco L. Pereira de Sousa1,2, Diana M. Morales Prieto1, Stephanie Ospina Prieto1, Wittaya Chaiwangyen1, Nelson Sass2, Silvia Daher2, Udo R. Markert1 1 University Hospital Jena, Department of Obstetrics, Placenta Laboratories, Jena, Germany, 2University Federal de Sao Paulo, Department of Obstetrics, Sao Paulo, Brazil

Maja Weber1, Anja Weise2, Kristin Mrasek2, Mario Párraga San Román3, Levon Khachaturyan1, Diana M Morales2, Thomas Liehr1, Udo R Markert1, Justine S Fitzgerald1 1 Placenta-Lab, University Hospital Jena, Jena, Thuringia, Germany, 2Institute for Humangenetics, University Hospital Jena, Jena, Thuringia, Germany, 3Biomedical Research Centre, School of Medicine, Universidad de Valparaiso, Valparaiso, Chile

Background: Migration and trophoblast invasion are controlled functionally along with the active participation of cytokines and growth factors. Two important intracellular signaling pathways are the Janus kinase/signal transducer and activator of transcription (JAK-STAT) and extracellular regulated kinase1/2 (ERK1/2). These pathways have been associated with the regulation of gene expression, cellular proliferation, differentiation, angiogenesis, embryo development and invasion in tumor and trophoblast cells. The aim of our study is to characterize and analyze the regulation and crosstalks of STAT1 and ERK1/2 in trophoblast cells and the identification of activating cytokines. Methods: The trophoblast derived cell line HTR-8/svneo and a choriocarcinoma cell line (JEG-3) were stimulated with interleukin-6 (IL-6), IL-11, granulocyte-macrophage colony-stimulating factor (GMC-SF), leukemia inhibitory factor (LIF) or oncostatine M (OSM). The the expression and phosphorylation of STAT1(tyr705) and ERK1/2 were analyzed by gel electrophoresis and Western blotting. Expression of STAT1 was inhibited by administration of 50 mM Fludarabine (2-Fluoro-ara-AMP) for 2, 4, 8, 24, 48 or 72 h or by using small interfering RNA (siRNA). Results: LIF and OSM induce STAT1 and ERK1/2 phosphorylation in HTR-8 and JEG-3 cells. Fludarabine inhibits the so induced phosphorylation of STAT1 when administered 48 or 72 h before stimulation. Simultaneously, ERK phosphorylation increases. In contrast, silencing of STAT1 by application of specific siRNA induces reduction of ERK1/2 phosphorylation. Conclusion: STAT1 in trophoblast cells can be activated by placental cytokines. Suppression of STAT1 by Fludarabine or siRNA influences activity of ERK1/2 which indicates a crosstalk between both pathways. Current studies will clarify the reason for the different effects on ERK1/2 in trophoblastic cells.

Objective: The HTR8/SVneo cell line are non-cancerogenic cells derived from human extavillous trophoblast (EVT) that were transfected with a plasmid containing a large T-antibody (TAG) from “simian virus 40(SV40)”. HTR8/SVneo share many characteristics with EVT (invasion, morphology), but are proliferative and immortal. HTR8/SVneo model EVT functionally, but are still genetically anonymous. Our aim was to characterize the HTR8/SVneo cytogenetically in order to better understand functional alterations, but also to distinguish these cells from others. Furthermore, we wished to establish constitutive Signal Transducer and Activator of Transcription 3 (STAT3) expression is in this cell line, since this is found in several tumors, in SV-transfected- and in embryonic stem cells (ESC), and is considered responsible for immortality, excessive proliferation and invasion of these cells. Methods: HTR8/SVneo were karyotyped through Multiplex-Fluorescence In Situ Hybridisation (M-FISH). In short, chromosomes were arrested in metaphase with colcemid and stained with specific colours for specific chromosomes. Chromosomal aberrations were identified on 20 metaphases of a single passage with ISIS software. STAT3 expression was identified per Western blot and cytoimmunohistochemistry. Results: The composite karyotype demonstrates relatively stabile, clonally-recurring aberrations found in the hypotriploid cell line (3n-) HTR8/SVneo. STAT3 is constitutively expressed in HTR8/SVneo cells to an intensive degree. This molecule is found here both in tyrosine and serine phosphorylated forms. Conclusion: This karyotype can function as an identification marker for HTR8/SVneo. The structural and numerical aberrations are typical for immortal cell lines. Physiological trophoblast cells become hyperdiploid after they leave the cell cycle and differentiate into the invasive phenotype, which is also congruent with our findings. Constitutive expression of STAT3 in HTR8/SVneo is in line with SV-transfection, but since STAT3 is one marker for ESC characteristics, it might be questioned whether HTR8/ SVneo have acquired ESC characteristics through a SVneo-transfection mediated back-differentiation into a trophoblast stem cell.

Abstracts / Placenta 32 (2011) A1–A149

P1.49 ADAM12 AND DYSFERLIN IN HUMAN PLACENTA Maja Weber1, Sebastian San Martin2, Udo R. Markert1, Justine S. Fitzgerald1 Placenta-Lab, University Hospital Jena, Jena, Thuringia, Germany, 2 Biomedical Research Centre, School of Medicine, Universidad de Valparaiso, Valparaiso, Chile 1

Objectives: Cell-cell fusion is the main biological event driving the formation of syncytia, such as skeletal muscle, osteoclasts and the synccytiotrophoblast (STB). ADAM12 (a disintegrin and metalloproteinase domain 12; meltrin a) plays a role in this regulation. Dysferlin, a transmembrane protein, is associated with the development of certain muscular dystrophies, due to its involvement in the repair of damaged skeletal muscle cell membranes. Dysferlin is also concentrated in STB and in cytotrophoblast (CTB) undergoing spontaneous fusion. Our aim was to investigate the expression profile of these two proteins in placental samples of problem pregnancies associated with altered trophoblast invasion and fusion: choriocarcinoma, mola, intrauterine growth retardation (IUGR). Methods: We analyzed 10 each of samples derived from above pathologies and control (normal term pregnancy placentae). Immunoperoxidase staining was accomplished to detect ADAM12 or dysferlin, and double stained with HLA-G. Results: Both proteins were detected in all samples. ADAM12 expression is more intensive in thick compared to thin areas of STB and in trophoblast of normal compared to IUGR placentae. The ADAM12 expression pattern in mola appears heterogenous, while in choriocarcinoma homogenous. ADAM12 was detected in trophoblastic cells in the process of invasion and in cells surrounding decidual vessels. Dysferlin staining morphology differed between trophoblast subsets. As described elsewhere, dysferlin appears line-formed along the apical membrane of fused STB. In invading non-fused trophoblast, dysferlin is expressed without the line formation. The line-formation reappears in areas surrounding decidual vessels. Conclusion: ADAM12 is widely expressed in normal and pathological placentae. Differences of expression intensity may be involved in trophoblast disorders that are associated with trophoblast invasion. The pattern of expression for both proteins in areas surrounding decidual vessels indicate that fusion events are taking place here. Further experiments are needed to verify if and why trophoblast fusion is occurring in the decidua.

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P1.50 BLOOD PRESSURE CONTROL ACROSS PREGNANCY IS CONCEPTUS REGULATED B. Anne Croy, Michael Adams, Valerie Barrette Queen’s University, Kingston, ON, Canada Objectives: Mean arterial pressure (MAP) has a normal pattern of fluctuation across pregnancy in mice and women. In mice, pattern changes align with stages of placental development [Burke SD et al Hypertension 55:729]. Because we saw hypertension in mice with late fetal deaths and stable MAP in vas-mated, non-decidualized pseudopregnant females, we hypothesized that gestational MAP is regulated by conceptuses not ovarian hormones. To advance this hypothesis, the role of endometrial decidualization which recruits uterine Natural Killer cells and induces decidual vascular changes, had to be excluded as regulating the early postimplantation drop in MAP. Methods: Randombred CD1 females were surgically implanted with PAC10 radiotransmitters (DSI, St. Paul MN). After 10 days recovery and copulation with a normal or vasectomized male, continuous arterial recording began. At gestation-equivalent day (ged) 2, vas-mated females, were transcervically infused with Concanavalin-A-coated agarose beads to induce decidualization. Data were collected until ped 11; then animals were euthanized to confirm decidualization. Recordings from decidualized pseudopregnant females were compared with normal pregnant and nondecidualized pseudopregnant females. Results: As previously reported for normal, inbred females, pregnant randombred CD1 females had a statistically declining MAP from gestation day (gd) 7 to 10. MAP then rose. The nadir in MAP occurred at gestation day 9 and was -11.77mmHg  2.34 compared to pre-pregnancy baseline. For vas-mated females with or without confirmed endometrial decidualization, no changes in were seen MAP from pre-pregnancy values. MAP at ged9 was -2.86mmHg  1.9 for decidualized and -1.77mmHg  4.03 for non-decidualized vas-mated females. Conclusions: Decidualization in the first half of pregnancy does not affect maternal blood pressure control. From implantation to mid pregnancy, the conceptus overrides its mother’s mechanisms for blood pressure control and depresses MAP by as yet undefined pathways. Supported by NSERC, CIHR, CFI and the CRC Program. [email protected].

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Abstracts / Placenta 32 (2011) A1–A149

P1.51 VASODILATOR EFFECT OF HYDROGEN SULPHIDE (H2S) IN THE PERFUSED HUMAN PLACENTA Tereza Cindrova-Davies1, Emilio Herrera2, YG Niu1, Dino Giussani1, Graham Burton1 1 Centre for Trophoblast Research, University of Cambridge, Cambridge, 2 Programa de Fisiopatologia, Universidad de Chile, Santiago, Chile Background: Alongside nitric oxide and carbon monoxide, novel H2S is the third gaseous signalling transmitter, which modulates physiological function. Endogenous H2S production is catalysed by cystathionine b-synthase (CBS) and cystathionine g-lyase (CSE) through desulphydration of cysteine1, 2. H2S can hyperpolarise cell membranes by targeting KATP channels3, and thus relax smooth muscle cells to act as a vasodilator agent. However, the role of endogenous or exogenous H2S has not been studied in the fetoplacental circulation or complications of pregnancy. Methods: Perfusion pressure and flow (Transonic Systems Inc., USA) were measured in 8 caesarean-delivered human placentae. Single lobes were perfused with equilibrated (95% O2/5% CO2, pH 7.4) Earle’s bicarbonate buffer (containing dextran, bovine serum albumin, L-arginine and heparin) by cannulating the chorionic artery and vein of the lobe. Following preconstriction with the thromboxane mimetic U46619 (10-7 mol/L), increasing doses of NaHS solution (10-12 to 10-6 mol/L) were infused. Glibenclamide (10-5 mol/L) was administered to block KATP channels, and LNAME (10-5 mol/L) to inhibit endogenous nitric oxide synthesis. In addition, protein and RNA were extracted from 6 Caesarean-delivered, 6 preeclamptic and 6 IUGR placentae to determine the expression of CBS and CSE under normal and pathological conditions. IHC determined the placental localisation of CBS and CSE. Results: Administration of the H2S donor NaHS led to significant concentration-dependent reductions in perfusion pressure and vascular resistance. The vasodilator effect of NaHS (AUC arbitrary units: 3977) was significantly diminished by L-NAME (3326) and further reversed by glibenclamide (2665; P<0.05). This implies that the primary vasodilator actions of H2S in the human placenta are mediated via KATP channels, and an additional interaction between H2S and NO. IHC confirmed the presence of CBS and CSE in the syncytiotrophoblast and vascular smooth muscle cells, respectively. CBS protein and RNA levels remained unchanged, whilst those of CSE were significantly down-regulated in preeclamptic and IUGR placentae. Conclusion: We show for the first time that H2S is a potent vasodilator in the human placenta and that the enzymes responsible for its synthesis are down-regulated in placentae from pathological pregnancies associated with impaired placental blood flow and fetal growth restriction. Supported by the Wellcome Trust.

1. Bukovska, G., Kery, V. & Kraus, J.P. Expression of human cystathionine beta-synthase in Escherichia coli: purification and characterization. Protein Expr Purif 5, 442-8 (1994). 2. Erickson, P.F., Maxwell, I.H., Su, L.J., Baumann, M. & Glode, L.M. Sequence of cDNA for rat cystathionine gamma-lyase and comparison of deduced amino acid sequence with related Escherichia coli enzymes. Biochem J 269, 335-40 (1990). 3. 3. Reiffenstein, R.J., Hulbert, W.C. & Roth, S.H. Toxicology of hydrogen sulfide. Annu Rev Pharmacol Toxicol 32, 109-34 (1992).

P1.52 ULTRASOUND CHARACTERIZATION OF HEMODYNAMIC AND CARDIAC ADAPTATIONS IN PREGNANT MICE LACKING SPIRAL ARTERIAL MODIFICATION Jianhong Zhang, Michael A. Adams, B. Anne Croy Queen’s University, Kingston, Ontario, Canada Objective: Uterine vascular remodelling is a key feature of pregnancy. Human pregnancies deficient in gestational spiral arterial (SA) remodelling are associated with pre-eclampsia, implantation site hypoxia and fetal growth restriction and/or loss, complications not seen in mice lacking SA remodelling. The aim of this study was to identify the murine cardiovascular adaptations that protect mothers and conceptuses from adverse outcomes in pregnancies with deficient SA remodelling. Methods: Micro-ultrasound scans were used to evaluate hemodynamic and cardiac functions in virgin, gestation day (gd)2, 4, 7, 9, 10, 12, 14, 16, 18 and postpartum BALB/c (WT) mice and BALB/c-Rag2-/-/Il2rg-/- mice, an immunodeficient strain that does not experience SA remodelling. At each time point, 3-6 females were imaged and for each gd10-18 pregnancy 2-4 implantation sites were imaged. Results: In comparison to WT dams with SA remodelling, Rag2-/-/Il2rg-/dams had normal SA flow velocities, greatly elevated uterine artery flow velocities between gd10-16 and less placental vascular flow. These alterations were accompanied by transient maternal cardiac hypertrophy with greater cardiac output and by cardiovascular changes in conceptuses. Conceptus alterations included higher flow velocities in the umbilicalplacental circulation between gd10-16 and heart rate depression that persisted to term. Conclusion: Pregnant alymphoid mice appear to compensate for absence of SA modification by transient changes in maternal heart structure and function and by modifications of the fetal-placental circulation. These cardiovascular adaptations normalize placental hemodynamics and placental size by late gestation and support successful fetuses that grow into larger than normal adults. Similar responses to incomplete human SA modification rather than hypertension may be the fundamental mechanisms that underlie postpartum gains in cardiovascular disease risk reported in affected women and their offspring. Supported by NSERC, CIHR, CFI & CRC Awards.

Abstracts / Placenta 32 (2011) A1–A149

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P1.53 CHARACTERISATION OF POTASSIUM (KD) CURRENTS IN CHORIONIC PLATE ARTERIAL SMOOTH MUSCLE CELLS

P1.54 LEPTIN EXPOSURE IMPAIRS PLACENTAL CHORIONIC PLATE ARTERY CONSTRICTION

Melissa F. Brereton, Mark Wareing, Susan L. Greenwood Maternal and Fetal Health Research Centre, The University of Manchester, M13 9WL, England, United Kingdom

Christina E. Hayward, Elizabeth J. Cowley, Tracey A. Mills, Colin P. Sibley, Mark Wareing The University of Manchester, Manchester, United Kingdom

Objective: Appropriate control of chorionic plate arterial (CPA) tone is important for regulating fetoplacental blood flow and a successful pregnancy. Smooth muscle cell (SMC) Kþ channels participate in excitationcontraction coupling and regulate arterial tone. We previously demonstrated that inhibition of the voltage-gated Kþ channel Kv1.5 enhance CPA constriction. Here we test the hypothesis that CPA SMCs express Kv1.5 channels. Methods: SMCs were isolated from CPAs of normal term placentas using methods developed in this laboratory. Whole-cell patch clamp was used to record Kþ currents and channel function assessed by extracellular application of; 4-AP (Kv inhibitor; 5mM), TEA (Kv þ KCa inhibitor; 5mM), DPO-1 (Kv1.5 inhibitor; 3mM), charybdotoxin (ChTx; BKCa þ IKCa inhibitor 100nM), iberiotoxin (IbTx; BKCa inhibitor; 100nM). Kv1.5 and BKCa protein expression was assessed in isolated SMCs with immunocytochemistry. Results: Whole-cell Kþ currents (IK) were outwardly-rectifying at potentials greater than þ50mV. TEA inhibited whole-cell Kþ currents (IK) at potentials positive to þ50mV with a 665% decrease observed at þ80mV (n¼11 cells, N¼6 placentas). However, TEA predominantly inhibited KCa channels as similar effects were seen with ChTx (n¼4, N¼2) and IbTx (n¼6, N¼3). 4-AP and DPO-1 significantly inhibited IK between 0mV and þ40mV (pA/pF at þ40mV: control 4.7 0.8; 4-AP 2.90.4, P<0.05; n¼11, N¼8; control 2.8 0.6, DPO-1 1.50.5, P<0.05; n¼9, N¼7). Isolated SMCs (N¼3) displayed positive immunostaining for Kv1.5 and BKCa. Conclusion: Previous observations that 4-AP and DPO-1 increase CPA constriction, coupled with inhibition of IK current by 4-AP/DPO-1 and the expression of Kv1.5 protein in SMCs, support a role for Kv1.5 in regulating CPA tone. Furthermore, the inhibitory effects of TEA, ChTx and IbTx on IK indicate BKCa channel activity in CPA SMCs. SMC Kv1.5 and BKCa channels may control fetoplacental blood flow in normal pregnancy and could be targets to improve blood supply in fetal growth restriction.

Maternal obesity (body mass index; BMI 30) triples the risk of preeclampsia (PE)1, a condition characterised by vascular endothelial dysfunction2. PE and obesity are associated with increased plasma leptin3,4. In the placenta, chorionic plate artery (CPA) blood flow is influenced by tone oscillations; these are disrupted in PE5 but the mechanism remains unidentified. Objectives: To test the hypothesis that leptin alters vascular function in CPAs in normal (non-obese) pregnancy. Methods: Term placentas (N¼12) were collected post-delivery (vaginal or Caesarean section) following uncomplicated pregnancies (maternal BMI 18.5-24.9). CPAs were mounted on a wire myograph, normalised at 0.9L5.1kPa and equilibrated (20min; 37 C; 5%O2/5%CO2). CPA contractility was assessed with 120mmol KCl, following which, a dose-response curve to U46619 (thromboxane A2 mimetic; 0.1-2000nM) was performed. Post-wash, tone, amplitude and frequency of oscillations produced by a sub-maximal (30nM) U46619 concentration were assessed in the presence/absence of bradykinin (endothelial-dependent vasodilator; 1mM). Experiments were repeated after 60 minutes incubation with leptin (100ng/ml) and responses compared with time-matched controls. Data are meanSEM. Results: Consistent with previous reports, U46619-induced tone oscillations (tone 4.60.5kPa, amplitude 2.30.3kPa and frequency 0.001 0.0001Hz) were observed in 58% of CPAs. Exposure to leptin resulted in a downward shift in the U46619 dose-response curve towards reduced constriction, compared with pre-treatment controls (p¼0.05; repeated measures two-way ANOVA). Leptin had no effect on tone, oscillation amplitude or frequency. Conclusion: Agonist-induced constriction of CPAs was impaired by leptin, consistent with previous reports of vasodilatory effects6. This may contribute to the impaired vascular function observed in obesity and PE; however, tone oscillations and endothelial function were unaffected. Further studies will investigate whether other adipokines affect vascular function and promote endothelial dysfunction. This study was funded by the British Heart Foundation. 1 Bodnar 2005 Ann Epidemiol;15:475; 2Ashworth 1997 BJOG;104:1152; 3 Ramsay 2002 JCEM;87:4231; 4Anim-Nyame 2000 Hum Reprod;15:2033; 5 Sweeney 2008 Placenta;29:356. 6Lembo 2000 Diabetes;49:293

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Abstracts / Placenta 32 (2011) A1–A149

P1.55 EFFECTS OF CIRCULATING AND LOCAL ANGIOTENSIN II ON MATERNAL AND FETAL HEARTS DURING PREGNANCY

P1.56 EXTRACELLULAR MATRIX COMPONENTS OF THE HUMAN UTERINE SPIRAL ARTERY WALL DIFFER IN A SPATIAL AND TEMPORAL MANNER

Lydia Hering1, Florian Herse1, K. Bridget Brosnihan2, Dominik N. Muller1, Ralf Dechend1 1 Experimental and Clinical Research Center, Berlin, Germany, 2Wake Forest University School of Medicine, Winston-Salem

Gendie E. Lash, Andrew Robson, Barbara A. Innes, Stephen C. Robson, Judith N. Bulmer Newcastle University, Newcastle upon Tyne, United Kingdom

Objectives: We hypothesize that activation of renin-angiotensin system (RAS) influences fetal and maternal heart hypertrophy during pregnancy in a rat model for preeclampsia. We therefore tested the influence of circulating plasma and local uteroplacental Angiotensin II on heart hypertrophy and fibrosis markers. Methods: A transgenic (hAogenxhRenin) rat model developing high blood pressure and proteinuria during pregnancy (PEþ) was compared with the opposite transgenic cross (hRenxhAogen) (no hypertension and no proteinuria (PE-)) and with a chronic ANGII infusion model (1 mg/kg/min) developing high blood pressure and proteinuria during pregnancy (ANGþþ). Angiotensin II concentration was determined in maternal plasma and the uteroplacental unit using radioactive immunoassay technology. Marker for heart hypertrophy and fibrosis were evaluated by quantitative RT-PCR in maternal and fetal hearts; on protein level fibrosis was detected by sirius red staining. Results: In the PEþ model we found elevated circulating and local Angiotensin II levels, in the PE- rat solely local Angiotensin II was increased whereas the ANGþþ model showed exclusively high circulating Angiotensin II levels. Maternal ANP was highly elevated in PEþ and elevated in PE- and ANGþþ whereas maternal BNP as a chronic heart hypertrophy marker was elevated in all groups. Fibronectin, collagen 1 and connectice tissue growth factor expression were increased in PEþ maternal hearts. PEþ and PE- fetal hearts showed heart pathologies as assessed by ANP and BNP dysregulation, but not by heart fibrosis. ANP, BNP and fibrosis markers were actually depressed in ANGþþ fetal hearts. Conclusion: Maternal and fetal hearts showed pathologies, independent of hypertension, indicating an important role for the local RAS activation, which is also observed in the uteroplacental unit.

Objectives: Uterine spiral artery (SpA) remodeling is required for successful pregnancy. Initial stages of SpA remodeling are characterised by vascular smooth muscle cell (VSMC) separation and extracellular matrix (ECM) breakdown before loss of VSMCs, fibrinoid deposition and extravillous trophoblast cell (EVT) invasion. This study examined ECM components of SpA in non-pregnant and pregnant uteri throughout the menstrual cycle and in early pregnancy. Methods: Immunohistochemistry was performed on non-pregnant endometrium/myometrium from three menstrual cycle stages (proliferative (P), early (ES) and mid/late (M/LS) secretory) and placental bed biopsies (8-16 weeks gestational age) for fibronectin, laminin, collagen IV and elastin. Quickscore assessment of immunostaining in myometrial, basal endometrial and functional endometrial/decidual SpA was performed. In pregnancy samples SpA were split into three categories; nontransformed and partially transformed  EVT. Results: SpA remodeling did not alter expression intensity for any of the factors. Fibronectin showed spatial and temporal variation in expression: expression in myometrium was reduced in pregnancy compared to M/LS stage while in functional endometrium/decidua it was increased. Fibronectin and collagen IV expression was greater in functional endometrium than in myometrium or basal endometrium. In myometrium collagen IV expression increased from P to ES and M/LS stages and into pregnancy. Laminin expression was low in SpA in all non-pregnant samples and in pregnant myometrium but was increased in decidual SpA in early pregnancy. In myometrium and basal endometrium all vessels had an internal elastic lamina but only w50% also contained an external elastic lamina; neither elastic lamina was observed in functional endometrium. Conclusion: ECM components of SpA show both spatial and temporal changes but do not alter in early stages of remodeling irrespective of the presence of EVT. Most strikingly, SpA in functional endometrium differ from those in myometrium and basal endometrium.

Abstracts / Placenta 32 (2011) A1–A149

P1.57 FETAL GENDER DETERMINES DIFFERENCES IN PROLIFERATION AND ANGIOGENESIS OF HUMAN PLACENTAL ARTERIAL ENDOTHELIAL CELLS Silvija Cvitic, Ursula Hiden, Luciana Lassance, Heidi Midel, Gernot Desoye Department of Obstetrics and Gynecology, Medical University of Graz, Graz, Austria Objectives: It is now established that males and females differ in susceptibility and response to diseases that are characterized with endothelial dysfunction. Male gender seems to be a risk factor by itself for differential outcome in normal pregnancies and pregnancy disorders. However, little is known about the underlying molecular mechanisms. We hypothesized that gender influences fundamental processes of the placental endothelium, such as proliferation and angiogenesis. To test this we examined primary human arterial endothelial cells (AEC) isolated from placentas of male and female fetuses. Methods: Male and female AEC were cultured with 5% FCS without supplements under 12% and 21% oxygen for 24, 48 and 96 hours. Proliferation was determined by counting viable and dead cells. In vitro angiogenesis was studied in media containing FCS (2% and 5%) or 2% cord blood serum (normal and diabetic) under 21% oxygen and quantified (branching points, meshes, tube length). Gender dependent biological processes were identified by Panther software fed with differentially expressed genes (microarray). Results: Under both oxygen concentrations in low density culture (24h and 48h) male AEC had higher number of viable cells, but after 96h, being more confluent, female AEC had significantly overgrown male AEC, showing higher proliferation and lower death rate. With FCS (2% and 5%) male AEC had more branching points, meshes and higher total tube length than female cells. Similar results were obtained with normal cord blood serum. However, diabetic serum reduced the angiogenic potential of male AEC at 21% oxygen. Microarray analysis showed upregulation of angiogenesis- and apoptosis-related genes in male AEC. Conclusion: Male and female AEC proliferate at different rates. This may be due to gender-dependent response to cell-cell contacts. Male AEC have higher angiogenesis potential in FCS and normal cord blood serum, but seem to be more sensitive to diabetic serum than female AEC. P1.58 NOTCH2 AND THE TRANSCRIPTIONAL CO-REPRESSOR TLE3 ARE ESSENTIAL FOR PROPER DEVELOPMENT OF MATERNAL VASCULAR SYSTEM IN THE MOUSE PLACENTA. Malgorzata Gasperowicz1, Florian Otto2, James C. Cross1 1 Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada, 2Tumorzentrum ZeTuP, Center for Tumor Detection, Treatment and Prevention, CH-9006 St. Gallen, Switzerland Objectives: The proper functioning of maternal vascular system in placenta is essential for development of healthy offspring. Unlike usual mammalian blood vessels, which are lined by endothelial cells, the placental maternal blood spaces in rodents are lined with specialized trophoblast cell types called trophoblast giant cells (TGC). The unusual ability of TGC to form vascular spaces without participation of endothelial cells is termed pseudovasculogenesis. Our aim is to get insight into molecular mechanisms governing this process. TLE3 is a general transcriptional co-repressor acting downstream of the Notch signaling pathway. Both NOTCH2 receptor and TLE3 are highly expressed in mouse placenta. Methods: To investigate the role of TLE3 in development, we generated a mouse strain deficient in the Tle3 gene. We performed a detailed analysis of wildtype, Tle3 deficient and Notch2 deficient placentas at different developmental stages. Results: Tle3 mutants die in utero and their death is associated with underdevelopment of the placenta, which shows a strong defect in maternal blood vasculature. We showed that maternal blood is carried out of the labyrinth via channels, which are formed by a novel TGC subtype channel-TGCs. Our data demonstrate that Tle3 and Notch2 are co-expressed in those TGC and both Tle3 and Notch2 mutants show a defect in differentiation of TGC-lined channels and lacunae that take maternal blood out of the placenta. Conclusion: Notch2 and Tle3 are key factors in establishing of functional maternal vascular system in mouse placenta.

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P1.59 TROPHOBLAST MIGRATION IN A LOW SHEAR STRESS CO-CULTURE MODEL: CONSEQUENCES FOR SPIRAL ARTERY REMODELLING IN THE FIRST TRIMESTER OF HUMAN PREGNANCY Joanna James1,2, Guy Whitley1, Darrel Greenhill3, Andreas Hoppe3, Judith Cartwright1 1 St. George’s, University of London, London, United Kingdom, 2University Of Auckland, Auckland, New Zealand, 3Kingston University, Surrey, United Kingdom Objectives: In early human pregnancy trophoblast migrate along uterine spiral arteries (SA) remodeling them into high-flow low-resistance vessels. Inadequacies in SA remodelling have been associated with pre-eclampsia and IUGR. Until 10-12 weeks of gestation trophoblast plug SA, preventing maternal blood flow into the intervillous space, resulting in slow, highresistance flow in these vessels. Our aims were to examine the consequences of these low shear stress conditions on trophoblast migration, adhesion molecule expression and attraction to chemotactic factors. Methods: SGHPL-4 trophoblast cells were cultured alone or on endothelial cells (ECs) for 6-12hrs under 0.5-6dyne/cm2 of shear stress using a BioFlux200 system, and imaged by time-lapse microscopy. Computer-based imaging algorithms were developed to quantify the speed and direction of migration. Adhesion molecule expression was determined by immunohistochemistry and confocal microscopy. Chemotaxis assays were run using parallel flow in BioFlux channels. Results: Trophoblast cultured either alone or on ECs did not undergo directional migration in 0.5 and 2dyne/cm2 cultures, however in 4 and 6dyne/cm2 trophoblast were stimulated to migrate with the direction of flow (n¼4, p<0.001). Low shear stresses did not affect the speed of trophoblast migration, or the expression or distribution of the adhesion molecules E-selectin, a4, b1 or avb3 integrin.Trophoblast cultured on ECs migrated into media containing IL-8, MCP-1 or RANTES (n¼5, p<0.05). Conclusions: This work challenges the dogma that trophoblasts migrate down spiral arteries retrograde to flow by demonstrating that as shear stress increases trophoblasts are stimulated to migrate in the same direction as flow. The low shear stresses generated by trophoblast plugging of SA in the first trimester may favour SA remodelling by preventing the migration with flow seen at higher shear stresses, allowing trophoblast to migrate down the arteries in response to alternate stimuli such as uterine or endothelial cell derived chemotactic factors.

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Abstracts / Placenta 32 (2011) A1–A149

P1.60 3D CONFOCAL IMAGING OF MOUSE PLACENTAL VASCULATURE AFTER LYCOPERSICON ESCULENTUM(TOMATO) LECTIN INJECTIONS INTO MATERNAL AND FETAL CIRCULATIONS Stephanie Lang, Helen Jones, Khaled Omar, Foong-Yen Lim, Sundeep Keswani, Timothy Crombleholme, Mounira Habli Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio Objectives: 3D reconstruction of the placental vasculature is obtained by use of either vascular casts or computer-assisted reconstruction after microtomy. A limitation of both techniques is lack of in situ visualization of placental vasculature. The objective of this research is to develop a technique to visualize and reconstruct in situ 3D-architecture of maternal and fetal compartments of whole mount placenta. Methods: At gestational day 20, the uterine artery, supplying one uterine horn, was ligated using 3-0 silk at the ovarian and cervical ends. Using a 31-gauge-insulin needle, 500ug/ml DyLight594-Lycopersicon Esculentum(Tomato) Lectin (TL) was injected into the uterine artery and allowed to perfuse for 10 minutes. Placenta and fetal sacs were removed en bloc from the uterus. 20ug/ml Fluorescein-TL was injected into the fetal circulation via heart and allowed to circulate for 10 minutes before harvesting the placenta. Placentas were fixed in 4% paraformaldehyde overnight at 4oC, washed in PBS, and underwent serial dehydration to 100% ethanol then methanol. Placentas were cleared using 1:1 solution of methanol:Murray’s Clear(2:1 Benzyl-Benzoate:Benyl-Alcohol)(MC) and incubated until the placentas appeared translucent, then two changes of 100%MC. Cleared placentas were visualized using Zeiss Confocal Microscopy(Zen9.0). Results: Our results demonstrate that DyLight594-TL(red) selectively binds maternally to the spiral arteries of the decidua, and the syncytiotrophoblast lining the maternal blood sinuses. Fluorescein-TL(green) selectively binds fetal endothelial cells. Clearing the placenta enables confocal microscopy to simultaneously visualize and reconstruct the in situ 3D-architecture of maternal and fetal placental vascular compartments. Conclusions: Confocal microscopy of cleared TL injected placentas is a novel technique to visualize both the fetal and maternal placental vasculature in situ whilst the placental vascular tree architecture is preserved. This technique can be used to quantify vessel density in order to study the differential response of the fetal and maternal vasculature in models of placental pathologies.

P1.61 AORTIC INTIMA MEDIA THICKNESS IN TWINS: A NEW MODEL Silvia Visentin, Vincenzo Zanardo, Stefania Vedovato, Daniele Trevisanuto, Francesco Cavallin, Silvia Chiarelli, Erich Cosmi University of Padua, Padua, Italy Objective: To assess aotic intima media thickness (aIMT), a sign of endothelial function, by ultrasonography in twin fetuses with intrauterine growth restriction, small for gestational age and appropriate for gestational age, as previously studied in singleton pregnancies. Methods: This was a longitudinal study performed between January 2009 and January 2011. Fetuses were classified as having IUGR if the estimated fetal weight (EFW) was below the 10th percentile and umbilical artery pulsatility index was greater than 2 standard deviations; SGA if the EFW was below the 10th percentile and appropriate for gestational age (AGA) if the EFW was between the 10th and the 90th percentiles. Abdominal aortic intima media thickness was measured in each twin fetus at a median gestational age of 32 weeks. Results: One hundred forty six twins were enrolled in the study: 20 IUGR and 20 SGA of one of the two, and 80 AGA. Aotic intima media thickness median values were significantly higher in IUGR and SGA than in AGA (0.9 mm compared with 0.7 mm, p¼0.003; 0.9 mm compared with 0.5 mm, p<0.0001) and was higher in SGA than in control twins (0.7 mm compared with 0.5 mm, p¼0.003). Conclusion: Aortic intima media thickness in twins with IUGR and SGA shows differences with respect to those who were AGA, reflecting also in twin pregnancies a correlation between impaired growth in utero, Doppler abnormalities and early signs of vascular dysfunction.

Abstracts / Placenta 32 (2011) A1–A149

P1.62 WHOLE BLOOD THROMBOELASTOMETRY NORMAL PREGNANCY.

(ROTEMÒ)

PROFILE

IN

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P1.63 CHARACTERIZATION OF NORMAL PREGNANCY

CIRCULATING

MICROPARTICLES

DURING

Silvia Visentin, Luca Spiezia, Claudia Radu, Maria Bon, Elena Campello, Cristiana Bulato, Erich Cosmi, Paolo Simioni University of Padua, Padua, Italy

Silvia Visentin, Claudia Radu, Luca Spiezia, Sonila Dhima, Elena Campello, Omar Anis, Erich Cosmi, Paolo Simioni University of Padua, padua, Italy

Objectives: Whole blood ROtation ThromboElastoMetry analyser (ROTEMÒ; Pentapharm, Germany) is a laboratory tool for studying the simultaneous and integrated effects of different blood components involved in the dynamic process of clot formation and lysis. Aim of the study was to analyse changes in coagulation during normal pregnancy. Methods: We compared ROTEMÒ coagulation profile in 35 healthy nonpregnant women (mean age 4514 yrs) and 45 women during first (T1, n. 15; mean age 286 yrs), second (T2, n. 15; mean age 324 yrs) and third (T3, n. 15; mean age 335 yrs) trimesters of normal pregnancy. The INTEM and EXTEM represent assays in which the intrinsic and the extrinsic coagulation pathways are triggered. NATEM was used to assess clot formation in the absence of activation of the clotting cascade other than recalcification. The FIBTEM assay was used for the assessment of the role of fibrinogen following inhibition of the platelets. Parameters considered were: Clotting Time (CT), the time from the beginning of the coagulation analysis until increase in amplitude of 2 mm; Clotting Formation Time (CFT), the time between an increase in amplitude from 2 to 20 mm; Alfa angle, the tangent to the clotting curve through the 2 mm point; Maximum Clot Firmness (MCF), the maximum amplitude; Area Under The Curve (AUC) defined as the area under the velocity curve. Results: Pregnant women compared to healthy controls showed a significant increase in MCF, alfa-angle, and AUC in all the four tests considered. Moreover, a significant and progressive reduction of CT (in NATEM, and FIBTEM) and CFT (in EXTEM, NATEM, and FIBTEM) during pregnancy was observed [Table 1]. Conclusions: ROTEMÒ analysis showed a marked increase in coagulability during normal pregnancy. Further studies are needed to investigate the role of ROTEMÒ in the risk of thrombosis or haemorrhage in pregnancy.

Objectives: Microparticles (MPs) are microvesicles of diameter 0,1-1 mm, shed from plasma membranes of different cell types, upon activation or apoptosis. Pregnancy is characterized by an hypercoagulant and inflammatory state with increased incidence of thromboembolic events. The knowledge about the role of circulating MPs during pregnancy is still unknown. Aim of the study: Investigate the quantitative and qualitative characteristics of MPs during the three trimester of pregnancy. Methods. We measured, by flow cytometry, total MPs (AnnexinV), CD61/ CD62P for platelets (PMP), anti-CD62E for endothelial (EMP) and TFbearing MPs. Furthermore, for each sample, we determined the procoagulant activity of plasmatic phospholipids (PPL). Results. Total MPs and PMPs levels decrease from I to II trimester (p ¼ 0.001; p ¼ 0.033) and increase from II to III trimester (p ¼ 0.001; p ¼ 0.025). Activated EMPs and EMPs-Annexin Vþ increase from I to II trimester (p ¼ 0.0045; p ¼ 0.296) and decrease from II to III trimester (p ¼ 0.001; p ¼ 0.424).TF-MPs decrease from I to II trimester (p ¼ 0.127) and increase in the III trimester (p ¼ 0.000). Concerning PPL activity, in I and II trimester it was higher compared to III trimester (p>0.05). Conclusions. Our study confirms the presence of elevated levels of MPs during pregnancy. During the I trimester we observed elevated MPs AnnexinV þ, in according to the literature which explain that the mechanism regulating syncytial fusion includes early apoptotic events and changes in the organization of the membrane phospholipids. We observed high levels of MPs expressing TF in early pregnancy and during the III trimester. The changes of the coagulation balance toward a procoagulant state is an important protective factor before delivery. These knowledge could be used in future as an approach for early diagnosis and possible therapy for various diseases in pregnancy. Circulating MPs are found in physiologic and pathophysiological conditions and play an important role in coagulation and inflammation.

Table 1 ROTEMÒ coagulation profiles.

CT (sec.)

INTEM EXTEM NATEM FIBTEM CFT (sec.) INTEM EXTEM NATEM INTEM Alfa angle ( ) EXTEM NATEM FIBTEM MCF INTEM EXTEM NATEM FIBTEM AUC (mm*100) INTEM EXTEM NATEM FIBTEM

Healthy T1 women n[35 (n[15)

T2 (n[15)

T3 (n[15)

17527 6014 684103 6723 8321 9927 25167 744 705 489 666 574 575 486 134 5589541 5524537 4787562 1278378

16834 5312 45019 4915 6712 7921 12131 773 755 675 726 656 656 606 2210 6534541 6441633 6048576 2129944

14641 5620 49878 508 6510 7115 11823 772 763 674 716 664 673 604 215 6594349 6616311 6160467 2048462

17331 548 472150 5010 7514 8318 14340 753 744 668 734 634 645 595 174 6278359 6362436 5875420 1710351

MPs/ mL

I Trimestre

Annexin Vþ CD61þ/CD62Pþ CD62Eþ/ AnnessinaVþ CD62Eþ / Annexin VMPs TFþ

7495,10  4100,86 3747,05  2055,16 9492,72  5003,85 674,45  531,10 377,50  295,01 616,94  296,96 182,10  126,95 210,41  116,12 186,78  87,48

II Trimestre

III Trimestre

2271,60  676,24

3156,77  1699,74 1509,06  531,99

106,90  34,24

91,50  31,33

165,39  73,51

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Abstracts / Placenta 32 (2011) A1–A149

P1.64 ANALYSIS OF EARLY POST-IMPLANTATION MOUSE VASCULAR DEVELOPMENT USING WHOLE MOUNT IN SITU STAINING B. Anne Croy1, Shawn Murphy2, Edith Lord2, Scott Gerber2 Queen’s University, Kingston, Ontario, Canada, 2University of Rochester Medical Center, Rochester, NY, United States

1

Objectives: Decidual angiogenesis is a dynamic, carefully regulated, multistep process in successful hemochorial placentation. Detailed understanding of early steps, potential vessel heterogeneity and roles of decidual leukocytes are lacking. A whole-tissue mount technique was assessed for visualization and phenotyping of vessels and their spatial relationships with leukocytes and conceptuses. Methods: At gestation days (gd)6-9, deciduas from normal C57Bl/6 (WT), interferon gamma null (Ifng-/-) and alymphoid (ALY; genotype Rag2-/-/Il 2rg-/-) mice were dissected to remove the uterine wall except at its mesometrial attachment. Deciduas were halved longitudinally and placed into medium containing Fc-blocking and multiple fluorescent probeconjugated antibodies. After incubation, tissues were mounted onto slides and examined by epi-fluorescence microscopy [Gerber et al, Br. J. Canc. 88: 1453, 2003]. Results: Whole-mount staining visualized vessel structure with minimal disturbance of spatial relationships. Four distinct WT decidual vascular zones were seen - two anti-mesometrial layers with finer web-like vessels in the primary decidual zone and a less dense outer vascular rim in the secondary decidual zone, lateral decidual vessels that appeared to be unique and receiving invading trophoblast cells and basal decidual vessels. Decidual vessels were positioned within clouds of CD45þ cells that occupied a greater area in Ifng-/-. Many CD45þ cells expressed CD31 (PECAM-1) but not the uNK cell marker NK1.1. At gd6, angiogenic sprouts were present anti-mesometrially; by gd7, vessels showed a more mature phenotype. In ALY, vessels of decidua basalis differed and large numbers of stellateshaped cells present throughout the decidua were more easily recognized. Conclusions: Whole-mount vascular staining comprehensively defines early implantation site vasculature, including capillaries. Its application to genetically-modified or therapeutically-manipulated mice will be important for understanding the influences of cytokines and other molecules in promotion of pregnancy. Supported by NSERC, CIHR, CFI and the CRC Program (AC) and NIH awards to SPM, EML and to SAG [email protected]

P1.65 TGF BETA-RECEPTOR-DEPENDENT ANGIOSTIMULATION THROUGH THE HYPERGLYCOSYLATED ISOFORM OF HUMAN CHORIONIC GONADOTROPIN. Sarah Berndt1,5, Julien Detilleux1, Silvia Blacher1, Sophie Perrier d’Hauterive1,2, Christel Péqueux1, Carine Munaut1, Ilpo Huhtaniemi4, Thierry Fournier5, Danièle Evain-Brion5, Agnès Noël1, Jean-Michel Foidart1,2 1 University of Liège, Laboratory of Tumor and Developmental Biology, Groupe Interdisciplinaire de Génoprotéomique Appliquée-Research (GIGA-Cancer), Liège, Belgium, 2University of Liège, Department of Gynaecology and Obstetrics, Liège, Belgium, 3University of Liège, Center of Immunology, Liège, Belgium, 4Department of Surgery and Cancer, Imperial College London, Hammersmith Campus, London, United Kingdom, 5Université Paris Descartes, Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 767, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, France Objectives: Hyperglycosylated hCG (H-hCG) produced early in pregnancy by invasive trophoblast cells may contribute to the angiogenic process occurring at the foeto-maternal interface at the time of implantation. Methods: To test this hypothesis, we purified H-hCG from Jeg-3 choriocarcinoma cell line conditioned medium. The models used to investigate the angiogenic effect were: the aortic ring assay applied to wild type and LH/hCG Receptor (LHR)-deficient mice, HUVEC and AoSMC proliferation and migration assays. Transient transfection studies with a luciferase reporter gene assay showed genomic activation of SMAD responsive elements after H-hCG treatment. In vitro and ex vivo angiogenesis and western blotting experiments were conducted with or without hyperglycosylated hCG treatment associated or not with TGFb inhibitors (SB431543, TGFb-receptor (TGFb-R) type 2 blocking antibody). The presence of TGFb-R type 2 was assessed by western blotting on our study cell lines and activation of second messenger SMAD 2 was evidenced by western blotting directed towards the phosphorylated isoform in comparison to its unphosphorylated one. Results: We first demonstrated a potent angiogenic effect of Jeg-3 conditioned medium and purified H-hCG (100 mIU/ml). By using LHR-deficient mice, we showed that this effect was independent of the classical hCG signaling pathway. Implication of TGFb-R type 2 signaling pathway was confirmed on HUVEC and AoSMC proliferation and migration. A synthetic inhibitor and a TGFb-R type 2 blocking antibody completely abolished the effect observed under H-hCG treatment in these experiments. Activation of the endothelial and mural TGFb-R type 2 pathways was demonstrated by SMAD 2 phosphorylation and genomic activation of SMAD responsive elements. Conclusions: H-hCG induces a strong angiogenic effect on endothelial and mural cells through TGFb-R type 2 signaling pathway. This underlines the pivotal role of this hCG glycoform in the success of human embryo implantation.

Abstracts / Placenta 32 (2011) A1–A149

P1.66 INCREASED EXPRESSION OF HOMEOBOX GENE TGIF-1 CONTRIBUTES TO ABERRANT FETO-PLACENTAL ANGIOGENESIS IN HUMAN IDIOPATHIC FETAL GROWTH RESTRICTION. Padma Murthi1, Tilini Gunatilake1, Simon Chua1, Guy Whitley2, Judith Cartwright2, Rosemary Keogh1, Shaun Brennecke1 1 University of Melbourne Department of Obstetrics and Gynaecology and Department of Perinatal Medicine Pregnancy Research Centre, Royal Women’s Hospital, Parkville, Victoria, Australia, 2Centre for Developmental and Endocrine Signalling, Division of Basic Medical Sciences, St. George’s, University of London, London, United Kingdom Objectives: Aberrant angiogenesis and endothelial cell (EC) dysfunction are associated with major pregnancy disorders such as fetal growth restriction (FGR) 1. Homeobox genes are known regulators of endothelial cell development and angiogenesis in the cardiovascular system2. Targeted disruption of homeobox gene, TGIF-1 (Transforming growth factor induced factor) in mouse models resulted in placental dysfunction3. However, the role of TGIF-1 in human placental angiogenesis is largely unknown. Therefore, the aims of this study were to determine the expression of TGIF1 in FGR-affected placentae compared with gestation-matched control (GMC), to investigate the functional role of TGIF-1 in feto-placental EC angiogenesis and to identify down-stream targets of TGIF-1. Methods: Placentae (n¼25) from idiopathic FGR-affected placentae and in GMC were collected and TGIF-1 mRNA expression was determined by realtime PCR. An immortal cell line SGH1E7 derived from the human umbilical vein endothelial cell (HUVEC) was utilized to determine the role of TGIF-1 in angiogenesis. Following inactivation of TGIF-1 using siRNA transfection (S1 and S2 and a non-specific control, NC) in SGH1E7 cells, angiogenesis was measured for 24h using a network formation assay. Down-stream target genes of TGIF-1 were identified following TGIF-1 inactivation using pathway specific angiogenesis array (Applied Biosystems). Results: TGIF-1 mRNA expression was significantly increased in FGR compared with GMC [1.38 0.27, FGR (n¼25) vs. 1.080.21, control (n¼25), t-test, p<0.05]. Following siRNA treatment, the SGH1E7 cells exhibited a greater angiogenic potential (1.01 0.01 (NC) vs. 1.38 0.17 (S1), 1.51  0.35 (S2), n¼4, p<0.01, ANOVA). Down-stream target genes of TGIF-1 following siRNA treatment in SGH1E7 were identified as integrinaV, Tie- 1, angiopoietin-1 and Neuropilin. Conclusion: These results suggest that TGIF-1 is an important regulator of feto-placental angiogenesis in human FGR.

References : 1. Kingdom, J. et al., 2000. Eur J Obstet Gynecol Reprod Biol 92, 3543. 2. Gorski, D.H and Walsh, K. 2000. Circ Res 87, 865-872. 3. Bartholin L et al. Develpmental Biology, 2008; 319 (2): 285-97.

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P1.67 LEPTIN STIMULATES TUBE FORMATION IN FIRST TRIMESTER TROPHOBLASTIC CELLS, HTR-8/SVNEO Sanjay Basak, Asim Duttaroy Dept of Nutrition, Faculty of Medicine, University of Oslo, Oslo, Norway Leptin, a 16-kDa-peptide hormone, appears to play an important role in female reproductive biology. Leptin has been suggested to be involved in fetal and placental development during pregnancy. Maternal plasma leptin concentrations undergo large changes during pregnancy and may affect feto-placental growth and development. The main purpose of this study was to investigate the effect of leptin on early placentation process such as tube formation and lipid metabolism. We also examined the influence of leptin on expression of lipid metabolic genes in these cells. Methods: In order to study the effects of leptin on fatty acid uptake a first trimester trophoblast cell line, HTR-8/SVneo was used. These cells were pre-stimulated in presence of leptin (25ng/ml or 50ng/ml) for 24h prior to the radiolabelled fatty acid (Arachidonic, eicosapentaenoic, docosahexaenoic acids at 100mM concentration). After 3h incubation, the uptakeradiolabelled fatty acids was determined. Under similar experimental conditions the effects of leptin on gene expression was determined by RTPCR. The tube formation assay (as a measure angiogenesis) was performed on matrigel in a 96-well plate. Result: Leptin at 25ng/ml increased uptake of DHA by 48% compared with control whereas it had no effect on the uptake of ARA and EPA by these cells. Leptin also stimulated tube formation process by increasing tube length as well as numbers compared with the control (320065 vs 89020 pixel). The use of VEGF inhibitor decreased the tube formation significantly. Leptin stimulated expression of FABP4 and ACSL5 genes after 24 hours incubation in these cells. Conclusion: Leptin may stimulate early placentation by increasing the tube formation process in first trimester trophoblast cells. Increase in the uptake of DHA by leptin may also positively modulate angiogenesis. Tube formation by leptin may be associated with VEGF in these cells.

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Abstracts / Placenta 32 (2011) A1–A149

P1.68 HYPOXIA INDUCES ELECTRON TRANSPORT CHAIN DYSFUNCTION IN HUMAN PLACENTAL MITOCHONDRIA Francesca Colleoni1, Hong-wa Yung1, Nisha Padmanabhan1, Irene Cetin2, Martha C. Tissot van Patot3, Graham J. Burton1, Andrew J. Murray1 1 Department of Physiology, Development & Neuroscience, University of Cambridge, Cambridge, UK, 2Unit of Obstetric and Gynecology, Department of Clinical Sciences, L. Sacco; University of Milan, Milan, Italy, 3 Department of Anesthesiology, University of Colorado Denver Health Sciences Center, Aurora, Colorado, USA Objectives: Fetal growth is critically dependent on energy metabolism in the placenta, with oxidative phosphorylation at the inner mitochondrial membrane providing the majority of ATP requirements. Oxygen levels in the placenta are therefore vitally important, and studies of high altitude pregnancies have suggested that chronic hypoxia during pregnancy contributes to impaired fetal growth. However, the molecular mechanism by which O2 deficiency might impair growth has not yet been elucidated. Therefore, the aim of this study was to determine the effect of different O2 concentrations on mitochondria in a placental cell line, a primary placental cell culture and intact human placentas. Methods: Human JEG-3 cells (a trophoblast-like cell line) and primary cultures of human placental fibroblasts were grown at 21%, 10% and 1% atmospheric O2 concentrations for 4 days. Mitochondrial O2 consumption was measured using appropriate substrates and inhibitors to investigate the function of complexes I, II and IV of the respiratory chain. In addition, we measured protein levels of complexes I-IV and ATP-synthase by western blotting and mitochondrial DNA (mtDNA) levels by qPCR. Finally, protein levels of complexes I-IV and ATP-synthase were measured in human placentas from pregnancies at sea level and 3100 m. Results: In both JEG3 cells and fibroblasts cultured at 1% O2, mitochondrial respiration via complexes I and IV was impaired, alongside loss of protein levels of complexes I and IV in JEG3 cells, and of complex IV in fibroblasts. No differences in mtDNA levels were measured in either JEG3 cells or fibroblasts at 1% O2. In high altitude placentas, levels of complex I were lower than in sea level placentas of the same gestational age. Conclusion: This study has, for the first time, demonstrated altered mitochondrial function in placental cells in response to hypoxia. Specific changes at complexes I and IV might compromise energy metabolism and thus underlie impaired fetal growth.

P1.69 HYPOXIA DECREASES MATRIX METALLOPROTEINASE(MMP)-2 ACTIVATION WITHOUT INCREASING HYPOXIA INDUCED FACTOR(HIF)1ALPHA IN EARLY HUMAN TROPHOBLAST. Taihei Tsunemi, Akira Onogi, Katsuhiko Naruse, Masayoshi Akasaki, Toshiyuki Sado, Taketoshi Noguchi, Yoshihiko Yamada, Hidekazu Oi, Hiroshi Kobayashi OB/GYN, Nara Medical University, Kashihara, Nara, Japan Objectives: Extravillous trophoblast cells (EVT) invasion in early pregnancy occurs in low-oxygen environment. Matrix metalloproteinase (MMP) is a key enzyme in the cell invasion process and usually increased by hypoxia induced factor (HIF). However, there have been few studies about the influence of low oxygen environment on HIF and MMP activation of primary trophoblast in early first trimester. In this study, we investigated HIF-1alpha and MMP2 secretion by primary EVTs in normal or hypo/ hyperoxia condition, to clarify the pathway of low oxygen on proteases that may contribute to the fair placental development. Methods: Placental samples (5-15 weeks gestation) were obtained from women undergoing elective surgical termination of pregnancy with written informed consent. Cytotrophoblast was separated with Percollbased method and cultured on Matrigel for 24hrs. Expression of HIF1alpha protein was semi-quantified with Western Blot. Activity and expression of MMP2 was investigated by gelatin gel zymography and ELISA. The culture was performed under 20% oxygen, 5%, and 5% that repeated three times of hypoxic stimulation for one hour (0.1%). All data were shown as a comparison of 20% or 0.1% with 5%. Results: The amount of HIF-1alpha protein on the cell membrane fraction was not alters after 0.1% oxygen stimulation. Concentration of latent form MMP2 in EVTs cultured supernatant was increased in both 0.1% and 20% oxygen condition. However, activity of MMP2 was decreased in 0.1%, instead of increasing in 20%. Conclusion: These results suggested that hypoxia might inhibit MMP2 activation of EVT cells in early first trimester of pregnancy, and HIF was not a crucial factor for the MMP activation. The appropriately low oxygen environment may suppress the excessive production and activity of proteases including MMP2, which seems to be necessary for fair invasion of trophoblast in the first trimester of human pregnancy.

Abstracts / Placenta 32 (2011) A1–A149

P1.70 Stacy Zamudio1, Michael Stark2, Nicholas Illsley3, Abdulla Al-Khan1, Vicki Clifton2 1 Hackensack University Medical Center, Dept. Ob/Gyn, Hackensack, NJ, United States, 2Robinson Institute, University of Adelaide, Adelaide, SA, Australia, 3UMDNJ-New Jersey Medical School, Newark, NJ, United States Introduction: Placental 11b-HSD2 protects the fetus from elevated maternal glucocorticoids by converting cortisol to inactive cortisone. Inhibition of 11b-HSD2 reduces fetal growth and contributes to programming effects on adult health (hypertension, hyperglycemia, insulin resistance); this is similar to the effects of fetoplacental hypoxia in experimental animals. In humans high altitude (HA) pregnancies result in placental and fetal hypoxia and diminished fetal growth. However decrement in birth weight (BW) is less in natives of HA than more recent migrants. We hypothesized that HA would reduce 11b-HSD2 activity, and would differ between natives (Andean Native Americans) vs. migrants (EH, European-Hispanic). Methods: Villous core samples were flash frozen in liquid N2 immediately after C-section. 11b-HSD2 activity was measured using thin-layer chromatography (n¼64 at 400 m [LA] n¼63 at 3600 m). Analysis was by ANOVA with Tukey’s multiple comparisons.

Ă

Results: HA reduced BW by 235 g in Andeans vs. 428 g in E-H (p<0.0001). Girls had greater BW reduction than boys (-488 g girls vs -309 g boys E-H; -302 g girls vs -163 g boys Andeans p<0.05). 11b-HSD2 activity was similar in both ethnic groups at LA. E-H girls had higher 11b-HSD2 than boys (Figure) and both sexes had an increase at HA. 11b-HSD2 did not change at HA in Andean girls, but decreased among boys (p<0.05). As a result 11bHSD2 activity levels were >50% greater in migrants than natives at HA. BW was not related to 11b-HSD2 activity, nor to indicators of hypoxia (erythropoietin, sFlt-1, PaO2). Conclusion: They hypothesis concerning ethnic differences at HA was supported, but not the hypothesis that 11b-HSD2 levels would be reduced. Only Andean boys had a decrease in 11b-HSD2 activity under conditions of chronic hypoxia. The magnitude and direction of changes in each group does not support a direct relationship with fetal growth. We speculate that increased 11b-HSD2 may protect the fetus against hypoxia-associated elevation in maternal stress hormones in non-adapted HA migrants. Support: NIH HD0427327, TWO744, HD46892, NSF BNS 0138567

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P1.71 EVIDENCE OF PROTEIN SYNTHESIS INHIBITION IN HIGH-ALTITUDE PLACENTAS: IMPLICATIONS FOR SMALL FOR GESTATIONAL AGE BABIES Hong wa Yung1, Mathew Cox1, Francesca Collenoi1, Martha Tissot van Patot2, Graham Burton1 1 University of Cambridge, Cambridge, Cambridgeshire, UK, 2University of Colorado Denver Health Sciences Center, Aurora, Colorado, USA Objective: To test for evidence of protein synthesis inhibition in highaltitude placentas. Pregnancy at high altitude is associated with small for gestational age (SGA) babies. Altitude and fetal weight are negatively correlated, with a reduction of 100g in birth weight per 1000m increase in altitude. Although hypobaric hypoxia is the obvious key factor, the molecular mechanisms behind the aetiology are still unclear. AKT-mTOR signalling is the central regulatory pathway for protein translation, cell metabolism, survival, growth and proliferation. In pathological intrauterine growth restriction, loss of AKT-mTOR signalling and protein synthesis inhibition has been demonstrated to be one of the possible mechanisms underlying the pathogenesis of the disorder. Methods: Snap-frozen samples from placentas delivered by non-laboured elective caesarean section from normotensive non-smoking women at sea level and 3100m (Leadville, Colorado) were used to analyse the growth and proliferation signalling pathways. Human choriocarcinoma cell lines and primary placental fibroblasts were cultured under 1% and 10% O2 to test whether activation of signalling pathways observed in high-altitude placentas resulted from hypoxia. Results: Samples from high altitude exhibited increased phosphorylation of eukaryotic initiation factor 2 a-subunit, and reduction of AKT signalling through decreased AKT phosphorylation at both the Threonine 308 and Serine 473 residues. mTOR complex 1 signalling, which regulates protein synthesis, was compromised as there was a reduction of Raptor and decreased phosphorylation of 4E binding protein 1 (4E-BP1). Increased of P-eIF2a and reduced AKT signalling were also observed in both choriocarcinoma cell lines and placental fibroblasts cultured under 1% O2. Conclusion: Down-regulation of AKT and mTOR signalling with increased P-eIF2a provides evidence of protein synthesis inhibition in high–altitude placentas, and can be mimicked in in vitro cell culture through hypoxia. Reduction of protein synthesis will limit growth of the placenta, which in turn restricts fetal growth. Supported by Wellcome Trust.

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Abstracts / Placenta 32 (2011) A1–A149

P1.72 REGULATION OF FETAL ANTIGEN EXPRESSION IN THE HUMAN PLACENTA BY HYPOXIA Caitlin Linscheid1, Lei Qui1, Herbert Hodes2, Margaret Petroff1 1 University of Kansas Medical Center, Kansas City, KS, United States, 2 Center for Women’s Health, Overland Park, KS, United States Objectives: Pregnancy is a unique immunological situation in which the immune system of the mother must accommodate a semi-allogeneic fetus. Trophoblast cells express a number of antigens that stimulate the maternal immune system, including the minor histocompatibility antigen, HMHA1. In this study we detailed the spatial and temporal expression of HMHA1 in the human placenta, and investigated whether oxygen levels could influence its expression in trophoblast cells. Methods: We analyzed mRNA and protein from normal first, second and term placental tissues. RT-PCR was used to determine if HMHA1 mRNA could be found in whole placental lysate, purified cytotrophoblast cells and fetal cord blood, and immunohistochemistry to map the cellular localization of HMHA1 protein. To ask whether oxygen regulates HMHA1 mRNA expression, term cytotrophoblast cells were cultured under 2%, 8% and 21% oxygen, or in the presence of the hypoxia mimetic cobalt chloride. Results: HMHA1 mRNA was found in whole placental lysate, purified cytotrophoblast cells and fetal cord blood. HMHA1 protein was found in extravillous trophoblasts, fetal macrophages and leukocytes across gestation and, in the first trimester placenta, in the syncytiotrophoblast. Culturing purified cytotrophoblast cells in 2% oxygen strongly upregulated HMHA1 mRNA as compared with 8% and 21% oxygen. Similarly, treatment of these cells with cobalt chloride increased HMHA1 mRNA as compared with untreated controls. Collectively, these results suggest that low oxygen delivery to the first trimester placenta may influence the expression of HMHA1. Conclusion: HMHA1 mRNA and protein are expressed in the human placenta and appear to be regulated by oxygen in trophoblast cells. This may be of importance in disease states such as preeclampsia, in which areas of the placenta are exposed to fluctuating levels of oxygen, thus potentially altering the fetal antigenic load encountered by the maternal immune system.

P1.73 PEROXYNITRITE STIMULATES 86RUBIDIUM EFFLUX FROM CYTOTROPHOBLAST CELLS: A ROLE FOR INTERMEDIATE CONDUCTANCE CALCIUM-ACTIVATED POTASSIUM CHANNELS Paula Diaz, Colin P. Sibley, Susan L. Greenwood Maternal and Fetal Health Research Centre, University of Manchester, Manchester Objectives: Nitrative stress in pre-eclampsia is thought to dysregulate trophoblast renewal and function. We previously reported that peroxynitrite (ONOO-) increases 86rubidium (86Rb) efflux, a marker of potassium (Kþ) permeability, from placental villous fragments. Here we explore which Kþ channels are activated by ONOO- using cytotrophoblast cells in primary culture. Methods: Kþ channel activity was assessed indirectly by measuring 86Rb efflux from cytotrophoblast cells isolated from normal term placentas (n¼4). At 66h of culture, cells were incubated with 86Rb (2h). After washing, 86Rb efflux was measured at 1min intervals over 15min (basal 86 Rb efflux) and/or exposed to various treatments over 5-15min (experimental period). % 86Rb efflux/min was expressed as (86Rb effluxt/86Rb in cellso)x100 and data were analyzed by a two-way ANOVA. Results: 86Rb efflux was stable over the experimental period. Basal 86Rb efflux was increased 2.8 fold (p<0.0001) by 10-4M ONOO-. This increase was significantly reduced by 5mM Ba2þ (Kþ channel blocker; 20%, p<0.01) and 10mM TRAM34 (intermediate conductance Ca2þ-activated Kþ channel blocker; IKCa; 40%, n¼2). Exposing cells to a hyposmotic buffer (145mOsm/ Kg/H2O) increased 86Rb efflux 3.6 fold (p<0.0001). This effect was enhanced by ONOO- (1.4 fold; p<0.0005) and inhibited by 50% with Ba2þ (p<0.0001) and by 70% with TRAM34 (n¼2). Opening IKCa channels with 100mM EBIO (IKCa channel opener) increased 86Rb efflux 8.4 fold which was completely blocked (>90%) by TRAM34 (n¼2). Conclusion: ONOO- stimulates both basal 86Rb efflux, and efflux following altered cell volume status, in part through an effect on IKCa channels. Inappropriate activation of these channels could compromise cell volume homeostasis, trophoblast turnover, nutrient transport and endocrine function in pre-eclampsia, a pregnancy condition associated with nitrative stress. Supported by CONICYT-Becas Chile 72090593 and Action Medical Research.

Abstracts / Placenta 32 (2011) A1–A149

P1.74 IMMUNOHISTOCHEMICAL LOCALIZATION OF BILIRUBIN OXIDATION IN HUMAN PLACENTA Michiko Suzuki, Takashi Nikaido, Rie Kawaguchi, Aikou Okamoto, Seiji Isonishi, Kazuhiko Ochiai, Tadao Tanaka Jikei University School of Medicine, Tokyo, Japan Objectives: Pregnancy is a state of oxidative stress, and in certain pathologic pregnancies such as those complicated with pregnancy-induced hypertension (PIH) oxidative stress has been recognized to be heightened. However, the response in human placenta is not fully clarified. Bilirubin is an intrinsic antioxidant, produced from heme through biliverdin which is catalyzed by heme oxigenase-1 (HO-1), and generates oxidative metabolites called biopyrrins (BPns) as a result of the reaction with reactive oxygen species. BPns are immediately excreted in urine and can reflect the severity of oxidative stress. Then in this study, to elucidate whether the oxidative stress may affect placental physiology and function, this heme catalytic pathway and oxidative response in human placenta was morphologically investigated. Methods: Placental tissues from ten patients complicated with PIH and ten normal pregnant women were examined immunohistochemically, using anti-bilirubin (24G7) and anti-HO-1 (EP1391Y) monoclonal antibodies. Myocardial infarction tissue was used as a positive material control.All specimens were fixed with formalin and VENTANA automatic immunostainer was used. Results: Of EP139Y, immune-localizations were demonstrated around decidual spiral arteries, especially with atherosis, in the area of infarction and villous stroma accompanied with peri-villous fibrin deposits, and in the syncytial knot. 24G7 -localizations were identified around the area of infarction and decidual vasculopathy. These proteins were stained with more diffuse and high intense degree in PIH than normal placenta. Conclusions: This is the first report to demonstrate the localization of bilirubin metabolism-related factors, HO-1 and BPns immunohistochemically, in human placenta. These factors related with bilirubin oxidation were strongly immunostained in PIH placenta than normal one. These results suggest that the oxidative stress implicates impaired placental micro-circulation and the occurrence of PIH. In addition, it may be conceivable that the urinary level of BPns become a possible biomarker of PIH in clinical application.

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P1.75 HIGH RESOLUTION RESPIROMETRY AND MTDNA CONTENT IN PLACENTAL CELLS AND TISSUES OF PREECLAMPSIA AND INTRAUTERINE GROWTH RESTRICTION Chiara Mandò1, Miriam Figus1, Clara De Palma2, Maria Antonella Marino1, Francesca Parisi1, Michela Pacei3, Daria Trabattoni3, Emilio Clementi2, Irene Cetin1 1 University of Milan_Dept. Clinical Sciences_Unit of Obstetrics and Gynecology and Center for Fetal Research Giorgio Pardi, Milan, Italy, 2University of Milan_Dept. Clinical Sciences_Unit of Pharmacology, Milan, Italy, 3University of Milan_Dept. Clinical Sciences_Unit of Immunology, Milan, Italy Objectives: Oxidative stress is likely occurring in placental cells in clinical conditions associated with placental insufficiency, such as preeclampsia (PE) and IUGR. We previously demonstrated higher mitochondrial DNA (mtDNA) levels in IUGR placentas. Here we investigate mtDNA in PE placentas and we present an innovative technique, High Resolution Respirometry (HRR), on cytotrophoblast cells (CTC) from PE, IUGR and normal placentas (NP), measuring cell oxygen consumption, thus the respiratory chain efficiency. Methods: mtDNA was measured by Real-Time PCR in 20 PE placentas, with (n¼14) or without (n¼6) IUGR, and 45 NP. CTC were isolated from 3 PE, 2 IUGR and 4 NP and characterized by cytofluorimetry. Substrates and inhibitors of different respiratory chain complexes were sequentially administered. Data were normalized by Citrate Synthase (CS) activity and CTC mtDNA content. Results: The mtDNA content was significantly decreased in mild PE (without IUGR) (p¼0.03), and increased in PEþIUGR (p¼0.02) vs NP. Both CS activity and mtDNA were decreased in PE and increased in IUGR CTC vs NP. Raw data (accounting for the cell global respiration) showed increased oxygen consumption both for PE and IUGR CTC vs NP. Normalized data (both by CS activity and mtDNA) indicated higher oxygen consumption in PE, and equal oxygen consumption in IUGR CTC, vs NP. However, IUGR presented a reduced complex IV activity. Conclusions: Our study addresses the hypothesis that in PE and IUGR placentas two different types of mechanisms occur: in PE, mitochondria attempt to compensate their lower number, consuming higher oxygen levels. Conversely, IUGR show increased mitochondrial biogenesis, leading to a higher respiratory capacity, likely as an attempt to compensate for other aspects of placental insufficiency, such as decreased nutrient availability. Moreover, in IUGR the mechanism leading to cytochrome-C reduction (complex IV activity) seems to be altered. Further data are needed to confirm these preliminary results.

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Abstracts / Placenta 32 (2011) A1–A149

P1.76 HYPOXIA/REOXYGENATION MAY ALTER THE EXPRESION OF AQUAGLYCEROPORINS IN HUMAN PLACENTA. THEIR ROLE IN APOPTOSIS

P1.77 PLACENTAL INTERFERON-g PRODUCTION CONTRIBUTES TO THE ANTIOXIDANT ACTIONS OF N3-LCPUFAS

Natalia Szpilbarg1, Valeria Dietrich1, Mauricio Castro Parodi1, Mariana Farina3, Elsa Zotta2, Alicia Damiano1 1 Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina, 2Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina, 3CEFyBO- CONICET, Buenos Aires, Argentina

Michael Stark, Vicki Clifton, Nicolette Hodyl The Robinson Institute, University of Adelaide, Adelaide, South Australia, Australia

It has been proposed that intermittent placental perfusion, secondary to deficient trophoblast invasion of the endometrial arteries, leads to an ischemia-reperfusion [hypoxia-reoxygenation (H/R)] type insult in preeclamptic placentas (PE). Such variations in oxygenation can further alter the syncyciotrophoblast transport functions. We observed that, in preeclamptic placentas, the molecular expression of aquaporin-3 (AQP3) decreases, while that corresponding to aquaporin-9 AQP9 increases. However, a lack of water transport activity mediated by AQPs was observed. It has also been proposed that H/R leads to an increase in apoptosis in human placenta. Apoptosis starts with the decrease on cell volume (AVD). Although the role of AQPs on apoptosis is related with the AVD, the exact mechanism that triggers the changes in cell volume is still unknown. Objectives: To establish the effect of H/R on AQP9 and AQP3 expression in human placenta and their possible role in the programmed cell death processes observed. Methods: Explants from normal placenta were cultured in normoxia, hypoxia, and H/R. Some explants were treated with HgCl2, a general inhibitor of AQPs. AQP3 and AQP9 expression was analyzed by semiquantitative RT-PCR, Western blot, and immunohistochemistry. Apoptosis was evaluated by DNA fragmentation and Bax expression. Results: In explants cultured under H/R conditions we observed that AQP9 expression increased significantly while AQP3 decreased compared with the control, a similar pattern to that observed in PE placentas. Furthermore, in all cases DNA fragmentation was inhibited after treatment with HgCl2. Even though Bax expression increased under H/R treatment it decreased dramatically after AQP blocking. Conclusions: Our findings show that changes in oxygenation may alter AQPs expression and increase apoptosis indexes in human placenta. We also suggest that AQPs are involved in this event and their inhibition may prevent apoptosis processes.

Objectives: Clinical evidence supports the anti-inflammatory actions of omega-3 (n3) fatty acids, such as docoahexanioc acid (DHA) and eicosapentanoic acid (EPA). However, the relative action of each and concerns about their role in lipid peroxidation and oxidative DNA damage remain unanswered. With the placenta a source of reactive oxygen species causally implicated in pathological processes affecting the fetus and neonate, we investigated the effects of DHA and EPA on LPS induced placental oxidative stress focusing on the potential for altered pro-inflammatory cytokine production and antioxidant capacity. Methods: Placental explants (n¼8) obtained from non-labour caesarean sections were pre-treated with DHA or EPA (1mM & 10mM) prior to Lipopolysacchride (LPS) (1ng) exposure or co-exposed to LPS. Malondialdehyde (MDA) 8-hydroxy-2-deoxy Guanosine (8-OH-dG), TNFa, interferon (IFN)-g, and total anti-oxidant capacity were measured by ELISA. Results: DHA increased MDA and 8-OH-dG production (p¼0.01) with lipid peroxidation, but not oxidative DNA damage, lower than LPS exposure (p<0.05). EPA increased 8-OH-dG production (p<0.05), with both MDA and 8-OH-dG levels lower than LPS exposure (p<0.01). MDA and 8-OH-dG production was lower with EPAþLPS (p¼0.01) but not DHAþLPS cotreatment. Pre-treatment with DHA and EPA inhibited LPS induced MDA and 8-OH-dG production (p¼0.01) with the reduction in 8-OH-dG levels greater for EPA (p<0.05). LPS resulted in higher TNFa and IFNg production and reduced total anti-oxidant capacity. IFNg but not TNFa production was reduced with co- and pre-treatment of DHA and EPA (p<0.01) with levels comparable for DHA and EPA. Conclusions: Pre-treatment with n3-PUFAs limits oxidative stress with EPA exerting a greater effect. Reduced placental production of IFNg may mediate these actions but does not explain the n3-PUFA specific differences in antioxidant effect. Further characterisation of the mechanisms through which n3-PUFAs exert biological actions in the placenta is central to understanding the basis for their beneficial effects on neonatal outcome.

Abstracts / Placenta 32 (2011) A1–A149

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P1.78 FORMALDEHYDE EXPOSURE OF PRIMARY CULTURES OF HUMAN TROPHOBLASTIC CELLS AFFECTS THEIR OXIDATIVE STATUS AND DIFFERENTIATION

P1.80 REDUCED LEVELS OF PLACENTAL HYPOXIA INDUCIBLE FACTOR 1a (HIF 1a) AND ANTIOXIDANT ENZYME ACTIVITY IN FIRST TRIMESTER PREGNANCIES AT HIGHER RISK OF DEVELOPING PREECLAMPSIA.

Guillaume Pidoux1,2, Pascale Gerbaud1,2, Danièle Sophie Gil1,2 1 Inserm U767, Paris, France, 2PremUP, Paris, France

Evain-Brion1,2,

Karin Leslie1, Basky Thiliganathan2, Guy Whitley1, Judith Cartwright1 1 St Georges University, London, United Kingdom, 2St Georges Hospital, London, United Kingdom

Objectives: : The exposure to chemical environmental contaminants is responsible for the development of human pathologies. Bathing in the maternal blood, the syncytiotrophoblast is a main target of polluants which might affect its essential functions during pregnancy. The aim of this study was to evaluate the effect of formaldehyde, a major polluant on human villous trophoblastic cells differentiation. Methods: Biochemistry experiments were performed on human trophoblastic cells purified from placentas obtained after caesarean sections. Cultured of human cytotrophoblastic cells were used to follow in vitro cellcell fusion and the formation of a multinucleated syncytiotrophoblast. Results: Concentrations of formaldehyde above 10 mM were cytotoxic on trophoblastic cells. Using a fusion assay, we observed that a 24h-exposure to 10 mM formaldehyde increased trophoblast fusion by 20%. Using a quantitative RTPCR array we observed that in the mean time formaldehyde exposure largely modified the oxidative status of these cells. Conclusion: We have clearly demonstrated the toxicity of formaldehyde on primary cultures of human trophoblastic cells and its impact on cell fusion and differentiation by profoundly modifying the oxidative status of these cells. This study suggests the toxicity of formaldehyde as xenobiotic on human placental development and thus during pregnancy.

Objectives: The first trimester placenta develops in a low oxygen environment. Until 10-12 weeks the maternal spiral arteries are plugged by trophoblast and there is little blood flow into the intervillous space. Premature onset of maternal blood flow and defective placentation have been associated with early pregnancy loss, preeclampsia and IUGR. Uterine artery Doppler provides a non invasive proxy measure of successful placentation. High resistance flow can identify those pregnancies at highest risk of developing preeclampsia. This study aimed to investigate whether first trimester pregnancies characterised as being at highest risk of developing preeclampsia showed evidence of alterations in the oxygen environment and oxidative stress. Methods: First trimester placental tissue was obtained with informed consent and ethical approval from women undergoing termination of pregnancy. Doppler ultrasound was performed prior to surgery: high resistance was defined as mean RI0.85 (95th centile). Protein expression was quantified by western blotting and antioxidant enzyme activity assayed. Results: In pregnancies with high resistance indices compared with normal resistance;

P1.79 PLACENTAL ISCHEMIC INJURY IN MILD AND SEVERE PREECLAMPSIA AS CHARACTERIZED BY CD39 AND CD73 ACTIVITY Marijke Faas1, Maria van Pampus2, Theo Borghuis3, Annemarie Bolt2, Monica Wong2, Winston Bakker3 1 Div of Medical Biology, Dept of Pathology and Medical Biology, University Medical Center Groningen, Groningen, Netherlands, 2Dept of Obstetrics and Gynecology, University Medical Center Groningen, Groningen, Netherlands, 3Div of Pathology, Dept of Pathology and Medical Biology, University Medical Center Groningen, Groningen, Netherlands Objectives: Vascular ischemic injury is a hallmark of preeclampsia (PE). The ecto-enzymes CD39 (ectoapyrase/ENTPD1) and CD73 (‘5nucleotidase/E5NT) are parameters of vascular ischemic injury: decreased CD39 activity and/or increased CD73 are associated with oxidant stress induced ischemic injury. We compared placental biopsies from women with severe early and mild late onset PE and healthy pregnant individuals for activity of these enzymes. Methods: We included severe early onset preeclamptic patients (EO-PE; n¼10) and healthy pregnant women (n¼10) delivering by elective caesarean section as well as late onset mild preeclamptic patients (LO-PE; n¼7) and healthy pregnant women (n¼12) delivering vaginally. Placental biopsies were immediately snap frozen in liquid nitrogen and prepared for enzyme-histochemical staining (standard techniques) to evaluate the activity of CD39 and CD73. Results: Similar CD39 activity was observed along the apical site of the villous syncytiotrophoblast in all groups. Endothelial cells of almost all fetal vessels showed CD39 activity in both control groups and LO-PE. However, fetal vessels of placentas from EO-PE showed decreased CD39 activity, with many fetal vessels being negative. CD73 activity was observed along trophoblastic sites in all biopsies. However, increased CD73 activity along the syncytiotrophoblast was observed in biopsies from both groups of PE, especially along the basal site. Increased CD73 activity also occurred in fetal vessels and in villous stroma of placental biopsies from both groups of PE patients. Conclusion: Although it is well known that ischemic injury occurs in the placentas from women with EO-PE, the present findings support the notion that toxic oxygen products may not only play a major role in the induction of placental and endothelial damage in EO-PE, but also in LO-PE. Ischemic damage in severe EO-PE was more severe, as judged by decreased CD39 expression in villous fetal vessels of this group as compared with LO-PE.

1) Levels of placental HIF1a protein were significantly lower. (p¼0.022, n¼35) 2) Placental glutathione peroxidase activity was significantly lower. (p¼0.007, n¼20) 3) There was no evidence of any alteration in placental tissue markers of oxidative stress (nitrotyrosine residues, 4- hydroxy nonenal , malondialdehyde, heat shock protein 70) (n¼34). Conclusions: The reduced level of HIF-1a may indicate that pregnancies at increased risk of preeclampsia are exposed to higher first trimester oxygen levels than normal, which could have consequences for angiogenesis and placental development. Although high resistance pregnancies had lower levels of antioxidant enzyme activity this did not result in higher levels of markers of oxidative stress. Lower antioxidant defences may contribute to the development of oxidative stress in the second and third trimester or influence the ability of the placenta to react to changes in oxygen tension.

Abstracts / Placenta 32 (2011) A1–A149

P1.81 CELLULAR SITES OF ATP-BINDING CASSETTE TRANSPORTER A1 FUNCTION IN HUMAN PLACENTA: POSSIBLE LINKS TO PLACENTAL PHYSIOLOGY Liudmila Nikitina1, Fabian Wenger1, Marc Baumann2, Daniel Surbek2, Meike Koerner3, Christiane Albrecht1 1 Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland, 2Department of Obstetrics and Gynecology, University Hospital, University of Bern, Bern, Switzerland, 3Institute of Pathology, University of Bern, Bern, Switzerland Objectives: The ATP-binding cassette transporter A1 (ABCA1) is involved in the transport of cholesterol, phospholipids, and other lipophilic molecules across cellular membranes. Knockout mice studies provide evidence of its crucial role in placental development and fertility. Based on its known transporter function, a role of ABCA1 in cholesterol transport across the placenta has been suggested. However, the specific mechanism underlying ABCA1 function(s) in the placenta has not been proven so far and its exact cellular localization in the human placenta has yet to be clarified. To determine the cellular sites of ABCA1 actions, we analyzed the cellular and subcellular localization of ABCA1 in human placental tissues and isolated primary cells. Methods: Paraffin embedded placental tissue samples from 1st trimester and term placentas were analyzed by immunohistochemistry and subsequent light or immunofluorescence confocal microscopy. Cytotrophoblast cells, amnion epithelial cells, villous macrophages (Hofbauer cells), and mesenchymal cells from chorionic membrane and placental villi were isolated from fresh placental tissues and ABCA1 localization was analyzed using immunocytochemistry and subsequent confocal or immunofluorescence microscopy. Results: In placental tissues, ABCA1 was localized in diverse compartments, including the villous trophoblast, mesenchymal cells, macrophages and endothelial cells of villi, amnion epithelial cells and mesenchymal cells of the chorionic plate, as well as in decidual cells. In isolated primary amnion epithelial cells, placental macrophages and mesenchymal cells, ABCA1 was predominantly found at the cell membrane and in cytoplasmic compartments partially corresponding to the endoplasmic reticulum. Cultivated primary cytotrophoblast cells showed after 12 hours intensive membrane and cytoplasmic staining for ABCA1 which was markedly decreased after 24 hours, i.e. with progressive syncytium formation. Conclusion: Localization of ABCA1 in placental cells from diverse functional placental compartments suggests that this protein may not only participate in transplacental lipid transport but could have additional regulatory functions.

P1.82 2D PLACENTAL SONOGRAPHIC ASSESSMENT INCREASES ULTRASOUND PREDICTION OF FGR IN HIGH RISK PREGNANT WOMEN Edward D Johnstone, Suzanne Thomas, Tracey Mills, Clare Tower University of Manchester, Manchester, United Kingdom Objectives: Fetal growth restriction (FGR) is associated with evidence for abnormal placental development resulting in reduced placental size which can be identified by second trimester placental biometry (Viero et al 2004). Previous studies have demonstrated that placental biometry combined with other songraphic and biochemical parameters improves prediction of pregnancy complications including FGR (Toal et al 2007. Here, we examined which combination of placental ultrasound measurements differentiated most reliably between normal and FGR infants. Methods: Data were obtained following a retrospective analysis of 78 women attending a high risk antenatal clinic (the Manchester Placenta Clinic) in 2010. Placental biometry at 23 weeks gestation is performed on women at risk of FGR. Placental biometry was collected in 3 planes, 2 at 90 degrees to each other in the longitudinal plane and 1 from the thickest point of inner to outer placental surface (the depth plane). Measurements were combined to produce different placental size models. Placental index (PLI) was calculated by the formula diameter x diameter /depth). Individualised birth weight centiles (IBC) were calculated using Bulk centile 6.4 UK. Infants were classified as FGR if their IBC was <10th centile. Statistical analyses were performed using Graphpad PRISM (vers.4). Results: Infants with FGR had significantly smaller placental diameters (p¼0.0004) and PLIs (p¼0.001) than appropriately grown babies; increased placental depth was not significantly different (p¼0.94). Receiver operator characteristic curves were calculated for each biometric with the PLI having the highest area under curve 0.8 (Fig.1). 100

Sensitivity %

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Diameter PLI Depth

75 50 25 0 0

10 20 30 40 50 60 70 80 90 100

1-Specificity % Fig. 1. ROC curve of different placental biometrics.Ă

Conclusions: Women who are destined to have infants with FGR have abnormal placental characteristics detectable at 23 weeks on ultrasound scan, much earlier than the clinical diagnosis of FGR. In this study calculated PLI is better at detecting FGR than maternal history (Llurba et al 2009) or uterine artery Doppler’s (Papageorghiou et al 2008) alone in high risk pregnant women.

Abstracts / Placenta 32 (2011) A1–A149

P1.83 OSTEOPONTIN EXPRESSED AT THE UTERINE-PLACENTAL INTERFACE INCREASES ION TRANSPORT ACROSS THE PIG PLACENTA Greg A. Johnson1, James W. Frank1, Xilong Li2, Kayla J. Bayless3, Robert C. Burghardt1, Fuller W. Bazer2, Guoyao Wu2 1 Department of Veterinary Integrative Biosciences, Texas A&M University, Texas, United States, 2Department of Animal Science, Texas A&M University, Texas, United States, 3Department of Molecular & Cellular Medicine, Texas A&M Health Science Center, Texas, United States Objectives: Osteopontin is a secreted extracellular matrix protein that binds to cell surface integrins. Osteopontin has prominent expression at the uterine-placental interface of pigs, sheep, goats, rabbits, mice, and humans. In pigs, osteopontin is induced just prior to implantation, and expression is maintained through term. We recently discovered that pig placentae release a factor(s) that increases ion transport across the placenta. We hypothesize that osteopontin is released by pig placentae to increase ion transport from mother to embryo/fetus. Methods: Day 60 pig chorioallantois was cultured at room temperature for 2h, conditioned medium was dialyzed and 0.2ml added to Day 60 chorioallantoic membranes inserted into Ussing chambers, followed by measurement of transepithelial voltage. In similar experiments, conditioned medium was replaced by osteopontin purified from cows milk or by conditioned medium in which osteopontin was removed by immunoprecipitation. Results: Addition of placental conditioned medium doubled the transepithelial voltage and current (measure of ion flux) across Day 60 chorioallantois. Osteopontin from cows milk dose- and time-dependently increased ion transport, and maximum transepithelial voltage plateaued at levels similar to placental conditioned media. Depletion of osteopontin from placental conditioned medium completely ablated the ability of placental conditioned media to increase ion transport across chorioallantoic membranes. Conclusions: Osteopontin expressed at the uterine-placental interface binds integrins on chorion to activate transport of ions and nutrients, e.g., glucose and amino acids, across the pig placenta. A role for osteopontin and integrins in ion transport across epithelial barriers, and the chorion in particular, has not been investigated. Results of these studies could have major implications for understanding ion transport across the placenta and other epithelia known to express high amounts of osteopontin including the alveolar epithelia of mammary glands, the epithelium of tubules of kidney, and the epithelium of intestinal villi.

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P1.84 EFFECT OF OXYGEN CONCENTRATIONS ON EXPRESSION OF SODIUMIODIDE SYMPORTER, HCG AND IODIDE UPTAKE IN HUMAN CHORIOCARCINOMA BEWO CELLS Huika Li1, Kelly Landers1, Jatin Patel1,2, Kerry Richard1,2, Robin Motimer1,2 Conjoint Endocrine Laboratory, Pathology Queensland, Royal Brisbane and Women’s Hospital, QLD, Australia, 2The Department of Medicine, The University of Queensland, QLD, Australia, 3The Department of Endocrinology, Royal Brisbane and Women’s Hospital, QLD, Australia 1

Objectives: Normal fetal development requires thyroid hormone from conception. Until 16 weeks gestation, the human fetus depends on of maternal thyroxine supplied by placental transfer. Subsequently the fetal thyroid synthesises thyroid hormones, requiring a supply of maternal iodide. Trophoblast iodide transfer is mediated by apical Sodium Iodide Symporter (NIS). During placental development, increases in oxygen concentration within placenta and its involvement in placental implantation are well recognized. However, effects of oxygen levels on placental materno-fetal exchange in trophoblast are poorly understood. The aim of this study was to investigate the regulation of changes in oxygen concentrations on sodium-iodide symporter (NIS) and its regulatorhuman chorionic gonadotropin (hCG), and iodide uptake in choriocarcinoma BeWo cell line. Methods: Choriocarcinoma BeWo cells were cultured in either 1%, 8% and 21% O2 incubator;  0.2 mM of desferrioxamine (DFO), or 2500IU/L of hCG. Quantitative Real-Time PCR was used to examine the mRNA expression of NIS and hCG. Expression of NIS membrane protein was detected by Western Blot and hCG secretion in culture medium was measured by immunoassay. NIS functional study conducted by radio iodide (I125) uptake. Results: The results indicate that expression of NIS and hCG mRNA and protein was significantly increased when oxygen levels rose from 1%, to 8% and 21% with accompanying I125 uptake. DFO, an iron chelator that mimics hypoxia, decreased NIS and hCG expression and I125 uptake in BeWo cells. Addition of hCG to BeWo cells cultured in 1% oxygen did not increase NIS expression and I125 uptake. Conclusion: Increases in oxygen concentration in BeWo cells is associated with increased NIS expression and iodide uptake. This suggests that vascularisation of the placenta and rising oxygen concentrations facilitate materol-fetal iodide transport at a time when fetal iodide requirements are increasing.

A54

Abstracts / Placenta 32 (2011) A1–A149

P1.85 THE NEONATAL IGG FC-RECEPTOR (FCRN) IS EXPRESSED IN THE SYNCYTIOTROPHOBLAST, FETAL ENDOTHELIAL CELLS AND HOFBAUER CELLS OF HUMAN TERM PLACENTAL CHORIONIC VILLI Martin Schepelmann1, Andreas Heindl1, Hana Uhrova1, Vivienne Pohl1, Radu Rogojanu2,1, Alexander Seewald3, Hanns Helmer4, Isabella Ellinger1 1 Dept. Pathophysiol. & Allergy Res., Med.Univ.Vienna, Vienna, Austria, 2 TissueGnostics GmbH, Vienna, Austria, 3Seewald Solutions, Vienna, Austria, 4Dept. Obstet. & Gynecol., Med.Univ.Vienna, Vienna, Austria Objectives: Human offspring acquire passive immunity by transplacental transport of maternal Immunoglobulin G (IgG), which reaches the fetal circulation by transport across the syncytiotrophoblast (STB) and fetal endothelial cells (FEC). IgG transcytosis in the STB is mediated by the neonatal IgG Fc-receptor (FcRn). However, the mechanism of IgG-transport across FEC is still unclear due to contradictory data on FcRn expression and the presence of other Fc-receptors (FcgRIIb). For engineering of antibodybased therapeutics or interference with transfer of pathogenic antibodies, a more detailed knowledge on placental FcRn expression is required. Methods: Tissue from central, mid and peripheral regions of uncomplicated term placentas (n¼5) was HOPE-fixed, and paraffin-embedded sections (4 mm) were stained for immunofluorescence microscopy. FcRn was detected using three affinity-purified rabbit antisera prepared against peptides corresponding to two extracellular and one cytoplasmic region of hFcRn a-chain. STB, FEC and Hofbauer cells (HC) were identified by antibodies against cytokeratin7, CD31 and CD68, respectively, and stem villi by expression of g-smooth muscle actin (gsm-actin). 6x6 adjacent fields of view per placental region were acquired using the automated epifluorescence microscope TissueFAXSTM (TissueGnostics GmbH) and the stitched FOVs were analysed for FcRn localization. Results: In all placentas and regions therein, FcRn localized to STB, FEC and, additionally, to HC, but the fluorescence signals were much higher in HC and FEC than in STB. All FcRn-expressing cell types were found in stem villi (gsm-actinþ) as well as in small terminal villi (gsm-actin-). The same pattern of FcRn expression was obtained irrespective of the applied antiFcRn antibodies. Conclusion: FcRn is expressed by the STB and FEC, which comprise the placental barrier. This suggests an involvement of FcRn in IgG-transcytosis across STB and FEC, and/or participation in recycling/salvaging of IgG present in the fetal circulation. The expression in HC could facilitate processing and presentation of immune complexes.

P1.86 ATP-BINDING CASSETTE TRANSPORTER A1 EXPRESSION IN THE VILLOUS TROPHOBLAST IN GESTATIONAL DISEASES Fabian Wenger1, Liudmila Nikitina1, Marc Baumann2, Daniel Surbek2, Christiane Albrecht1, Meike Körner3 1 Institute of Biochemistry and Molecular Medicine, Bern, Switzerland, 2 Departement of Obstetrics and Gynecology, Bern, Switzerland, 3Institute of Pathology, Bern, Switzerland Objectives: The ATP-binding cassette transporter A1 (ABCA1) plays a role in the transport of cholesterol and other lipophilic molecules. It shows a high expression in the human placenta with a wide distribution in diverse cell types, but its functions in this organ are unclear. In animal models, loss of ABCA1 results in severe disruption of placental and fetal development. In humans, preliminary and conflicting data suggest expressional changes of placental ABCA1 in gestational diseases. The aim of this study was to characterize the ABCA1 expression in placentas from common gestational diseases. Methods: Ninety third-trimester placentas (12 from normal and 78 from pathological gestations, including preeclampsia, antiphospholipid syndrome, idiopathic intrauterine growth restriction, gestational diabetes, cholestasis of pregnancy, and placental abruption) were semi-quantitatively assessed for their ABCA1 expression with immunohistochemistry. Results: Changes of ABCA1 expression levels in pathological compared with normal pregnancies were found in the villous syncytiotrophoblast, but not in other placental compartments. The villous syncytiotrophoblastic ABCA1 expression was significantly more extensive in pre-term placentas of 30 to 36 gestational weeks compared with placentas of later and earlier gestational ages(p¼0.01 and p¼0.02, respectively). Moreover, it was reduced in preeclamptic placentas and showed a trend towards increase in idiopathic intrauterine growth restriction compared with normal controls (both statistically not significant). Finally, it also showed a trend for upregulation in placentas with low weight or evidence of fetal occlusive vasculopathy (both statistically not significant). Conclusion: A subset of the conditions suggested to exhibit an abnormal syncytiotrophoblastic ABCA1 expression, including preeclampsia and fetal occlusive vasculopathy, are characterized by intraplacental hypoxia; hypoxia has been previously shown to regulate trophoblastic ABCA1 levels. The data suggest that placental ABCA1 is differentially regulated in the third trimester, which may depend on various factors such as gestational age and hypoxia.

Abstracts / Placenta 32 (2011) A1–A149

A55

P1.87 TRANSFER OF 3-O-METHYLGLUCOSE AND L-GLUCOSE ACROSS THE ISOLATED PERFUSED HUMAN PLACENTA

P1.88 THE CONTRIBUTION OF GLUT3 TO GLUCOSE UPTAKE BY BEWO CHORIOCARCINOMA CELLS

Priscilla Day, Jane Cleal, Emma Field, Mark Hanson, Rohan Lewis University of Southampton, Southampton, United Kingdom

Nicholas Illsley1, Kelecia Brown1, Stacy Zamudio2 1 UMDNJ-New Jersey Medical School, Newark, NJ, United States, 2Hackensack University Medical Center, Hackensack, NJ, United States

Objectives: The factors which limit placental glucose transfer are relevant for both fetal growth restriction and macrosomia. This study aimed to investigate the competitive inhibition of 3H-3-o-methylglucose (3H-3MG) transfer by D-glucose in order to determine the concentrations at which placental glucose transport becomes saturated. Methods: Tracer concentrations of 3H-3MG and 14C-L-glucose (paracellular transport marker) were perfused into the maternal (n¼5) or fetal (n¼5) artery of the isolated perfused human placenta. In the maternal experiments maternal and fetal arterial glucose concentrations were maternal:fetal (mmol/l); 0:0, 3:0, 6:0, 9:0, 9:3, 6:3, 3:3, 0:3, 0:6, 3:6, 6:6, 9:6 and 12:6. In the fetal side experiments maternal and fetal arterial glucose concentration were maternal:fetal (mmol/l); 0:3, 3:3, 6:3, 9:3, 9:6, 6:6, 3:6, 0:6, 0:9, 3:9, 6:9, 9:9, 12:9, 15:9, 15:20. Maternal and fetal venous samples were analysed by liquid scintillation counting. Results: The maternal artery to fetal vein ratios for D-glucose 3H-3MG and 14 -C-L-glucose were 2.7, 3.0 and 12.6 respectively indicating that the placenta was a barrier to the transport of these substances. Neither 3H3MG nor 14C-L-glucose transfer were inhibited by increasing D-glucose concentrations. Maternal to fetal 14C-L-glucose transfer was 27% of 3H-3MG transfer while fetal to maternal 14C-L-glucose transfer was 39% of 3H-3MG transfer suggesting that paracellular diffusion provides a significant route for placental glucose transfer. Higher fetal to maternal diffusion may reflect pressure differences in the perfused placenta. Conclusion: The maternal to fetal glucose gradient indicates that the placenta provides a barrier to glucose transfer. However the absence of competitive inhibition of 3H-3MG transport by D-glucose suggests very high glucose transporter capacity. This raises the question as to whether transporter capacity or some other factor (transporter affinity, blood flow or diffusion) is rate limiting. Using this data to mathematically model placental glucose transfer may help shed light on these questions.

Objectives: We have shown that the GLUT3 glucose transporter is present in human syncytial cells across gestation, however the contribution made by GLUT3 to glucose uptake is unknown. In these studies we determined the extent to which GLUT3 contributes to glucose uptake in the BeWo choriocarcinoma cell line. Methods: GLUT3 was knocked down in BeWo cells utilizing a pool of 4 siRNA sequences directed against human GLUT3 (SMARTpool siRNA; Dharmacon) compared to a Non Targeting Control (NTC) siRNA. Cells were incubated with siRNA (25 nM) and a transfection reagent (Dharmafect, 0.4 mg/ml) for 6 hr then cells were returned to growth medium for a further 42 hr. Knockdown was assessed by Western blotting using a specific polyclonal antibody (IBL). Glucose transport was determined by measurement of the uptake of [3H] 2-deoxy-D-glucose ([3H] 2DG) in the presence and absence of 10 mM cytochalasin B, a transport inhibitor. Results: GLUT3 protein expression in GLUT3 siRNA-treated cells, normalized to b-actin expression, was reduced by 80  6% (n¼3; p < 0.05, paired t test) compared to expression in cells treated with NTC siRNA. Experiments examining glucose uptake showed that 10 mM cytochalasin B reduced [3H] 2DG uptake by 71  3% compared to cells treated with the NTC siRNA (n¼3; p< 0.01, ANOVA) whereas GLUT3 siRNA treatment of BeWo decreased uptake by 41  3% (p < 0.05). Conclusions: Using the value for GLUT3 knockdown calculated from Western blotting data, we calculate that GLUT3 is responsible for w50% of glucose uptake in BeWo cells. We have shown previously that GLUT3 expression is confined to the apical membrane of BeWo cells. As BeWo are derived from first trimester trophoblast, this may be a reasonable first estimate of the role of syncytial GLUT3 early in gestation. (Supported by NIH HD46982, NPI) P1.89 SIZE IS EVERYTHING; SEARCHING FOR AN EARLY ULTRASOUND MARKER WHICH PREDICTS THE TERM SMALL FOR GESTATIONAL AGE (SGA) BABY. Sally Collins1,2, Gordon Stevenson1, Alison Noble1, Lawrence Impey2 1 University of Oxford, Oxford, 2John Radcliffe Hospital, Oxford Introduction: Fetal growth restriction is one of the major causes of stillbirth. Evidence is growing that the biochemical markers used for trisomy 21 screening may predict the small for gestational age (SGA) baby. We have sought to develop an ultrasound marker, which in combination with these analytes, might lead to a screening test for growth restriction. This may prevent some stillbirths and facilitate better targeting of NHS resources. Methods: We prospectively recruited women with a singleton pregnancy and a BMI of 35. A 3D ultrasound scan was performed using a Voluson E8 (GE Medical systems) between 11 and 13þ6 weeks gestation. The placental volume, total placental surface area and placental basal plate area were calculated using our previously validated technique. These were used to calculate placental shape estimates including the novel measure of placental functional quotient (PFQ) and sphericity. SGA was defined as <10th customised birthweight centile. The data were analysed with linear regression using SPSS (SPSS inc). Results: 65 women were recruited to the study, all had an ultrasound scan between 11 weeks and 13þ6 weeks gestation. Seven women had SGA babies (birth-weights between <1st and 8th centile). Linear regression analysis with correction for gestation showed that SGA babies had significantly smaller placentas (Volume p¼0.04; Placental Surface Area p¼0.04; Basal Plate Area p¼0.05). However, no significant difference could be found for any estimates of placental shape (sphericity p¼0.73; PFQ p¼0.92). Discussion: The size of the placenta at the time of the combined test might predict SGA neonates.To the best of our knowledge this is the first time that the functional area of the placenta (the basal plate) has been measured in-utero. The shape at this gestation does not appear to correlate with birthweight. This finding may aid in the development of a screening test for growth restriction.

A56

Abstracts / Placenta 32 (2011) A1–A149

P1.90 CHARACTERIZATION OF ADHESION MOLECULE EXPRESSION OF TROPHOBLAST CELL LINES AND POSSIBLE IMPLICATIONS FOR TROPHOBLAST HOMING TO THE ENDOTHELIUM

P1.91 EXPRESSION OF REGULATORS OF TROPHOBLAST INVASION IN PLACENTA ACCRETA, INCRETA PERCRETA. AN IMMUNOHISTOLOGICAL STUDY

Tanja Groten1, Ekkehard Schleussner1, Martina Klas1, Udo Markert1, Berthold Huppertz2 1 University hospital Jena, Friedrich Schiller university Jena, Jena, Germany, 2 Institute of Cell Biology, Histology & Embryology, Medical University of Graz, Graz, Austria

Sabine Dekan1, Beatrix Studierach2, Gunda Poschalko-Hammerle3, Sandra Haider2, Kinga Chalubinski3, Martin Knöfler2 1 Institute of Clinical Pathology, Medical University of Vienna, Vienna, Austria, 2Department of Obstetrics and Fetal-Maternal Medicine, Reproductive Biology Unit, Medical University of Vienna, Vienna, Austria, 3 Department of Obstetrics and Fetal-Maternal Medicine, Medical University of Vienna, Vienna, Austria

Objectives: Trophoblast homing to maternal spiral arteries is mandatory for successful placentation. Cell-cell adhesion molecules are regulating this process and adhesion molecule expression is altered in impaired placentation. We aimed to characterize cell-cell adhesion molecule expression on commonly used trophoblast cell lines in order to identify molecules involved in trophoblast-endothelial interaction. Methods: Expression of adhesion molecules involved in homing of immune cells to the endothelium like VCAM, PECAM, ICAM and selectin L, P and E and their corresponding ligands CD34, CD162 und CD147 were investigated using flow cytometry and RT-PCR in HUVEC and the trophoblast cell lines HTR-8, JEG-3, ACH3P and AC1M32. Results: Results are listed table 1.

Objectives: The aim of this study was to gain more knowledge about the trophoblastic invasion of placenta accreta, increta percreta. The role of different proteases like MMP-2, uPA and its inhibitors PAI-1/2 as well as the influence of the Wnt-pathway on in physiological trophoblast invasion in has been shown while its role in development of placenta accreta, increta percreta is unkown. Methods: We investigated 27 placentas, accreta (4), increta (3) percreta (8) and 11 controls, using immunohistochemistry to determine the expression of MMP-2, uPA, PAI-2 and the critical regulator of Wnt signalling, b -catenin. The distribution of the trophoblast within the maternal tissue was explored by cytokertin 7 stainings. We used a scoring system based on the intensity and count of stained trophoblasts to quantify the expression. Results: The distribution of trophoblast showed a compact band in the control group, whereas trophoblasts in placenta creta were arranged less tightly in maternal tissue. No significant differences in expression of PAI-2 in extavillous trophoblasts (EVTs) and in villous trophoblasts (VTs) between normal and abnormal placentae were found. The expression of MMP-2 in VTs between healthy controls and pathologic placentas did not differ neither, but was reduced in EVTs of accreta, increta percreta. Also, we found considerable differences in the expression of uPA and b-catenin. Whereas uPA was increased both in VTs and EVTs of pathological placentae, ß-catenin was reduced in both cell types. Conclusion: We found that the expression of uPA and b -catenin, differ in healthy controls and pathological samples of placenta accreta, increta percreta Increased invasiveness of pathological trophoblasts may correlate with elevated uPA production and diminished expression of membranebound ß-catenin, potentially correlating with activated Wnt signalling. Further investigations with larger numbers of cases re required to confirm the present statistical signficance in protein expression.

Conclusions: Cell-cell adhesion molecules mediating lymphocyte recruitment to the endothelium are possible candidates regulating trophoblast recruitment to maternal spiral arteries. HUVEC express ICAM and E-selectin, all trophoblast cell lines express the corresponding ligands ICAM and CD147. Furthermore, HUVEC are positive for VCAM, P-selectin and L-selectin mRNA. The corresponding ligands VCAM, CD162 and CD34 are exclusively expressed in the extravillous cell line HTR-8. Thus, specific interaction between HUVEC and the extravillous trophoblast cells might be mediated by these adhesion molecules. This hypothesis is currently evaluated in co-culture experiments investigating alterations in HUVEcHTR-8 cell interaction after knock down of VCAM, CD162 or CD34.

Table 1 Adhesion molecule protein and mRNA expression in trophoblast cell lines and HUVEC. HUVEC

HTR-8

JEG-3

ACH3P

AC1M32

mRNA

Protein

mRNA

Protein

mRNA

Protein

mRNA

Protein

mRNA

Protein

ICAM-1 PECAM VCAM

D D D

D D D

D D

D D

D -

D -

D -

D -

D -

D -

E-selectin CD147 P-selectin CD162 L-selectin CD34

D D D D D D

D D D D

D D D

D D D

D -

D -

D -

D -

D D -

D -

Abstracts / Placenta 32 (2011) A1–A149

P1.92 ANTIPHOSPHOLIPID ANTIBODIES TRIGGER THE PATHOLOGICAL ACTIVATION OF TROPHOBLASTIC CELL VIA TOLL-LIKE RECEPTOR 2 Tess Marchetti1, Nathalie Satta2, Philippe de Moerloose2, Olivier Irion3, Marie Cohen1,3 1 Faculty of Medicine, Geneva, Switzerland, 2Division of Angiology and Hemostasis, University Hospital of Geneva and Faculty of Medicine, Geneva, Switzerland, 3Department of Obstetrics and Gynaecology, Maternity, University of Geneva, Geneva, Switzerland Objectives: Antiphospholipid Syndrome (APS) is an acquired thrombophilia characterized by arterial or venous thrombosis and/or pregnancy morbidity and by the presence of antiphospholipid antibodies (aPL). aPL are implicated in recurrent foetal losses and preterm labor, associated with preeclampsia. aPL pathogenicity is still not completely understood. Tolllike receptors (TLR), mainly TLR2 and TLR4, have been implicated in the pathological activation of endothelial cells, monocytes and platelets by aPL. The aim of our study was to investigate the role of TLR2 and TLR4 in trophoblast cell activation by aPL. Methods: We first determine the expression of TLR2 and TLR4 in first trimester and term trophoblastic cell by RT-PCR and cell-ELISA. We then investigate whether aPL modify the invasive properties of trophoblastic cells or induced their apoptosis via TLR2 or TLR4, by using specific blocking antibodies. Results: Both TLR2 and TLR4 are present throughout pregnancy, but their expression levels increase from first trimester to term. aPL induce a proapoptotic response and inhibit about 30% the invasive properties. In the presence of anti-TLR2 antibody, this effect is reversed and invasion is restored. Anti-TLR4 antibodies have no effect. Conclusion: aPL trigger trophoblast apoptosis via TLR2. This might explain miscarriage or preterm birth in P1.93 N-ACETYLGLUCOSAMINYLTRANSFERASE IVA MAY REGULATE CELL INVASION IN CHORIOCARCINOMA Kaoru Niimi, Eiko Yamamoto, Kanako Shinjo, Sawako Fujiwara, Yukio Mano, Seiji Sumigama, Tomomi Kotani, Fumitaka Kikkawa Nagoya University, Nagoya, Japan Objectives: Human chorionic gonadotrophin (hCG) is mainly a product of placental syncytiotrophoblasts and it can also be secreted by trophoblastic neoplasms. Trophoblastic neoplasms secret hyperglycosylated hCG, with N-linked carbohydrate chains, which is different from regular hCG. Nacetylglucosaminyltransferase IVa (GnT-IVa) is one of glycosyltransferases to attach abnormal biantennary N-linked sugar chains to hCG. We reported that GnT-IVa expressed in all choriocarcinoma cell lines and that immunohistochemistry revealed strong GnT-IVa expression in choriocarcinoma and invasive mole, but not in hydatidiform mole. In this study, we investigated the role of GnT-IVa in regulation of choriocarcinoma proliferation, migration, and invasiveness. Methods: Choriocarcinoma Jar cells were transfected with two validated GnT-IVa siRNA. Scrambled siRNA sequences were used as negative control. (1) We confirmed suppression of GnT-IVa expression in siRNA transfectants compared to wild type (WT) and control cells using Western blot. (2) Cell proliferation was measured by MTS assay. (3) Migration assay and invasion assay were performed. (4) We investigated MMP activity by zymography, (5)hCG and hyperglycosylated hCG secretion by EIA, (6) and glycosylation level of hCG by immunoprecipitated and lection blot. Results: (1) Expression level of GnT-IVa protein in siRNA transfectants were significantly reduced compared to WT and controls at 24 and 48 hours after transfection. (2) MTS assay showed no affection on cell proliferation in Jar after GnT-IVa siRNA transfection for 72 hours. (3) Suppression of GnT-IVa reduced significantly migration and invasive abilities by 50% and 32%, respectively (p<0.001). (4) MMP-2 and MMP-9 activitywere not affected by GnT-IVa knockdown. (5)(6) GnT-IVa knockdown reduced only b1-4GlcNAc branching on hCG, but not hyperglycosylated hCG or hCG secretion. Conclusion: These results suggest that GnT-IVa may play an important role in the regulation of invasiveness of choriocarcinoma with specific glycosylation to hCG.

A57

P1.94 THE LOSS OF ENDOGLIN PROMOTES THE INVASION OF EXTRAVILLOUS TROPHOBLASTS BY THE INDUCTION OF UROKINASE-TYPE PLASMINOGEN ACTIVATOR Yukio Mano, Tomomi Kotani, Kiyosumi Shibata, Hiroko Matsumura, Hiroyuki Tsuda, Seiji Sumigama, Eiko Yamamoto, Akira Iwase, Takeshi Senga, Fumitaka Kikkawa Nagoya University Graduate School of Medicine, Nagoya, Japan Objectives: Endoglin is a co-receptor for transforming growth factorb (TGF-b), which is expressed in syncytiotrophoblasts. Soluble endoglin (sEng) has been observed to increase in the serum of preeclamptic patients. Several studies have shown that endoglin is involved in cancer invasion. However, the role of endoglin in human extravillous trophoblasts (EVTs), which have an invasive phenotype, remains unknown. The present study was designed to investigate the role of endoglin in human EVTs. Methods: The expression and localization of endoglin in the placenta and villous explants were confirmed by immunostaining. Next, we evaluated the regulation of endoglin expression by TGF-b in human EVT cell line, HTR-8/SVneo, using quantitative RT-PCR (qRT-PCR). The stable knockdown of endoglin was performed by lentivial short hairpin RNA transfection into the HTR-8/SVneo, and proliferation and motility was investigated by MTS assay and migration/invasion assay, respectively. Key molecules that affect EVT invasion were examined by qRT-PCR and zymography. We determined secreted sEng from HTR-8/SVneo by ELISA, and migration assay was also conducted with exogenous sEng. Results: We found that endoglin was mainly expressed on cytotrophoblasts within the cell column during the first trimester and its expression decreased in the EVTs by immunostaining. The expression of endoglin significantly increased with TGF-b1 and 3 as detected by qRT-PCR. Although proliferation was not affected, the motility of the HTR-8/SVneo cells significantly increased by the endoglin knockdown. Both the mRNA expression and secretion of urokinase-type plasminogen activator (uPA) significantly increased in endoglin knockdown cells. The secretion of sEng was very low in HTR-8/SVneo, and the treatment of sEng had no effect on their migration ability. Therefore, the suppression of sEng was not involved in the increased motility of endoglin knockdown cells. Conclusion: These results suggested that EVTs increased their invasive function via uPA as a result of decreasing expression of transmembrane endoglin.

A58

Abstracts / Placenta 32 (2011) A1–A149

P1.95 REGULATION OF INVASION BY OXYGEN IN PRIMARY EXTRAVILLOUS TROPHOBLAST AND CHORIOCARCINOMA CELL LINE

P1.96 ESTABLISHMENT OF A NEW MODEL SYSTEM FOR EARLY PLACENTATION

Katsuhiko Naruse, Akira Onogi, Taihei Tsunemi, Masayoshi Akasaki, Toshiyuki Sado, Taketoshi Noguchi, Yoshihiko Yamada, Hidekazu Oi, Hiroshi Kobayashi OB/GYN, Nara Medical University, Kashihara, Nara, Japan

Culture chamber for simultaneous co-culture of tissues under different oxygen concentrations Gerit Moser, Martin Gauster, Monika Siwetz, Veronika M. Berghold, Berthold Huppertz Medical University of Graz, Graz, Austria

Objectives: During early pregnancy, cytotrophoblast differentiate into extravillous trophoblast (EVT) cells and invade into decidua, myometrium and uterine spiral arteries. It is well recognized that trophoblastic villi is exposed to low pO2 values also in successful pregnancy. Although a low oxygen environment is reported to effect on the invasiveness of each cancer cell lines and it might be similar to the EVT cells, there have been few study reports with primary EVT cells on the culture in low oxygen environment. In this study, we investigated the invasiveness of primary EVTs compared to JAR, choriocarcinoma cell line in normal or hypo/ hyperoxic condition, to show the behavior of the cells in normal and pathological pregnancies. Methods: Placental samples (5-15 weeks gestation) were obtained from women undergoing elective surgical termination of pregnancy with written informed consent. Cytotrophoblast was separated with Percollbased method and cultured on Matrigel coated invasion assay chamber for 24 hours. The culture was performed under 20% oxygen, 5%, and 5% that repeated three times of hypoxic stimulation for 1 hour (0.1%). Since 5% oxygen environment is regarded as similar to normal placental condition in 5-10 weeks gestation, all data were shown as a comparison of 20% or 0.1% with 5% in this study. Results: Cell viabilities between each oxygen concentration showed no difference by MTT assay. Invasion of primary EVTs was significantly increased in 20% and decreased in 0.1%. In contrast, that of JAR was significantly increased in 0.1% and decreased in 20%. Conclusion: The primary trophoblast and cell lines displayed different responses to culture period in hypoxia or hyperoxia with respect to invasion. Hypoxia reduced invasive capacity of EVT cells, unlike cancer cell lines including trophoblastic disease origin. These results suggested that comparatively low oxygen environment suppresses an excessive invasion of primary EVT cells in early pregnancy.

Objectives: The process of human implantation and subsequent early placentation is still a black box in research. Various in vitro co-culture models with placental tissues have been developed as useful tools to study processes during early placentation and trophoblast invasion. However, the fact that during the first trimester fetal and maternal tissues are under different physiological oxygen concentrations has not been addressed with any model system up to now. Hence, our aim is to develop a co-culture model system to mimic the physiological gradient in oxygen concentrations during early placentation. Methods: Pieces of decidua parietalis (6-10 weeks gestation) are confronted directly with villous explants from the same pregnancy. Confrontation co-cultures are harvested after 72h, cryosectioned and processed for immunostaining. Results: In cooperation with a plastic processing company a prototype for a tissue culture chamber was designed and constructed. The split culture chamber consists of an upper and lower compartment. The decidual tissue is placed on a porous membrane and fixed by a ring with funnel-shaped openings. Villous tissue is placed in these funnel shaped openings in direct contact to the decidual tissue. Culture media equilibrated with different oxygen concentrations is pumped separately through the two chambers. Conclusions: So far there is no system available worldwide, which allows co-culture of two tissues in direct contact under different oxygen concentrations. Thus, our novel system comes closer to the physiologic condition during early pregnancy than any former system. The culture chamber will be used to study effects of varying oxygen gradients and concentrations on placental tissues and on trophoblast invasion. The system is not limited to placental tissues; it may also draw attention of researchers from other fields.

Abstracts / Placenta 32 (2011) A1–A149

A59

P1.97 DESCRIPTIVE AND FUNCTIONAL ANALYSIS OF ADAM-12 DURING EXTRAVILLOUS TROPHOBLAST INVASION

P1.98 ROLE OF INTERLEUKIN-1 BETA DURING EXTRAVILLOUS TROPHOBLAST INVASION - REVISED

Jürgen Pollheimer, Valerie Fock, Martin Knöfler Medical University of Vienna, Vienna, Austria

Nicole Prutsch, Jürgen Pollheimer, Martin Knöfler Medical University of Vienna, Vienna, Austria

Objectives: Recent data of our laboratory, describing differential gene expression profiles of invasive and non-invasive trophoblasts, suggest high expression levels of the protease ADAM-12 (a disintegrin and metalloproteinase 12) during EVT invasion. ADAM-12 exists in two naturally occurring splice variant forms, the transmembrane ADAM-12L and the secreted ADAM-12S. Based on the above-mentioned preliminary data, we here show expression pattern of ADAM-12L/S during extravillous trophoblast (EVT) differentiation and present first evidences supporting a role for ADAM-12S in trophoblast invasion. Methods: Protein and mRNA were isolated from placental villi, decidual tissue and fibroblasts, primary first trimester cytotrophoblasts and EVTs as well as trophoblast cell lines to perform RT-PCR and Western Blot analyses. Moreover, using specific antibodies recognizing either of the two forms, we analyzed expression patterns of ADAM-12L/S in tissue sections of healthy and complete hydatiform mole (CHM) placentae. Functional assays were performed by titrating blocking antibodies into invasion assays and differentiating villous explant cultures. Results: Descriptive analyses including RT-PCR, immunohistochemistry and Western blotting revealed high expression levels of ADAM-12L in placental tissue, primary isolated EVTs, trophoblast cell lines and placentaderived fibroblasts. Interestingly, ADAM-12S was found to be exclusively expressed and secreted from primary trophoblast cultures and its expression increased during EVT differentiation in vitro. Moreover, blockage of ADAM-12S by a specific antibody decreased the invasiveness of first trimester explant cultures as well as isolated primary EVTs. In addition, we found that various ADAM-12 antibodies revealed a prominent staining in EVTs of CHMs being clearly stronger than the ADAM-12 expression found in age-matched healthy placenta sections. Conclusion: In summary, we propose that placenta-associated ADAM-12S expression is restricted to trophoblasts, suggesting a prominent role for the particular protease in trophoblast invasion. In addition, we provide first evidence that ADAM-12 may be markedly overexpressed in CHM.

Objectives: In the past, it was reported that autocrine production of interleukin-1b (IL-1b) induces trophoblast invasion via enhanced production of MMP-9 in vitro. However, contrasting data suggested either that the cytokine has no effect on motility of a trophoblast cell line or indirectly induces motility of HTR-8 cells by triggering IL-8 secretion from fibroblasts. We therefore sought to re-evaluate the initially predicted effects of IL-1b on trophoblast invasion by studying its effect on different primary trophoblast model systems. Methods: Supernatants and mRNA were isolated from 1st trimester primary extravillous trophoblasts (EVTs) to perform real-time PCR and Western Blot analyses, respectively. Human recombinant (rhu) IL-1bmediated effects on human trophoblast invasion were assessed by either studying migration of primary trophoblasts through fibronectin-coated transwells or outgrowing differentiation of villous explant cultures on collagen-I. In addition, proliferation was evaluated by BrdU incorporation in primary trophoblast cultures in the presence or absence of rhuIL-1b. Results: Quantification of outgrowth in villous explant cultures and invasion of primary EVTs revealed that IL-1b induces trophoblast motility. In parallel, evaluation of BrdU labelling did not indicate changes of trophoblast proliferation in the presence of rhuIL-1b. Moreover, the particular cytokine strongly activated mRNA expression and secretion of various proteases including MMP-3/9 and uPA in primary trophoblasts. Conclusion: IL-1b induces in vitro invasion of primary trophoblasts presumably via activation of various proteolytic systems. This result is in accordance with reports demonstrating high concentration of the particular cytokine in the human decidua and a markedly induction of its receptor, IL-1R1, during EVT differentiation. P1.99 EXPRESSION OF N-CADHERIN AND RNAI-INDUCED DIMINUTION OF MYOFERLIN AND DYSFERLIN IN HTR-8/SVNEO CELLS: MODEL FOR EXTRAVILLOUS TROPHOBLAST CELL MOTILITY AND INVASION Taryn Summerfield, Ruth Li, Leo Volakis, William Ackerman, Douglas Kniss Ohio State University, Columbus, OH, United States Objective: Trophoblast migration into the uterine decidua and invasion into the myometrium are hallmarks of interactions between future maternal-fetal interface. Although many extracellular cues may participate in trophoblast invasion, there is little mechanistic detail permitting a thorough understanding of how the delicate low-resistance uteroplacental circulation is established. Our group has demonstrated that dysferlin and myoferlin, two proteins involved in membrane fusion and endocytic events, may be involved in cytotrophoblast fusion into syncytiotrophoblast and potentially remodeling of the maternal-fetal interface that are critical for successful placentation. We employed lentivirus-driven RNAi-mediated diminution of myoferlin and dysferlin to examine the individual effects of these proteins in extravillous trophoblast. Methods: HTR-8/SVneo extravillous-like trophoblasts were cultured in DMEMþ10% FCS and grown to w 50% confluence. Lentiviral vectors (containing drug-selectible markers) harboring a 21 nt shRNA sequence directed against either myoferlin or dysferlin were transduced into HTR-8 cells. After selection with puromycin, HTR-8 cells were probed for the presence of ferlins by immunoblotting. Results and Conclusion: shRNAs reduced the expression of either myoferlin or dysferlin, but not both, indicating selective inhibition of only the targeted ferlin. Moreover, the HTR-8 cells expressed the migratory N-cadherin but not E-cadherin, suggesting a mesenchymal rather than epithelial phenotype in this trophoblast-like cell. Given the findings from our laboratory suggesting a prominent role for dysferlin in cytotrophoblast fusion and previous work by others indicating ferlins’ roles in vesicular membrane trafficking, including cell motility, these newly-modified cell lines will useful for studies of extravillous trophoblast motility and invasion.

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Abstracts / Placenta 32 (2011) A1–A149

P1.100 T-CELL FACTOR 4 IS A CRITICAL REGULATOR OF HUMAN TROPHOBLAST INVASION

P1.101 PATHOLOGICAL INVESTIGATION OF PLACENTA PREVIA ACCRETA: THE SITE OF UTERINE WALL AND THE WIDTH/DEPTH OF VILLOUS INVASION

Gudrun Meinhardt, Peter Haslinger, Sandra Haider, Martin Knöfler Medical University of Vienna, Department of Obstetrics and Fetal-Maternal Medicine, Reproductive Biology Unit, Vienna, Austria

Seiji Sumigama, Tomomi Kotani, Yukio Mano, Yuka Hattori, Sawako Fujiwara, Eiko Yamamoto, Tetsurou Nagasaka, Fumitaka Kikkawa Nagoya Graduate University School of Medicine, Nagoya, Japan

Objectives: Transcription factors committing and differentiating progenitor cells into invasive trophoblasts are largely unknown. Here, we analyzed expression pattern and function of T-cell factor 4 (TCF-4) which we recently identified as an extravillous trophoblast (EVT)-specific gene using comparative gene chip analyses. In general, TCF-4 is known to suppress transcription upon interaction with Transducin-like enhancer of split (TLE) proteins, but gets activated upon Wingless (Wnt)-dependent, nuclear recruitment of active b-catenin. Methods: Localization of TCF-4, other TCF family members (TCF-1, TCF-3), TLE1, 2, 3, 4 and active b-catenin was analysed in first trimester placentae and differentiating villous explant cultures using immunofluorescence. Activation of the canonical Wnt-pathway in explant cultures was studied by using a lentiviral GFP reporter harbouring TCF binding sites. Gene silencing of TCF-4 in SGHPL-5 cells and primary extravillous trophoblasts was achieved by lentiviral transduction of TCF-4 shRNAmir constructs and verified by Western blotting. Invasion assays of SGHPL-5 cells and primary EVT through fibronectin-coated transwells were performed in the absence or presence of a recombinant Wnt ligand. Proliferation was measured by analyzing cumulative cell numbers. Results: TCF-3 and TCF-4 are expressed in KI67-positive, proliferative cell column trophoblasts and p57KIP2-positive, non-proliferating extravillous trophoblasts, respectively. TLE1-4 are abundantly expressed in both trophoblast subtypes, whereas active b-catenin is predominantly localized in nuclei of invasive trophoblasts. Analyzes of the lentiviral reporter in explant cultures revealed GFP expression in some cell column trophoblasts as well as in differentiated EVT. Gene silencing of TCF-4 in SGHPL-5 cells reduced basal as well as Wnt-stimulated invasion, but did not affect proliferation. Conclusions: Expression data and functional studies suggest a major role of TFC-4 in invasive trophoblast differentiation. The transcription factor is specifically expressed and activated in EVT and controls basal as well as Wnt-induced trophoblast invasion. TCF-3 could be a regulator of cell column proliferation. This work was supported by a grant from the Austrian Science Funds (P22687-B13).

Objectives: Abnormal villous invasion is found when decidua is defective and this condition is associated to previous uterine operations including caesarean sections. The question now arises: Is the villous invasion limited to sites of uterine scar? To clarify this hypothesis, we compared the width and depth of villous invasion between uterine anterior wall (caesarean scar can be exist) and posterior wall (caesarean scar cannot be exist). Methods: We picked up 16 placental previa accreta cases and reviewed hysterectomy specimens. Fifteen cases had history of 1-2 caesarean sections, 5 cases had 1-3 curettages and 3 cases had 1-2 vaginal deliveries. Ten cases had no history of uterine operation except caesarean (posterior wall must be intact). Numbers of pathological sections were 4-22/case, including 1-10 anterior wall sections/case and 2-6 posterior wall sections/ case. There were total 63 sections of anterior and 52 sections of posterior. We reviewed existence of decidua, villous invasion into myometrium and thicknesses of remaining myometrium. To examine the width of invasion, the percentages of sections in which invasion was observed on anterior/ posterior were calculated. The remaining myometrium thickness was compared anterior with posterior. Results: The villous invasion was observed in all cases on anterior and in 14/16 cases on posterior. The percentages of invasion were 76.2% on anterior sections and 57.7% on posterior sections. The remaining myometrium thicknesses were anteriorposterior in 2 cases. In 10 cases that had only caesarean history, 50% sections of posterior wall showed villous invasion. Conclusion: The villous invasion was wider and deeper on anterior than posterior wall. It is quite likely that the existence of caesarean scar and lack of decidua lead on the invasion of villi. However, the fact that high invasion was also observed on scar-less posterior wall suggests another mechanism of villous invasion to the myometrium.

Abstracts / Placenta 32 (2011) A1–A149

P1.102 ISOLATED EXTRAVILLOUS TROPHOBLAST CELLS FROM BASAL PLATE OF TERM PLACENTA: EXPRESSION OF INTEGRINS AND INVASIVENESS ACTIVITY Silvana Sandri1, Alexandre Borbely2, Isabella Fernandes3, Karen Prado2, Simone Correa-Silva2, Renata Albuquerque1, Patricia Cristina Beltrão Braga3.4, Ana Campa1, Estela Bevilacqua2 1 Departamento de Análises Clínicas e Toxicológicas- Faculdade de Ciências Farmacêuticas - Universidade de São Paulo, São Paulo, Brazil, 2Departamento de Biologia Celular & Desenvolvimento - Instituto de Ciências Biomédicas - Universidade de São Paulo, São Paulo, Brazil, 3Faculdade de Medicina Veterinária e Zootecnia - Universidade de São Paulo, São Paulo, Brazil, 4Escola de Artes, Ciências e Humanidades - Universidade de São Paulo, São Paulo, Brazil Objectives: The extravillous trophoblast cells (EVT) are essential for the placental growth and correct attachment, as well as for the successful gestation. Herein, we isolated EVT cells directly from basal plate of term placenta and evaluated their intrinsic invasive potential. Methods: Fragments from the term placenta basal plate were carefully separated from villi and digested. EVT cells were isolated by Percoll gradient centrifugation and cultured in standard conditions. The isolated cells were characterized for cytokeratin 7, HLA-G and vimentin by immunofluorescence, flow cytometry and PCR assay. In time course, cell death was assessed by TUNEL reaction; integrity of mRNA was evaluated in agarose gel and gene expression by RT-PCR for housekeeping genes. Invasive ability of term placenta EVT cells were determined in transwell invasion assays using Matrigel-coated upper compartments and by immunofluorescence for integrins (a1, a5 and b1) in the presence or absence of serum in the culture medium. Results: Isolated cells were >92% positive for cytokeratin-7, 20% positive for HLA-G and <2% vimentin positive cells. Cell death was around 5 % at 24 and 48 h, but it increased significantly at 72 h of culture (w46%). The integrity of total RNA and expression of GADPH and cyclophilin mRNA was maintained up to 72 h of culturing. The majority of cells showed immunoreactivity for a5 and b1 integrins and few for a1 integrin. In addition, these cells were able to invade Matrigel; the invasion indexes were higher in the presence of 10% serum. Conclusions: We showed that obtaining EVT cells from basal plate term placenta is an alternative method in addition to the villous explant-derived EVT cells. These cells still maintain intrinsic invasive properties, and may be an approach to investigate molecular mechanisms in invasivenesschanged placental pathologies.

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P1.103 THE OXYGEN-SENSITIVE CCN3 PROTEIN REGULATES BOTH, TROPHOBLAST MIGRATION / INVASION AND PROLIFERATION Alexandra Gellhaus1, Jessica Wagener1, Wei Yang1, Nadine Wolf1, Caroline E. Dunk2, Markus Schmidt3, Rainer Kimmig3, Stephen J. Lye2, Elke Winterhager1 1 Molecular Biology, University Hospital, Essen, Germany, 2Dept. Obstetrics and Gynecology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada, 3Gynecology, University Hospital, Essen, Germany Objectives: In preeclampsia migration and invasion properties of extravillous trophoblast (EVT) cells are impaired leading to insufficient invasion of EVT into the decidua and as a consequence to reduced fetal oxygen and nutrition support. We focused on key proteins in the trophoblast invasion and differentiation process, the matricellular CCN proteins CCN1 (CYR61) and CCN3 (NOV) which are all upregulated in the invasive trophoblast of placental explants and in trophoblast cell lines by low oxygen (1-3% O2) and found to be deregulated in early-onset preeclampsia which is characterized by chronic hypoxia. We identified a direct role of HIF-1a and TGFb3 for the elevation of the CCN proteins under low oxygen. Here we focussed on the regulation properties of CCN3 in trophoblast cell migration, invasion and proliferation. Methods And Results: To elucidate the role of CCN3 in trophoblast differentiation we used Jeg3 trophoblast cells and performed transwell migration and invasion assays and analysed proliferation by counting cell numbers. The increase in CCN3 levels by low oxygen enhanced migration and invasion properties of Jeg3 cells combined with an elevation of MMP-2 and MMP-9 activity but decreased proliferation capacities. Analysis of the CCN3-mediated signalling pathways showed that CCN3 stimulated migration properties by activating two different signal transduction pathways, the PI3 kinase/Akt and the MAPK cascade and these two signalling cascades worked independently from each other. Moreover we detected that CCN3-stimulated trophoblast cell migration - but not the antiproliferative capacity of CCN3 - was mediated by Integrin a5b1 using RGD peptides and siRNA transfection. Conclusion: Our data showed that changes in CCN3 levels are associated with changes in trophoblast function which may provide insights in the pathogenesis of preeclampsia in respect to an impaired balance in proliferation and migration/invasion properties of trophoblast cells.

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Abstracts / Placenta 32 (2011) A1–A149

P1.104 COMPARATIVE EXPRESSION OF TWO IODIDE TRANSPORTERS, NIS AND PENDRIN IN HUMAN VILLOUS AND EXTRAVILLOUS TROPHOBLAST Severine Degrelle1,2, Francoise Galland1, Ludovic Lacroix3, Jean-Michel Bidart3, Daniele Evain-Brion1,2, Thierry Fournier1,2 1 INSERM, U767, Université Paris Descartes, F-75006 Paris, France, 2PremUP Foundation, Faculté des Sciences Pharmaceutiques et Biologiques, F-75006 Paris, France, 3Department of Nuclear Medicine and Endocrine Oncology, Institut Gustave Roussy, F-94800 Villejuif, France Objectives: From a previous study, we know that in situ in term placenta and in cultured villous trophoblastic cells (VCT) two iodide transporters: sodium/iodide symporter (NIS/SLC5A5) and pendrin (SLC26A4) are expressed. We therefore analyze the comparative expression of NIS and Pendrin in VCT and extravillous cytotrophoblastic cells (EVCT) isolated from 1rst trimester human placentas. Methods: Human placental tissues were obtained from first trimester placentas(8-10th week of pregnancy) after legal abortion. VCT and EVCT (n¼7) were isolated by sequential trypsin digestion and purified on percoll gradient, then plated on plastic (VCT) or matrigel-coated (EVCT) culture dishes and cultured for 72h (VCT) or 48h (EVCT) in DMEM/F12 10% FBS. Cells were snap-frozen or fixed with cold methanol. The expression of NIS and Pendrin mRNA were quantified by real-time PCR and protein expression and localization were analysed by western blot and immunofluorescence. Results: Compared to VCT, EVCT expressed a higher level of NIS and Pendrin mRNA. We observed a decreased expression of NIS and an increased expression of Pendrin during in vitro syncytiotrophoblast formation or matrigel invasion. Western Blots and immunofluorescence showed that only EVCT expressed NIS and pendrin at the cellular membrane. Furthermore, NaI treatments showed (1) a significant increase in Pendrin mRNA expression but no effect on NIS, (2) a 2-fold higher invasion index as compared to controls. Conclusion: Only EVCT expressed high levels of NIS and Pendrin mRNA and showed a membrane localization of these receptors. As NaI treatments induced an increase in Pendrin mRNA expression and cell invasiveness, iodide may have a stimulating effect on EVCT invasion.

P1.105 INHIBITION OF TROPHOBLAST INVASIVENESS AND MIGRATION BY HUMAN CYTOMEGALOVIRUS INVOLVE IMPAIRED ACTIVATION OF PAR4 BY THE PROTEASE PAPP-A Benjamin Rauwel1, Christian Davrinche1, Danièle Evain-Brion2,3, N Vergnole1, N. Cénac1, Thierry Fournier2,3 1 Inserm U563, Toulouse, France, 2Inserm U767, Paris, France, 3Paris Descartes University, Paris, France Human Cytomegalovirus (HCMV) primary infection during the first trimester of pregnancy could lead to miscarriages, IUGR and neurological sequelae in newborns. We demonstrated that HCMV induced activation of the nuclear receptor PPARg for its de novo replication, thanks to the presence of functional DNA binding sequences for PPARg into the distal part of the MIEP (Major Immediate-Early Promoter). According to the role of PPARg in trophoblast invasion, we showed that HCMV impaired migration and invasiveness of infected cytotrophoblasts by activating cellular PPARg for its own replication. Studies of sustained mechanisms allowed us to identify potential targets involved in trophoblast invasion, amongst which the thrombin receptor PAR (Protease-Activated Receptor). Objectives: Because PAPP-A (Pregnancy-Associated Plasma Protein A) is a protease secreted in large amount by the invasive trophoblast and a PPARg-target gene, we hypothesized that: i) PAPP-A regulates trophoblast invasion through PAR signalling, and ii) PAR activation by PAPP-A is impaired by HCMV infection. Methods: Primary cultures of extravillous cytotrophoblasts (EVCTs) and EVCT cell line HIPEC was either incubated with HCMV, recombinant PAPPA, synthetic agonist peptide of PARs (AYP for PAR4), and inhibitor of PAR4 signalling (pepducin). PARs were immunodetected in vitro in EVCTs, HIPEC and in situ in chorionic villi. Cell migration was analyzed using wound healing test and activation of PAR signalling by measure of intracellular Ca2þ release. Results: Interestingly and for the first time, we demonstrated that PAR-4 was expressed in EVCTs and was specifically activated by PAPP-A that did not activate PAR2 nor PAR3. HCMV infection decreased PAR-4 expression in the placenta and incubation of trophoblast in vitro with PAPP-A or AYP abrogated the HCMV-mediated inhibition of cell migration. Conclusion: PAPP-A as a new activator of PAR4 signalling regulates human trophoblast migration and impaired activation of PAR-4 by infection may participate to the HCMV inhibition of trophoblast invasion.

Abstracts / Placenta 32 (2011) A1–A149

P1.106 LYSYL OXYDASE IN VITRO

REGULATES

HUMAN

TROPHOBLAST

INVASION

Nadine Segond1,2, Elodie Clouqueur1,2, Katalin Csiszar3, Josette Badet1,2, Daniele Evain-Brion1,2, Thierry Fournier1,2 1 Inserm U767, Paris, France, 2Paris Descartes University, Paris, France, 3 University of Hawaii, Hawaii, France Objectives: Among up-regulated genes in PPARg-activated extravillous cytotrophoblasts (EVCT) with reduced invasiveness, we examine the expression and role of lysyl oxidase (LOX) family. LOX is considered as a tumor suppressor but is also involved in metastatic cell invasion. Its expression and activity depends on oxygen levels. We hypothesized that LOX might regulate human trophoblast invasion. Methods: The RNA expression of the five LOX isoforms in PPARg-activated primary EVCT cultures isolated from 8-9 WG placentae following 24h rosiglitazone (1mM) treatment was compared to controls using relative QPCR. RNA copies, protein expression and cellular localization of the three rosiglitazone-regulated isoforms (LOX, LOXL, LOXL2) in chorionic villi and primary EVCT cultures were studied by Q-PCR, western-blot, confocal analysis and immunohistochemistry. EVCT treated or not with b-aminopropionitrile (BAPN 100, 200mM), a specific irreversible inhibitor of LOXs activity, and/or rosiglitazone were cultured on Matrigel-coated transwell to test the invasiveness of cytotrophoblasts. We calculated the percentage of invasive cells and pseudopods number (CK7 immunostaining) to total nuclei (DAPI) in treated versus control cells. Results: Only LOX, LOXL and LOXL2 RNA were increased (2-3 fold) in rosiglitazone-treated cells. LOXL2 was more expressed than LOX and LOXL in EVCT. Mature LOXL was stronger expressed than LOX and localized in nuclei and nucleoli, while LOX was cytoplasmic. The mature LOXL2 was undetectable in our experimental conditions. In BAPN treated cells, EVCT invasion was 90% increased. The inhibitory effect (40%) of rosiglitazone on EVCT invasion was reversed by BAPN treatment. Conclusion: LOX, LOXL and LOXL2 were regulated by PPARg in primary EVCTs. Weak cytoplasmic localization of mature LOX suggests that it is secreted and presence of mature LOXL in nucleoli a recapture by the cells after secretion. The BAPN effect showed that LOX inhibited EVCT invasion and was responsible in part for the PPARgmediated inhibition of EVCT invasion.

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P1.107 ENDOGLIN KNOCKDOWN ATTENUATED THE SUPPRESSIVE ROLE OF ANGIOTENSIN LL ON EXTAVILLOUS TROPHOBRAST INVASION Yuka Hattori, Tomomi Kotani, Seigi Sumigama, Yukio Mano, Eiko Yamamoto, Sawako Fujiwara, Kaoru Niimi, Fumitaka Kikkawa Nagoya University Graduate School of Medicine, Nagoya, Japan Objectives: Preeclampsia causes maternal and fetal morbidity and mortality. Recently, the angiotensinII typeIreceptor agonistic autoantibody, has emerged as a contributor. Furthermore, soluble VEGF receptor 1(sFlt1) and soluble endoglin are also prominent components. We investigated whether endoglin would be related in the failure of placentation by angiotensin II, which is the first step of preeclampsia. The relationship between other angiogenic factors and endoglin was also examined. Methods: HTR-8/SVneo cells, immortalized human extravillous trophoblasts (EVT), were stably infected with a lentivirus expressing short hairpin RNA targeting endoglin gene (shEng) or with a non-targeting shRNA vector (NTV). Those cell invasion were examined with or without 10-6 M of angiotensinIItreatment using the matrigel chamber. Their concentrations of VEGF, sflt-1 and PlGF were also evaluated with ELISA. RESULT: Invaded cells of both shEng and NTV cells are decreased by anigotensin II, but the reduction rates of NTV cells were higher than those of shEng cells. Then, the VEGF-A and PlGF secretion of shEng cells were higher than that of NTV cells (135.8 11.0 vs.36.8 15.5 pg/ml, and 15.40.38 vs. 9.92.12 pg/ml, respectively, P < 0.05). However, the difference of the sFlt-1 secretion in between shEng and NTV cells (35.3 5.8 vs. 49.011.2 pg/ml) was small. Conclusion: Our results show that endoglin knockdown attenuated the suppressive role of angiotensin II on EVT invasion. In addition, endoglin knockdown increased VEGF-A and PlGF secretion. Those suggest that reduction of endoglin would be important for the therapy of preeclampsia. P1.108 ELASTIN-DERIVED PEPTIDES STIMULATE TROPHOBLAST INVASION: A POSITIVE FEEDBACK LOOP TO ENHANCE SPIRAL ARTERY REMODELLING? Michelle Desforges, Lynda Harris, John Aplin University of Manchester, Manchester, United Kingdom Objectives: Incomplete uterine spiral artery (SpA) remodelling in early pregnancy is associated with pre-eclampsia (PE). We hypothesise that a key event facilitating vasodilatation is breakdown of vascular wall elastin by invading extravillous trophoblast (EVT). Elastolysis generates elastinderived peptides (EDP) which have multiple biological functions including stimulation of invasion and migration. We therefore tested the hypothesis that EDP enhance first trimester (FT) EVT migration and elastase activity. Methods: SGHPL4 cells (EVT-derived cell line) were treated with 1ug/ml scrambled (control) or active EDP (VGVAPG). Prior to treatment, a scratch wound was created in the culture plate. The number of cells in the wound site after 24 hours was counted as a measure of migration. SGHPL-4 invasion through Matrigel-coated transwells was also assayed. Total elastase activity was determined by applying elastase substrate to SGHPL4 lysates 48 hours post-treatment and measuring absorbance at 405nm of the cleaved substrate. EVT outgrowths from FT (<8 weeks gestation) placental villous explants were measured using an image analysis protocol following 24 hour treatment with VGVAPG or vehicle control (0.1% DMSO). Image Pro-Plus software was used to calculate increase in outgrowth area. Results: Active EDP increased SGHPL4 cell migration by 2.4-fold (mean, N¼4) and invasion by 2.5-fold (mean, N¼5). Elastase activity was significantly higher in VGVAPG-treated SGHPL4 cells compared to controls (p<0.05, Mann Whitney U test. N¼5). VGVAPG also stimulated EVT outgrowth from FT explants by 5.5-fold (mean, N¼3). Conclusion: EDP stimulate EVT migration and elastase activity in vitro suggesting induction of elastolysis in SpA may orchestrate a positive feedback loop that promotes EVT invasion and further elastin breakdown. There is evidence for reduced SpA elastolysis in PE and generation of EDP could be reduced as a consequence. This would compromise further elastin catabolism and could contribute to incomplete SpA remodelling in PE. Funded by the Medical Research Council.

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Abstracts / Placenta 32 (2011) A1–A149

P1.109 TROPHOBLAST MIGRATION IS ACTIVATED VIA CHEMOKINE RECEPTOR 1 AND 3 Ilka Knoefler, Carolin Roehler, Sebastian Hoelters, Justine S. Fitzgerald, Diana M. Morales Prieto, Ekkehard Schleussner, Udo R. Markert University Hospital Jena, Department of Obstetrics, Placenta Laboratories, Jena, Germany Background: Cytotrophoblast cells invade the decidua, manly towards the myometrium and maternal blood vessels. The responsible (chemo-) attractants are not completely identified. Methods: We used the immortalized first trimester trophoblast cell line HTR8/svneo to form spheroids with a diameter of approximately 700 mm and confronted them to human placental tissue explants of similar size from different regions. Before confrontation, spheroid cells were stained with fluorescent green Mito Tracker, blood vessels were stained after confrontation red with anti-CD31 antibodies. Finally, a part of spheroids were incubated with the specific blocker chemokine receptor 1 (CCR1) and CCR3 UCB 35625. A total of 200 different confrontation products were analyzed after 10, 24 and 48 h by using a laser scanning microscope. Results: After 10 h, HTR8/svneo cells start to leave spheroids, which after 48 h are completely disaggregated. Cells invade decidual tissue and cover villi. These effects are completely inhibited when blocked CCR1/3 is blocked. Conclusion: Approximately 10 different possible CCR1/3 candidate ligands are known to be present in human placenta. At least one chemokine which binds to CCR1/3 is responsible for directed trophoblast migration and invasion in the decidua.

P1.110 INCREASED PRODUCTION OF MICROPHAGE MIGRATION INHIBITORY FACTOR AND OTHER PRO-INFLAMMATORY CYTOKINES BY PREECLAMPTIC PLACENTAL MESENCHYMAL STEM CELLS: NEW INSIGHTS INTO THE ETIOPATHOGENESIS OF PREECLAMPSIA Alessandro Rolfo1, Anna Maria Nuzzo1, Domenica Giuffrida2, Katia Mareschi3,4, Annalisa Piazzese2, Tullia Todros1 1 Department of Obstetrics and Gynaecology, University of Turin, Turin, Italy, 2O.I.R.M. S. Anna Hospital, Turin, Italy, 3Stem Cell Transplantation and Cellular Therapy Unit; Regina Margherita Children’s Hospital, Turin, Italy, 4 Department of Pediatrics, University of Turin, Turin, Italy Objectives: Placenta-derived Mesenchymal Stem Cells (PDMSCs) possess unique immunomodulatory properties and anti-phlogistic activity. Preeclamptic (PE) placentae are characterized by exacerbated inflammation accompanied by increased production of Macrophage migration Inhibitory Factor (MIF) and other pro-inflammatory molecules. Herein, we characterized the expression and secretion of 80 different cytokines, included MIF, by PDMSCs isolated from normal and PE placentae, in order to define their role in the anomalous phlogistic reaction typical of PE. Methods: PE (n¼12) and control (n¼12) placentae were collected. PDMSCs were isolated and characterized for HLA-I, HLA-II, CD133, CD166, CD45, CD14, CD20, CD34, CD73, CD90 and CD105 expression by FACS analysis to exclude trophoblast contamination. Control and PE PDMSCs were plated at 3x105 cells/ml for 72h. Conditioned media (CM) was collected and cells were treated for mRNA isolation. CM was processed by Cytokine Array Assay to detect levels of released cytokines. Results: Normal and PE-PDMSCs showed proper mesenchymal phenotype without trophoblast contamination. Cytokine array revealed increased release of pro-inflammatory molecules by PE-PDMSCs vs controls. In particular, increased levels of MIF (1.81 Fold increase) and TNF-a (3.5 Fold increase), negative regulator of trophoblast integration into maternal endothelial network, were found. Data were confirmed at the gene level by Real Time PCR. To confirm PE-PDMSC pro-inflammatory activity, physiological villous explants (n¼48) were treated with normal or PE-PDMSCs CM for 72h and MIF mRNA expression was analyzed. Explants conditioned with PE-PDMSCs CM showed significantly increased MIF mRNA expression (p¼0.022, 2.6 Fold increase) relative to controls. Conclusion: In stark contrast with normal PDMSCs anti-phlogistic properties, herein we reported elevated production of pro-inflammatory cytokines by PE-PDMSCs. Increased secretion of MIF and TNF-a suggest a pivotal role of PE-PDMSCs in mediating PE aberrant placentation. Increased MIF expression in normal villi treated with PE-PDMSCs CM further corroborates our findings.

Abstracts / Placenta 32 (2011) A1–A149

P1.111 EXTRAVILLOUS TROPHOBLAST (EVT) FROM HUMAN TERM PLACENTA MAY NOT BE GOOD CANDIDATES FOR USING IN CELL THERAPY PROTOCOLS Isabella Fernandes1,2, Fabiele Russo1,2, Graciela Pignatari1,2, Alexandre Borbely3, Silvana Sandri3, Maria Miglino1,2, Estela Bevilacqua3, Patricia Beltrão-Braga1,2 1 Stem Cell Lab, Departament of Surgery - FMVZ / USP, São Paulo, São Paulo, Brazil, 2National Institute of Stem Cell- INCT, Ribeirão Preto, São Paulo, Brazil, 3Trophoblast Research Lab, Department of Cell and Developmental Biology - ICB I / USP, São Paulo, São Paulo, Brazil, 4Department of Obstetrics, School of Arts, Sciences and Humanities/ USP, São Paulo, São Paulo, Brazil The placenta is an extra-embryonic tissue that has attracted great interest as a source of fetal stem cells for regenerative medicine due to phenotypic plasticity of some of the various cell types isolated from this tissue. Besides, placental tissue is discarded after birth. One of placental cells, called Extravillous Trophoblast (EVT) are involved in maintaining the tolerance of the fetus by the mother, because it contains components that have immunomodulatory properties, working as immunotolerance inducers. Objectives: In this project our aim was to culture and to characterize EVT cells, in order to localize and explore the use of EVT stem cells in allogenic cell therapy protocols. Methods: This project was approved by the ethics committee on human research. Pieces of tissue were fixed in paraformaldehyde 4% for histological characterization of placental tissue to determine the exact place for collect EVT cells. The EVT cell culture was made using cotyledon fragments from human term placenta (38-40 weeks of gestation). The tissue was subjected to enzymatic digestion and cells were submitted to density gradient centrifugation. The cultured EVT cells were characterized to cytokeratin 7, human leukocyte antigen-G (HLA-G), Nanog, Oct4 and vimentin. Results: EVT cells were positive for cytokeratin 7, HLA-G and Nanog and Oct4, confirming trophoblastic origin and stem cell characteristics. However, cell cultures showed that the EVT cells have low rate of cell proliferation according to MTT assay, a characteristic that limits their use in cell therapy protocols. The EVT cells also could not survive more than one week in cell culture. Conclusion: According to our findings, we conclude that EVT cells are short time culturing, show low rate of proliferation and present some stem cell markers. Therefore, we believe it would be interesting reprogramming these cells to check if they increase their proliferative capacity and still keep their immunomodulation profile. Keywords: human term placenta, extravillous trophoblast; immunomodulation; HLA-G.

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P1.112 PARACRINE EFFECTS OF AMNIOTIC MEMBRANE AND AMNION-DERIVED CELLS Anna Cargnoni, Lorenzo Ressel, Daniele Rossi, Marta Magatti, Ornella Parolini Centro Ricerca E.Menni - Fondazione Poliambulanza, Brescia, Italy Objectives: Since the placenta was first recognized as a valuable source of stem/progenitor cells, scientists have continued to study the properties of these cells, with the aim of exploiting their potential for transplantation. In this context, considering the well-documented anti-inflammatory and anti-scarring features of the amniotic membrane (AM) and the immunomodulatory properties of AM-derived cells, we are currently investigating the potential of AM and its cells in pre-clinical models of diseases involving inflammatory and fibrotic mechanisms. We have previously demonstrated that AM patching promotes ischemic rat heart repair, and that transplantation of fetal membrane-derived cells reduces lung fibrosis in bleomycin-challenged mice. These positive outcomes were observed despite low or absent levels of donor cells in host tissues, suggesting that AM-derived cells may act by secreting paracrine soluble factors. Consistent with these studies, here we report the efficacy of AM fragments in counteracting liver fibrosis induced by bile duct ligation (BDL), as well as positive effects of conditioned medium generated from AM-derived cells (AM-CM) in bleomycin-injured mouse lungs. Methods: Preparation of AM fragments and AM-CM. Transplantation of these materials into animal models. Histological evaluation of fibrosis. Results: AM patching onto livers of rats with BDL resulted in confinement of fibrosis to portal/periportal regions, reduced collagen deposition and delayed progression of the ductular reaction in comparison to control rats. No donor cells were found in host livers. Injection of AM-CM in bleomycin-challenged mice reduced both the extent and severity of lung fibrosis compared to controls, and also prevented fibrosis progression. Conclusion: These results support the concept that AM-derived cellmediated repair takes place via release of paracrine factors. The identity and mode of action of these factors remain to be elucidated, however it is plausible that cell-free treatments might become an alternative/complementary therapy to cell transplantation in the future.

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Abstracts / Placenta 32 (2011) A1–A149

P1.113 IPLASS: ADVANCING HUMAN PLACENTAL CELL RESEARCH FOR REGENERATIVE MEDICINE

P1.114 HUMAN PLACENTAL MULTIPOTENT MESENCHYMAL STROMAL CELL MAY MODULATE TROPHOBLAST MIGRATION VIA RAP1 ACTIVATION

Ornella Parolini1, Cesario V. Borlongan2 1Centro di Ricerca E.Menni - Fondazione Poliambulanza, Brescia, Italy, 2Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa

Chie-Pein Chen1,2, Jian-Pei Huang1, Yi-Hsin Wu1,2, Chia-Yu Chen1,2, Tzu-Yun Chu1,2 1 Division of High Risk Pregnancy, Mackay Memorial Hospital, Taipei, Taiwan, 2Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan

Objectives: Recently, the placenta has attracted wide attention as an easily procured, ethically acceptable source of stem/progenitor cells. Placentaderived cells (PDCs) display multi-lineage differentiation capacity and immunomodulatory properties, and secrete biomolecules with potentially multifaceted activities. Attempts to take advantage of these properties have led scientists to investigate PDC-based therapeutic approaches in animal models and clinical applications. The International Placenta Stem Cell Society (IPLASS) was founded in June, 2010, with the aim of creating a network of researchers, skills and ideas to promote research on all aspects concerning knowledge, experimentation and clinical use of PDCs, to thereby foster translation of placental cell research into clinical therapies. Methods: Cooperative research activities between IPLASS members; promotion of staff mobility; organization of a biannual scientific meeting; support of young researchers’ education; maintenance of a web site (www. iplass.net) to promote connection among members and foster new collaborations. Results: Scientists have long been fascinated by the placenta due to its roles during fetal life (e.g. nutrition, protection, participation in fetomaternal tolerance). Nowadays, however, the placenta has also proved to be an appealing source of cells for regenerative medicine. Cross-exchange of knowledge and competencies fostered by IPLASS between basic immunologists has increased understanding of PDCs’ immunomodulatory capabilities, further clarifying not only the placenta’s role in feto-maternal tolerance, but also, key mechanisms for successful transplantation. Meanwhile, cooperation between researchers in the field of cell therapy has revealed beneficial effects of PDCs in animal disease models, particularly for pathologies involving inflammatory and fibrotic degeneration. Conclusion: We believe that IPLASS will grow as a scientific association and foster the increase and dissemination of knowledge on PDCs, with the final aim of elucidating the mechanisms underlying their therapeutic effects. This will be possible through increased membership, partnerships with other scientific societies, and support from companies for sustaining basic and academic science.

Objective: We hypothesize that human placental multipotent mesenchymal stromal cells (hPMSCs) produce hepatocyte growth factor (HGF) which may regulate trophoblast cell migration during placentation. Methods: hPMSCs were isolated from term placentas and conditioned medium were collected after 2 days of culture in hypoxia (PO2<1%) and controls (PO2 20%). HGF recombinant protein, selective agonists and inhibitors or siRNA for protein kinase A (PKA) and exchange protein activated by cAMP-1 (Epac1) were used to investigate the roles of the cAMP effectors in trophoblast cell Rap1 and integrin b1 activation by Rap1-GTP pull down assay, flow cytometry, adhesion and migration assay. Alterations of hPMSC number and HGF level in preeclamptic placentas compared with gestational age-matched controls were further assessed by flow cytometry and ELISA. Results: HGF induced cAMP production and Rap1 activation in trophoblasts, which activated the cell integrin b1. The HGF, PKA activator 6-BnzcAMP and Epac1 activator 8-pCPT-2’-O-Me-cAMP significantly induced trophoblast Rap1 activation with increased trophoblast adhesion and migration. These changes were significantly inhibited by PKA inhibitor H89 and Epac1 siRNA. HGF expression in preeclamptic placentas was decreased. hPMSCs expressed HGF and the expression was reduced in hypoxia. The hPMSC number was decreased in preeclamptic placenta compared to controls (0.680.1% vs.1.320.5%; P¼0.026). hPMSC conditioned medium activated trophoblast Rap1 and enhanced trophoblast migration, which was inhibited by c-Met blocking antibody. Furtherrmore, trophoblast migration was reduced by hPMSC conditioned medium cultured in hypoxia. Conclusion: hPMSCs express HGF and increase trophoblast cAMP production. cAMP effector PKA and Epac1 act in concert to modulate adhesion and migration of trophoblast via signaling to the Rap1 and integrin b1. These results suggest that hPMSC may involve in trophoblast migration and pathogenesis of preeclampsia.

Abstracts / Placenta 32 (2011) A1–A149

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P1.115 SECRETION OF ANGIOGENIC FACTORS BY AMNION-DERIVED MESENCHYMAL STROMAL CELLS

P1.116 PLACENTAL MESENCHYMAL STEM CELLS MIGRATE IN A PLACENTAL VESSEL PERFUSION MODEL

Julia König1, Gregor Weiß1,2, Berthold Huppertz1, Achim Lass2, Ingrid Lang1 Institute of Cell Biology, Histology and Embryology; Medical University of Graz, Graz, Austria, 2Institute of Molecular Biosciences; Karl-FranzensUniversity Graz, Graz, Austria

Batla Al-Sowayan1,2, Rosemary Keogh1,2, Mohamed Abumaree3, Dr Mohammed Al Jumah3, Shaun P Brennecke1,2, Bill Kalionis1,2 1 University of Melbourne Department of Obstetrics and Gynaecology, Parkville, Victoria, Australia, 2Department of Perinatal Medicine Pregnancy Research Centre, Royal Women’s Hospital, Parkville, Victoria, Australia, 3 King Abdullah International Medical Research Center, King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Riyadh, Saudi Arabia

1

Objectives: Bone marrow-derived mesenchymal stromal cells (MSC) are multipotent cells with the ability to promote angiogenesis by paracrine effects. MSC isolated from the amnionic membrane (hAMSC) are a promising alternative source of MSC, as cells can be isolated non-invasively and are immunologically well tolerated. Therefore, we investigated the secretion of angiogenic factors by hAMSC and assessed the effect of hAMSCconditioned medium (CdM) on endothelial cells. Methods: hAMSC and placental endothelial cells were isolated from human term placentas according to the protocols of Soncini et al. [1] and Lang et al. [2]. CdM was prepared by incubating confluent hAMSC with EGM-2 for 48h. Effect of CdM on viability and network formation of endothelial cells was determined using LDH and Matrigel assay, respectively (n¼4). For detection of angiogenic proteins, CdM was analysed with an angiogenesis array kit (n¼3). Non-conditioned medium was used as control. Results: Culture of placental endothelial cells in the presence of CdM significantly reduced the activity of LDH in the culture supernatants to 48.714.9% of control (n¼4, p<0.001). As LDH is released into the culture medium by damaged cells, endothelial cell viability was clearly enhanced. In addition, CdM promoted the formation of network-like structures of placental endothelial cells in the Matrigel assay. While networks formed by endothelial cells in control medium started to disintegrate after 24h, cells cultured with CdM were still stable at 72h. Protein analysis of CdM revealed secretion of pro-angiogenic factors such as angiogenin, angiopoietin-1&2, and IL-8 by hAMSC. Conclusion: hAMSC secrete factors that enhance viability and support network formation of endothelial cells. These results indicate angiogenic properties of hAMSC which might be beneficial for a therapeutic application in vascular biology. [1] Soncini M et al. J Tissue Eng Regen Med 2007; 1:296-305. [2] Lang I et al. Differentiation 2008; 76:1031-1043.

Objectives: We hypothesised that placental mesenchymal stem cell (pMSCs) migration can be assessed using a placental vessel ex vivo perfusion model. To test this hypothesis, placental vessels were perfused using pMSCs labelled with live-cell fluorescent tracker dye and cells migrating across the endothelium were counted. Method: Term placentae from uncomplicated pregnancies were obtained with informed consent. A stem cell line (PRCFP) was created from primary, term pMSCs by hTERT transformation (Prof. S Gronthos, Adelaide). We verified that PRCFP preserves important mesenchymal stem cell characteristics. PRCFPs were >95% positive for surface markers CD105, CD73, and <5% positive for CD45, as expected. PRCFPs were differentiated into adipocytes and osteocytes (n¼5 each), and PRCFPs were able to migrate in scratch assays (22þ/- 2% scratch closure in 8 hours, n¼5). Live-cell staining with Cell Tracker orange (CMTMR; Invitrogen, CA, USA) was carried out after optimising for the strongest cell fluorescence, whilst maintaining cell viability and proliferation. The perfusate contained 1.6x106 – 4.1x106 CMTMR-labelled PRCFPs and incubation times ranged between three hours to four days in 37oC. The perfused vessels were then washed, cryopreserved and sectioned (5mm). Multicolour immunofluorescence was carried out. Endothelium was detected with a fluorescein conjugated antivWF antibody (Meridian Life Science, USA). Nuclei were detected by DAPI staining. Our criterion for migration was the detection of CMTMR-labelled PRCFPs that had crossed the endothelial barrier. Results: Six vessels were perfused. Qualitative assessment of immunofluorescence in sections revealed CMTMR-labelled PRCFPs had crossed the endothelium barrier in five of the six perfusions. Preliminary results from one of the vessels (n¼19, 5mm sections) showed an average of 1.05 cells/ 5mm section crossed the endothelial barrier (i.e. 210 PRCFPs/cm of vessel). Conclusion: The placental vessel ex vivo perfusion model can be used to assess pMSC migration.

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Abstracts / Placenta 32 (2011) A1–A149

P1.117 SEX-SPECIFIC REGULATION OF GLUCOCORTICOID EXPOSURE HUMAN PRETERM PREGNANCIES BY P-GLYCOPROTEIN

IN

Nicolette Hodyl, Vicki Clifton, Michael Stark University of Adelaide, Adelaide, Australia Objective: Fetal glucocorticoid exposure is tightly regulated across gestation by the placental glucocorticoid barrier. This is comprised in part by the cortisol inactivating enzyme 11beta hydroxysteroid dehydrogenase (11bHSD2) together with multidrug resistant transmembrane efflux proteins, such as P-glycoprotein (P-gp), located on the syncytiotrophoblast. Observations of a greater prophylactic effect of the synthetic glucocorticoid betamethasone in female preterm births compared to males have been demonstrated. We questioned whether this was due to sex-specific alterations in placental P-gp. The aim of this study was to assess placental P-gp together with 11bHSD2 activity in placenta from preterm pregnancies to understand glucocorticoid regulation. Methods: Placental samples were collected from women who delivered preterm (24-36 weeks n¼42) or at term (n¼11). P-gp mRNA was measured by qRT-PCR and 11bHSD2 activity by radiometric conversion assay. Betamethasone exposure was classified as delivery <72 or >72 hours after maternal steroid administration. Results: Placental P-gp expression increased exponentially with advancing gestation in the preterm infants (R2¼.364, p¼0.018), but expression returned to low levels at term. P-gp expression was inversely correlated with 11bHSD2 activity in females (r¼-.664, p¼0.018) but not in males. P-gp was not affected by betamethasone exposure. Conclusion: Antenatal betamethasone does not alter expression of P-gp in preterm placenta. However, P-gp appears to play a strong role in maintaining fetal glucocorticoid homeostasis across gestation. This is particularly evident with a female preterm infant, where our results suggest increased glucocorticoid efflux from the placenta by P-gp when 11bHSD2 activity is at its lowest. This study supports previous observations of greater placental sensitivity and adaptation in the female preterm neonate compared to male.

P1.118 THE INVOLVEMENT OF SPHINGOSINE-1-PHOSPHATE IN HUMAN LABOR ONSET VIA INDUCTION OF COX-2 Tomomi Kotani, Yuka Hattori, Yukio Mano, Seiji Sumigama, Sawako Fujiwara, Eiko Yamamoto, Fumitaka Kikkawa Nagoya Graduate University of Medicine, Nagoya, Japan Objectives: Sphigosine-1-phosphate (S1P) has been reported to be involved in inflammation of several tissues. Therefore, we investigated the involvement of S1P in labor onset, which is similar to inflammation, and examined the effect of S1P on the other molecules such as cox-2 and MMP9, which are well known to regulate labor, using human amnion. Methods: Amnions were obtained with informed consent from normal pregnancies following elective caesarean deliveries (labor (þ)) and vaginal deliveries (labor (-)). The expression of sphingosine kinase (SK), which produces S1P, was compared between amnion tissues from labor (þ) and those form labor (-) by Western blotting. In addition, cox-2 expression was also examined in them. Immunohisochemistry was performed to identify the localization of SK in placenta. Human amnion cells were cultured using amnions from elective caesarean deliveries and the expressions of subtypes of S1P receptor were determined by RT-PCR. Then, the expressions of cox-2 and MMP-9 were examined under 0-1000nM S1P treatment of amnion cells. Results: SK was expressed in more highly amnion tissues from labor (þ) than those form labor (-). In addition, the expressions of cox-2 and MMP-9 were also increased in amnion of labor (þ). SK was strongly expressed in amniotic epithelial cells, and weak in deciduas and placenta. Primary cultured human amnion cells were expressed mainly S1P receptor 1-3, which was coupled with Gi protein. The treatment of S1P induced cox-2 and MMP-9 expression in primary cultured amnion cells and the induction was suppressed by Gi inhibitor. Conclusion: In this study, we firstly showed that SK expression was increased in amnion tissues from labor (þ) and S1P increased cox-2 and MMP-9 expression in human amnion. Our results suggest that S1P might play a role in human labor onset via induction of cox-2 and MMP-9.

Abstracts / Placenta 32 (2011) A1–A149

P1.119 EPIGENETIC REGULATION OF MATRIX METALLOPROTEINASE-9 IN PRIMARY AMNION CELLS Martha Lappas1,2, Nicole Dellios3, Murray Mitchell3, Greg Rice1,3 1 University of Melbourne, Heidelberg, Victoria, Australia, 2Mercy Hospital for Women, Heidelberg, Victoria, Australia, 3University of Queensland Centre for Clinical Research, Herston, Brisbane, Australia Introduction: Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade the extracellular matrix (ECM) thus leading to the rupture of the fetal membrane. In keeping with this, amniotic fluid concentration of MMP-9 is higher in patients with preterm prelabour rupture of membranes (PPROM) than in those with preterm labour and intact membranes. Aberrant DNA methylation patterns and histone modifications have been associated with dysfunction and disease. There is, however, a paucity of data concerning the role of DNA methylation and histone modification in the process and consequences of human labour. Objective: To determine the effects of the inhibitor of DNA methylation 5aza-2-deoxycytidine (AZA) and the inhibitor of histone deacetylation trichostatin A (TSA) on IL-1b-stimulated MMP-9 activity secreted form primary amnion epithelial cells. Methods: Primary amnion epithelial cultures (n¼5) were isolated from term amnion and treated with 5 mM AZA or 300 nM TSA in the absence or presence of 10 ng/ml IL-1b for 24 h. Media was analysed for MMP activity by gelatin zymography. Results: IL-1b treatment significantly increased the release of MMP-9 from primary amnion cells. Both AZA and TSA treatment significantly reduced IL-1b induced MMP-9 release. There was, however, no effect of AZA and TSA on basal MMP-9 release. Discussion: Our data demonstrate that the release of MMP-9 from human primary amnion cells is inhibited by AZA and TSA treatment following IL1b stimulation. These preliminary data suggests a role for epigenetic regulation of pro-inflammatory mediators through histone modifications and inhibition of DNA methylation.

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P1.120 HOMOCYSTEINE-INDUCED PREMATURE DELIVERY IN MICE REQUIRES THE NIACIN RECEPTOR GPR109A Srinivas Sonne1, Varun Bhalla1, Stefan Offermanns2, Vadivel Ganapathy1 Georgia Health Sciences University, Augusta, GA, United States, 2University of Heidelberg, Heidelberg, Germany

1

Objective: Mice with heterozygous deletion of cystathionine-b-synthase (Cbs) have increased levels of homocysteine and deliver pups 3 days earlier than wildtype mice. This process is associated with increased expression of cyclooxygenase-2 (Cox-2) in uterus and placenta. Pharmacological inhibition of Cox-2 in pregnant Cbsþ/- mice reverses the premature delivery. GPR109A is a G-protein-coupled receptor for niacin. Therapeutic use of niacin produces undesirable side effects such as flushing, caused by induction of Cox-2 in epidermal dendritic cells. This suggested that deletion of Gpr109a might prevent Cox-2 induction in Cbsþ/- mice and consequently reverse premature delivery. Here we investigated the role of Gpr109a in homocysteine-induced premature delivery in Cbsþ/- mice. Methods: Cbsþ/-/Gpr109a-/- mice were obtained by crossing Cbsþ/- mice and Gpr109a-/- mice, and the gestational period was compared between Cbsþ/-/Gpr109aþ/þ mice and Cbsþ/-/Gpr109a-/- mice. Uterine expression of Gpr109a was compared between wildtype and Cbsþ/¼ pregnant mice. The influence of homocysteine on GPR109A expression was assessed in the human myometrial cell line ULTR. Results: Cbsþ/-/Gpr109aþ/þ mice delivered pups on gestational day (GD) 16.6  0.12 whereas Cbsþ/-/Gpr109a-/- mice and wildtype mice delivered pups on GD 20  0.5 and 20  0.17 respectively. Gpr109a was expressed in uterus from wildtype pregnant mice, demonstrable at mRNA and protein levels. The expression of Gpr109a in uterus was higher in Cbsþ/- pregnant mice than in wildtype pregnant mice. Treatment of ULTR with homocysteine induced the expression of GPR109A. Conclusions: Elevated levels of homocysteine cause premature delivery in mice and functional Gpr109a is obligatory for this process, indicating that homocysteine-induced premature delivery in Cbsþ/- mice is mediated through Gpr109a. Homocysteine induces Gpr109a expression in uterus, but whether homocysteine or its metabolites directly activates the receptor remains to be determined. (Supported by a grant from the Child Health Discovery Institute at GHSU)

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Abstracts / Placenta 32 (2011) A1–A149

P1.121 DIFFERENT SERUM INSULIN-LIKE GROWTH FACTOR (IGF) LEVELS ARE ASSOCIATED WITH CHANGES IN THE LOCAL GALECTIN EXPRESSION IN THE BOVINE NEAR TERM UTERUS AND PLACENTA

P1.122 THE RELATIONSHIP BETWEEN ANTIPHOSPHOLIPID ANTIBODIES AND THE OVARIAN RESERVE IN PATIENTS WITH TRANSITIONAL CONDITION OF REPRODUCTIVE FAILURES

Rebecca Froehlich1, Nina Hambruch1, Marion Piechotta2, Herbert Kaltner3, Hans-Joachim Gabius3, Heinrich Bollwein2, Christiane Pfarrer1 1 Department of Anatomy, University of Veterinary Medicine Hannover, Hannover, Germany, 2Clinic for Cattle, University of Veterinary Medicine Hannover, Hannover, Germany, 3Department of Biochemistry, LMU Munich, Munich, Germany

Eri Takahashi, Yuki Nakada, Atsuko Kato, Rie Kawaguchi, Tomoko Hashimoto, Takayuki Haino, Nozomu Yanaihara, Kouhei Sugimoto, Tadao Tanaka Jikei University, School of Med., Tokyo, Japan

Objectives: It has been demonstrated that lower levels of serum IGF-1 are associated with elevated inflammation markers in the endometrium and subsequently higher incidence of postpartum metritis. As galectins (gal) were shown to have a modulatory influence on the local immune response of the uterus our objective was to examine whether or not the local expression of galectins can be related to serum IGF levels. Methods: 17 clinically healthy, pregnant cows were divided according to their IGF levels prepartum into an IGF high (n¼10) and IGF low (n¼7) experimental group. From each animal two placentomes (P) and interplacentomal uterine tissues (IP) were sampled during a caesarean section performed on day 275 after artificial insemination. Gene expression of gal-1, -3, -4, -9 and -15 was assessed by RT-qPCR and immunohistochemistry. Results: On mRNA level gal-1 in P, gal-3 and gal-13 in IP and gal-9 in P and IP showed a higher expression in IGF high animals. For gal-13 this difference was statistically significant (p¼ 0.05). Gal-4 in both P and IP and gal13 in P were more abundantly expressed in IGF low animals. No difference in expression occurred for gal-1 in IP and for gal-3 in P. Gal- 1 protein was localized in stroma, whereas gal-3 appeared in epithelia of IP. In P gal-1 stained all cells except mesenchyme and TGC. Gal-3 was restricted to mesenchyme and maternal epithelium. Gal-4 and gal-9 occurred in epithelia and stroma of both IP and P, while in P gal- 9 showed granular staining in TGC and a stronger immunoreaction in maternal stroma and vessel walls of IGF high animals. Conclusion: Differences in the gal expression between IGF high and low animals lead to the assumption that a systemic imbalance of the IGF system may influence the local gal expression pattern. Funded by Pfizer Inc.

Objectives: Pathogenic antiphospholipid antibodies (aPLs) has been found approximately 10% in circulating blood of patients with recurrent spontaneous abortion (RSA). However, the incidence has been a matter of controversy in patients with unexplained infertility (UI). Moreover, pathomechanisms of aPLs have been elucidated to some degree in RSA, but still not clarified in UI. Then in the clinical practice, it is not a little case who have experienced RSA become to suffer UI thereafter, and this is the other way around, called transitional condition (TC). So we discuss the impact of aPLs to these reproductive failures focusing on ovarian reserve to investigate the difference and contribute to appropriate clinical management. Methods: Thirty-nine women with RSA, 8 with TC, and 12 with UI without any episode of pregnancy were analyzed retrospectivey. The blood level of aPLs including lupes anticoagulant, anti-cardiolipin (CL) antibody, CLbeta2-GP1, anti-phosphatidylserine antibody and antiphosphatidylethanolamine antibody, and LH, FSH, estradiol, antiMullerian hormone (AMH) were measured. Statistical probability level of p ¼ 0.05 was taken as the limit of significance. Results: Incident of any aPL was 43.8% in RSA, 62.5% in TC and 50.0% in UI. Concerning about the incidence of plural aPLs in RSA, TC and UI showed 23.1%, 12.5% and 33.3%, respectively, without significant difference. As for LH, FSH, and estradiol, these were within normal level in three groups, while the incidence of low revel AMH was significantly higher both in RSA with 28.2% and TC with 37.5% than 0% in UI. Conclusion: This is the first report lighting to TC, a particular condition of reproductive failure between RSA and infertility. The existence of aPLs is the possible risk factor of both TC and UI like RSA, however, aPLs may not affect the ovarian reserve in UI. These results suggest the different pathomechanisms may exist among these reproductive failures.

Abstracts / Placenta 32 (2011) A1–A149

P1.123 REGULATION OF TROPHOBLAST IN THE DEVELOPING HUMAN PLACENTA BY INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-4 Laura Goodfellow, Karen Forbes, Melissa Westwood, Edward Johnstone Maternal and Fetal Health Research Group, University of Manchester, Manchester Objective: Insulin-like growth factors (IGFs) are known to enhance cytotrophoblast proliferation and therefore expansion of the human placenta in first trimester. IGF bioavailabity and function are regulated by a family of IGF-binding proteins (IGFBPs). IGFBP-1 and 3 have been shown to inhibit IGF-stimulated cytotrophoblast proliferation. IGFBP-4 is also known to be present at the maternal-fetal interface at term, however it’s role in regulating early placental development has yet to be investigated. Methods: The expression and localisation of IGFBP-4 in human first trimester placental tissue was assessed by immunohistochemistry (IHC). The effect of IGFBP-4 (100ng) on IGF-II (10nM)-stimulated cytotrophoblast proliferation was assessed by IHC analysis of BrdU (100mM) incorporation 24h after incubation of serum-starved placental explants (n¼5) with hormone(s). Statistical analysis was performed using Graphpad PRISM software (ver. 4). Results: IGFBP-4 was present in both syncytio- and cytotrophoblasts of first trimester placenta. IGF-II treatment of explant cultures increased cytotrophoblast proliferation by 84% relative to control (p<0.05), in agreement with previous studies. Addition of IGFBP-4 did not significantly alter IGF-II induced cytotrophoblast proliferation, suggesting that this IGFBP may have a different role in the placenta. Conclusion: The presence of IGFBP-4 in early placenta supports a role in regulating IGF function. In contrast to work in other tissues, our data suggest that in cytotrophoblast, IGFBP-4 does not attenuate the proliferative response to IGF-II. IGFBP-4 is a substrate for pregnancy associated plasma protein A (PAPPA), a protease that is abundantly expressed by first trimester trophoblast, thus it is possible that IGFBP-4’s actions are moderated by post-translational cleavage. Interestingly, low maternal PAPPA levels in the first trimester are associated with adverse pregnancy outcome thus the role of this system in regulating early placental development warrants further investigation.

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P1.124 DYSREGULATION OF PROTEIN SYNTHESIS DISRUPTS PLACENTAL ENDOCRINE BIOACTIVITY: IMPLICATIONS FOR INTRAUTERINE GROWTH RESTRICTION Hong wa Yung1, Claire Senner2, Randal Kaufman3, Myriam Hemberger2, Stephen Charnock-Jones4,5, Graham Burton1,5 1 Centre for Trophoblast Research, University of Cambridge, Cambridge, Cambridgeshire, UK, 2The Babraham Institute, Cambridge, Cambridgeshire, UK, 3Howard Hughes Medical Institute, University of Michigan, Michigan, USA, 4Department of Obstetrics and Gynaecology, University of Cambridge, Cambridge, Cambridgeshire, UK, 5Cambridge Comprehensive Biomedical Research Centre, Cambridge, Cambridgeshire, UK Objective: To determine whether endoplasmic reticulum (ER) stress impacts on placental and fetal growth. All membrane and secretory proteins are synthesized and processed in the ER. We recently demonstrated evidence of ER stress in the syncytiotrophoblast of growth restricted human placentas. We hypothesise that ER stress leads to abnormal glycosylation and loss of function of placental hormones and growth factors. Methods: Mouse embryonic fibroblasts were prepared from mice in which Ser51 of the translation initiation factor eIF2a had been mutated to Ala (eIF2a A/A MEF), causing elevated protein synthesis. Conditioned media (CM) were prepared from wild-type and mutant MEF. Using lectin column purification, we tested for a change in glycosylation pattern of secreted proteins. Conditioned media were applied to mouse trophoblast stem (TS) cells, and human umbilical vascular endothelial cells (HUVEC) as bioassays for secreted growth factors. Results: eIF2a A/A MEF exhibited ER stress as phosphorylation of PKR-like ER kinase (PERK) was increased under normal culture conditions. eIF2a A/ A MEF displayed a w50% reduction in cell proliferation and decreased Akt signalling. A change in glycosylation pattern in the CM was observed, while the TS bioassay revealed loss of function of the secreted proteins. In particular, there was an accumulation of pro-IGF2 in the eIF2a A/A CM, while cellular pro-IGF2 level was unchanged. An additional VEGF isoform was found in the eIF2a A/A CM, which was neither the result of alternative mRNA splicing nor a change of cellular VEGF level. Crucially, eIF2a A/A CM failed to activate VEGF receptor 2 in the HUVEC bioassay. Conclusion: ER stress in vitro affects glycosylation and post-translational modifications of secreted proteins,, and is associated with loss of bioactivity. This has profound implications for autocrine and paracrine placental signalling, for eIF2a A/A mice display both placental and fetal growth restriction. Supported by Wellcome Trust.

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Abstracts / Placenta 32 (2011) A1–A149

P1.125 MESENCHYMAL PARACRINE FACTORS OVERCOME DEFECTIVE TRISOMY 21 TROPHOBLAST FUSION AND DIFFERENTIATION IN VITRO

P1.126 EARLY DEXAMETHASONE (DEX) EXPOSURE HAS LASTING EFFECTS ON GLUCOCORTICOID RECEPTOR ALPHA (GRA) IN SHEEP PLACENTOMES

Pascale Gerbaud1,2, Guillaume Pidoux1,2, Niroshani Pathirage3, Jean Guibourdenche1,2, Jean-Marc Costa4, Josette Badet1, Jean-Louis Frendo1, Padma Murthi3, Danièle Evain-Brion1,2 1 Inserm U767, Paris, France, 2PremUP, Paris, France, 3University of Melbourne, Melbourne, Australia, 4CERBA, Cergy Pontoise, France

Thorsten Braun1,2, Hongkai Shang1, Wenbin Meng1, Deborah M. Sloboda3, Shaofu Li4, Andreas Plagemann1,2, Joachim W. Dudenhausen1, John P. Newnham4, John R.G. Challis5 1 Charite University Berlin Department of Obstetrics, Berlin, Germany, 2 Charite University Berlin Division of Perinatal Programming, Berlin, Germany, 3Liggins Institute University of Auckland and The National Resaerch Centre for Growth and Development, Auckland, New Zealand, 4 School of Women’s and Infants’ Health, The University of Western Australia, Perth, Australia, 5Michael Smith Foundation for Helath Research, Vancouver, Canada

Objectives: Placental development is markedly abnormal in trisomy 21 (T21) pregnancies. We hypothesized that abnormal paracrine crosstalk between the fetal mesenchymal core and the trophoblast might be involved in the defect of syncytiotrophoblast (ST) formation and function. Methods: In a large series of primary cultured human cytotrophoblasts isolated from second-trimester control (n ¼ 44) and T21 placentae (n ¼ 71), abnormal trophoblast fusion and differentiation was observed in more than 90% of T21 cases. We then isolated and cultured villous mesenchymal cells from control (n ¼ 10) and T21 placentae (n ¼ 8) and confirmed their fetal origin. Results: Conditioned medium of control mesenchymal cells overcome the abnormal trophoblast fusion of T21 cytotrophoblasts in a cAMPindependent manner but stimulating the TGFb signaling, as shown by the phosphospecific protein microarray analysis and the use of TGFb signaling pathway antagonists. Using protein arrays, we further analyzed the cytokines present in the conditioned medium from control and T21 mesenchymal cells. Activin-A was strongly secreted by cells from both sources, but at a significantly (p < 0.01) lower level in the case of T21 mesenchymal cells. Recombinant activin-A stimulated T21 trophoblast fusion. Blocking activin-A antibody inhibited the fusion induced by conditioned medium and exogenous activin-A. Conclusion: The defective trophoblast fusion and differentiation associated with T21 can be overcome in vitro, and reveal the key role for fetal mesenchymal core in trophoblast differentiation in this process.

Objectives: We hypothesized that GRa may play an important role in mediating the effects of endogenous cortisol on binucleate cell function, and that early DEX exposure, as a model for early stress, modifies this response. Methods: Pregnant ewes were randomized to control (2ml saline/ewe) or DEX treated groups (i.m. injections of 0.14 mg/kg ewe weight per 12h) at 40–41 days of gestation (dG). The localization of GRa immunopositive BNCs (GRa-BNC) was analyzed at 50, 100, 125, 140dG according to placentome subtype (A,B,C,D). Results: Three different GRa-BNC were found: BNCs with two GRa positive nuclei (þþ), BNCs lacking any nuclear GRa staining (–) and BNCs with one positive and one negative GRa nucleus (þ-). (þþ) was the most frequent, (–) the least frequent cell-type. The proportion of (þþ) decreased from 100 to 140dG, the proportion of (–) increased towards term. In males, the number of GRa-BNCs was decreased at 100 and 140dG compared to controls. At 100dG, these changes were due to a decrease in all three celltypes in A’s; at 140dG due to a decrease in (þþ) and (þ-) in C’s. In females, DEX decreased the number of (þþ) at 125dG in Bs compared to controls. The proportion of (þþ) after DEX increased from 50 to 100dG, whereas the proportion of (þ-) and (–) decreased. Conclusions: Early DEX exposure altered the number of GRa-BNCs; changes were sex- and cell type- dependent. We speculate that 3 functional cell-types of BNCs exist; (þþ) as the active form, (þ-) as the intermediate form and (–) as the inactive form and that the activity of BNCs is determined by the distribution of these 3 cell-types. GRa may play an important role in mediating the effects of cortisol on BNCs function, converting BNCs from an active to inactive form.

Abstracts / Placenta 32 (2011) A1–A149

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P1.127 REGULATION OF LEPTIN EXPRESSION BY HCG AND CAMP IN PLACENTAS FROM NORMAL AND PATHOLOGICAL PREGNANCIES

P1.128 CO-CULTURE OF HUMAN ADRENOCORTICAL AND TROPHOBLAST CELLS TO STUDY THE REGULATION OF FOETO-PLACENTAL STEROIDOGENESIS

Julieta Maymó1, Antonio Pérez-Pérez2, Bernardo Maskin3, Juan Carlos Calvo1, Víctor Sánchez Margalet2, Cecilia Varone1 1 Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina, 2Departamento de Bioquímica Médica y Biología Molecular. Universidad de Sevilla, Sevilla, Spain, 3Hospital Nacional Profesor Alejandro Posadas, Provincia de Buenos Aires, Argentina

Andrée-Anne Hudon-Thibeault, Cathy Vaillancourt, J Thomas Sanderson INRS-Institut Armand Frappier, Université du Québec, Laval, Quebec, Canada

Objective: Leptin, the 16000 MW protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta, where it was found to be expressed. Recently, we have demonstrated that leptin promotes proliferation and survival of trophoblastic cells. We also showed that leptin expression is tightly regulated by different important molecules in pregnancy, such as hCG, cAMP and estradiol. In different pathologies associated with reproduction, leptin expression level changes dramatically, but little has been described on its mechanisms of action and regulation. In the present work we aimed to study the regulation of leptin expression by hCG and cAMP in normal and pathological placentas. Methods: JEG-3 cells, cultured under standard conditions, as well as human placenta explants were used. Western blot analyses were carried out to detect leptin expression as well as the phosphorylated form of the ERK1/2 protein. Real time-PCR was also used to detect leptin expression. Results: We observed by Western blot and qRT-PCR that leptin expression is increased in pathological placentas compared with normal ones. Moreover we determined that the regulation of leptin expression by hCG (0-100 IU/ml) and cAMP (0-1mM) is lost in placentas from different pathologies (gestational diabetes, IUGR, preeclampsia). In these cases, leptin expression was significantly lower in pathological placentas than in normal placentas at term. Evenmore, ERK 1/2 phosphorylation is stimulated by hCG in normal placental explants and this effect disappears in placentas from pathological pregnancies. Conclusion: These results improve the comprehension of leptin function during pregnancy and further support the importance of leptin in the biology of reproduction.

Objectives: To develop an in vitro co-culture system of human BeWo (trophoblast) and H295R (foetal adrenocortical) cells to study the regulation of steroidogenesis of the foeto-placental unit. To validate our system, specific objectives are to determine the effect of the co-culture on BeWo and H295R cell phenotypes and the capacity of the co-culture to produce estrogens (aromatase activity). Methods: H295R cells (bottom of wells) and BeWo cells (in the insert) were cultured in plates with transwell permeable polycarbonate inserts (0.4 mm pores) in a modified co-culture medium: DMEM/F-12 HAM without phenol red, supplemented with 1.2 g/L NaHCO3, 2 mg/L pyridoxine-HCl, 1% FBS, 2.5% Nu-serum and 1% ITSþ1. Cell proliferation and doubling time were evaluated with the xCELLigenceÔ system (Roche) a technology that enables continuous cell monitoring. Aromatase activity was determined by tritiated water-release assay and human chorionic gonadotropin (hCG) production was determined by ELISA. Results: The xCELLigenceÔ system demonstrated that the co-culture medium did not affect proliferation of the two cell lines. H295R cell doubling time was reduced in the co-culture medium (29.6  0.7 h) compared to the normally recommended cell culture medium (34.4  1.2 h). The co-culture medium had no impact on hCG production in BeWo cells suggesting that their differentiating capacity was not affected. Aromatase activity was four times higher in BeWo than in H295R cells, but was not significantly altered in the presence of co-culture medium. Conclusion: These results demonstrate that a BeWo and H295R co-culture is suitable to study the regulation of foeto-placental steroidogenesis. This co-culture model provides a unique and valuable research tool to evaluate effects of drugs and toxic compounds on the foeto-placental unit and consequently on pregnancy and foetal development.

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Abstracts / Placenta 32 (2011) A1–A149

P1.129 IMPACT OF MONOCARBOXYLATE TRANSPORTER 8 DEFICIENCY ON FETOPLACENTAL GROWTH IN MICE.

P1.130 LEPTIN RECEPTOR RECRUITS SAM68, AN RNA-BINDING PROTEIN, TO INTRACELLULAR SIGNALING IN HUMAN TROPHOBLASTIC JEG3 CELLS

Elisavet Vasilopoulou1, Heike Heuer2, Marija Trajkovic2, Laurence Loubière1, Christopher McCabe1, Jayne Franklyn1, Mark Kilby1, Shiao Chan1 1 University of Birmingham, Birmingham, United Kingdom, 2Leibniz Institute for Age Research-Fritz Lipmann Institute, Jena, Germany

Flora Sánchez-Jiménez1, Antonio Pérez-Pérez1, Carmen González-Yanes1, Cecilia L. Varone2, Víctor Sánchez-Margalet1 1 Department of Clinical Biochemistry. Virgen Macarena University Hospital, University of Seville, Seville, Spain, 2Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina

Objectives: Monocarboxylate transporter 8 (Mct8) is a plasma membrane thyroid hormone (TH) transporter expressed in the human placenta from 6 weeks of gestation. We examined the role of Mct8 in fetoplacental growth and development in vivo using a Mct8 knockout mouse model. Methods: Heterozygous females were mated with male knockout (Mct8-/y) or wild-type (Mct8þ/y) mice. Mct8þ/y and Mct8-/y fetoplacental tissues were collected before (E14.5) and after (E18.5) the onset of fetal TH production. Mct8 protein was localised in the mouse placenta by immunohistochemistry. The volume fractions of the labyrinthine (Lz) and the junctional (Jz) zones of the placenta were estimated by stereology. Cyclin D1 (proliferation) and cleaved Caspase 3 (apoptosis) protein expression was assessed by immunohistochemistry and Western blotting. Results: At both E14.5 and E18.5, Mct8 protein was present in the decidua, in spongiotrophoblast and glycogen cells in the Jz, in trophoblast giant and syncytiotrophoblast cells in the Lz, in the wall of chorionic blood vessels and in the chorionic membrane. Fetal:placental weight ratios were decreased in Mct8-/y fetuses at E18.5 compared with Mct8þ/y (25%, P<0.05; n¼8 litters) but there were no differences at E14.5. This was accompanied by a reduction in the volume fraction of the Lz (10%; P<0.05; n¼2 litters) with no difference in the absolute volume. Cyclin D1 was predominantly expressed in endothelial cells within the Lz and its expression was reduced in Mct8-/y placentae at both E14.5 and E18.5, whilst there was no difference in the expression of cleaved Caspase 3. Conclusion: The localisation of Mct8 expression suggests a role for Mct8 in TH-responsive placental development and in the transplacental transport of TH from the mother to the fetus. Lack of Mct8 results in reduced placental efficiency, which may be associated with a reduction in the proliferation of endothelial cells within the Lz.

Leptin is produced in placenta where it functions as an important autocrine signal for human trophoblastic growth during pregnancy, promoting antiapoptotic and trophic effects, by activating leptin receptor signaling. Sam68 is an RNA-binding protein that has previously been implicated in leptin signaling in other systems. More recently, Sam68 has been found to play an important role in fertility. Objectives: In this study we aim to investigate the possible role of Sam68 in leptin receptor signaling and leptin action in trophoblastic cells (JEG-3 cells). Methods: We studied leptin-mediated Sam68 phosphorylation by immunoprecipitation and immunoblot with anti-phosphotyrosine antibodies. Leptin effect on Sam68 RNA binding properties was assessed by poly-U affinity precipitation. The pharmacological inhibitor of JAKs, AG490, was used to assess the JAK-2-dependency of the Sam68 phosphorylation. Antisense strategy was used to knockdown Sam68 expression. Leptin receptor signaling pathways activation were analysed by specific immunoblot using antibodies against the phosphorylated forms of MEK, MAPK, PKB, GSK-3, RPS6KB1, EIF4E, EIF4EBP. Leptin effect on proliferation and protein synthesis were assessed by 3[H]-Thymidine and 3[H]Leucine incorporation respectively. Quantitative PCR and immunoblot were used to study both leptin receptor and Sam68 expression. Results: Leptin dose-dependently stimulated Sam68 tyrosine phosphorylation in JEG-3 cells, thus inhibiting its RNA binding capacity, as previously observed in other systems. We have also found that leptinstimulated Sam68 tyrosine phosphorylation is dependent on JAK-2 activity. Moreover, leptin dose-dependently promoted an increase in Sam68 expression. Consistently, knockdown of Sam68 expression decreased leptin receptor expression. Even though leptin actvation of JAK2 was preserved, the inhibition of Sam68 expression prevented leptin stimulated activation of STAT, MAPK and PI3K signaling pathways as well as its proliferative and trophic action. Conclusion: These results strongly suggest the participation of Sam68 in leptin receptor signaling and action in human trophoblastic cells, also mediating the trophic effects of leptin in placenta.

Abstracts / Placenta 32 (2011) A1–A149

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P1.131 HUMAN VILLOUS AND EXTRAVILLOUS TROPHOBLAST CELLS PRODUCE SEROTONIN DE NOVO

P1.132 GLUCOCORTICOID MEDIATED SUPPRESSION OF GROWTH FACTORS MAY UNDERLIE IMPAIRED PLACENTAL CELL TURNOVER

Kathy Deroy, J. Thomas Sanderson, Cathy Vaillancourt INRS-Institut Armand Frappier, Université du Québec, Laval, Québec, Canada

Justine L. Nugent, Alka Shah, Colin P. Sibley, Rebecca L. Jones Maternal and Fetal Health Research Centre, University of Manchester, Manchester, United Kingdom

Altered maternal serotonin levels are observed in pregnancy complications associated with trophoblast alterations, suggesting a role for this neurohormone in placental function and development. Serotonin and its receptors and transporter have been found in placental villi and trophoblast. However, whether human placenta synthesizes serotonin or depends on circulating serotonin has never been studied. Objective: We propose that the human placenta produces serotonin de novo. To verify this hypothesis, the expression and activity of tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin synthesis, was determined. Methods: Placentas were obtained with informed consent from uncomplicated pregnancies following vaginal delivery (term) or from therapeutic abortion (1st trimester). Immunohistochemical analyses determined the tissue sublocalization of TPHs. Primary cultures of term (villous) and first trimester (villous and extravillous) trophoblasts were isolated and purified using a magnetic cell sorter. TPH1 and TPH2 expression was quantified by real-time RT-PCR and immunoblot analyses. Serotonin secretion by primary trophoblast cells was measured by LC-MS/MS. Results: Immunohistochemical analyses showed that villous cytotrophoblast, syncytiotrophoblast, foetal capillaries (from both 1st trimester and term placentas) and extra-villous cytotrophoblast express TPH1 and TPH2. The expression (mRNA and protein) of both TPHs by villous and extravillous cytotrophoblast and syncytiotrophoblast was confirmed using primary trophoblast lysates. Moreover, de novo production of serotonin in trophoblast cells occurred and serotonin secretion was increased along with differentiation of term villous cytotrophoblast (68.9  6.3 pg/h/mg protein) into syncytiotrophoblast (461.2  22.2 pg/h/mg protein). Conclusion: This study demonstrates for the first time that the human trophoblast expresses both peripheral TPH1 and brain-specific TPH2 and produces serotonin de novo. Serotonin production by the human trophoblast suggests an important autocrine/paracrine role for this neurohormone in placental function and development and, consequently, in pregnancy and foetal well-being.

Objective: Excessive exposure to synthetic or endogenous glucocorticoids (GC) is associated with reduced fetal growth. Animal studies reveal a key role for the placenta in mediating this effect. Treatment of placental explants from human pregnancies with dexamethasone (DEX) reduces cytotrophoblast proliferation and increases trophoblast apoptosis. The mechanisms involved in GC-induced placental growth restriction are unknown. We hypothesised that GCs suppress the expression of growth factors important for placental development. Methods: First trimester explants (n¼10) were cultured with vehicle or DEX (1mM) for 48 hours prior to RNA extraction and transcriptomic analysis using Affymetrix genechips. The expression patterns of selected candidate genes were examined by RT-qPCR. The effect of treatment with selected candidate growth factors on placental explant cell turnover (n¼5), in the presence and absence of DEX, was examined by immunohistochemistry for Ki67 and cytokeratin M30. Results: 271 genes were found to be inhibited by DEX by gene array, including 10 genes known to regulate cell turnover. These changes were validated by RT-qPCR for: transforming growth factor-b3 (TGF-b3), heparin binding epidermal growth factor (HB-EGF), fibroblast growth factor-2 (FGF-2), hepatocyte growth factor (HGF), leukaemia inhibitory factor (LIF) and Bcl2a (p<0.05 for all). Treatment of explants with TGF-b3 (10nM) or HB-EGF (25nM) alone had no significant effects on placental cell turnover. Co-treatment with DEX and either TGFb3 or HB-EGF ameliorated the stimulatory effect of DEX on apoptosis, but did not prevent DEX-inhibition of proliferation. Conclusions: These studies identify a number of growth factors and cell turnover regulators that are suppressed by DEX in early pregnancy placentas. Two of these growth factors, TGFb-3 and HB-EGF, were selected for further analyses and were found to rescue DEX-induced trophoblast apoptosis. Further analyses of GC-sensitive growth factors will lead to a greater understanding of the adverse effects of GCs on placental development.

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P1.133 LOSS OF ENDOTHELIAL CELLS IN SPIRAL ARTERY REMODELING: MYTHS VERSUS FACTS

P1.134 WHY IS THERE VARIETY IN PLACENTAL SHAPE: EARLY INFLUENCES VS TROPHOTROPISM?

Nicola Ellis, Barbara A. Innes, Gendie E. Lash, Stephen C. Robson, Judith N. Bulmer Newcastle University, Newcastle upon Tyne

Carolyn Salafia1,2, Michael Yampolsky3, Nadav Schwatrz4, Oleksandr Shlakhter5, Danielle Mandel1 1 Placental Analytics, LLC, Larchmont, NY, 2Institute For Basic Research, Staten Island, NY, 3University of Toronto, Toronto, ON, Canada, 4University of Pennsylvania, Philadelphia, PA, 5Rotman School of Management, Toronto, ON, Canada

Objectives: Uterine spiral artery (SpA) remodeling is a key feature of successful pregnancy. Extravillous trophoblast cells (EVT) invade decidua and myometrium via two distinct routes; interstitial and endovascular, and aid SpA remodeling. During SpA remodeling medial vascular smooth muscle cells are lost and replaced by fibrinoid material containing intramural EVT. A common belief is that the vessel lumen is lined by EVT that take on endothelial cell (EC) characteristics. During our extensive studies of placental bed biopsies from 6-20 weeks gestational age we have observed EC in SpA throughout the remodeling process, except when endovascular EVT is present within the vessel lumen. The aim of this study was to quantify these observations. Methods: Placental bed biopsies were immunostained for cytokeratin 7, CD31, CD34 and Von Willebrand factor, and double labelled for cytokeratin 7/CD34. Images were taken of SpA at different stages of remodeling, with and without a notable endovascular EVT plug. Using the Image J measurement tool the total luminal circumference of each vessel and the length of positive staining for the particular EC marker was measured. The percentage of vessel circumference covered by EC was then determined. Results: In SpA that did not contain an endovascular EVT plug 100% of the vessel luminal circumference was covered by EC, irrespective of the degree of remodeling (n¼150). In SpA that contained an endovascular EVT plug (n¼28) the percentage of vessel luminal circumference covered by EC ranged from 18% - 95% (mean 77.6 19.1%) dependent on the size of the EVT plug. Conclusions: EC are lost only transiently during SpA remodeling and only in the presence of an endovascular EVT plug where it has contact with the vessel wall. Once the EVT plug has dissipated and a vessel is fully remodeled the lumen of that vessel is then fully lined by ECs.

Aims: Our modeling of placental vascular growth suggests that many common abnormalities of placental shape, such as marginality of umbilical cord insertion, or multi-lobate shape are results of processes which influence early placental growth, and not trophotropism. We test this hypothesis by considering the data gathered from 3D scans of the placentas in the end of the first trimester of pregnancy, followed up by a set of placental measurements after delivery. Materials and methods: During the nuchal translucency exam, the transabdominal probe (GE Voluson E8) was used to obtain a 3D volume sweep of the placenta, with measurement of maternal surface diameters, cord location, and placental thickness (see Figure). Post delivery, the placenta was weighed, and its surface was digitally photographed. A total of 93 placentas were thus measured. Cross-sections of the surface and measurements of placental disk thickness were recorded in 50 of the cases. The modeling of placental vascular growth was carried out using a stochastic growth model based on Diffusion Limited Aggregation (DLA), employing the software package DLA-3D-Placenta, which we have developed. Results and discussion:

1. Strong and significant correlations are observed between the umbilical cord marginality at 11-14 weeks and at delivery. 2. Placental thickness is correlated with cord marginality when both measurements are taken both at 11-14 weeks and at delivery. 3. A marginal cord insertion at 11-14 weeks, as well as at delivery, strongly correlates with lowered chorionic vascular density at delivery. Consistent with our modeling, placentas at 11-14 weeks are typically not round, and the early non-roundness does not influence the regularity of the shape at delivery. In our previous work (Yampolsky et al, 2008) we have shown that bi-lobate placentas have normal vascular efficiency. Our empirical modeling suggests that bi-lobate shape is associated with irregularly shaped early vasculogenic zone; this is consistent with increase in number of bi-lobate placentas in IVF (c.f., Jauniaux et al, 1990). Our findings support the conclusions of empirical DLA modeling: common irregularities of placental surface shape such as marginal umbilical cord insertion and multi-lobate shape result from early influences, and not trophotropism.

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P1.135 17. ALPHA-HYDROXYPROGESTERONE CAPROATE (17-P) AND PLACENTA AT NEW YORK METHODIST HOSPITAL (NYMH)

THE

Lindsy Mastrine, Carolyn Salafia New York Methodist Hospital, Brooklyn, NY, USA Objectives: To review the indications and outcomes of patients treated with 17-P at NYMH. Methods: This retrospective chart review included patients who received 17-P from May 2008 - September 2010. The treated group included those with a history of preterm delivery and those with a shortened cervix. The control group was comprised of the next person who delivered at NYMH of the same gender, gestational age (GA), and without progesterone therapy. Primary outcomes were placental weight and presence and type of placental microscopic pathology including evidence of intraamniotic infection or poor maternal or fetal blood flow, and birth weight. Categorical variables were compared using contingency tables and continuous variables were compared in cases v controls using ANOVA and MannWhitney U tests when variable distributions were non-normal. Results: Comparing groups, there were no significant differences in birth or placental weight (2716 þ914 g v 2719þ779 g, and 430þ 130 g v 408 þ117g, respectively). Those treated with 17-P were significantly more likely to have a diagnosis of uteroplacental vascular injury to placental villi (8/42 vs. 1/41, p¼ 0.011). These findings were the same when the 17-P treated group was split by indication. Conclusion: Patients given 17-P have no difference in placental weights compared to untreated, but histologically their placental villi significantly more frequently show injury typically attributed to effects of poor maternal uteroplacental perfusion. This finding held for those given 17-P for previous preterm birth indication and those given 17-P for shortened cervix in the current pregnancy. These changes may be less than they would have been without the addition of 17-P. Future work includes examination of placentas from prior preterm births, and increased sample sizes to determine whether there is truly no difference between the two treatment groups.

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P1.136 VARIABLE PLACENTAL THICKNESS AFFECTS PLACENTAL FUNCTIONAL EFFICIENCY INDEPENDENT OF OTHER PLACENTAL SHAPE ABNORMALITIES M. Yampolsky1, CM. Salafia2,3, O. Shlakhter1, DP. Misra4, D. Haas2, B. Eucker5, J. Thorp5 1 University of Toronto, CA 2Placental Analytics, LLC, USA, 3New York Methodist Hospital, USA, 4Wayne State University, USA, 5University of North Carolina, Chapel Hill, Chapel Hill, North Carolina. Background: Our previous work suggests that stressors that impact placental vascular growth result in a deformed chorionic surface shape, which in turn reflects an abnormal placental three-dimensional shape. We propose to use variability of placental disk thickness as a reflector of deviations in placental vascular growth at the finer level of the fetal stems. We hypothesize that increased variability of thickness is associated with abnormal chorionic surface shape, but will be a predictor of reduced placental functional efficiency (smaller baby for a given placental weight) independent of shape. These measures may shed light on the mechanisms linking placental growth to risk of adult disease. Materials and Methods: The sample was drawn from the Pregnancy, Infection, and Nutrition Study. 94.6% of the cohort consented to placental examination. Of the 1023 delivered at term, those previously sectioned by the Pathology Department were excluded, leaving 587 (57%) cases with intact placentas that were sliced and photographed. The chorionic surface shape and the shape of a central randomly oriented placental slice were analyzed and measured compared using correlation. Results: Lower mean placental disk thickness and more variable disk thickness were each strongly and significantly correlated with deformed chorionic plate shapes; depending on the measure of irregularity, p ranged from <0.0001-0.007. More variable disk thickness was strongly correlated with reduced placental efficiency independent of abnormal chorionic surface shape or cord insertion (p¼0.002, r¼0.13). Conclusions: Variability of placental disk thickness, simple to measure in a single randomly oriented central slice, may be an easily acquired measure that is an independent indicator of lowered placental efficiency which may in turn program the infant and result in increased risk for development of adult diseases.

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P1.137 TREE TOPOLOGY OF PLACENTAL VASCULATURE R.-K. Seong1, C. M. Salafia2, and D. D. Vvedensky1 1 The Blackett Laboratory, Imperial College London, UK, 2Placental Analytics LLC, New York, USA Goal/Background: The vascular system of a placenta has a branching structure within the chorion which resembles a tree. The origin of the tree is the umbilical cord insertion in the placenta. In 2010, we introduced a quantitative measure of the branching structure of a placenta, and presented its relation to medical parameters. The project here aims to support this idea with a large sample set of placentas. Materials/Methods: The vasculature and its branching points are represented by edges and vertices respectively. An edge connects a nth generation vertex to a (nþ1)th generation vertex. Two edges are sides of a triangle if its corners are a nth generation vertex and two (nþ1)th generation vertices. A function called the entropy (S) is introduced to measure the distribution of triangle areas in the tree. A high area distribution relates to a higher symmetry in the branching structure. By varying the distribution parameter £], one is able to weight generations of the network differently. Results: Panel (a) shows the means of the area distribution functions for 45 placentas divided into sets corresponding to low, normal and high birth weights of the baby. Panel (c) shows the means of the area distribution functions for 28 placentas divided into more constrained sets. Panels (b) and (d) show the corresponding distributions of the birth weights in the chosen placenta sample. Panels (a) and (c) indicate that the distribution of triangle areas and hence the branching symmetry in a normal birth weight

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placenta is higher closer to the umbilical cord insertion point (large f Ò) than for the abnormal cases. The area distributions of all three types of placenta converge by moving away from the umbilical cord insertion point (small f Òf w. Conclusions: The results confirm that it is possible to distinguish between normal and abnormal placentas by analysing only the topology of the placenta vasculature.

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P1.138 THE SHAPE OF THE CHORIONIC PLATE AND PLACENTAL VASCULATURE R.-K. Seong1 ,C. M. Salafia2, and D. D. Vvedensky1 1 The Blackett Laboratory, Imperial College London, UK 2Placental Analytics LLC, New York, USA Goal/Background: The chorionic plate has varying shapes ranging from a circle to an ellipse. The chorion also contains the vasculature of veins and arteries with the branching structure resembling a tree. The umbilical cord insertion point is the origin of the tree and is not necessarily the geometrical centre of the chorionic plate. The goal of this project is to use a quantitative measure of the branching structure of the tree introduced by us in 2010, and to relate it to the shape of the chorionic plate. In doing so, we compare between a model of simulated tree networks and a set of real placental vasculatures. Materials/Methods: We generate sets of 300 model trees which are bounded by an ellipse with the ratio between the semi-minor and semimajor axes given by e. The tree origin is taken to be the geometric centre of the ellipse, and the area of the ellipse is kept fixed. The tree is generated by randomly distributing 100 points within the boundary, and creating nearest neighbour connections starting from the origin. Not every

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point results in a branching point as shown in Panels (a) to (d) corresponding to ratios e¼0.30, 0.53, 0.83 and 1.00 (circle) respectively. In comparison, we have 28 real placenta divided into sets of low, normal and high birth weights of the baby. For both cases, the tree is divided into triangles made each up of two tree edges connecting one nth generation to two (nþ1)th generation branching points. The distribution of triangle areas is measured by a function S called the entropy. A high triangle area distribution relates to a high branching symmetry. By varying the entropy function parameter â, one is able to weight different parts of the tree network. Results: Panel (e) shows the mean area distribution functions for each value of e of generated trees, and indicates that the area distribution is higher for more circular boundaries closer to the tree origin (large â). Panel (f) shows the mean area distribution functions for each birth weight category of real placentas, and indicates that the area distribution is higher for normal birth weight placentas closer to the umbilical cord insertion point (large â). Conclusions: The comparison between real placentas and simulated tree networks shows that a high area distribution function closer to the tree origin is associated to both normal birth weight placentas and trees restricted by near circular boundaries.

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P1.139 PRO-COAGULANT CAPACITY OF PLACENTAL MICROPARTICLES (PMPS)

C. Bonnke2, M. Sossdorf2, A. Brückmann1, L. Seyffarth1, E. Schleußner1, W. Lösche2, J.S. Fitzgerald1 1 Obstetrics Department, University Hospital Jena, Jena, Germany 2 Department of Anethesiology and Intensive Medicine, University Hospital Jena, Jena, Germany Background: Preeclampsia (PE) is a dire pregnancy disorder accompanied by endothelial dysfunction as distinguished through vasoconstriction and platelet activation. When PE exacerbates, the Hemolysis Elevated Liver enzymes Low Platelet (HELLP) Syndrome is often the result. The release of placental microparticles (pMPs) into the maternal circulation is a characteristic of severe forms of PE. Much research has been dedicated to the effects of MPs on vessels; however, their thrombogenic potential is not well characterized. Aim of this study was to investigate the pro-coagulant activity of pMPs. Methods: pMPs from placenta perfusates of different concentrations were immobilized on an ELISA-based test that allows measurement of procoagulant activity as seen through the concentration of negatively charged phospholipids on the surface of pMPs and through the creation of fibrin after supplementation with normal plasma. Furthermore, turbidimetric analyses were accomplished to assess extrinsic and plasmatic coagulation. Results: pMP surfaces appear rich in phosphatidylserine and this seems to promote the generation of thrombin in pMP-rich perfusate. A tendency towards a swifter course of extrinsic coagulation was observed in pMP-rich perfusates, but did not reach significance. The rate of ADP-induced platelet aggregation was significantly elevated after addition of pMP-rich perfusate (p¼0,028). Conclusion: pMPs seem to possess a pro-coagulatory feature. Proof of negatively charged phospholipids on pMP surfaces indicates the procoagulant potential of pMPs. A tendency to quicken the actions within the extrinsic cascade was observable. pMPs seem to directly provoke significant acceleration of platelet aggregation, which is one of the hallmarks of PE/ HELLP- Syndrome. One possible explanation could be pMP expression of tissue factor. Future investigations are underway to analyse the expression and activity of tissue factor on patient-derived pMPs.

P1.140 DIFFERENTIAL RESPONSE OF PLACENTAL ARTERIAL AND VENOUS ENDOTHELIAL CELLS TO EXTRACELLULAR MATRICES IS FOCAL ADHESION KINASE (FAK)-MEDIATED AND MODULATED BY OXYGEN Luciana Lassance1, Ursula Hiden1, Heidi Miedl1, Gernot Desoye1 Medical University of Graz, Graz, Austria

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Objectives: Endothelial cell adhesion to extracellular-matrix (ECM) components can activate intracellular signaling resulting in differential responses in processes such as proliferation, apoptosis and angiogenesis. In order to examine the ECM contribution for placental endothelium growth and formation, human placental venous (VEC) and arterial (AEC) endothelial cells were grown in different matrices. Cell viability, cell cycle, apoptosis, integrin expression and focal adhesion kinase (Fak) activation were measured. Methods: Primary AEC and VEC were grown in collagen I (rat) and IV (mouse; 3 mg/cm2), bovine fibronectin (1 mg/cm2), mouse laminin I (1mg/ cm2), 1% gelatin or uncoated-plates for 48 hours in 12% and 21% oxygen. Proliferation was determined by counting of viable and dead cells, cell cycle distribution (PI incorporation) and apoptosis (cleaved-caspase-3) measured by FACS. Integrin expression was screened by microarray and confirmed by real-time-PCR. Fak phosphorylation was determined by immunoblot. Results: AEC viability was ECM or oxygen independent, while the effect of ECM on VEC viability was only observed under 21%O2 with higher number of viable cells in collagen I and IV and fibronectin as compared to laminin, gelatin or without ECM. Fak phosphorylation showed the same pattern among the matrices and oxygen conditions. There was a higher proportion of cells in S-phase on collagen as compared to gelatine or non-coated plates, but this was not oxygen dependent. In the presence of ECM apoptosis was reduced in VEC, but not in AEC compared to uncoatedplates. Expression of angiogenesis-related integrins was higher by up to 300-fold in AEC than in VEC. Conclusion: Placental AEC and VEC respond differently to ECM components, collagen always being the better matrix for the cellular processes studied. The data suggest that cell interaction with the ECM triggers a survival mechanism. The preference of AEC and VEC for collagen may reflect the placental microenvironment around the blood vessels.

Abstracts / Placenta 32 (2011) A1–A149

P2.2 TROPHOBLASTIC DEBRIS UNDERGOES SECONDARY NECROSIS IF NOT RAPIDLY PHAGOCYTOSED LEADING TO ENDOTHELIAL CELL ACTIVATION

Poster session 2 (P2.1 – P2.143) P2.1 ABNORMAL PLACENTAL PREECLAMPSIA

FOXO1

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EXPRESSION

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SEVERE Qi Chen1,2, Sophie Gormack1, Ting Peng2, Peter Stone1, Larry Chamley1 The University of Auckland, Department of Obstetrics & Gynaecology, New Zealand, 2Fudan University, The Hospital of Obstetrics & Gynaecology, China 1

Rachel Sheridan, Jerzy Stanek, Stuart Handwerger Cincinnati Children’s Hospital Medical Center and University of Cincinnati, Cincinnati, Ohio, USA Objectives: The transcription factor FOXO1, which regulates cellular proliferation, apoptosis and other cellular functions, is expressed in amnion, chorion and decidua, with the highest expression in the latter. Total FOXO1 mRNA and protein expression has been shown to be normal in severe preeclamptic (SPE) term placentae, but the relative amounts of FOXO1 in individual cell types of the SPE placenta were not examined. In this study, we examined the expression of FOXO1 in specific placental cell types of SPE and normal placentas by immunohistochemistry. Methods: Sections from archived formalin-fixed and paraffin-embedded chorionic discs and placental membranes of 12 placentas from pregnancies with SPE and 12 gestational-age matched controls (mean: 31.75 weeks, range 26-39 weeks) were double immunostained with antibodies against FOXO1 and E-cadherin, the latter to distinguish between cytotrophoblasts and syncytiotrophoblasts. Ten high power fields from each section (a total of 120 SPE and 120 control fields) were examined blindly by two independent observers. Percentages of cytoplasmic and nuclear immunostaining for FOXO1 were assessed semiquantitatively in various placental cell types. Statistical differences were determined by Yates c2 test. Results: FOXO1 localization was predominantly cytoplasmic in the decidua and amnion, and nuclear in villous and extravillous trophoblasts, villous endothelial cells and stromal cells. In SPE, the nuclear expression of FOXO1 was decreased in membrane EVT (p¼0.004) and villous syncytiotrophoblasts (p¼0.001), while cytoplasmic accumulation was increased in chorionic disc extravillous trophoblasts (p¼0.008) and decidua basalis (p¼0.0001). There were no statistically significant differences in FOXO1 immunostaining in the amnion, villous endothelium or stroma. Conclusion: Our results show significant differences in the expression of FOXO1 in the nuclei and cytoplasm of specific placental cell types. Abnormal placental expression of FOXO1 in SPE may contribute to the decreased trophoblastic oxidative stress resistance, increased apoptosis, and abnormal decidualization and placentation in this complication of pregnancy.

Introduction: It is unclear what causes preeclampsia but it seems that a placental factor(s) triggers endothelial cell activation leading to preeclampsia. One possible placental trigger is trophoblastic debris that is shed into the maternal blood. In normal pregnancy, trophoblast debris is thought to be shed as the result of an apoptosis-like “safe” cell death process and the debris is rapidly and safely cleared from the maternal circulation. We have shown that endothelial cells can phagocytose trophoblastic debris. Phagocytosis of necrotic debris induces endothelial cell activation whereas; phagocytosis of apoptotic debris does not. However, when apoptotic cells are not cleared rapidly they may undergo secondary necrosis and their clearance could induce endothelial activation. Since the amount of trophoblastic debris is increased in preeclampsia, we undertook this study to examine whether trophoblastic debris undergoes secondary necrosis if it is not rapidly phagocytosed. Methods: Trophoblast debris was collected from first trimester placental explants. This “apoptotic” debris was exposed to endothelial cells for 24 hours. Control debris was incubated in culture dishes without endothelial cells. Unphagocytosed trophoblast debris was washed from the endothelial cells and exposed to fresh endothelial cells for 24 hours. Expression of cell-surface ICAM-1 by the fresh endothelial cells was measured by ELISA. Fresh trophoblast debris or unphagocytosed trophoblast debris was stained for the M30 neoeiptope of cytokeratin. Results: The phagocytosis of trophoblast debris by fresh endothelial cells, after the debris had been exposed to, but not phagocytosed by, other endothelial cells, resulted in increased expression of ICAM-1 by the fresh endothelial cells. In contrast, control debris did not increase endothelial ICAM-1. The M30 neoepitope was decreased in the unphagocytosed trophoblast debris compared to fresh trophoblast debris. Conclusion: Our data suggests that, if the maternal clearance system becomes overburdened, trophoblastic debris may undergo secondary necrosis leading to endothelial cell activation.

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P2.3 PLACENTAL MARKERS OF FOLATE-RELATED ONE-CARBON UNITS METABOLISM IN PREECLAMPSIA Maria Obolenskaya1, Olga Martsenyuk1, Csila Mislanova2, Bernhard Huppertz3 1 Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv, Ukraine, 2Slovak Medical University, Bratislava, Slovakia, 3 Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Graz, Austria Etiology and degree of clinical symptoms of preeclampsia depend on genotypic and phenotypic maternal and trophoblast factors, and elevated level of plasma homocysteine (Hcy) is one of the pathogenetic factors of preeclampsia. Objectives: To assess folate-related metabolism in human placenta in normotensive (NTP) and preeclamptic pregnancies (PREP) in compliance with 677 C/T polymorphism of methylene tetrahydrofolate reductase (MTHFR) and the impact of hyperhomocysteinemia .on the vital placental functions – proliferation and apoptosis. Methods: The placental samples at term from NTP (40) and PREP (28) pregnancies were used in the study. The total content of folate, methionine (Met), homocysteine (Hcy) and related cysteine (Cys) and glutathione (GSH) in compliance with the genotype of MTHFR were analysed by microbiological test, HPLC-coulochemical detecttion and PCR with subsequent restriction analysis, relatively. Results: The prevalence of MTHFR genotypes was not significantly different between the two groups. The polymorphism of MTHFR was not connected unambiguously with the content of total placental Met, Hcy and related Cys and GSH either in both groups. By contrast, the combination of the heterozygous MTHFR genotype with folate deficiency in the samples from PREP was characterized by a statistically significant decrease of the Met content, a trend towards increased Hcy levels and a tight association between metabolically directly and indirectly related compounds, e. g. positive relation between Hcy vs. Cys and folate vs. GSH, and negative relation between folate vs. Hcy and both Hcy and Cys vs. GSH. Cultivation of placental explants in the presence of elevated concentrations of Hcy revealed accumulation of Cys, down- adn up-regulation of proliferation and apoptotic indices, correspondingly. Conclusion: The disturbance of folate-related metabolism and hyperhomocysteinemia associated with confused proliferation and apoptosis in human placenta make their input in the pathogenesis of preeclampsia.

P2.4 IMMUNOHISTOCHEMICAL DISTRIBUTION OF CELL CYCLE PROTEINS PCNA, KI67, CYCLIN D3, P27 AND P57 IN NORMAL AND PREECLAMPTIC PLACENTAS Gozde Unek1, Asli Ozmen1, Mehmet Simsek2, Inanc Mendilcioglu2, Emin Turkay Korgun1 1 Akdeniz University Medical Faculty Histology and Embryology Department, Antalya, Turkey, 2Akdeniz University Medical Faculty Obstetrics and Gynecology Department, Antalya, Turkey Objectives: We hypothesize that cell cycle machinery is disrupted in preeclamptic placentas. In order to understand the role of cell cycle regulators in preeclamptic placentas we aimed to determine the immunohistochemical distribution of cell cycle regulators PCNA, Ki67, cyclin D3, p27 and p57 in preeclamptic and normal human term placentas. Methods: Term placentas were obtained from women diagnosed with preeclampsia and from normal pregnancies with informed consent following caesarean deliveries. Following tissue processing, placental tissues were studied by immunohistochemistry for PCNA, Ki67, cyclin D3, p27 and p57 antibodies. The intensities of PCNA, Ki67, cyclin D3, p27 and p57 immunoreactivities were evaluated semi-quantitatively using HSCORE. Results: Staining intensities of the proliferative markers PCNA, Ki67 and cyclin D3 statistically significantly increased in villous parts while statistically significantly decreased in the basal plates of preeclamptic placentas compared to control. For proliferative markers, there were no statistically significant difference between the chorionic plates of preeclamptic and control placentas. In addition, staining intensities of cell cycle inhibitors p27 and p57 significantly increased in all parts of preeclamptic placentas compared to control. Conclusion: We demonstrated altered expressions of cell cycle promoter proteins and CKIs (cyclin dependent kinase inhibitors) in preeclamptic placentas compared to control. Moreover, we found a relationship between the immunoreactivities of these proteins and the sizes of placentas. Our results indicate that proliferation, cell cycle arrest or apoptosis mechanisms’ differences might affect the placental sizes.

Abstracts / Placenta 32 (2011) A1–A149

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P2.5 THE ROLE OF NATRIURETIC PEPTIDE PRECURSOR B GENE (TTTC)N MICROSATELLITE POLYMORPHISM IN PRE-ECLAMPSIA

P2.6 FOLATE TRANSPORTER MRNA AND PROTEIN EXPRESSION DECREASES THROUGHOUT PREGNANCY AND IN PRE-ECLAMPSIA

Szabó Gábor, Molvarec Attila, Stenczer Balázs, Rigó János, Nagy Bálint First Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary

Paula Williams, Linda Morgan University of Nottingham, Nottingham, United Kingdom

Objectives: There is a variable tandem repeat (TTTC) polymorphism in the 5’-flanking region of the natriuretic peptide precursor B gene (NPPB), which shows association with essential hypertension. Our aim was to identify this polymorphism in samples of pre-eclamptic patients and healthy controls. We also compared the natriuretic peptide B (BNP) concentrations. Methods: Blood samples were collected from healthy pregnant normotensive women (n¼235) and women with pre-eclampsia (n¼220). DNA was isolated and fluorescent PCR and DNA fragment analysis was performed for the detection of (TTTC) repeats. The plasma BNP concentration was measured by fluorescence immunoassay method using Triage BNP (Alere, San Diego). Results: We detected 12 different repeats on the NPPB gene. The overall distribution of alleles and genotypes was significantly different between the control and pre-eclamptic groups. The number of 10-repeat genotype carriers showed significantly lower frequency in pre-eclamptics than in the healthy pregnant controls (p¼0.032). After adjustment for confounding factors - prepregnancy BMI, maternal age, primiparity and smoking the calculated odds ratio (OR) was 0.19 (95% CI: 0.04-0.87). Similarly 12repeat genotype carriers showed a significantly lower frequency in preeclamptics than in the healthy pregnant controls (p¼0.037); adjusted OR: 0.53 (95% CI: 0.29-0.96). In contrast the 11-repeat genotype carrier frequency was significantly higher in the pre-eclamptic group (p<0.001, adjusted OR 2.91 (95% CI: 1.75-4.84)). The concentration of the BNP was 9.75 pg/ml in the healthy controls and 32.40 pg/ml in the pre-eclamptic group (p<0.0001). The 11/11 genotype carriers had significantly higher BNP levels in the pre-eclamptic group. Conclusions: The NPPB gene (TTTC) microsatellite polymorphism in the 5’-flanking region showed a significant difference in the distribution of alleles and genotypes between healthy pregnant controls and preeclamptic patients in an ethnically homogeneous population. The concentration of the BNP was higher in pre-eclamptic women, and it showed association with the genotypes.

Objectives: In addition to its role in the prevention of neural tube defects, folate has many other physiological functions, including cell proliferation, DNA replication, antioxidant protection and angiogenesis. The transport of folate across the placenta involves a number of different receptors including folate receptor-alpha (FR-a), reduced folate carrier (RFC) and proton coupled folate transporter (PCFT). The ATP-dependent transporters ABCB1, ABCC2 and BCRP (ABCG2) are also involved in folate transport. The aim of the current study was to characterise the placental mRNA and protein expression of these folate transporters throughout gestation and also to see if expression is altered in pre-eclampsia. Methods: Placental samples were obtained from 10 early (8–10 weeks gestational age [GA]) and 10 late (12–14 weeks GA) pregnancy terminations (TOP) and from women undergoing elective Caesarean section following normal pregnancy (n¼10; 39 weeks GA) and with pre-eclampsia (n¼10; 37 weeks GA). To investigate mRNA expression quantitative real time PCR was used with gene specific oligonucleotide primers to FR-a, RFC, PCFT, ABCB1, ABCC2, BCRP and the housekeeping gene YWHAZ. Protein expression was also characterised using immunohistochemistry of paraffin embedded placental tissue. Results: Protein and mRNA expression of all transporters examined decreased as gestation increased, ranging from 2 to 8 fold reductions. There was a 5 fold reduction in FR-a and 2 fold reduction in PCFT mRNA and protein expression in pre-eclampsia compared with normal term pregnancy. Conclusions: The higher levels of expression of FR-a, RFC, PCFT, ABCB1, ABCC2 and BCRP in early pregnancy indicate that these transporters may have an important role in the establishment and development of the placenta, with expression reducing in preparation for parturition. Reductions in FR-a and PCFT in pre-eclampsia may be a mechanism involved in the pathogenesis of pre-eclampsia by limiting placental folate uptake resulting in reduced levels of angiogenesis, cell proliferation and antioxidant protection.

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P2.7 PLACENTAL GROWTH FACTOR QUANTIFIED ON RAPID ASSAY AS A DIAGNOSTIC TEST FOR EARLY-ONSET PRE-ECLAMPSIA Samantha Benton1, Yuxiang Hu1, Fang Xie1, Kenneth Kupfer2, Seok-Won Lee2, Laura Magee1, Peter von Dadelszen1 1 University of British Columbia, Vancouver, British Columbia, Canada, 2 Alere International, London, United Kingdom Background: Placental growth factor (PlGF) levels in maternal circulation are altered in pre-eclampsia. Recent advances in technology now allow for rapid quantification of biomarkers such as PlGF at the bedside. We sought to determine if a positive PlGF test measured on the Triage PlGF rapid assay (Alere, San Diego) differentiates early-onset pre-eclampsia from normal pregnancy. Methods: In this case-control study, plasma was collected from women with early-onset pre-eclampsia (<35 weeks, n¼25) and uncomplicated pregnancies (n¼47). Cases were matched to controls based on maternal age, parity and gestational age at the time of sampling. Plasma free PlGF was quantified using the Triage assay according to the product insert. Briefly, this immunoassay uses fluorescently labelled murine monoclonal antibodies against PlGF for quantification. PlGF results are available in approximately 15 minutes in pg/mL. A positive test was defined as PlGF concentration <5th percentile for gestational age in uncomplicated pregnancy. Sensitivity and specificity of a positive PlGF test to differentiate earlyonset pre-eclampsia from uncomplicated pregnancy were determined. Results: Higher blood pressure, more proteinuria, and a trend towards lower platelets were seen in cases compared with controls. Gestational age at delivery was lower, pre-term birth and small for gestational age fetuses more common in cases. Women with early-onset pre-eclampsia had lower median levels of PlGF (12.0 pg/mL [IQR: 12.0, 17.1]) compared with controls (334.3 pg/mL [IQR: 203.5, 688.6], p-value<0.0001). All 25 women with early-onset pre-eclampsia had a positive PlGF test while 45/47 controls had a negative test. A positive PlGF test identified early-onset preeclampsia from normal pregnancy with 100% [95% CI: 86, 100] sensitivity and 96% [95% CI: 85, 99] specificity. Conclusion: These data suggest that a positive PlGF test using Triage may aid in the clinical diagnosis of early-onset pre-eclampsia.

P2.8 PLACENTAL EXPRESSION AND SECRETION OF CYTOKINES AND ANGIOGENIC FACTORS IN FIRST TRIMESTER PREGNANCIES AT INCREASED RISK OF PRE-ECLAMPSIA Amanda Host, Judith Cartwright, Guy Whitley, Karin Leslie St. George’s, University of London, London Objectives: This study aimed to profile cytokines and angiogenic factors produced by placental villous tissue from first trimester pregnancies characterised by their risk of developing pre-eclampsia (PE) if the pregnancy had progressed. Methods: Uterine artery Doppler ultrasound scans were carried out on women attending clinic for elective surgical termination of pregnancy. Pregnancies were defined as those at most risk of PE (>25%) or at least risk of PE (<1%) based on the mean uterine artery resistance index and the presence or absence of bilateral notching. Placental tissue was either immediately snap-frozen and homogenised or cultured for 24 hours after which the medium was replaced and cultured for a further 48 hours and collected. Levels of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), activin A, vascular endothelial growth factor (VEGF), interleukin-8 (IL-8) and interleukin-6 (IL-6) were quantified by ELISA or by multi-analyte profiling (Luminex). Results: Activin A, Ang-1, IL-8 and IL-6 secretion did not differ between placental villous culture supernatants from low risk versus high risk placentae. Secretion of Ang-2 and VEGF from high risk placentae was significantly greater than low risk placentae; however, no difference in Ang-2 or VEGF expression was observed in tissue lysates. When samples were analysed by gestational age, there was a significant decrease in Activin A and Ang-2 secretion from low risk but not high risk placentae over time. In contrast, there was a significant increase in IL-8 secretion from high risk but not low risk placentae. Conclusion: This study demonstrated differences in the secretion of angiogenic factors between tissue obtained from first trimester pregnancies classified as at least or at most risk of developing PE. Differences in placental secretory characteristics between the two groups may contribute to the imbalance of angiogenic factors found in maternal serum of preeclamptic pregnancies.

Abstracts / Placenta 32 (2011) A1–A149

P2.9 ESTABLISHMENT OF A NORWEGIAN PREECLAMPSIA FAMILY BIOBANK Linda T. Roten1, Liv Cecilie Vestrheim Thomsen2, Astrid Solberg Gundersen1, Maria Lisa Odland1, Kristin Melheim Strand1, Mona Høysæter Fenstad1, Heiko Leuschner3, Per Solberg4, Christian Tappert5, Ingvill Lyslo6, Kjersti Tollaksen6, Line Bjørge2, Rigmor Austgulen1 1 Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway, 2Haukeland University Hospital, Bergen, Norway, 3Namsos Hospital, Namsos, Norway, 4Levanger Hospital, Levanger, Norway, 5St. Olavs Hospital, Trondheim, Norway, 6Stavanger University Hospital, Stavanger, Norway Objectives: To establish a biobank consisting of clinical data and biological material from families with increased occurrence of preeclampsia in order to study genetic predisposition for this disease. Methods: Exemption from the patient confidentiality was obtained in order to identify the potential participants and review their hospital records prior to invitation. Index women with a familial disposition for preeclampsia, defined as preeclamptic women with a mother, daughter or sister registered as having the disease, were identified by linking data from the Medical Birth Registry of Norway with information from The National Registry. Women who gave birth at St.Olavs Hospital, Haukeland University Hospital, Stavanger University Hospital, Levanger Hospital or Namsos Hospital during the period 1967-2006 were included. Diagnostic inclusion criteria for preeclampsia were minimum two measurements of blood pressure  140/90 mmHg combined with proteinuria occurring after 20 weeks of pregnancy. The preeclampsia diagnosis was confirmed or rejected based on information from hospital records. After exclusion of nonconfirmed individuals and segregation, the dataset contained only “true cases”. These index women were invited to attend the study together with their family members. Clinical information and blood samples were collected from all the participants. Results: In total, 1006 women were identified as preeclamptic having a first degree relative with preeclampsia where both women had given birth at one of the involved hospitals. The preeclampsia diagnosis was validated for 637 (63%) of these women, resulting in a total number of 426 women fulfilling all our inclusion criteria. So far, 445 individuals are included in the study of which 221 are index women. Conclusion: The process to establish this Norwegian preeclampsia family biobank has been challenging and time consuming. The fact that all families in the biobank are identified with two “true” preeclamptic women strengthens the potential to uncover genetic factors contributing to development of preeclampsia.

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P2.10 EVALUATION OF A NEW, SIMPLE AND RAPID PLACENTAL GROWTH FACTOR TEST FOR THE EVALUATION OF HYPERTENSIVE DISORDERS IN PREGNANCY Balazs Stenczer, Nora Gullai, Gabor Szabo, Gergely Fugedi, Brigitta Hanuszka, Attila Molvarec, Janos Rigo Jr. Semmelweis University, 1st Department of Obstetrics and Gynecology, Budapest, Hungary Objectives: Hypertensive disorders are the most common complications of pregnancy, frequently leading to an adverse outcome. Maternal circulating placental growth factor (PlGF) concentrations are decreased in these women, in correlation with disease severity. There is a clinical need to more accurately diagnose those forms of the disease that have an increased likelihood of progressing to an adverse fetal or maternal outcome. The aim of this study was to test a new test for measuring PlGF levels in maternal circulation, and to evaluate its clinical utility in various forms of hypertensive disorders in pregnancy. Methods: Thirty-one pregnant women with chronic hypertension (CHTN), 33 with gestational hypertension (GHTN), 40 with preeclampsia (PE), 24 with HELLP-syndrome, 24 with superimposed preeclampsia (SIPE) and 40 healthy pregnant controls were enrolled in our case-control study. Blood draw occurred between the 22nd and 36th completed gestational weeks. Plasma was analysed for PlGF by the Alere TriageÒ PlGF assay using fluorescently-labelled monoclonal antibodies against PlGF. Results: For hypertension forms conjoined with proteinuria occurring before 35 weeks the sensitivity of the test was the following: 95.7% for PE, 95.0% for HELLP-syndrome and 82.4% for SIPE. The negative predictive value of the test in exclusion of hypertension forms conjoined with proteinuria was 93.9%. A higher prevalence of preterm delivery associated with severe disease conditions (placental insufficiency, severe hypertension, etc.) was shown in CHTN and GHTN groups in the case of a positive PlGF test [11/26 (42.3%) vs. 3/38 (7.9%), p<0.001, OR: 8.6 (2.1-35.1)]. A strong association was demonstrated between PlGF concentrations and the remaining time interval from the blood draw until the delivery (R ¼ 0.65, p<0.001) in hypertensive patients. Conclusions: The new TriageÒ PlGF test can provide useful additional information to inform clinical decisions in pregnancy-associated hypertensive disorders, especially before the 35th completed gestational week.

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P2.11 THE ROLE OF ANGIOGENIC, ANTIANGIOGENIC AND AT1 RECEPTORS IN PRE-ECLAMPTIC AFRICAN WOMEN

P2.13 THE EFFECT OF OXYGEN ON PLACENTAL CONNEXINS 43 AND 46 AND ITS CONTRIBUTION TO PREECLAMPSIA

Premjith Gathiram, Lucinda Govender, Irene Mackraj, Jagidesa Moodley University of KwaZulu-Natal, Durban, KwaZulu-Natal, South Africa

Alexandra Gellhaus1, Teresa Otto1, Nadine Wolf1, Jan Scheidler1, Kerstin Boengler2, Caroline E. Dunk5, Markus Schmidt3, Rainer Kimmig3, Klaus Lennartz4, Stephen J. Lye5, Elke Winterhager1 1 Molecular Biology, University Hospital, Essen, Germany, 2Pathophysiology, University Hospital, Essen, Germany, 3Gynecology, University Hospital, Essen, Germany, 4Cell Biology, University Hospital, Essen, Germany, 5Department of Obstetrics and Gynecology, Mount Sinai Hospital, Toronto, Canada

Objectives: To show that AT1 receptor upregulation, angiogenic and antiangiogenic factors may have varying roles in the aetiology of early and late onset pre-eclampsia in Black South African women. Methods: 58 women at term ,were recruited after obtaining informed consent and divided into: normotensive (n¼30) (N), chronic hypertensive (n¼9) (CH), (3), early- (n¼10) (EO) and late-onset (LO) pre-eclamptic groups (9). Blood samples were taken prior to delivery and post-partum central placental samples were obtained. All study material were stored at -70 C until used. Serum sFlt-1, VEGF and PlGF levels were determined using the ELISA technique and placental mRNA levels of sFlt-1, VEGF, PlGF and the AT1 receptors were determined using real-time PCR. The latter was used as an indicator of AT1 receptor regulation. Results: Compared to the N group, serum sFlt-1 level was significantly elevated in the EO group with a concomitant significant reduction in VEGF and PlGF levels. The mean placental mRNA expression levels of sFlt-1 were similar in the EO and N groups.. The VEGF mRNA expression levels in the LO (1.059  0.4338) and CH (0.682  0.113) groups were significantly lower (0.47 and 0.3 fold, resp.) than in the N group (p<0.05,). The VEGF mRNA expression level in the EO group (0.943  0.281) compared with the N group had a p value of (p ¼ 0.051. Amongst all 4 groups the LO group (23.20  13.36) had the highest level of AT1 mRNA expression (7.86  3.34). Notably the difference in AT1 receptor mRNA expression between the EO and LO groups showed a p value very close to significance (p ¼ 0.0592, ). Conclusion: This study suggests that role of the angiogenic and antiangiogenic factors and AT1 receptors differ in the late and early onset preeclamptic groups in our population. Further experiments are warranted. P2.12 SEXUAL DIMORPHISM IN PLACENTAL FUNCTION WITH PREECLAMPSIA Sribalasubahini Muralimanoharan, Alina Maloyan, Evelyn Miller, Leslie Myatt Department of OB/GYN, University of Texas Health Science Center, San Antonio, TX, United States Objective: Preeclampsia (PE) affects 5-10% of pregnancies worldwide and is associated with subsequent long-term health complications for mother and offspring. The presence of a male fetus increases the risk of developing PE by 1.2 fold and also affects other pregnancy complications including preterm delivery and asthma. Our aim was to determine the effect of sexual dimorphism in the placenta from pregnancies complicated by PE on expression of markers of inflammation, hypoxia, apoptosis and angiogenesis. Methods: Villous tissue was collected from normotensive pregnancies or those complicated by PE with male (MPE) and female (FPE) fetuses (n¼4 each) after c-section at term. Inflammatory markers (TNFa, IL-6 and IL-8) were measured in placental homogenates using ELISA. Markers for hypoxia (NFkB p65 and HIF-1a), angiogenesis (VEGF), and apoptosis (active caspase 3 and Bcl-2) were measured using Western blotting and TUNEL assay. Results: Concentrations of inflammatory markers TNFa (p<0.001), IL-6 (p<0.03) and IL-8 (p<0.001) were significantly increased in MPE but not FPE placentas compared to controls. In MPE, both p65 (p<0.001) and HIF-1a (p<0.02) were significantly increased while in FPE, HIF-1a was increased compared to controls. The expression of VEGF was reduced by 2-fold (p<0.02) in MPE vs. controls. The apoptotic index was elevated in MPE vs. FPE and controls as evidenced by TUNEL, a 3-fold increase in caspase-3 cleavage (p<0.004), a 2-fold increase in p53 expression (p<0.01), and a decrease in Bcl2 expression (p<0.02). The electrophoretic mobility shift assay revealed a significant increase in NFkB DNA binding activity in MPE compared to either FPE or controls indicating that NFkB pathway is activated in MPE and could be the signaling route regulating the inflammatory response. Conclusion: In pregnancies complicated by PE, the placenta from a male fetus exhibited significantly higher expression of inflammatory as well as hypoxia/apoptosis markers but reduced expression of angiogenic markers compared to female.

Objectives: Gap Junction proteins play a pivotal role in placental development. While Cx43 regulates fusion between cyto- and syncytiotrophoblast which is responsible for the feto-maternal exchange, Cx40 is characteristic for the proliferation of the extravillous trophoblast (EVT) which migrates into the decidua. In the placental disorder preeclampsia migration and invasion properties of the EVT are disturbed resulting in placental insufficiency and chronic hypoxia. Here we tested whether the expression of placental connexins changes upon low oxygen. Methods and Results: Our data showed an increase of Cx43 and Cx40 in early-onset preeclamptic placentas compared to matched controls analysed by quantitative RT-PCR and immunoblotting. In addition we found for the first time Cx46 expressed in the placenta, a connexin which is predominantly detected in lens and is important for the survival of cells under hypoxia. To resolve if hypoxia is responsible for the change in connexin levels invasive JAr trophoblast cells were cultivated under different O2 concentrations. 1% O2 levels increased Cx43 and Cx46 but not Cx40. Interestingly, upon hypoxia the localisation of the connexins 43 and 46 shifted from the cell membrane to the cytoplasm. In contrast reoxygenation resulted in an even enhanced return of Cx43 and Cx46 to the cell membrane. Cell coupling properties analysed by calcein transfer using flow cytometry showed a significant decrease in cell coupling under hypoxia. Thus though we found an upregulation of Cx43 and Cx46 expression levels in JAr cells upon hypoxia the coupling properties via connexin channels were decreased due to the translocation to the cytoplasm. Conclusion: To summarize, this study showed that placental connexins Cx43 and Cx46 are hypoxia-regulated and are associated with loss in function which could contribute to the pathomechanisms in preeclampsia.

Abstracts / Placenta 32 (2011) A1–A149

P2.14 ENDOPLASMIC RETICULUM STRESS IN DECIDUAL TISSUE FROM PREGNANCIES COMPLICATED BY PRE-ECLAMPSIA AND FETAL GROWTH RESTRICTION Ingrid Lian1, Mari Løset1, Siv Mundal1, Mona Fenstad1, Matthew Johnson2, Katherine Freed2, Irina Eide3, Line Bjørge4, John Blangero2, Eric Moses2, Rigmor Austgulen1 1 Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway, 2Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, Texas, United States, 3Central Norway Regional Health Authority, Stjørdal, Norway, 4Department of Obstetrics and Gynaecology, Haukeland University Hospital, Bergen, Norway Objectives: Endoplasmic reticulum (ER) stress, secondary to ischemiareperfusion insults, has been ascribed a role in development of preeclampsia (PE) and fetal growth restriction (FGR). ER stress is characterised by activation of three signalling branches: 1) PERK and pEIF2a, 2) ATF6, and 3) splicing of XBP1(U) into XBP1(S). However, the contribution of ER stress in the pathogenesis of PE relative to FGR has not previously been evaluated. To investigate this, we compared the levels of ER stress markers in pregnancies complicated by PE and/or FGR. Methods: Whole-genome transcriptional profiling of decidual tissue from women with PE and/or FGR and controls was used for pathway- and targeted transcriptional analyses of ER stress markers. The expression and localisation of ER stress markers in decidual tissue was assessed by immunofluorescence analyses, and protein levels were further examined by Western blot analysis. Results: Transcriptional and pathway analyses revealed increased ER stress in FGR and PEþFGR, whereas ER stress was less evident in isolated PE. Scattered cytoplasmic and nuclear staining of ATF6 and XBP1 was detected in most (>80-90%) extravillous trophoblasts, decidual cells and macrophages. The proportion of ATF6 or XBP1 positive cells were the same in all groups examined, while Western blot analyses revealed increased levels of ATF6 and pEIF2a in FGR and PEþFGR, whereas elevated levels of XBP1(U) was observed in PE. Conclusion: Decidual tissue from women with FGR and PEþFGR is characterised by activation of the PERK-pEIF2a and ATF6 ER stress signalling branches. In PE, increased levels of XBP1(U) was observed, which may reflect a potentially protective mechanism, as XBP1(U) has been proposed as a negative regulator of ER stress. Disparities in the ER stress response between PE and FGR/PEþFGR may reflect disparate ongoing pathophysiological mechanisms that could in part account for the clinical differences between case groups.

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P2.15 THE IMPORTANCE OF CATECHOL-O-METHYLTRANSFERASE (COMT) IN PRE-ECLAMPSIA – PLACENTAL INITIATED OR WHOLLY SYSTEMIC? Ian Crocker1, Yu Zhang1, Joanna Stanley2, Mark Wareing1, Philip Baker2 1 Maternal and Fetal Health Research Centre, University of Manchester, Manchester, United Kingdom, 2Dept. of Obstetrics & Gynecology, University of Alberta, Edmonton, Canada Objectives: COMT metabolises estradiols to 2-Methoxyestadiol (2-ME). COMT and 2-ME are elevated in the third trimester, with lower levels/ activity in pre-eclampsia. Knockout COMT-deficient mice show a preeclampsia-like phenotype and concomitant exaggeration in placental HIF-1a and sFlt-1, ameliorated by administered 2-ME. Consequently, disturbed redox signalling in COMT/2-ME deficient placentas is a proposed pre-eclamptic trigger. We investigated this suggestion, and alternatively the direct impact of COMT/2-ME on maternal endothelial dilatation. Methods: Term placental explants from uncomplicated pregnancies (N¼6) were cultured in normoxic (6%O2) or hypoxic (1%O2) conditions with/ without 2ME-2(0-100mM). Tissue necrosis, apoptosis, proliferation, hCG production, HIF-1a expression and sFlt-1 liberation were recorded. Human uterine resistance arteries, (N¼7, uncomplicated pregnancies) were exposed to 2ME-2 using wire myography. Dose responses to 2ME-2, U46619 (thromboxane mimetic) and bradykinin (BK) were conducted, with/without nitric oxide synthase inhibition (L-NNA, 10mM). The impact of 2ME-2 on NO was further measured in vitro in human uterine microvascular endothelial cells using the Griess reaction. Results: Hypoxia reduced placental explant viability; increasing necrosis (p<0.05), apoptosis (p<0.05), and inhibiting proliferation (p<0.05). It also reduced hCG secretion (p<0.05), but failed to elevate HIF-1a expression or sFlt-1. 2ME-2 failed to influence these markers. Relaxation of uterine vessels was stimulated by 2ME-2 (3mM) (p<0.01) and pre-incubation (10mM, 1 hour) significantly enhanced relaxation in response to BK (p<0.05). These effects of 2ME-2 were blocked with L-NNA. The direct addition of 2ME-2 (1mM, 20hrs) to uterine endothelial cells encouraged NO production (N¼6, p<0.01). This again was abolished by L-NNA. COMT inhibitors, Ro41-0960 and OR-486 (5mM), restricted NO production in 17bestradiol (100nM) stimulated endothelial cells (35.4 and 50.5%, respectively p<0.01), suggesting COMT involvement. Conclusion: 2ME-2, delivered in vitro, had no impact on placenta tissue but acutely relaxed maternal uterine arteries via an NO/endothelialdependent mechanism. These studies question placental importance in COMT-driven pre-eclampsia; instead proposing its direct vascular involvement.

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P2.16 MOLECULAR CHANGES OF CELL ADHESION AND INVASION MARKERS IN TWO MOUSE MODELS OF PREECLAMPSIA Bei Xu1,3, Gabriele Bobek1, Laura Surmon1, Angela Makris2,3, Annemarie Hennessy1,3 1 School of Medicine, University of western Sydney, NSW, Australia, 2Renal Unit, Liverpool Hospital, NSW, Australia, 3Heart Research Institute, University of Sydney, NSW, Australia Objectives: It has been postulated that shallow trophoblast invasion in early pregnancy results in reduced placental perfusion which leads to the symptoms of preeclampsia. Our previous in vitro data has shown that proinflammatory TNF-a interferes with the interaction between trophoblast cells and maternal endothelial cells by affecting cell adhesion and invasion molecules. An increase in VE-cadherin and decreases in integrin a1ß1, matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were observed, consistent with trends described in human preeclampsia. We have implemented two mouse models of hypertension in pregnancy to study the mechanisms of preeclampsia; the TNF-a induced hypertension mouse model and the Reduced Uterine Perfusion Pressure (RUPP) mouse model. In this study we examined whether these invasion and adhesion molecules VE-cadherin, E-cadherin, MMP-2, MMP-9, TIMP-1, plasminogen activator inhibitor (PAI)-1, integrins (a1, a6, ß1 and ß4) and hypoxia inducible factor (HIF)-1a were changed in vivo and how they differ between the two mouse models. Methods: Placentas were obtained from RUPP-induced (gestational day (gd) 11) and early (gd11) or late (gd14) TNF-a induced hypertensive mice. Total RNA was extracted from the placental homogenates. Complementary DNA was reverse transcribed using iSCRIPT. Quantitative PCR was performed to analyse the mRNA expression of VE-cadherin, E-cadherin, MMP2, MMP-9, TIMP-1, PAI-1, Integrins (a1, a6, ß1 and ß4) and HIF-1a. Results: An increase of VE-cadherin, MMP-9, Integrin a1ß1, and HIF-1a mRNA expression were found in both early TNF-a-induced and RUPP mice. There was an increase of integrina6 and MMP-2 in early TNF-a induced mice, which was not seen in RUPP induced mice. Conclusion: The diversity of the change in mRNA expression of cell adhesion and invasion molecules in the placentas from two different mouse models suggest that hypertension induced by TNF-a and RUPP involves different pathways and are dependent on the time of the placenta insult.

P2.17 MATERNAL SERUM OF WOMEN WITH PRE-ECLAMPSIA INDUCES PLACENTAL OXIDATIVE STRESS: EVIDENCE FOR AN INCREASED SENSITIVITY TO THE SOLUBLE VEGF RECEPTOR SFLT-1 Renu Dhingra, Betsy Varughese, Kalpana Luthra, Neerja Bhatla, Rani Kumar All India Institute of Medical Sciences, New Delhi, India Introduction: Preeclampsia is one of the leading and mystical causes of maternal, fetal and neonatal mortality worldwide. Although the exact etiology of preeclampsia remains unclear; oxidative stress possibly secondary to abnormal placentation may play a major role in the pathophysiology of preeclampsia. Abnormal trophoblast invasion of the spiral arteries leading to hypoxic placenta may then induce the production of a circulating toxin soluble fms-like tyrosine kinase-1 (sFlt-1) which may bind to free Vascular Endothelial Growth Factor (VEGF) leading to endothelial dysfunction. This may also lead to an exaggerated state of oxidative stress in the placenta. Our previous findings also indicate higher serum levels of sFlt-1in preeclamptic patients. Objective: The linkage between the generation of circulating sFlt-1 and the placental oxidative stress in preeclamptic patients remains unidentified. Therefore we aimed to analyze the association of circulating sFlt-1 and placental oxidative stress in vitro. Methods: Trophoblast cell lines (JAR cells) were cultured with (i) preeclamptic sera, (ii) normotensive sera, (iii) preeclamptic sera with recombinant VEGF and (iv) normotensive sera with recombinant sFlt-1.The oxidative stress markers like malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD) were estimated in these trophoblastic cell lines. Results: The levels of MDA were significantly increased in JAR cells treated with preeclamptic sera as compared to the JAR cells treated with control sera. The levels of GSH and SOD were significantly decreased in JAR cells treated with preeclamptic sera as compared to the JAR cells treated with control sera. This effect was reversed in JAR cells treated preeclamptic sera after the addition of recombinant VEGF and JAR cells treated control sera after the addition of recombinant sFlt-1. Conclusion: Increased serum sFlt-1 may lead to the increased levels of oxidative stress in trophoblast cell lines (JAR) as observed by the stress markers suggesting an association of circulating sFlt-1 and placental oxidative stress in preeclampsia. The increased production of reactive oxygen species like lipid peroxides and the dysfunction of antioxidants may result in damage to the placenta and hence the progression of the disease.

Abstracts / Placenta 32 (2011) A1–A149

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P2.18 THE EFFECT OF PREECLAMPTIC SERA ON TROPHOBLAST VIABILITY: AN IN VITRO STUDY

P2.19 PODOCYTURIA IN THE EARLY DETECTION OF PRE-ECLAMPSIA- A PILOT STUDY

Rani Kumar, Betsy Varughese, Kalpana Luthra, Neerja Bhatla, Renu Dhingra All India Institute of Medical Sciences, New Delhi, India

T Naicker1, D Ramsuran1, T Dauth1,2, J Moodley1 1 University of Kwa Zulu Natal, KwaZulu Natal, South Africa, Laboratories, KwaZulu Natal, South Africa

Introduction :Preeclampsia is a pregnancy-related hypertensive disorder diagnosed in the latter half of pregnancy. Abnormal placentation, suggested to be possibly the key feature of this disorder, has been attributed to the presence of a circulating ’toxin’ soluble fms-like tyrosine kinase-1 (sFlt-1). Although sFlt-1 is generated and secreted by the placenta, it may act as a major inhibitory factor on trophoblast cell proliferation and differentiation. However, the binding of this toxin with Vascular Endothelial Growth Factor (VEGF) in circulation and the effect of this toxin on trophoblast cell proliferation are not clearly understood. Objectives: To estimate the levels of sFlt-1and VEGF in preeclamptic sera and to test whether this sera affects the viability of trophoblast cells. We also aimed to investigate whether this effect could be the result of impaired or altered VEGF/ sFlt-1 interaction. Methods: The serum levels of sFlt-1 and VEGF in forty preeclamptic pregnant women and forty normal pregnant women were analyzed by ELISA. The effect of preeclamptic sera on trophoblast cell proliferation were studied by treating trophoblast cell lines (JAR cells) with (i) preeclamptic sera, (ii) normotensive sera, (iii) preeclamptic sera with recombinant VEGF and (iv) normotensive sera with recombinant sFlt-1. Cell proliferation was determined by the MTT assay. Results: The levels of sFlt-1 were significantly increased and the levels of free VEGF were significantly decreased in the sera of preeclamptic women compared to the sera of normal pregnant women. Serum from preeclamptic women showed reduced trophoblast viability, and this effect was enhanced by the treatment with recombinant VEGF. In contrast, serum from normal pregnant women showed enhanced trophoblast cell viability and this effect was reversed by the treatment with recombinant sFlt-1. Conclusion: Increased levels of sFlt-1 in the preeclamptic sera may induce the cytotoxicity of JAR cells which appears to be related to the changes in trophoblast sensitivity to sFlt-1 suggesting that circulating sFlt-1 may have a role in the pathogenesis of preeclampsia.

2

Lancet

Objective: In South Africa, hypertensive diseases of pregnancy are the commonest direct cause of maternal deaths, accounting for 15.7% of all deaths in 2005-2007. Preeclampsia accounts for the majority (83%) of deaths. Detecting preeclampsia early with timeous delivery will prevent complications associated with this disorder. Urinary podocyte excretion occurs concurrently with proteinuria and may be a useful marker for preeclampsia. The objective of this study was to establish an effective method for assessment of podocyturia as a risk indicator for preeclampsia, this pilot study compared viable podocyte excretion vs total podocyturia in urine samples. Method: Midstream urine samples were collected from 18 normotensive healthy primigravidae at approximately 20 weeks gestation. Two methods of preparation were performed. Method 1: Urine was aliquoted in 4 chamber collagen coated tissue culture slides and incubated overnight at 37oC in 5% CO2. Method 2: Urine was cytospun onto poly-l-lysine coated slides. Samples from both methods were immunostained with antipodocalyxin, anti-podocin, anti-synaptopodin and anti-nephrin and conjugated to a fluorescein isothiocynate-labelled secondary antibody. Results: Podocytes in cytospun sediments and culture were recognized as either nucleated or multinucleated cells expressing the antibody panel by immunofluorescence. Podocyte viability was confirmed by their ability to grow in culture. 44% of samples grew podocytes in culture whilst 66% of cytospun samples were immunopositive for the podocyte markers. Podocalyxin was the most sensitive and specific marker. Conclusion: Six% of patients developed preeclampsia. The cytospin method was the more sensitive and less costly method of assessing podocyturia. Urinary detection of podocyte injury by podocyte number count and by podocyte-specific molecule measurement may serve as a non-invasive test with diagnostic value for preeclampsia. Future research involves extrapolation of this data to a larger sample size to confirm the high specificity and sensitivity of podocyturia as a marker for preeclampsia.

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P2.20 IMMUNO-ELECTRON LOCALIZATION OF SFLT1 IN TERM PLACENTAE FROM HIV INFECTED WOMEN WITH PREECLAMPSIA N Govender1, T Naicker2, J Moodley3 1 Department of Basic Medical Sciences, Durban University of Technology;, Durban, South Africa, 2Optics and Imaging Centre,University of KwaZulu Natal, Durban, South Africa, 3Womens Health and STD Research Unit, University of KwaZulu Natal, Durban, South Africa Objective: The preeclamptic, hypoxic placenta releases excess antiangiogenic factors, viz., sFlt1 and sEndoglin into the maternal circulation. This results in widespread endothelial dysfunction characteristic of preeclampsia. Therefore this study examined the ultrastructural localization of the anti-angiogenic factor sFlt1 in placentae of HIV infected and non infected normotensive and preeclamptic pregnancies at term. Methods: A total of 25 placentae were collected (HIV–ve normotensive, n¼6; HIV–ve preeclamptic, n¼5; HIVþve normotensive, n¼8; and HIVþve preeclamptic, n¼6). Post conventional immuno-electron processing was conducted. Ultra-thin sections (50-60 nm) were labeled with a polyclonal anti-human Flt1conjugated to anti-goat 10nm gold IgG particles. Sections were post-fixed, counter-stained with uranyl acetate and Reynolds lead citrate and viewed on a Jeol 1011 Transmission Electron Microscopy. Results: sFlt1 was localized within the cyto- and syncytiotrophoblast cell populations as well as within the endothelial cells. Immunolocalization of sFlt1 was on the microvilli and the basal pole of the syncytiotrophoblast. Subcellular immuno-localization for all regions was predominantly within the ER and mitochondria. Additionally, 10nm gold particles were observed in the perinuclear region of both the syncytio and cytotrophoblasts. No detectable differences in distribution intensity of sFlt1 between HIV infected and non-infected pregnant women at term were observed. Conclusion: This is a novel study demonstrating the subcellular distribution of sFlt1 within the placenta. Qualitative assessment between the HIV–ve and HIV infected women revealed no noticeable differences in the distribution patterns of Flt1. This study supports the role of excess production of sFlt1 by trophoblastic tissue in the aetiology of preeclampsia. Future research will be directed to a quantitative morphometric ultrastructural analysis of placental-derived sFlt1.

P2.21 PLACENTAL AND SERUM LEVELS OF PRO AND ANTI-ANGIOGENIC FACTORS IN HUMAN IMMUNODEFICIENCY VIRUS (HIV) ASSOCIATEDPREECLAMPSIA IN SOUTH AFRICA N Govender1, T Naicker2, J Moodley3 1 Department of Basic Medical Sciences, Durban University of Technology, Durban, South Africa, 2Optics and Imaging Centre, University of KwaZulu Natal, Durban, South Africa, 3Womens Health and STD Research Unit, University of KwaZulu Natal, Durban, South Africa Objective: Preeclampsia complicates 16% of pregnancies in KwaZulu Natal, South Africa, the epicenter of the HIV pandemic. The balance between pro and anti-angiogenic factors necessitate effective vasculogenesis, angiogenesis and placental development during normal pregnancy. Preeclampsia is associated with an anti-angiogenic state. However in HIV associated preeclampsia, this balance may be offset. We hypothesized that the immune insufficiency stimulated by HIV possibly reduces a predisposition to immune hyperreactivity, averting preeclampsia development. The objective of this study was to correlate the expression of VEGF & PlGF vs sFlt1 & sEng in normotensive, preeclamptic pregnancies compared to those compromised by HIV infection. Methods: 76 term pregnancies (HIV–ve normotensive, n¼20; HIV–ve preeclamptic, n¼19; HIVþve normotensive, n¼21; and HIVþve preeclamptic, n¼16) were assessed. Serum and placental tissue were quantitatively evaluated using ELISA and Real-Time Polymerase Chain Reactions respectively. Results: Placental sFlt1 revealed a significant difference between HIVþve preeclamptic versus HIV-ve and HIVþve normotensive groups (0.00070.001 vs 0.00040.001, vs 0.00020.0004, p<0.05), the HIV–ve preeclamptic versus HIV–ve and HIVþve normotensive groups (0.00040.0003 vs 0.00040.001, vs 0.0002 0.0004, p<0.05). Similarly sEng was significant different between the HIVþve preeclamptic versus HIV–ve normotensive groups and the HIV–ve preeclamptic versus HIV–ve normotensive groups (p<0.05). However, serum sFlt1 and sEng were significantly different for HIV–ve preeclamptic and HIVþve normotensive groups respectively (154596353 vs 97365356, p<0.05; 27.9421.5 vs 11.417.1, p<0.05). No significant differences were evident for VEGF and PlGF. sEng expression were elevated in both placenta and serum, whilst placental sFlt1 differed from serum. Correlation analyses between serum and placental VEGF, PlGF, sFlt1 and sEng was negative across all study cohorts. Conclusion: This study demonstrates elevation of both sFlt1 and sEng in HIV associated preeclamptic pregnancies. HIV association does not seem to affect the role of the anti-angiogenic factors causing endothelial dysfunction that characterizes preeclampsia.

Abstracts / Placenta 32 (2011) A1–A149

P2.22 SERUM LEVELS OF SFLT1, SENDOGLIN, PLACENTAL GROWTH FACTOR AND TRANSFORMING GROWTH FACTOR b1 IN HUMAN IMMUNODEFICIENCY VIRUS (HIV) INFECTED WOMEN WITH PREECLAMPSIA N Govender1, T Naicker2, J Moodley3 1 Department of Basic Medical Sciences, Durban University of Technology, Durban, South Africa, 2Optics and Imaging Centre, University of KwaZulu Natal, Durban, South Africa, 3Womens Health and STD Research Unit, University of KwaZulu Natal, Durban, South Africa Objective: Balance between the angiogenic factors, viz., placental growth factor (PlGF) and transforming growth factor beta 1 (TGF-b1) with their receptors, Flt1 and endoglin (Eng) is essential for normal pregnancy. Elevated placental production of the anti-angiogenic factors, sFlt1 and sEng has been implicated in preeclampsia. We hypothesised that the immune insufficiency stimulated by HIV infection may lessen a predisposition to the immune hyper-reactivity of preeclampsia. The objective of this study was to evaluate the expression of PlGF, TGFb1, sFlt1 and sEng in HIV-ve normotensive and preeclamptic compared to HIVþve preeclamptic term pregnancies. Methods: Serum from HIV–ve normotensive, n¼27; HIV–ve preeclamptic, n¼27; HIVþve normotensive, n¼31; and HIVþve preeclamptic, n¼25 were quantitatively evaluated for the angiogenic and anti-angiogenic factors using Elisas (R&D Systems). Results: A significant difference for sFlt1, sEng and PlGF (p< 0.05) was noted across all cohorts. sFlt1 was significantly different between HIV–ve preeclamptic and HIV–ve normotensive groups (156056454 vs 105714448, p¼0.0061), compared to HIV–ve preeclamptic and HIVþve normotensive groups (156056454 vs 102105458, p¼0.0061). A significant difference for sEng was evident between HIVþve preeclamptic and HIVþve normotensive groups (30.6522.25 vs 12.036.8, p¼0.0017), compared to HIV–ve preeclamptic and HIVþve normotensive groups (25.1022.61 vs 12.036.8, p¼0.0017). For PlGF, a significant difference was observed between HIV–ve preeclamptic and HIV–ve normotensive groups (341.2385.5 vs 616.2463.9, p¼0.02), compared to HIV–ve normotensive and HIVþve normotensive groups (616.2463.9 vs 255.8219, p¼0.02). TGFb1 remained unchanged across all groups (p¼0.359). Ratio analysis (sEng:TGFb1) indicate a significant difference (p<0.05) for HIVþve preeclamptic and HIVþve normotensive groups (0.950.74 vs 0.350.2, p¼0.002) compared to HIV–ve preeclamptic and HIVþve normotensive groups (0.850.7 vs 0.350.2, p¼0.002). Conclusion: Quantification of the angiogenic/anti-angiogenic factors in HIV associated preeclampsia is a novel study. The increase in sFlt-1 and sEng contributes to the reduced blood flow of preeclampsia.

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P2.23 INVESTIGATION OF THE ACTIN SCAVENGING SYSTEM IN PREECLAMPSIA Dionne Tannetta, Christopher Redman, Ian Sargent University of Oxford, Oxford, United Kingdom Objectives: Actin, the most abundant cell protein, has fundamental roles in morphology, mobility and apoptosis. Cell injury leads to release of large amounts of actin, causing clotting activation, vessel obstruction and inhibited clearance of released DNA. The actin scavenging system comprises gelsolin and vitamin D binding protein (VDBP), which together depolymerise and clear cell free actin. Impaired actin clearance is associated with several diseases and correlates with poor clinical outcome. The actin scavenging system was investigated in preeclampsia (PE), a procoagulant state associated with placental and endothelial damage. Methods: Plasma gelsolin and actin free vitamin D binding protein (AFVDBP) were measured in PE (early onset <33wks; late onset >35þ6wks), matched normal pregnant (normP) and non-pregnant (NP) women, using ELISAs. Longitudinal samples from normP and women who subsequently developed PE were also analysed. Gelsolin levels were verified using semi-quantitative western blotting. Finally, the presence of actin-gelsolin and actin-VDBP complexes in NP, normP and PE was determined by co-immunoprecipitation and actin western blotting. Results: Plasma gelsolin fell significantly during pregnancy (p<0.0002), with a concomitant significant rise in AFVDBP (p<0.0008). No differences were seen between normP and PE in gelsolin or AFVDBP, However, gelsolin concentrations were significantly lower in established late (p<0.01) and early onset PE (p<0.05) compared to NP, a result verified using western blotting. Finally, both actin-gelsolin and actin-vitDBP complexes were present in plasma samples from NP, normP and PE groups, with the normP having the lowest levels. Conclusions: We show that circulating gelsolin levels fall significantly and confirm that AFVDBP rises during normal pregnancy. We also show an increase in actin-gelsolin and actin-vitDBP complexes in PE, presumably reflecting the release of actin from damaged tissues. However, significantly lower plasma gelsolin, in established PE compared to NP, suggests that this is a consequence rather than cause of PE.

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Abstracts / Placenta 32 (2011) A1–A149

P2.24 LEPTIN LEVELS IN HIV INFECTED PREECLAMPTIC AFRICAN WOMEN Firoza Haffejee1, J Moodley2, M Singh3, T Naicker4 Department of Basic Medical Science, Durban University of Technology, Durban, South Africa, 2Woman’s Health and HIV Research Group,University of KwaZulu-Natal., Durban, South Africa, 3Biochemistry, University of KwaZulu-Natal., Durban, South Africa, 4Optics & Imaging Centre; University of KwaZulu-Natal., Durban, South Africa 1

Objectives: We hypothesise that leptin is implicated in the aetiology of HIV positive preeclamptic African patients. We therefore assessed serum leptin levels at term in HIVþ and HIV- normotensive and preeclamptic women. Methods: Following informed consent demographic profile, anthropometric data and venous blood samples were collected at term. The groups were HIVþ normotensive (n¼30), HIVþ preeclamptic (n¼30) and HIVnormotensive (n¼30), HIV- preeclamptic (n¼30). The serum was analysed for leptin by ELISA (R&D Systems test kit). Results: In the HIVþ normotensive group 23 women were on HAART and in the HIVþ preeclamptic women 27 were on HAART. In the normotensive cohort, leptin levels were significantly lower in HIVþ (13.9ng/ml1.8) compared to HIV- (24.6ng/ml1.0) group. In the preeclamptic cohort, leptin levels were elevated in HIV- group (28.3ng/ml2.2) compared to the HIVþ group (9.31ng/ml1.6). Whilst, leptin levels were not correlated with the BMI (r ¼ 0.15, p ¼ 0.51), they correlated with triceps skin-fold thickness (r ¼ 0.35, p < 0.01). There was no correlation between leptin levels and gestational age, birth weight and CD4 cell counts. Conclusion: To our knowledge, this is the first study demonstrating decreased leptin levels in HIVþ preeclamptic women. In HIV- preeclampsia, elevated leptin levels promote the release of cytokines which may cause endothelial dysfunction associated with preeclampsia. However, in HIV infection, wasting occurs. Patients who are on long term HAART may have lipodystrophy with peripheral lipoatrophy and thus a loss of subcutaneous adipose tissue. The low triceps skin fold thickness observed in our HIVþ groups indicates peripheral lipoatrophy. Since subcutaneous adipose tissue is the largest source of leptin, this will account for the decreased leptin in HIV-associated preeclampsia. We therefore conclude that leptin is not involved in the aetiology of preeclampsia in HIV positive African women.

P2.25 THE RELATIONSHIP BETWEEN LEPTIN AND INSULIN IN HIV ASSOCIATED PREECLAMPSIA Firoza Haffejee1, J Moodley2, M Singh3, T Naicker4 Department of Basic Medical Science, Durban University of Technology, Durban, South Africa, 2Woman’s Health and HIV Research Group,University of KwaZulu-Natal, Durban, South Africa, 3Biochemistry, Genetics, and Microbiology, University of KwaZulu-Natal, Durban, South Africa, 4Optics & Imaging Centre; University of KwaZulu-Natal, Durban, South Africa 1

Objectives: Preeclampsia is associated with elevation of proteins such as leptin and insulin. This study investigates these proteins in HIV-associated preeclampsia. Methods: Following informed consent, demographic profile, anthropometric data and venous blood samples were collected at term. The groups were normotensive HIVþCD4<200 (n¼30), normotensive HIVþCD4>200 (n¼30), normotensive HIV- (n¼30), preeclamptic HIVþCD4<200 (n¼30), preeclamptic HIVþCD4>200 (n¼30) and preeclamptic HIV- (n¼30). The serum was analysed for leptin and insulin by ELISA (R&D Systems and DRG Diagnostics test kits, respectively) and statistically evaluated. Results: 83% of the HIVþ (CD4<200) and 18.3% of the HIVþ (CD4>200) cohort were on HAART. Amongst normotensive pregnant women, leptin levels were significantly lower in the HIVþCD4<200 group (13.9ng/ml1.8) compared to the HIV- group (24.6ng/ml1.0;p<0.001). Leptin levels were raised in HIV- preeclamptic women (28.3ng/ml2.2) compared to all other study groups. Leptin levels were significantly lower in preeclamptic HIVþCD4<200 women (9.31ng/ml1.6) compared to the normotensive groups. However, it was not statistically different between HIV infected women with CD4 counts above and below 200 in both the normotensive and preeclamptic cohorts. Amongst normotensive pregnant women, insulin levels were significantly lower in HIVþCD4<200 women (2.51mU/l 0.5) compared to HIV- women (8.21mU/l 0.9; p<0.001). In the preeclamptic cohort, insulin levels were also significantly lower in HIVþCD4<200 women (1.33mU/l 0.3) compared to HIV- women (5.91mU/l 1.4; p¼0.03). Spearman’s correlation test revealed a significant correlation between insulin and CD4 counts (r¼0.2; p¼0.03) and between insulin and triceps skin-fold thickness (r¼0.52; p<0.001). The correlation between insulin and leptin was weak and not significant. Conclusion: In this study both leptin and insulin are lowered in HIV associated preeclampsia. Disease progression in HIV infection is associated with lower insulin possible due to the associated peripheral lipoatrophy. This would also account for the lower leptin levels in this group.

Abstracts / Placenta 32 (2011) A1–A149

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P2.26 THE ROLE OF CD35 AND CD55 IN HIV COMPROMISED PREECLAMPTIC PATIENTS

P2.27 PPAR-g REPRESSES SFLT-1 SECRETION IN FLOATING FIRST TRIMESTER PLACENTAL VILLI IN A GCM1-DEPENDENT MANNER

R Khan1, T Naicker1, F Haffejee1,2, S Reddy1, J Moodley1 1 University of KwaZulu Natal, KwaZulu Natal, South Africa, University of Technology, KwaZulu Natal, South Africa

Sascha Drewlo1,2, Fergus McCarthy4,1, Dora Baczyk1, Khrystyna Levytska2, John Kingdom3,2 1 Samuel Lunenfeld Research Institute, Toronto, Canada, 2University of Toronto, Toronto, Canada, 3Mount Sinai Hospital, Totonto, Canada, 4Cork University Maternity Hospital, Cork, Ireland

2

Durban

Objectives: We hypothesize that the two complement control proteins CD35 (complement receptor) and CD55 (decay accelerating factor, DAF) in HIV associated normal and pre-eclamptic pregnancies would be dysregulated. The altered CD35 and CD55 expression predisposes preeclamptic patients to an impaired ability to handle circulating immune complexes contributing to a systemic maternal inflammatory response, however, this may be altered in HIV infection. Methods: Post informed consent, blood was collected from a total of 150 participants viz., non-pregnant HIVþve (n¼25), non-pregnant HIV-ve (n25), normotensive HIVþve (n-25; CD4 <200), normotensive HIV-ve (n¼25), pre-eclamptic HIVþve (n¼25; CD4 < 200) and pre-eclamptic HIV-ve (n¼25) patients. Monoclonal antibodies to human CD35 and CD55 and CD49d were conjugated to FITC, PE and APC respectively. Absolute counts and percentages were assessed on whole blood lysed samples using multiparametric flow cytometry (LSR11 flow cytometer). Analysis was performed using FlowJo version 9.1. Results: The CD35 marker was significantly lower in the pregnant normotensive HIV-ve group (7731,41933.1) compared to the non pregnant HIVþve (19769.43122.8; p¼0.25), pregnant normotensive HIVþve (291306322.0; p¼0.03) and the preeclamptic HIV-ve group (18675.02199.0; p¼0.01). The CD55 marker was lower in the pregnant normotensive HIV-ve group (9187.81889.2) compared to the preeclamptic HIV-ve group (20208.12622.3; p¼0.02). CD49d was lower in the pregnant normotensive HIV-ve group (3348.4473.0) compared to the non pregnant HIVþve group (6105.3766.1; p¼0.04). There was no statistical difference between normotensive HIV-ve and preeclamptic HIVþve. CD49d was also lower in the non pregnant HIV-ve group (3385.8319.0) compared to the non pregnant HIVþve group (6105.3766.1; p¼0.03). Conclusion: Whilst CD35 and CD55 are raised in HIV-ve preeclampsia, these complement proteins are remain unchanged in HIVþve preeclampsia. Maternal insufficiency of HIV infection seems to counteract the hyperactivation of the complement proteins in HIV associated preeclampsia.

Objective: To determine the role of Peroxisome Proliferator Activated Receptor Gamma activation to the angiogenic response of the first trimester human placenta Methods: First trimester floating placental villi (8-12weeks) were exposed to the PPAR-g agonist rosiglitazone (10mM) in an in-vitro explant model (8% oxygen; n¼6-12). Tissue and culture media was harvested and wet weight recorded after 48h of cultivation. RNA and protein was isolated to assess sFLT-1 expression in both tissue and culture media using qRTPCR, Western blot, immuno-histochemistry and ELISA respectively. Glial cell missing 1 expression was assessed via qRT-PCR to monitor the effect of rosiglitazone on trophoblast differentiation. Results: Rosiglitazone treatment of first trimester placental villi resulted in significant alterations of trophoblast differentiation and the angiogenic response in this experimental model. sFLT-1 release / expression was reduced in the treatment group compared to both the vehicle and nontreatment control 386.5  41.6 versus 775.1 158 (p>0.01). and 556.8 79pg/ml*mg tissue (SEM). GCM1 mediated placental differentiation was accompanied by a significant increase in GCM1 expression in the villous explants. GCM1 mRNA levels were up-regulated by 5.4 and 3.2 -fold at 24h and 48h cultivation. Conclusions: In the previously published reduced uterine perfusion pressure (RUPP) rat model of preeclampsia PPAR-g activation was shown to ameliorate RUPP induced hypertension and endothelial dysfunction. Severe early preeclampsia is characterized by increased release of sFLT-1 into the maternal circulation as a result of impaired trophoblast turn over and reduced GCM1 mediated differentiation. Our data suggest a direct link between PPAR-g and human placental function by reducing sFLT-1 expression via the up-regulation of GCM1 in vitro. These findings are highly relevant for the pathogenesis of preeclampsia and suggest a potential novel therapeutic target for the treatment of this disease.

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Abstracts / Placenta 32 (2011) A1–A149

P2.28 PROTECTIVE EFFECTS OF HIGH DENSITY LIPOPROTEINS IN A MODEL OF PRE-ECLAMPSIA

P2.29 HIGH DENSITY LIPOPROTEINS RESTORE INTEGRIN SWITCHING IN A MODEL OF PRE-ECLAMPSIA

Francesca Charlton1, Bei Xu1,2, Angela Makris1, Kerry-Anne Rye1, Annemarie Hennessy1,2 1 The Heart Research Institute, Sydney, NSW, Australia, 2School of Medicine, The University of Western Sydney, Sydney, NSW, Australia

Francesca Charlton1, Bei Xu1,2, Angela Makris1, Kerry-Anne Rye1, Annemarie Hennessy1,2 1 The Heart Research Institute, Sydney, NSW, Australia, 2School of Medicine, The University of Western Sydney, Sydney, NSW, Australia

Objectives: Failure of the trophoblast to appropriately invade uterine spiral arteries is thought to be an initiating event in preeclampsia, a disorder associated with endothelial dysfunction. A dyslipidemia characterised by low plasma levels of high density lipoproteins (HDL) and elevated triglycerides has also been described in preeclampsia. The proinflammatory cytokine TNF-a inhibits trophoblast invasion of uterine endothelial cells. This study asks if HDL, which have anti-inflammatory properties, or its components phosphatidylcholine (PLPC) and apolipoprotein (apo) A-I, can protect against the deleterious effect of TNF-a on trophoblast-endothelial cell interactions. Methods: An in vitro trophoblast-uterine endothelial cell co-culture model was used to investigate the effect of discoidal reconstituted HDL containing PLPC and apoA-I, (A-I)rHDL, as well as lipid free A-I and small unilamellar phospholipid vesicles on trophoblast incorporation into endothelial tubules in the presence and absence of TNF-a. Uterine endothelial cells were pre-incubated with discoidal (A-I)rHDL or lipid free A-I (final apoA-I concentration 1 mg/mL) or PLPC vesicles (final phospholipid concentration 3mM), for 16 hrs prior to seeding on matrigel coated plates. Tubules formed within 4 hrs. Fluorescence-labelled JEG-3 (trophoblastderived) cells were then co-cultured with the endothelial cells TNFa (final concentration of 0.2 ng/mL). Bright field and fluorescent images were captured after 24 hrs. The effect of TNF-a on JEG-3 cell invasion was quantified with Image J software. Results: TNF-a inhibited JEG-3 cell integration into endothelial tubular structures by 28 9% (p<0.001). This effect was reversed when the endothelial cells were pre-incubated for 16 hrs with discoidal (A-I)rHDL (p<0.001 compared to non-incubated cells) and lipid free A-I (p<0.001 compared to non-incubated cells). This protective effect was not observed with PLPC vesicles. Conclusion: (A-I)rHDL and lipid-free apoA-I enhance trophoblastendothelial cell integration in the presence of a pro-inflammatory stimulus. A healthy lipid profile may affect pregnancy outcomes by priming endothelial cells in preparation for trophoblast invasion.

Objectives: Implicated in the cause of preeclampsia is the failure of the trophoblast to switch from integrin a6b4 to integrin a1b1 expression, and from E-cadherin to VE-cadherin expression in order to appropriately invade uterine spiral arteries. Preeclampsia is a disorder of pregnancy associated with endothelial dysfunction. A dyslipidemia (low plasma levels of high density lipoproteins (HDL) and elevated triglycerides) early in pregnancy is also associated with increased risk of preeclampsia. Previous work using an in vitro trophoblast-uterine endothelial cell (TrUEc) co-culture model has shown that the pro-inflammatory cytokine TNF-a inhibits trophoblast invasion. We have shown a protective effect of discoidal reconstituted HDL containing phosphatidylcholine and apolipoprotein (apo) A-I, (A-I)rHDL on trophoblast incorporation into endothelial tubules in the presence of TNF-a. We aim to identify the molecular changes in these cells when pre-incubated with (A-I)rHDL. Methods: The in vitro Tr-UEc co-culture model was used to investigate the effect of (A-I)rHDL on the markers of tissue invasion and trophoblast incorporation into endothelial tubules. Uterine endothelial cells were preincubated with discoidal (A-I)rHDL (final apoA-I concentration 1 mg/mL) for 16 hrs prior to seeding on matrigel coated plates. Tubules formed within 4 hrs. JEG-3 (trophoblast-derived) cells were then co-cultured with the endothelial cells TNF-a (final concentration of 0.2 ng/mL). Invasion was evaluated by performing quantitative PCR (qPCR) to analyse gene expression of matrix metalloproteinase (MMP)-2, tissue inhibitor of matrix metalloproteinase (TIMP)-I, integrins a1b1 and a6b4 plasminogen activator inhibitor (PAI)-I, E-cadherin and VE-cadherin. Results: qPCR analysis showed that when endothelial cells were preincubated with discoidal (A-I)rHDL the mRNA expression of integrin a6b4 and E-cadherin was downregulated, whilst MMP-2 expression was upregulated. No significant changes in PAI-I, TIMP-I, integrin a1b1 or VEcadherin were observed. Conclusion: HDL confers a protective effect on trophoblast-endothelial cell interactions by restoring the switch to integrin a1b1 and VEcadherin expression.

Abstracts / Placenta 32 (2011) A1–A149

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P2.30 MATERNAL SERUM RESISTIN IN FIRST TRIMESTER NORMAL AND PREECLAMPSIA PREGNANCIES

P2.32 MOLECULAR KARYOTYPING OF BLOOD FROM PREECLAMPTIC WOMEN REVEALS GROSS COPY NUMBER VARIATIONS

Michael Christiansen1, Paula L. Hedley1,2, Sophie Placing1, Karen R. Wøjdemann3, Anting L Carlsen1, Anne-Cathrine Shalmi3, Line Rode3, Karin Sundberg3, Ann Tabor3 1 Statens Serum Institut, Copenhagen, Denmark, 2Stellenbosch University, Cape Town, South Africa, 3Rigshospitalet, Copenhagen, Denmark

Mona Nystad1,2, Vasilis Sitras3, Ganesh Acharya2,4 Division of Child and Adolescent Health, Department of Medical Genetics, University Hospital of North Norway, Tromsø, Norway, 2Women’s Health and Perinatology Research Group, Department of Clinical Medicine, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway, 3 Department of Obstetrics and Gynaecology, Akershus University Hospital, Lørenskog, Norway, 4Department of Obstetrics and Gynaecology, University Hospital of North Norway, Tromsø, Norway

Context: Resistin is a hormone synthesised by mononuclear cells and placenta. In rat models resistin is associated with increased insulin resistance (IR) which has been associated with pre-eclampsia (PE). Objective: To determine the ability of resistin to predict the later development of PE, and examine the association with clinical severity of PE. Design, Setting, Patients and Main Outcome Measures: Case control study. The maternal serum resistin concentration was determined in case pregnancies that developed PE and 285 control pregnancies which were matched for gestational age, parity and maternal age. Samples were from gestational week 10þ0 – 13þ6 and obtained from the Copenhagen First Trimester Screening Study. Main Outcome Measures: Differences in maternal serum resistin concentrations between PE and control pregnancies; the ability of resistin to identify PE pregnancies. Results: In controls, resistin decreased significantly with gestational age, (Spearman’s rho ¼ -0.225, p <0.001), but this was only apparent after week 12þ6. Maternal serum resistin was decreased in PE pregnancies, median 22.2 ng/mL (range: 7.5 – 58.0 ng/mL) compared to controls, median 25.4 ng/mL (range: 4.9 – 83.0 ng/mL) (p ¼ 0.002). There was a significant negative correlation between clinical severity of PE and maternal serum resistin, however, the ability of resistin to identify pregnancies developing PE is poor. Conclusions: The constant low level of resistin in PE pregnancies may reflect increased maternal insulin sensitivity compromising nutrient supply to placenta in first trimester. PE may be preceded – or caused - by a maternal metabolic disturbance. P2.31 TUMOUR NECROSIS FACTOR ALPHA IN FIRST TRIMESTER PREGNANCY SERUM: A PREDICTOR OF SEVERE PREECLAMPSIA Paula L. Hedley1,2, Sophie Placing1, Karen R. Wøjdemann3, Anne-Cathrine Shalmi3, Anting L Carlsen1, Line Rode3, Karin Sundberg3, Ann Tabor3, Michael Christiansen1 1 Statens Serum Institut, Copenhagen, Denmark, 2Stellenbosch University, Cape Town, South Africa, 3Rigshospitalet, Copenhagen, Denmark Context: Pre-eclampsia (PE) has been linked to inflammation e.g. in chronic malaria and rheumatoid arthritis. Tumour necrosis factor alpha (TNFa) is an inflammatory marker which is known to influence the synthesis of leptin which is increased in second and third trimester of pregnancies that develop pre-eclampsia (PE). Objective: To examine whether the first trimester maternal serum concentrations of TNFa in pregnancies that later develop PE differs from the concentrations in normal pregnancies. Design and Patients: Retrospective case control study using first trimester serum samples, 123 from singleton pregnancies that developed PE and 285 control pregnancies matched for gestational age, parity and maternal age, from the Copenhagen First Trimester Screening Study. Main Outcome Measure: Differences in maternal serum TNFa concentrations between PE and control pregnancies. Results: The fraction of pregnant women with measurable TNFa was significantly increased in severe PE and HELLP pregnancies. There was no significant difference between control pregnancies and mild PE. Conclusions: The difference between maternal serum TNFa concentrations in control/mild PE and severe PE/HELLP pregnancies in first trimester suggests that TNFa may be a very early marker of severe PE.

1

Objectives: Molecular karyotyping is a versatile method for detecting whole genome copy number variations (CNVs). In the present study, using array comparative genomic hybridisation (aCGH), we demonstrate several regions of losses and gains in severely preeclamptic women compared to their healthy controls. Methods: DNA extracted from peripheral blood of 16 severely preeclamptic women were pooled together and compared with pooled DNA from 16 healthy controls on 720 K arrays and 2.1 M arrays from NimbleGen. Individual patients with preeclampsia from this cohort were compared to normal controls on arrays. Results were confirmed by quantitative PCR. Regions of gains and losses were analysed using the genome browsers of University of California, Santa Cruz (UCSC) (http:// www.genome.ucsc.edu/), the National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) and Ensembl (EMBL, WTSI) (http://www.ensembl.org/index.html). The Database of Genomic Variants (DGV) (http://projects.tcag.ca/variation/) was used to identify structural variation identified in healthy control samples. Results: aCGH revealed several big regions of losses on chromosomes 1, 2, 7, 8, 9 and 15. Genes in these regions were POTEC, POTEB, RPS8P10, MIRN1268, VIT and ADAM5P. Different immunoglobin kappa genes and ribosomal protein genes were also deleted. Gains were found on chromosomes 6, 7, 8, 11, 14 and X. Genes included mainly POTEG, AGPAT4, PARK2 and POLR2J. Several different microRNA genes and some new genes were also included in the regions. Conclusion: Molecular karyotyping might have a role in elucidating the genetic factors associated with pathogenesis of preeclampsia.

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Abstracts / Placenta 32 (2011) A1–A149

P2.33 COMPARISON OF THE EXPRESSION OF KILLER IMMUNOGLOBULIN-LIKE RECEPTORS IN DECIDUAL AND PERIPHERAL LEUCOCYTES, IN PREECLAMPTIC AND NORMOTENSIVE WOMEN.

P2.35 INHERITED SUSCEPTIBILITY TO PREECLAMPSIA – ANALYSIS OF FIVE POLYMORPHISMS PREDISPOSING TO CARDIOVASCULAR DISEASE IN 279 WHITE AND 241 BLACK WOMEN

Elly Natty Sanchez-Rodriguez1,2, Janette Vega-Miranda1, C. Adriana Mendoza-Rodriguez1, Anne Croy2, Marco Cerbon1 1 National University Autonomous of Mexico, Mexico, DF, Mexico, 2Queen’s University, Kingston, Ontario, Canada Objectives: Previous results from our laboratory have shown the presence of decidual NK cells (dNK) at the end of the pregnancy. In order to understand better the role of NK cells, in the pathophysiology of preeclampsia, the aim of this work was to compare the expression of Killer Immunoglobulinlike Receptors (KIR) in decidual tissue and Peripheral Mononuclear Blood Cells (PMBC), from preeclamptic (PE) and normotensive women (controls). Methods: Decidual tissue samples were obtained during cesarean section from controls (n¼6) and PE (n¼6) women. Heparinised peripheral blood samples were obtained during the first hours of puerperium. Leucocytes were isolated using Ficoll-Hypaque gradient and total RNA was extracted following the Trizol protocol. KIR expression was performed using PCR-SSP. Results: Based on our previous results, dNK persist throughout pregnancy and they expressed the cell surface markers, CD3-, CD56þ, CD16- and CD9þ, as dNK in early gestation. The expression of KIR was different between decidua and PBMC samples. Some samples did not express the KIR 2DL4, previously reported as a receptor always expressed on NK cells. No differences were found in the expression of KIR between PE and control women. Conclusion: A significant percentage of dNK persists until the end of the pregnancy with a similar phenotype to those found during early pregnancy. Even though we didn’t find any difference in KIR expression between controls and PE groups, their expression differs between decidual tissue and PBMC.

Tanja Groten1, Thomas Lehmann3, Ekkehard Schleussner1, F.O. Danso4, Robert Zeillinger2 1 University hospital Jena, Friedrich Schiller university Jena, Jena, Germany, 2 Department of Obstetrics & Gynecology, University of Vienna, Vienna, Austria, 3Institute of Medical Statistics and Computer Science, University Hospital Jena, Jena, Germany, 4Department of Obstetrics & Gynecology University of Science and Technology Kumasi, Kumasi, Ghana

P2.34 THE DYNAMIC ROLE OF ACID CERAMIDASE IN PREECLAMPSIA. Megan Melland-Smith2,7, Reshef Tal1, Isabella Caniggia1,2, Martin Post5,6 1 Samuel Lunenfeld Research Institute, Toronto, Canada, 2Mount Sinai Hospital, Toronto, Canada, 3Department of Obstetrics and Gynaecology, Toronto, Canada, 4Department of Physiology, Toronto, Canada, 5Department of Pediatrics, Toronto, Canada, 6Hospital for Sick Children, Toronto, Canada, 7University of Toronto, Toronto, Canada Objective: Sphingolipids act as bioactive mediators in a variety of pathophysiological processes by regulating cell death and survival. Sphingolipid metabolism is tightly controlled by a balance between their synthesis and breakdown via the action of specific enzymes. In particular, the enzyme acid ceramidase (AC) regulates ceramides (CERs) turnover. The objective of this study was to examine CERs expression and function as well as the role of its regulatory enzyme AC in placentae from preeclamptic (PE) and pre-term control (PTC) pregnancies. Methods: Protein and mRNA expression levels of AC were assessed by Western Blot analysis (WB) and quantitative PCR (qPCR) in normal and preeclamptic placentae. Human villous explants and choriocarcinoma JEG3 cells were treated with C-16 ceramide and sodium nitroprusside (SNP: 2.5 and 5 mM), a nitric oxide donor known to mimic oxidative stress. In addition JEG-3 cells were treated with 2-oleoylethanolamine (2-OE), a specific inhibitor of AC activity. Markers of cell death and autophagy, including cleaved-caspase3, LC3B-II and lysosomal activity using lysotracker dye, were assessed. Lipid analysis in normal and pathological placental tissue and in SNP-treated cells was performed using high power liquid chromatography linked to tandem mass spectometry (MS/MS). Results: Lipid analysis revealed a significant increase in CERs levels in PE relative to PTC and this associated with a decrease in mRNA and protein expression of AC rate-limiting enzyme. Similarly, exposure of explants and JEG-3 cells to SNP increased CERs while decreasing AC expression levels. Moreover, C-16 ceramide and 2-OE treatments resulted in increased cleaved -caspase3 expression and this was accompanied by increased LC3B-II expression and lysosomal activity. Conclusions: Altered AC expression in preeclamptic placentae, likely induced by the oxidative stress status, is responsible for CERs accumulation that in turn may contributes to increased cell death typical of this disorder. (Supported by CIHR)

Objectives: With an incidence of 3-7% in all pregnant women preeclampsia is still the leading cause of fetal and maternal mortality. Clinical studies have documented a familiar tendency to develop preeclampsia. Additionally, patients with impaired endothelial health are at increased risk. We studied genetic polymorphisms associated to cardiovascular disease and impaired endothelial function in women with and without preeclampsia. Methods: 241 African and 279 Caucasian women were recruited in a two hospital-based case-control study for genetic testing of the polymorphisms epoxide hydrolase 1 (EPXH1) (Tyr113His), endothelial nitric oxide synthase (NOS3) on exon 7 (Glu298Asp) and variable nucleotide tandem repeats in intron 4 (NOS3I4a/b), angiotensinogen (AGT) (Met235Thr) and the estrogen receptor1 polymorphism in intron 1 at position T938C. Statistics were performed as logistic regression analysis. Results: Of 241 African women, 95 developed preeclampsia including 19 evolving eclampsia. Out of the 279 Caucasian women 81 had preeclampsia including 28 developing hemolysis, elevated liver enzymes and low platelets (HELLP)-syndrome. Carriers of the NOS3I4a/b polymorphism had a 1.7-fold increased risk to develop preeclampsia. There was no difference in distribution of the other tested polymorphism. Furthermore we could show a statistical significant fourfold reduction to encounter severe course of preeclampsia (defined as occurrence of HELLP-syndrome or eclampsia) in carriers of the EPHX1 polymorphism encoding histidine. Conclusions: Our data reveal a highly significant association of the NOS3I4a/b polymorphism with increased risk to develop preeclampsia in pregnancy. The performed logistic regression to evaluate genetic risk association and the inclusion of two diverse ethnic groups constitute the strength of our data. Furthermore, the shown association of EPXH1 polymorphism with the severity of preeclampsia strengthen the concept, that individual susceptibility determines the capability of the maternal organism to deal with the pregnancy derived agents causing preeclampsia.

Abstracts / Placenta 32 (2011) A1–A149

P2.36 CYTOCHROME P450 EPOXYGENASE 2J2 (CYP2J2) AND IT’S METABOLITES ARE UPREGULATED IN PREECLAMPSIA Florian Herse1, Carl A. Hubel2, Berthold Huppertz3, Babbette B. LaMarca4, Ian Sargent5, Friedrich C. Luft1, Wolf-Hagen Schunck1, Dominik N. Muller1, Anne Cathrine Staff6, Ralf Dechend1,7 1 Experimental and Clinical Research Center, Max-Delbrueck Center for Molecular Medicine and Medical Faculty of Charité, Berlin, Germany, 2 Magee-Womens Research Institute, Department of Obstetrics, Gynecology & Reproductive Sciences, University of Pittsburgh, Pittsburgh, United States, 3Institute of Cell Biology, Histology and Embryology, Center for Molecular Medicine, Medical University of Graz, Graz, Austria, 4 Department of Obstetrics and Gynecology, University of Mississippi Medical Center, Jackson, United States, 5Nuffield Department of Obstetrics and Gynecology, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom, 6Deptartment of Obstetrics and Gynecology, Oslo University Hospital, Oslo, Norway, 7Helios Clinic, Berlin, Germany Preeclampsia (PE) is a multisystem disorder of pregnancy, originating in the placenta. It presents after 20 weeks of gestation as hypertension and proteinuria. The cytochrome P450 (CYP) -system regulates vascular functions, inflammation, and angiogenesis that are mechanistically important in PE. In a microarray screening of placenta and decidua (maternal site of placenta) from preeclamptic (preecl.) women and controls (n¼23 each) we found CYP2J2 significantly upregulated. We further investigate the role of CYP2J2 and their metabolites, the epoxyeicosatrienoic acids (EETs), the dihydroxyeicosatrienoic acids (DHETs), and their secondary metabolite 5,6-epoxythromboxane (5,6 epTX) in the uteroplacental unit and the circulation of preecl. women. We could confirm upregulation of CYP2J2 in placental tissue (1.6 fold) and decidual tissue (3.9 fold) of preecl. women (p<0.001) by realtime RT-PCR. Immunohistochemistry of preecl. deciduas demonstrated an increased CYP2J2 protein expression. CYP2J2 was localized in trophoblast cells of villous and deciduas at gestational week 12 and at term. The 5,6-EETs,14,15EETs, and the corresponding DHETs were elevated in serum of preecl. women compared to controls in the 2nd and 3rd trimester and after delivery, measured by HPLC-MS-MS. We also detected EETs and DHETs in isolated primary trophoblasts and in syncytiotrophoblast microvillous membranes. In a rat model of preeclampsia with reduced uterine perfusion pressure (RUPP), we detected elevated EET and DHET serum level. Intervention with the CYP-Inhibitor MSPPOH reduced preeclamptic features. Stimulation of a trophoblast derived cell line (SGH-PL4) with the pro-inflammatory cytokine TNF-alpha enhanced CYP2J2 gene expression dose- and timedependently, which was confirmed on protein level. For functional analysis, we tested the invasion of SGH-PL4-cells in a fibrin gel. The invasive process, stimulated by epidermal growth factor, was decreased by a thromboxane analog, representing the 5,6 epTX. Our data demonstrate that the CYP P450 system is dysregulated in the uteroplacental unit and in circulation of preecl. women. The downstream metabolite of CYP2J2, 5,6 epTX, is a potential candidate for the disturbed uteroplacental remodeling, leading to hypertension and endothelial dysfunction.

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P2.37 DUAL SPECIFICITY PHOSPHATASE 9 (DUSP9) REGULATION IN SEVERE EARLY ONSET PRE-ECLAMPSIA AND IN A PLACENTAL VILLOUS EXPLANT MODEL Marie Czikk1, Sascha Drewlo2, Dora Baczyk2, S. Lee Adamson1,2, John Kingdom1,2 1 Department of Obstetrics and Gynecology, University of Toronto, Toronto, Canada, 2Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada Objectives: 1) To determine the effect of DUSP9 down-regulation in placentas of patients with severe pre-eclampsia (sPE) on downstream targets and whether this down-regulation is epigenetically mediated. 2) To silence DUSP9 expression in cell and tissue based models and study downstream targets. 3) To investigate the role of hypoxia on DUSP9 expression. Methods: Phosphorylation of target proteins ERK1/2 and p38 was assessed with ELISA (Pre-term (PT) controls N¼8, sPE N¼8, severe growth restriction N¼6). EpityperÔ was used to determine methylation status of the DUSP9 promoter in sPE and PT control placentas (N¼4/4). DUSP9 was silenced with siRNA in BeWo cells and in explanted first trimester placental villi. Expression was confirmed via qRT-PCR and phosphorylation of ERK1/2 was assessed via ELISA. Explants were subjected to hypoxia (3% O2) to determine the impact on DUSP9 expression. Results: DUSP9 is decreased in sPE placentas compared to those with severe growth restriction. We could not demonstrate hypermethylation and thus epigenetic mediation of this down-regulation. The ratio of phosphorylated to total ERK1/2 showed a trend to an increase in sPE, and no change for p38. In the tissue and cell based models DUSP9 expression was reduced (62/61%) in response to siRNA (N¼4/4). ERK1/2 phosphorylation was not significantly altered in response to DUSP9 silencing in either BeWo cells or in explanted placental villi. These explants did however show a significant decrease in DUSP9 expression by 7420% in response to hypoxia. Conclusion: DUSP9 dephosphorylates MAP kinase proteins thus reducing cellular proliferation, a process which is significantly altered in sPE. DUSP9 down-regulation could not be explained with epigenetic promoter analysis. Hypoxia did however prove to be a negative regulator of DUSP9 expression. This down-regulation might be related to local hypoxic mechanisms governing stability, or from patchy necrosis of the syncytiotrophoblast layer. Further study of cytotrophoblast proliferation in DUSP9 silenced explants is ongoing. Funding: CIHR and Department of Ob/Gyn, Mount Sinai Hospital, Toronto Canada

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Abstracts / Placenta 32 (2011) A1–A149

P2.38 MICRO-RNA EXPRESSION IN HEMOGLOBIN PERFUSED PLACENTAS Tina Rasmussen1, Karen May2, Henning Schneider3, Bo Åkerström4, Stefan R. Hansson1 1 of Obstetrics and Gynecology, Department of Clinical Sciences, Lund University Hospital, Lund, Sweden, 2Department of Clinical Pharmacology, Ernst Moritz Arndt University of Greifswald, Greifswald, Germany, 3 Department of Obstetrics and Gynecology, Insel Spital, University of Bern, Bern, Switzerland, 4Division of Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden Objectives: We have previously shown that free fetal haemoglobin (HbF) may play a role in the patophysiology of preeclampsia (PE). HbF causes functional and structural damage to the placenta and we hypothesize that there might be an altered micro-RNA (miRNA) expression. We therefore examined the miRNA profile in placental tissue perfused with free HbF and in PE placentas. Methods: Human term placentas, obtained from uncomplicated singleton pregnancies delivered by Caesarean section or vaginal delivery, were used for the perfusion experiments using the dual placental perfusion system. Tissue samples from the placentas were taken from an adjacent cotyledon before the perfusions were initiated and from the perfused cotyledon after completion of the perfusion. Perfusions were performed with medium only and with free Hb. Total RNA was extracted from the placenta samples and miRNA expression was determined using a miRNA specific real-time quantitative reverse transcriptase-polymerase chain reaction assay for nine selected miRNAs. Results: Placentas perfused with hemoglobin showed an increase in perfusion pressure as well as morphological changes, similar to what is seen in PE. Mir-205 showed a significantly increased expression in the Hb perfused placentas compared to controls. This tendency was also seen for mir-517a, mir-517b and mir-16, however the results were not significant. Conclusion: Free Hb cause oxidative stress and PE like damage to the placenta. Our results show that mir-205 and the placenta specific mir-517 are increased after perfusion with free Hb, both previously related to hypoxia and oxidative stress. Interestingly, elevation of mir-16 expression has been shown to causes increased synthesis of HbF, suggesting a positive feedback loop as a possible mechanism for the increased levels of HbF seen in PE.

P2.39 DOES MATERNAL PSYCHOLOGICAL DISTRESS IN SECOND TRIMESTER OF PREGNANCY AFFECT FETO-PLACENTAL VOLUME BLOOD FLOW IN THIRD TRIMESTER? Anne Helbig1, Anne Kaasen1, Ulrik Fredrik Malt1,2, Guttorm Haugen1,2 1 Oslo University Hospital, Oslo, Norway, 2Oslo University, Oslo, Norway Objectives: Maternal psychological distress in pregnancy has been linked to lower birthweight. Reduced placental circulation has been suggested as a mechanism, and timing of distress may play a role. We aimed to examine the effect of maternal distress in the first half of pregnancy on fetoplacental volume blood flow in third trimester. Methods: This prospective observational study was performed at a tertiary center. The population consisted of 62 pregnant women with a structural malformation in a previous child or fetus, but normal ultrasound findings in the current pregnancy. Psychological distress was assessed at 16 weeks gestational age (GA)(range 12–24) by standardized psychometric questionnaires: the Depression and Anxiety subscales of the General Health Questionnaire-28, Edinburgh Postnatal Depression Scale (EPDS), and Impact of Event Scale-22 (subscales Intrusion, Avoidance and Arousal). At 30 weeks GA, time-averaged maximum velocity and vessel diameter in the intra-abdominal portion of the umbilical vein were measured by ultrasound. Placental flow was calculated and normalized for fetal abdominal circumference (PFAC, mL/cm/min). Results: Distress measures were strongly intercorrelated, but not associated with GA. All but Avoidance correlated positively with PFAC (Spearman’s r 0.26–0.39, P-values 0.002–0.043). The mean difference between those above vs. below the median for the most strongly correlated measure (EPDS) was 1.28 mL/cm/min (95%CI 1.11,1.48; P¼0.001), corresponding to 1 SD. The covariates maternal age, parity, body mass index at 30 weeks GA, and smoking were explored in univariate analyses. They were not related to PFAC, and showed no significant effect in multiple regression analyses. Conclusions: A positive correlation between maternal distress in second trimester and feto-placental volume blood flow at 30 weeks GA was found in pregnant women with a previous child or fetus with malformation.

Abstracts / Placenta 32 (2011) A1–A149

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P2.40 MORPHOLOGICAL COMPARISON OF PLACENTAL STEM VILLI ARTERIES BETWEEN TERM AGA AND SGA NEWBORNS.

P2.41 THE EXPRESSION OF AKT AND ERK1/2 PROTEINS DECREASED IN INTRAUTERINE GROWTH RESTRICTED RAT PLACENTAL DEVELOPMENT

Chizuko Yaguchi, Hiroaki Ito, Toshiyuki Uchida, Kazunao Suzuki, Kazuhiro Sugihara, Naohiro Kanayama Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Japan

Asli OZMEN1, Gozde UNEK1, Dijle KIPMEN-KORGUN2, Emin Turkay KORGUN1 1 Akdeniz University Medical Faculty Histology and Embryology Department, Antalya, Turkey, 2Akdeniz University Medical Faculty Biochemistry Department, Antalya, Turkey

Objective: The purpose of this study was to compare morphology of placental stem villi arteries between small-for gestational age (SGA) and appropriate for gestational age (AGA) newborns among full-term singleton pregnancy. Methods: We studied the placentas from 20 singleton pregnancies delivered at the department of Obstetrics and gynecology at Hamamatsu university school of medicine between 2005 and 2009. Placentas were classified into 2 groups, SGA newborns (SGA, n¼11) and AGA group (n¼9). All cases had no complications. SGA newborns were defined as those whose birth weight was below the 10th percentile for that gestational age. The control group comprised the placentas of term pregnancies obtained from elective caesarean section and those newborns birth weight were appropriate for gestational age. For each case, clinical information was referred by clinical chart. The tissue section was stained with elastic tissue Elastica van Gieson (EVG) stains. The vessel wall thickness, luminal area and obstruction ratio were measured. Semiquantitive measurement of collagen fibers in the vessel wall and immunohistochemistory were performed. Results: There were significant differences between SGA and AGA newborns placenta in luminal area SGA: 604.6 þ/- 304.7mm2, control: 1291.9 þ/- 938.5 mm2 (p<0.05) and obstruction ratio SGA: 0.082 þ/- 0.026, control: 0.208 þ/- 0.078 (p<0.01). Pathological findings showed that SGA placenta was vacuolated in endothelial cells and those being thickened media without inflammation. Collagen fibers in the total vessel wall of SGA were significantly lower than those in AGA group (p<0.01). Immunohistochemistry showed strong staining for VEGF in media layer of stem villi arteries and 8Hydroxydeoxyguanosine (8-OHdG) which indicated oxidative stress, in villous stroma in SGA placenta. Conclusion: The stem villi arteries showed luminal narrowing and increased obstruction ratio in SGA placenta. These distinct differences between SGA and AGA placenta might be involved in the pathogenesis for SGA newborns.

Objectives: We aimed to investigate effects of maternally administered dexamethasone on Akt and ERK1/2 proteins during rat placental development. Therefore we studied the expression levels and spatio-temporal immunolocalizations of Akt, p-Akt, ERK1/2 and p-ERK1/2 proteins in normal and IUGR (Intrauterine Growth Restriction) placentas in rats. Methods: In order to compose IUGR group, pregnant Wistar rats were injected with 100mg/kg dexamethasone in 0,1 ml 10% ethanol on 12th day of gestation subcutaneously. Injections continued as 200mg/kg until they were sacrificed on 14th, 16th, 18th and 20th day of their gestation. For immunohistochemical studies, one part of the dissected placentas were fixed in Holland fixative and for Western blot analysis one part were frozen and stored in liquid nitrogen. Results: The IUGR group had significantly smaller embryos on day 20 of gestation and had smaller placentas on day 14, 16, 18 and 20 compared with control. Maternal dexamethasone treatment led to a significant decrease in Akt activation on day 16, 18, and 20. Total Akt protein expression was not significantly affected by the treatment. There was a significant decrease in ERK1/2 activation on day 18 in IUGR group; on the other hand there was a significant increase on day 16. Total ERK1/2 protein expression didn’t show any significant difference between groups. Phospho-Akt immunolabelings were remarkable in junctional zone in control groups and weaker in IUGR groups. Phospho-ERK1/2 immunolabelings were considerable in the junctional and labyrinth zones in the control groups and weaker in IUGR groups. Conclusion: Maternal dexamethasone treatment led to a decrease in Akt and ERK1/2 activation during rat placental development. The decrease in Akt and ERK1/2 activations may result with cell survival inhibition or apoptosis stimulation. Hence, dexamethasone induced placental and embryonal development abnormalities could be associated with reduction of Akt and ERK1/2 activation.

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Abstracts / Placenta 32 (2011) A1–A149

P2.42 CAN PLACENTAL GROWTH FACTOR IDENTIFY PLACENTAL INTRAUTERINE GROWTH RESTRICTION IN FETUSES WHO ARE ANTENATALLY IDENTIFIED AS BEING SMALL FOR GESTATIONAL AGE? Samantha Benton1, Yuxiang Hu1, Fang Xie1, Kenneth Kupfer2, Seok-Won Lee2, Laura Magee1, Peter von Dadelszen1 1 University of British Columbia, Vancouver, British Columbia, Canada, 2 Alere International, London, United Kingdom Background: In the group of fetuses identified antenatally as being small for gestational age (SGA), there would be clinical utility in differentiating those with intrauterine growth restriction (IUGR) due to placental origin from those who are constitutionally small. Placental growth factor (PlGF) levels in maternal circulation may provide this information. Studies have found decreased circulating concentrations of PlGF in women who deliver SGA fetuses; however, these studies failed to categorise cases based on the etiology of SGA (constitutional versus placental). We sought to determine if a positive PlGF test measured on the Triage PlGF rapid assay (Alere, San Diego) differentiates fetuses with placental IUGR from those who are constitutionally small. Methods: In this case-control study, maternal blood was collected from women identified as an SGA fetus (fetal abdominal circumference <10th percentile) on antenatal ultrasound. Plasma free PlGF levels were quantified using the Triage immunoassay according to the product insert. After delivery, the cause of SGA for each fetus was determined. Placental IUGR (n¼9) was defined as birthweight <10th percentile with abnormal placental pathology while constitutionally small fetuses (n¼7) were defined as birthweight <10th percentile with normal placental pathology. Women with uncomplicated pregnancies served as controls (n¼79). A positive PlGF test was defined as PlGF concentration <5th percentile for gestational age in uncomplicated pregnancy. Sensitivity and specificity of a positive PlGF test to differentiate placental IUGR from constitutional SGA were determined. Results: A positive PlGF test was found in 9/9 placental IUGR cases, 1/7 constitutional SGA cases and 4/79 controls (P-value<0.0001). PlGF differentiated placental IUGR fetuses from constitutionally small fetuses with 100% [95% CI: 66-100] sensitivity and 86% [95% CI: 42, 100] specificity (pvalue¼0.0009). Conclusion: These preliminary data suggest that a positive PlGF test using Triage may identify placental IUGR antenatally. Further investigation is warranted.

P2.43 PLACENTALLY-DERIVED FACTORS CAN PREDICT POOR PREGNANCY OUTCOME IN REDUCED FETAL MOVEMENTS Lynne Warrander, Philip Dutton, Josh Kroll, Susan Greenwood, Colin Sibley, Rebecca Jones, Alexander Heazell Maternal and Fetal Health Research Group, University of Manchester, Manchester, United Kingdom Objective: 20% of women presenting with reduced fetal movements (RFM) in the third trimester have a poor pregnancy outcome defined as preterm birth, intrauterine growth restriction (IUGR) or stillbirth. Therefore, women with RFM represent a group with increased pregnancy pathology. Currently, methods to accurately identify women who have a poor pregnancy outcome have insufficient sensitivity and specificity. As stillbirth and IUGR are intimately linked to placental dysfunction, we hypothesise that placentally-derived factors could be used as a screening tool for poor pregnancy outcome after RFM. Methods: 303 women experiencing RFM after 28 weeks gestation were recruited. Serum samples taken at time of presentation with RFM were analysed using ELISA for human chorionic gonadotrophin (hCG), human placental lactogen (hPL), placenta associated plasma protein-A (PAPP-A), alpha-fetoprotein (AFP), lactate dehydrogenase (LDH) and ischaemia modified albumin (IMA). Results: In women with poor pregnancy outcome after RFM (n¼67), there was a significant reduction in plasma concentrations of hCG (median 12843 vs 17362 iu/L; P<0.01) and hPL (median 308.1 vs. 376.1 mg/L; P<0.0001), compared to those with a normal outcome (n¼236). Levels of PAPP-A, AFP, LDH and IMA were not significantly different between the two groups. On logistic regression, both hCG and hPL had a strong relationship with the outcome of pregnancy (hCG p¼0.009, hPL p<0.001). Women with hPL levels <1SD below the mean for gestation were more likely to have poor outcome following RFM (odds ratio 7.58 [95% CI 2.820.3], specificity 95%, sensitivity 30%). Discussion: This preliminary study suggests that placentally-derived factors are useful markers for obstetric complications after RFM. Importantly, factors which may reflect systemic disturbance, such as LDH or IMA are not significantly different in normal and complicated pregnancies, reflecting the importance of placental function in determining pregnancy outcome. Dysregulation of placental factors may provide clues to the underlying pathologies.

Abstracts / Placenta 32 (2011) A1–A149

P2.44 THE ENP0 MOUSE: A NEW MODEL OF FETAL GROWTH RESTRICTION Mark Dilworth1, Laura Kusinski1, Bernadette Baker1, Lewis Renshall1, Philip Baker2, Miguel Constancia3, Susan Greenwood1, Mark Wareing1, Colin Sibley1 1 University of Manchester, Manchester, United Kingdom, 2University of Alberta, Edmonton, Canada, 3University of Cambridge, Cambridge, United Kingdom Objectives: To test the hypothesis that crossing two mouse models of fetal growth restriction (FGR), one with abnormal placental morphology and transport (placental specific Igf2 promoter P0 (P0þ/-) knockout mice) and one demonstrating aberrant uterine artery function (endothelial synthase knockout mice (eNOS-/-)), would result in a more severe FGR than either model alone. Methods: eNOS-/- dams were mated with P0þ/- males. All offspring were eNOSþ/- and had either the presence (eNP0 WT) or absence (eNP0 P0) of the P0 allele. Fetal and placental weights were measured at E18.5. Maternofetal clearance of 14C-MeAIB (Kmf) was estimated following administration to the pregnant dam and subsequent fetal radiolabel counts. Results: At E18.5, 89% of eNP0 P0 fetuses fell below the 5th centile of C57/ BL6J wild-type weights, defined as FGR in this study. Only 13% of eNP0 WT fetuses met this criterion. Placental weights (mg, mean  SEM) were significantly reduced (P < 0.01, one-way ANOVA with Tukey’s multiple comparison test) in eNP0 P0 mice (641) compared with both eNP0 WT (901) and C57/BL6J (843) mice. 14C-MeAIB Kmf (ml/min/g placenta; median, IQR) was significantly lower (P < 0.05, Kruskal-Wallis with Dunn’s multiple comparison test) in placentas from eNP0 WT (125, 107-156) compared with C57/BL6J (198, 163-230) and eNP0 P0 (168, 139-193) mice. There was no significant difference in 14C-MeAIB Kmf between eNP0 P0 and C57/BL6J mice. Conclusions: Crossing P0þ/- with eNOS-/- mice results in a growth restriction of similar magnitude to mice with deletion of P0 alone [1]. This suggests that there is no additive effect on fetal growth of deleting both genes. Deletion of P0 also appears to normalise 14C-MeAIB Kmf in the absence of maternal eNOS. The eNP0 mouse has potential as a tool in which to investigate the fetal signals involved in FGR. [1]. Constancia et al 2002. Nature. 417, 945-948.

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P2.45 INTRAUTERINE GROWTH RESTRICTION MEDIATED BY CHRONIC MATERNAL INFLAMMATION IN RATS IS LINKED TO DEFICIENT REMODELLING OF THE SPIRAL ARTERIOLES AND ALTERED UTERO-PLACENTAL HEMODYNAMICS Tiziana Cotechini, Shannyn K. Macdonald-Goodfellow, Charles H. Graham Queen’s University, Kingston, Ontario, Canada Objectives: Inadequate placental perfusion resulting from deficient trophoblast-mediated remodelling of the spiral arterioles is linked to the development of intrauterine growth restriction (IUGR). IUGR is also associated with abnormal maternal inflammation often linked to infiltration of activated macrophages in the uterus. In this study we determined whether chronic maternal inflammation in the rat restricts trophoblast invasion and spiral arteriole remodelling leading to altered utero-placental hemodynamics and IUGR. Methods: Pregnant rats were injected with daily low doses of lipopolysaccharide (LPS) on gestational days (GD) 13.5-16.5, the time during which trophoblast invasion and remodelling of the spiral arterioles occurs. Prior to euthanasia on GD17.5, Doppler ultrasound was performed to assess placental hemodynamics. Trophoblast and macrophage infiltration was assessed by immunohistochemistry for cytokeratin (CK) and CD68. To determine whether the effects of maternal inflammation on pregnancy outcomes are mediated by tumour necrosis factor a (TNFa), the TNFa inhibitor etanercept was injected to pregnant dams on GD 13.5 and 15.5, six hours prior to LPS administration. Results: Pups from rats receiving daily LPS injections were significantly growth-restricted compared to pups from saline-treated dams. Compared to control rats, LPS administration resulted in a significant reduction in extravillous trophoblast invasion into the mesometrial triangle, and increased infiltration of activated macrophages. Interestingly, macrophages in the uterus were only present in regions unoccupied by trophoblasts. Doppler ultrasound revealed significant decreases in spiral arteriole flow velocity in LPS-treated rats. Alterations in utero-placental hemodynamics resulting in IUGR were prevented by maternal administration of etanercept, implicating TNFa in the pathophysiology of inflammation-induced IUGR. Conclusion: This study links chronic maternal inflammation with impaired trophoblast invasion and remodelling of the spiral arterioles leading to altered utero-placental hemodynamics and IUGR. The findings provide a rationale for investigating a potential use of immunomodulatory agents in the prevention of IUGR.

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Abstracts / Placenta 32 (2011) A1–A149

P2.46 NITROGLYCERINE INHIBITS THE DEVELOPMENT OF INFLAMATIONINDUCED IUGR IN RATS BY PREVENTING ALTERATIONS IN UTEROPLACENTAL HEMODYNAMICS

P2.47 INCREASED EXPRESSION OF HOMEOBOX GENE MEIS2 IN HUMAN IDIOPATHIC FETAL GROWTH RESTRICTION CONTRIBUTES TO ABERRANT TGFb SIGNALLING AND TROPHOBLAST DYSFUNCTION.

Tiziana Cotechini, Shannyn K. Macdonald-Goodfellow, Ivraym B. Barsoum, Charles H. Graham Queen’s University, Kingston, Ontario, Canada

Padma Murthi, Niroshani Pathirage, Shaun Brennecke, Bill Kalionis 1 University of Melbourne Department of Obstetrics and Gynaecology and Department of Perinatal Medicine Pregnancy Research Centre, Royal Women’s Hospital, Parkville, VICTORIA, Australia

Objectives: Inflammation-associated intrauterine growth restriction (IUGR) is often linked to deficient trophoblast-mediated remodelling of the spiral arterioles leading to alterations in utero-placental hemodynamics, oxidative stress, and decreased placental nitric oxide (NO) bioavailability. In this study we tested the hypothesis that deficient NO signalling is an important aspect of the pathophysiology of inflammationassociated IUGR. Specifically, we determined whether administration of the NO mimetic nitroglycerin (GTN) prevents utero-placental hemodynamic alterations and IUGR induced by chronic maternal inflammation. Methods: To induce chronic inflammation during pregnancy, rats were injected with daily low doses of lipopolysaccharide (LPS) on gestational days (GD) 13.5-16.5, the time during which trophoblast invasion and remodelling of the spiral arterioles occurs. The effects of GTN on pregnancy outcomes induced by LPS were assessed following continuous maternal delivery of GTN via daily application of a transdermal GTN patch (releasing 25 mg/h) on GD12.5-16.5. Pregnancy outcomes were assessed on GD17.5 and included alterations in utero-placental hemodynamics (as determined by Doppler ultrasound) as well as fetal growth restriction. Results: Dams receiving daily LPS injections exhibited significant decreases in peak systolic spiral arteriole flow velocities associated with fetal growth restriction. Administration of GTN to LPS-treated rats prevented the inflammation-induced IUGR as well as the associated hemodynamic alterations. Conclusion: This study supports the hypothesis that a deficiency in NO signalling is causally linked to the pathophysiology of IUGR induced by inflammation. The findings provide a rationale for investigating a potential use of nitroglycerin in the prevention of IUGR.

Objectives: Abnormal trophoblast function is associated with human pregnancy disorders including fetal growth restriction (FGR) (1). Studies on mouse embryos have shown that the MEIS2 gene, a member of the TALE (three-amino-acid loop extension) family of homeobox transcription factor genes, is a critical regulator of retinoic acid synthesis and important for fetal growth (2). However, the role of MEIS2 in human placental development and in placental pathologies such as FGR is unknown. The aims of this study were to determine the expression of MEIS2 in idiopathic FGR-affected placentae compared with gestation-matched controls (GMC), to determine the functional role of MEIS2 and to identify the downstream target genes of MEIS2 in human trophoblasts. Methods: Placentae (n¼25) from idiopathic FGR, and GMC, were used to identify the spatial distribution of MEIS2 protein by immunohistochemistry and to measure MEIS2 mRNA by real-time PCR. The effect of siRNAmediated MEIS2 gene-inactivation on trophoblast differentiation marker expression (bhCG, 3bHSD, syncytin) and apoptosis marker expression (Caspase 3 and p57) was determined in BeWo cells. Down-stream target genes of MEIS2 were identified following siRNA-mediated gene inactivation using TGF-b signaling arrays, which included genes that regulate retinoic acid synthesis (Applied Biosystems). Results: MEIS2 mRNA was significantly increased in FGR-affected placentae compared with GMC (0.66  0.12 vs 0.55  0.10, p<0.01, n¼25). Immunohistochemistry revealed predominant localisation of MEIS2 protein in the nuclei of syncytiotrophoblast in third-trimester FGR and GMC (n¼6). Following MEIS2 inactivation, mRNA expression for bhCG, 3bHSD, syncytin, caspase 3 and p57 were significantly decreased in siRNAtreated cells compared with untreated control (n¼6, p<0.05). Downstream target genes following siRNA-mediated gene-inactivation for MEIS2 in BeWo cells were identified as decorin, activin R, inhibin a, retinoblastoma-like protein 2, p57 and TGFb-3. Conclusion: Increased MEIS2 expression observed in FGR may contribute to differential expression of TGFb signaling pathway and increased trophoblast differentiation and apoptosis in human FGR.

(1). IP Crocker et al. American Journal of Pathology. 2003;162:637643. (2). K Niederreither et al., Development 2000 ; 127, 75-85.

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P2.48 ANTAGONISM OF THE SPHINGOSINE-1-PHOSPATE SIGNALING PATHWAY AFFECTS PLACENTAL AND FETAL DEVELOPMENT Bryan White1, Kathrin Dunlap1, James Frank1, Carey Satterfield1, Guoyao Wu1, Robert Burghardt1, Fuller Bazer1, Greg Johnson1, Kayla Bayless2 1 Texas A&M University, College Station, Texas, United States, 2Texas A&M Health Science Center, College Station, Texas, United States Objectives: Pregnancy requires successful uterine and placental angiogenesis to support the growing fetus. The lysosphingolipid sphingosine-1phosphate (S1P) stimulates angiogenesis. We hypothesize that S1P signaling stimulates placental angiogenesis and that antagonism of S1P hinders placentomal and fetal development in sheep. Methods: Ewes pregnant with a single fetus were given daily intravenous injections of vehicle or S1P receptor antagonist FTY720 (1.5 mg/day) from Days 30 to 60 (STUDY 1; n¼6/treatment) or from Days 30 through 90 (STUDY 2; n¼11/treatment) of pregnancy to antagonize S1P signaling and assess effects on placental and fetal development. Results: In STUDY 1, FTY720 decreased area and branching of caruncular crypts, increased area of cotyledonary villi in placentomes, decreased crown-rump length of fetuses (p<0.05), and decreased abundance of select amino acids in allantoic and amniotic fluids. In STUDY 2, FTY720 decreased uterine weight, including the placenta, fetus, and amniotic and allantoic fluids (p<0.005). FTY720 treatment decreased placentome size (p<0.005), total placentome weight (p<0.001), and amniotic fluid volumes (p<0.05). Obvious branching differences in cotyledonary and caruncular tissues were no longer detected at Day 90 of pregnancy. However, western blotting revealed decreased placentomal Arp2, a regulator of actin polymerization and branching, and Daam1, a Wnt signaling, actin polymerization regulator. Immunofluorescence localized Arp2 to vascular smooth muscle and Daam1 to adjacent stroma. FTY720 also decreased fetal crownrump length (p<0.05) and total weights of body (p<0.005), lungs (p<0.05), heart (p<0.05) and liver (p<0.01). When normalized to body weight, fetal brain weights were significantly larger with FTY720 treatment. Conclusions: Antagonism of S1P signaling adversely affected placentomal and fetal development in both studies. Extending the duration of FTY720 treatment from 30 to 60 days exasperates dysfunction. We hypothesize that S1P is critical in mediating development of angiogenic networks in placentomes of sheep that provide hematotrophic support for the fetus.

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P2.49 INTRAPLACENTAL INJECTION OF ADENOVIRUS-MEDIATED HUMAN INSULIN-LIKE GROWTH FACTOR-1 MODULATES PLACENTAL HIF2a AND IGFBP-1 EXPRESSION IN A MOUSE MODEL OF PLACENTAL INSUFFICIENCY. Helen Jones, Khaled Omar, Chuck Klanke, Stephanie Lang, Foong-Yen Lim, Sundeep Keswani, Timothy Crombleholme, Mounira Habli Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio Objectives: Regulation of IGFBP-1 by hypoxia has been demonstrated in vitro and a hypoxia response element identified in the IGFBP-1 sequence. Previously, we reported that Ad-hIGF-1 restores fetal weight in a placental insufficiency(PI) model developed using specific uterine artery branch ligation(UABL). We hypothesize that the placental hypoxic response in our PI model leads to increased placental IGFBP-1, reducing IGF-1 levels and resulting in IUGR and that intra-placental injection of AdhIGF-1 restores fetal weight through modulating IGFBP-1 levels. Methods: At gestational day (GD) 18, animals were divided into three groups; Sham-operated controls, UABL, UABLþAd-hIGF-1(108 PFU). At GD20, placentas were harvested by C-section. Protein expression was evaluated by immunohistochemistry. For RNA analysis by qPCR the Labyrinth zone(Lz) of the placenta was separated from the Junctional and Decidual zones(JDz) using forceps under a dissecting microscope. Statistical analysis by ANOVA (p<0.05significant). Results: Placental mouse IGF-1 RNA levels were significantly reduced following UABL as compared to both Sham and Ad-hIGF-1 in Lz respectively(1.280.22vs.2.230.21vs. 2.090.18,n¼3,p<0.05) but not in JDz. IGF-2 RNA expression showed no changes in any of the placental zones. Placental IGFBP-1 RNA levels were significantly increased in the UABL as compared to Sham and Ad-hIGF-1 in all zones (Lz;4.290.21vs.2.740.35 vs.2.350.31. JDz;2.040.41vs.0.350.09,vs0.190.1,n¼3,p<0.01).Expression of IGFBP-1 was only seen in decidual zone and fetal endothelial cells in Lz. A significant increase in expression of IGFBP-1 seen in UABL as compared to sham and Ad-hIGF-1. HIF2a protein expression was increased in all zones in UABL placentas compared to both Sham and Ad-IGF-1. Conclusion: In this PI model, placental expression of mouseIGF-1 is reduced in the labyrinth zone and conversely IGFBP-1is increased, which may inhibit circulating fetal IGF-1 and result in IUGR. Ad-hIGF-1 decreases IGFBP-1 expression to sham levels which may represent a mechanism by which Ad-hIGF-1 corrects fetal growth restriction.

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Abstracts / Placenta 32 (2011) A1–A149

P2.50 CHARACTERIZATION OF CHANGES IN THE MOUSE PLACENTA IN RESPONSE TO STRESS DURING PREGNANCY Christina Schweitzer, Bryony Natale, David Natale University of Calgary, Calgary, AB, Canada Objective: To investigate the ability of, and mechanisms by which the mouse placenta responds to stress during pregnancy. Methods: Using a previously described model of reduced uteroplacental perfusion pressure (RUPP), in pregnant mice, uterine arteries were ligated at embryonic days (E) 14.5 and 16.5 to reduce blood flow to the placenta and developing fetus. Feto-placental units were dissected from RUPP or sham-operated controls two days following ligation. Fetuses and placentas were weighed, fixed, and placentas sectioned for histological staining, immunohistochemistry and in situ hybridization. Results: Uterine artery ligation at E14.5 and E16.5 resulted in different effects on fetal and placenta weights. Uterine artery ligation at E14.5 resulted in no change in average embryo weight but a 10% increase in average weight of placentas by E16.5 (n¼5 litters, p < 0.01). Uterine artery ligation at E16.5 resulted in a 13% reduction (n¼5 litters, p < 0.01) in average embryo weights but no change in placental weights by E18.5. Histological and molecular analyses were conducted on tissue sections from these placentas to assess changes in proliferation and the expression of markers of trophoblast sub-types. Importantly, we noted a significant increase in proliferating trophoblast cells, localized only within the labyrinth layer of placentas subjected to uterine artery ligation at E14.5. In addition, the architecture of the placentas in this group was different, displaying increased numbers of spongiotrophoblast and glycogen trophoblast cells within the labyrinth, which were localized close to the chorionic plate. Conclusions: Our results suggest that the ability of and mechanisms by which the mouse placenta responds to stress during pregnancy is dependent upon the timing of the stress. Furthermore, it appears to involve the recruitment of trophoblast cells to proliferate and differentiate in order to facilitate the remodelling of the labyrinth layer.

P2.51 A COMPARATIVE STUDY ON PLACENTATION IN THE SCALLOPED HAMMERHEAD SHARK, SPHYRNA LEWINI AND THE MANTA RAY, MANTA BIROSTRIS Hiroaki Soma1,2, Noriko Murai1, Kayoko Tanaka1, Hiroko Kokuba2, Koji Fujita2, Minoru Toda3, Senzo Uchida3, Shoichiro Mineo2 1 Saitama Medical School, Dept.of Obstetrics & Gynecology and Section of Electron Microscopy, Moroyama-cho, Saitama, Japan, 2Tokyo Medical University, Section of Electron Microscopy and Pathology, Shinjyuku-ku, Tokyo, Japan, 3Okinawa Churaumi Aquarium, Motobu-cho, Okinawa, Japan Objectives: Most sharks are oviparous, but some species are viviparous including members of Sphyrna and Carcharhinidae. This study sought to determine specific placentation of Hammerhead sharks(Sphyrna lewini) that can be nourished more than 20 pups during 1 year’s gestational period, in addition to investigation of the gravid uterus of the Manta ray(Manta birostris) that can be nourished one pup without placentation during one year’s gestational period. The objective of the study was to examine different reproductive behavior between both fishes. Methods: The placental tissues of pregnant scalloped hammerhead sharks obtained from Okinawa and Taiwan, and the gravid uterus of Manta ray caught by Okinawa Aquarium were investigated using histochemical and electronmicroscopical methods(SEM and TEM). Results:

1) In hammerhead shark, the yolk sac placental epithelial cells were stained positively for PAS, Cytokeratin, hCG, hPL, SP1 and pl.alphos. 2) The umbilical cord has a ductus vitellointestinalis in addition to one artery and one vein. In such ductus, a bundle of ciliar filaments protruded from the surface of cylindrical epithelial cells as dynein-forming microtubules. 3) The chorionic epithelial cells have nuclei and organelle similar to human cytotrophoblast. 4) The gravid uterus of Manta ray has thick villous strings called trophonemata which nourish the unborn pup: By SEM villous strings with flat surface include crypt formation and by TEM, they had vacuoles granules showing secretion of lipid stained with Sudan III and prolactin. Conclusions: The hammerhead shark placenta is formed to utilize yolk and nourish many pups during the relatively long gestational period. The Manta ray has the gravid uterus where secretes milk substance, thereby nourishes the pup.

Abstracts / Placenta 32 (2011) A1–A149

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P2.52 SIBLING AND FETAL MICROCHIMERISM IN HEALTHY AND PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS INFECTED DAMS. Uladzimir Karniychuk1, Wander Van Breedam1, Claire Rogel-Gaillard2, Nadine Van Roy3, Hans Nauwynck1 1 Ghent University, Laboratory of Virology, Ghent, Belgium, 2INRA, Laboratory of Animal Genetics and Integrative Biology, Jouy-en-Josas, France, 3 Ghent University Hospital, Center for Medical Genetics, Ghent, Belgium Objectives: Microchimerism is the presence of small numbers of foreign cells or DNA within the tissue or circulation of an individual. Naturally acquired microchimerism refers to the sibling, maternal and fetal microchimerism. PRRSV is the cause of severe reproductive problems in sows. How PRRSV passes from mother to fetuses remains unknown. PRRSV replication in the fetal implantation sites is restricted to Snþ and CD163þ macrophages. However, uterine epithelium and trophoblasts are not susceptible to PRRSV infection. Therefore, we hypothesize that PRRSV can use susceptible macrophages as a vehicle to cross uterine epithelium/ trophoblast layers and spread between mother and fetuses as well as between siblings. To support this hypothesis, it is important to verify if porcine cells can pass transplacentally. Methods: Six dams were inoculated intranasally with 105 TCID50 PRRSV 07V063 at 80-90 days of gestation and sampled at 10-20 days postinoculation (Table I). Three non-inoculated dams were included as negative controls. During sampling, a gender of individual fetuses was recorded, fetal blood from umbilical cords and fetal organs were collected. DNA was extracted from sera of all female fetuses and maternal sera and subsequently tested in a qPCR assay to detect the male sex determination region Y (SRY). Furthermore, cryosections from female and male fetal tissues were subjected to sex-typing fluorescence in situ hybridization (FISH). Results: Male SRY sequences were detected in female fetal sera of all PRRSV-positive and PRRSV-negative dams (Fig.1). The percentage of SRYpositive female fetuses varied from 20% to 100% among different dams. Male DNA was also detected in the maternal circulation of pregnant dams. FISH showed the presence of male cells in the female fetal organs and vice versa (Fig.2). Conclusion: The exchange of DNA and/or nucleated cells between porcine fetuses in utero has been demonstrated. Also, male DNA was detected in the maternal circulation during pregnancy.

Fig. 1. Diagrams schematically representing the porcine uteri of the nine dams included in the study and referred to as 1 to 9 on top of each scheme. L and R: left and right uterus horn, respectively. Each circle is an individual fetus. F and M: female and male fetuses. Plus and minus represent PRRSV-positive or -negative fetuses, respectively. Filled circles are female fetuses tested for the male sex-determining region Y (SRY) in sera. Red and green circles are SRY-positive or -negative fetuses, respectively. The mean number (range) of SRY GenEq cells/ml of serum is given in addition to the SRY-positive fetuses; *þ: SRY-positive samples with values below the quantification limit. The percentage of SRY-positive female fetuses among dams ranged between 20 and 100%. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table I Experimental design Dam

y

Parity

PRRSV inoculation at . gdy

Euthanizedat . gd

Number of fetuses Total

Female

Male

1 2 3 4 5 6

4 5 1 1 1 1

70 90 90 90 90 90

80 100 100 100 110 110

11 14 14 17 12 16

5 9 5 7 6 8

6 5 9 10 6 8

7 8 9

1 10 6

-

100 100 100

13 16 15

7 10 7

6 6 8

gd: gestation day; -: not inoculated.

Fig. 2. FISH analysis on female lungs and male liver using probes for the X (green signal) and Y (red signal) chromosomes. Arrows indicate microchimeric cells. Cell nuclei were counterstained with DAPI (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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P2.53 FETAL AND MATERNAL CELLS ARE NOT PRESENT IN THE PORCINE MYOMETRIUM AND FETAL PLACENTAS, RESPECTIVELY, AT THE LAST STAGE OF GESTATION. Uladzimir Karniychuk1, Wander Van Breedam1, Claire Rogel-Gaillard2, Nadine Van Roy3, Hans Nauwynck1 1 Ghent University, Laboratory of Virology, Ghent, Belgium, 2INRA, Laboratory of Animal Genetics and Integrative Biology, Jouy-en-Josas, France, 3 Ghent University Hospital, Center for Medical Genetics, Ghent, Belgium Objectives: The invasion of fetal macrophages into the myometrium has been proposed to trigger murine parturition. This issue was not previously studied in swine. Microchimerism is the presence of small numbers of foreign cells or DNA within the tissue or circulation of an individual. Bidirectional cell exchange between mother and fetus and cell trafficking from fetus to mother was recently shown within placentas of mice and human, respectively. The purpose of the present study was to determine if fetal cells invade the porcine endometrium and myometrium at the late stage of gestation (100 days of gestation). Furthermore, the presence of maternal cells within the fetal placentas was also examined. Methods: Twenty three samples of porcine uteri with placenta of 3-5 male fetuses from five dams were subjected to a sex-typing fluorescence in situ hybridization (FISH) analysis. Samples of the uteri with placenta of three female fetuses were used as controls to prove the specificity of the sextyping FISH. Results: In the present study, fetal cells were not found in the porcine myometrium. This observation is reminiscent of that reported in humans. Mechanisms of parturition triggering are most probably different in swine and mice. Furthermore, maternal female cells were not detected in the male fetal placentas. Male fetal cells were identified in the endometrium of almost every sample. Subsequently, a combination of FISH and immunofluorescent staining (IF) using CK7 antibody (Ab) was performed. CK7 Ab specifically reacts with the uterine epithelium but trophoblasts are negative. Male cells within the endometrium were trophoblasts surrounded by the female uterine epithelium (Fig. 1, C). Most probably these are artefacts originating from cutting of sections. Conclusion: Fetal cells are not present in the porcine myometrium at the late stage of gestation. In addition, maternal cells were not detected in the fetal placentas. Attention should be paid to the cutting artefacts in the porcine endometrium during FISH and IF result interpretation.

P2.54 GLYCOSYLATION AND ULTRASTRUCTURE OF THE PLACENTA IN THE TAMMAR WALLABY MACROPUS EUGENII. Carolyn Jones1, Marilyn Renfree2 University of Manchester, Manchester, United Kingdom, 2University of Melbourne, Victoria, Australia

1

Introduction: The tammar wallaby has a choriovitelline (yolk sac) placenta tightly apposed to the uterine epithelium for the last 8 days of the 26.5 day pregnancy. The placenta consists of a non-vascular (bilaminar) and a vascular (trilaminar) region. Uterine gland secretions (histiotrophe)

and exchange with the maternal circulation nourish the embryo until its birth after which it develops in the mother’s pouch. Objectives: To investigate both bi- and trilaminar regions of an “in situ” d24 placenta with respect to glycosylation of the component tissues. Methods: Full-depth pieces were embedded in resin and lectin histochemistry used to determine the nature of the glycans present; tissue with and without osmication was also examined at the ultrastructural level. Results: Electron-lucent vacuoles under the trophoblast microvillous membrane contained richly glycosylated material in both bi and trilaminar regions, bound by almost all the lectins used in the study, indicating the presence of most types of glycan. Unusually, a2,6-linked sialic acid was evident, a feature not common in mammalian placentae. In the bilaminar region, cells were taller and many contained accumulations of electron-dense granules which bound most lectins strongly. Heavy staining with Galanthus nivalis agglutinin suggested many of these were residual bodies; they were generally less densely distributed in the trilaminar areas. Evidence of biosynthetic activity was also observed. There was little reactivity with lectins in the endoderm while the vascular endothelial cells expressed few glycans on their surface and were, indeed, hard to detect at the light microscope level, unlike their eutherian mammal counterparts. The amount of secreted material present between the fetal and maternal tissues suggested that contact between the two was not close, unlike the situation seen in eutherian epitheliochorial placentation. Conclusion: The near-term tammar placenta showed features suggesting functional differences between bi-and trilaminar areas with some unusual patterns of glycosylation.

P2.55 THE INITIAL DEVELOPMENT OF THE VISCERAL YOLK SAC PLACENTA IN NECROMYS LASIURUS (RODENTIA, CRICETIDAE, SIGMODONTINAE) AND ITS INTERACTIONS WITH THE UTERINE WALL DURING PLACENTATION Phelipe Favaron1, Anthony Carter2, Andrea Mess1, Moacir Franco Oliveira3, Maria Angelica Miglino1 1 Department of Surgery, School of Veterinary Medicine, University of Sao Paulo, Sao Paulo, Brazil, 2Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark, Denmark, 3Department of Animal Science, Universidade Federal Rural do Semi-Árido, Mossoro, Rio Grande do Norte, Brazil Evolutionarily, the yolk sac (YS) is the only fetal membrane present in all vertebrate taxa. In rodents and other mammals this membrane results in the development of a YS placenta, which is responsible for maternal-fetal exchange. Necromys lasiurus is a common rodent, which is regarded as a reservoir of Hantaviruses and other diseases in South America. Moreover, related species have been used as an experimental model. Objectives: Herein, we show data on the early development of the YS and its relationship with the uterus in order to promote a better understand of sigmodont placentation. Methods: Implantation sites of Necromys were obtained from a breeding group at the UFERSA, Mossoró, Brazil. Samples were examined by means of histology and immunohistochemistry for cytokeratin (1:300), vimentin (1:200), and PCNA (1:800).

Abstracts / Placenta 32 (2011) A1–A149

Results: An inverted YS placenta was present closely attached to the labyrinthine region of chorioallantoic placenta. The mostly one-layered parietal YS covering was associated with a well-developed Reichert’s membrane. It was highly proliferative as demonstrated by PCNA labeling. In very early pregnancy the visceral YS was in places closely attached to the uterine wall. At these attachments the uterus possessed infolded structures associated with blood vessels and surrounded by a number of vimentin positive glands. Especially in early gestation the visceral YS near to the chorioallantoic placenta was highly villous with blood vessels inside with a vimentin positive endothelium. Where the YS was more distant from the chorioallantoic placenta and close to the uterine wall, it became compact in shape. A strong response to PAS including plenty of granular vesicles indicated secretory activity of the endodermal cells, such as release of histiotrophe. Conclusion: Data so far suggest that these characteristics may represent a derived character condition of cricetid rodents, since such an association has not been described in the mouse and related species.

P2.56

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CAN WE STUDY ANTIPHOSPHOLIPID ANTIBODIES AS A MATERNAL RISK FACTOR FOR PREECLAMPSIA IN A RAT MODEL? Sien Yee Lau1,2, Peter Stone2, Qi Chen2,3, Sarah-Jane Guild1, Carolyn Barrett1, Larry Chamley2 1 Department of Physiology, University of Auckland, Auckland, New Zealand, 2Department of Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand, 3Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China Objectives: Both maternal and placental factors contribute to preeclampsia but current animal studies frequently model only a maternal or a fetal pathogenic feature of the disease. Antiphospholipid autoantibodies (aPL) are a maternal factor which increase the risk of preeclampsia nine-fold while necrotic trophoblastic debris is a placental factor which may trigger preeclampsia. We have shown that rats administered necrotic trophoblasts developed hypertension relative to controls but, did not increase their blood pressure above pre-pregnant levels. We hypothesised that the ability of necrotic trophoblasts to induce relative, but not frank, hypertension was due to the absence of maternal factors in our model. Therefore we aim to add aPL as a maternal risk factor, to our rat model.

Fig.1. (A) Sex-typing FISH within the fetal implantation sites of male fetus. fpl: fetal placenta; tr: trophoblasts; uep: uterine epithelium; end: endometrium. Male fetal cells were identified in the endometrium. They were organized as rings and were surrounded by female XX cells (insert). Male cells were identified as having one Y (red signal) and one X (green signal) chromosome. Female cells were identified by the presence of two X chromosomes. Images were recorded with Olympus IX81 microscope connected with cell-M-live-cell imaging module, objective x10. (B) Magnification (objective x20) of male cells within the endometrium. Male cells (arrowheads) were organized as rings and surrounded by female XX cells (arrows). (C) Combination of the sex-typing FISH and cytokeratin IF staining. C1: sex-typing FISH; C2: cytokeratine 7 staining; C3: merged image of C1 and C2. CK7 Ab specifically react with the uterine epithelium (uep) but trophoblasts (tr) remain negative. Male cells within the endometrium were trophoblasts (arrowheads) surrounded by the female uterine epithelium (arrows). Most probably these are artefacts originated during cutting of sections. (D) Hematoxylin-eosin staining of the fetal implantation sites. fpl: fetal placenta; tr: trophoblasts; uep: uterine epithelium; end: endometrium. Staining revealed rings (arrows) within the endometrium. Rings histologically represent uterine epithelium and trophoblasts and appeared to be artefacts originated during cutting of sections. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Abstracts / Placenta 32 (2011) A1–A149

Since injection of aPL can cause fetal death in rodents, we undertook this preliminary investigation to determine whether we could inject rats with clinically-relevant levels of aPL without destroying the litters. Methods: Pregnant Wistar rats received intravenous injections of a monoclonal aPL on days 6 and 13 post coitum (pc), at a dose of 3.5mg/kg body weight. Control rats received an isotype-matched control antibody. Levels of aPL in the blood were monitored by ELISA. Fetal viability was examined by palpation and cardiac ultrasound on days 13 and 18 pc and by post-mortem observations following euthanasia on day 21 pc. Results: This protocol resulted in persistent, clinically-relevant, levels of circulating aPL. The aPL had variable effects on litter size ranging from, no effect to resorption of more than half the litter. However, pup numbers was not significantly different between the aPL and control groups (p>0.05). Conclusion: These results mirror the clinical presentation of women with aPL suggesting that aPL may be a suitable maternal risk factor to add to our rat model of the effects of necrotic trophoblast debris in preeclampsia. P2.57 PLACENTATION IN A SQUAMATE REPTILE (MABUYA SPP.: SCINCIDAE): STRUCTURE AND FUNCTION OF A HIGHLY SPECIALIZED PLACENTA Martha Patricia Ramírez Pinilla Universidad Industrial de Santander, Bucaramanga, Santander, Colombia Objectives: To describe the mature chorioallantoplacenta of the viviparous lizard Mabuya sp. and relate its complexity with functional differences in the transfer of nutrients required by the developing embryo. Methods: The oviducts of Mabuya pregnant females containing embryos in the incubatory chambers were dissected and processed for light (LM) and electron microscopy (EM); LM and EM sections were treated with antibody markers for immunolocalization of molecular transporters of nutrients. The net uptake of key nutrients for embryonic development was also determined by measuring the total dry mass, or the mass of separate nutrients, in recently ovulated eggs and in neonates. Results: The chorioallantoic placenta of Mabuya exhibits the highest placental complexity known in Reptilia. The placenta possesses areas functionally related to gas exchange and highly specialized structures related to nutrient transfer (placentome, paraplacentome, chorionic areolas, and absorptive plaques). Some of these morphological specializations are exclusive to Mabuya whereas others show an impressive convergence with those of eutherian mammals. Nevertheless, no mammal species has the variety of specializations observed in the placenta of Mabuya. Localization of the molecular transporters of nutrients showed that some molecules are widely distributed among placental structures whereas others have restricted distributions. Physiological analyses showed that a massive nutrient transfer (water, lipids, proteins, inorganic ions) occurs during the embryonic development, especially during the final stages of development when the chorioallantoic placenta is mature. Conclusions: Neotropical species of the lizard genus Mabuya produce microlecithal eggs, develop a specialized chorioallantoic placenta, and exhibit an obligate placentotrophy. The reduction of the egg size is consequence of a decrease in the quantity and a change in the proportions of yolk components. The morphological complexity of the chorioallantoic placenta of Mabuya is related to functional differences in the transfer of nutrients required by the developing embryo.

P2.58 MATERNAL WEIGHT AND FETO-PLACENTAL BLOOD FLOW IN BABOONS (PAPIO SPP) N. Schlabritz-Loutsevitch1, M. Tchirikov3, J. Samson1, M. Schenone2, P. Nathanielsz1, G. Mari1 1 University of Tennessee Health Science Center, Memphis, TN, USA, 2 University of Texas Health Science Center at San Antonio, San Antonio, TX, USA, 3University of Mainz, Mainz, Germany Introduction: Increased maternal weight is associated with an increased risk of stillbirths in both humans and non-human primates. The harmful effects of maternal obesity on the circulatory system of humans and adverse effects of a high fat diet in non-human primates on utero-placental blood flow have been demonstrated. The goal of this study: To evaluate Doppler parameters of feto-placental blood flow in obese and non-obese baboons. Material and methods: Animals were divided into two groups: obese (n¼5) and non-obese (n¼16) based on their weight at the time of ultrasound evaluation at 0.5 gestation. Fetal umbilical artery (UA), fetal middle cerebral artery (MCA) and maternal uterine arteries (UtA) were sampled in both groups using a GE LOGIQ 5 (USA) Doppler ultrasound system. The pulsatility index (PI), maximum blood flow velocity (PS), end-diastolic flow velocity (EDV), mean velocity (MV) of each vessel were analyzed. Data are presented as Mean þ SEM. Significance was set at p < 0.05 (Nonpaired Student’s t-test). Results: In the obese mothers UA PI, was lower than the non-obese (1.46  0.2 vs. 1.98  0.13 respectively) (p¼0.04). Fetal PS and MV of MCA were lower in obese pregnancies (23.3  0.3 cm/s and 10.9  0.8 cm/s respectively) compared to non-obese (44.4 7 cm/s and 23.15  1.8 cm/s) (p¼0.03 and p¼0.001). Conclusion: The data suggest that increased maternal weight might stimulate blood flow to the placenta and change fetal cerebral circulation in the baboon model. Further studies are merited in both human and experimental primate obesity to evaluate redistribution of blood in the maternal, placental and fetal circulations. The effect of constitutional obesity might differ from the isolated effect of the high fat diet.

Abstracts / Placenta 32 (2011) A1–A149

P2.59 WHAT DO PREGNANT WOMEN EAT? Vicki Clifton1, Penelope McLernon1, Lisa Wood2, Vanessa Murphy2, Nicolette Hodyl1 1 University of Adelaide, Adelaide, Australia, 2Hunter Medical Research Institute, Newcastle, Australia Objective: Diet and nutrition are essential for a normal healthy pregnancy and current evidence suggests that unbalanced consumption of fats, proteins or carbohydrates can impact on fetal growth with long term health implications for the offspring. We were interested in the dietary intake of a group of pregnant women to determine what nutrients may impact on abnormalities in placental function. Method: Women were prospectively recruited at 12 weeks gestation at the John Hunter Hospital antenatal clinic in Newcastle, Australia. They were all Caucasian and from a mixed socioeconomic background. We compared dietary intake of healthy pregnant women relative to an asthmatic population. At 18, 30 and 36 weeks gestation, participants completed a self assessed 24 hour food recall questionnaire. Data was analysed in the Foodworks (Xyris, Brisbane) database. Dietary intake questionnaire data from controls (n¼47) and asthmatic (n¼84) women were recorded. Asthmatic groups were compared based on severity (mild; n¼31, moderate/severe; n¼53). Data was analysed using SPSS. Results: There were no significant differences between the groups with respect to maternal age, body mass index (BMI) or total weight gain over pregnancy. All women were overweight based on a BMI >25. There were no significant differences in fetal growth parameters or neonatal outcomes. The mild asthmatic group (n¼25) consumed higher quantities of energy, protein, fats, carbohydrates, starch, thiamin, riboflavin, niacin equivalents, magnesium and phosphorous than the moderate/severe asthmatics (n¼34) or control population (n¼32). When comparing this data to the nutrient reference values for Australia and New Zealand for pregnant women in the third trimester, all women have below average intake of iron, calcium, magnesium, folate, retinol, and dietary fibre. Riboflavin, niacin, vitamin C and A, phosphorous and potassium intake were all above average. Intake of energy and protein were above average in all groups. The total fat intake contributed to 37% of the total energy consumed by the women and 15% of total energy was derived from saturated fats in all groups. Conclusion: These data indicate that pregnant women consume more nutrients than required for pregnancy and generally consume too much fat in their diet especially saturated fat. They have a significant deficiency in key nutrients derived from fruit and vegetables.

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P2.60 THE METABOLOME OF HUMAN PLACENTAL TISSUE: INVESTIGATION OF FIRST TRIMESTER TISSUE AND CHANGES RELATED TO PREECLAMPSIA IN LATE PREGNANCY Alexander Heazell3, Marie Brown1, Stephanie Worton3, Rebecca Jones3, Warwick Dunn1,2 1 Manchester Centre for Integrative Systems Biology, Manchester, United Kingdom, 2Centre for Advanced Discovery and Experimental Therapeutics, York Place (off Oxford Road), Central Manchester University Hospitals NHS Foundation Trust, Manchester, United Kingdom, 3Maternal and Fetal Health Research Centre, Manchester, United Kingdom Objective: Unique biochemical and physical challenges to mother and fetus are observed during human pregnancy and can lead to complications including preeclampsia (PE). Many pregnancy complications are associated with altered placental biochemistry and structure. We aimed to develop metabolomic tools to extract the tissue metabolome of placental tissue and describe changes in early and late first trimester and between normal term pregnancy and PE. Methods: 100mg of tissue (n¼6 each group) was snap frozen, defrosted, washed, then homogenised in a mixture of water, methanol and chloroform. The polar fraction was analysed by GC-ToF-MS and non-polar fraction by UPLC-LTQ-Orbitrap-MS. Data were deconvoluted and subsequently analysed by multivariate and univariate analysis. Results: There were significant changes in the metabolomes of placental tissue between early and late first trimester pregnancy. 156 unique and chemically identified metabolic features showed statistically significant differences (p<0.05). These included changes in tri- and diglycerides, phospholipids (including lysophosphatidylethanolamines, phosphatidylethanolamines and phosphatidylcholines), sphingolipids, fatty acids and fatty acid carnitines. A small number of changes were observed in tissue samples collected from different positions on the same term placenta (6 unique and chemically identified metabolic features) and from tissue collected from different types of delivery (vaginal delivery and caesarean section; 31 unique and chemically identified metabolic features). 86 unique identified metabolic features were significantly different (p<0.05) when comparing tissue from term uncomplicated pregnancies with PE; the changes include mitochondrial metabolism, vitamin D metabolism and in oxidative and nitrative stress. Conclusions: Changes in oxygen tension and nutrient availability in early placental development are associated with changes in metabolism, particularly in either initiation or switch to fatty acid beta-oxidation in the mitochrondria for ATP production. Similar changes are also evident in PE, indicating that energy usage may be different in PE. In PE, vitamin D metabolism merits further exploration.

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Abstracts / Placenta 32 (2011) A1–A149

P2.61 URINE LEVELS OF PHTHALATE METABOLITES IN NEWBORNS AND PREGNANT WOMEN AS A MEASURE OF PHTHALATE EXPOSITON DURING PREGNANCY Uta Enke1, Holger M. Koch2, Claudia Pälmke2, Ekkehard Schleussner1, Lydia Seyfarth1 1 Department of Obstretrics and Gynecology, Placentalabor, Jena University Hospital, Friedrich-Schiller-University Jena, Jena, Germany, 2Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr-University Bochum (IPA), Bochum, Germany Objectives: Phthalatic acid esters, also known as phthalates, have been widely used as plasticizers and therefore became ubiquitous in the developed countries. Many studies have reported different biological consequences of phthalate exposure, e.g. reproductive and developmental toxicity due to their action as endrocrine disruptors. Therefore, phthaltes are banned from products for babies and infants such as toys. 10 years ago, DEHP (lipophilic and characterized by long side chains) was the mainly used phthalate. Nowadays, new types of phthalates have also long but branched side chains, such as DiNP or DiDP. In contrast to the fast metabolized and excreted DEHP, only a low percentage of DiNP or DiDP is excreted immediately. What happens to those substances? Are they stored in the maternal lipid depots in the beginning of pregnancy and is there a release towards birth, when the fetal requirements for e.g. energy or essential fatty acids are highest? Methods: By applying LC-MS technique, we determined a wide range of phthalate metabolites (with special emphasis on DEHP, DiNP and DiDP) in urine of pregnant women during pregnancy (n¼ 16) as well as in urine of newborns at day 0 to 5 post partum after uncomplicated pregnancy at term (n total ¼ 28). Results and Conclusions: Metabolites of different long and short chain phthalates could be detected in all maternal and newborn urine samples with strong individual differences. Accumulation of all types of phthalates in fetal tissue obviously occurs already during pregnancy which is reflected by metabolite excretion in the first urine after birth. Distribution pattern of single phthalate metabolites differ between maternal and newborn urine which may rely on premature fetal/newborn metabolism or enhanced accumulation/transport of phthalate metabolites during pregnancy. Therefore, threshold values for phthalate exposition for adults are probably non-transferable during pregnancy and hence should be revised and re-evaluated.

P2.62 IMPACT OF FOLATE DEFICIENCY ON PLACENTAL APOPTOSIS AND AMINO ACID TRANSPORT IN TEENAGE PREGNANCY Fiona L. Mackie, Cara E. Taylor, Ainslee J. Garrod, Sanantha C. Lean, Susan L. Lonsdale, Rebecca L. Jones Maternal and Fetal Health Research Centre, University of Manchester, Manchester Objective. Pregnant teenagers are susceptible to folate deficiency, which is associated with an increased risk of delivering small-for-gestational-age infants. Folate is vital for DNA synthesis and DNA methylation. Previous studies have shown increased apoptosis of isolated cytotrophoblast cells cultured in folate-free conditions. However, little is known about the impact of folate deficiency on the placenta in vivo. We hypothesised that folate deficiency would be detrimental to placental development and function in teenage pregnancies. Methods. Teenagers (n¼80) and adults (n¼21) were recruited from 28 weeks gestation and their folate status assessed. To date, placentas from 35 teenagers, including 8 folate-deficient and 27 folate-adequate, have been studied. Cell proliferation and apoptosis was evaluated immunohistochemically using antibodies against Ki67 and cytokeratin M30 in fixed placental samples. Sodium-dependent 14C-methyl aminoisobutyric acid (MeAIB) uptake via the system A transporter was assessed as a marker of placental function. Results. Significantly more teenagers (30%) were folate deficient compared to the adults (9.5%) (p<0.01). Folate deficiency did not affect placental weight. There was an increased rate of trophoblast apoptosis in placentas from folate-deficient compared to folate-adequate teenagers (0.38 vs. 0.08%; p<0.01). No difference in placental cell proliferation was detected. Folate-deficient teenagers had lower placental system A transport activity compared to those with adequate folate status (p<0.05; Figure 1). Conclusion. Folate deficiency is associated with impaired placental cell turnover and reduced amino acid transport in teenage pregnancies. This may contribute to their susceptibility to poor pregnancy outcome. How folate deficiency causes these effects is unknown, but it may be due to aberrant DNA synthesis or altered methylation status of key placental genes. The effect of confounding factors, such as maternal smoking, on folate status and placental dysfunction requires further analyses. Figure 1. System A activity in placentas from folate deficient and folate adequate teenagers.

Abstracts / Placenta 32 (2011) A1–A149

P2.63 EFFECT OF FOLIC ACID DEFICIENCY AND SUPPLEMENTATION IN VITRO ON FIRST TRIMESTER AND TERM PLACENTAL CELL TURNOVER AND FUNCTION. Fiona L. Mackie, Sara Tarhini, Ainslee J. Garrod, Susan L. Greenwood, Rebecca L. Jones Maternal and Fetal Health Research Centre, University of Manchester, Manchester Objective. Folate is essential for cell function and DNA synthesis, and folate deficiency is associated with reduced fetal growth. Little is known about the effects of folate deficiency or folic acid supplementation on the placenta. This is important as high-dose folic acid (5-10mg/day) is prescribed to an increasing number of women throughout pregnancy. Methods. Explants from first trimester (n¼6) and term (n¼6) placentas were cultured for 4 days in folate-free conditions, with the addition of 0 (folate deficient), 25 (control ¼ physiological), 250 or 500nM folic acid. Proliferative and apoptotic indices were determined by quantification of immunostaining for Ki67 and cytokeratin M30. Placental function was assessed by hCG secretion and uptake of 14C-MeAIB via system A amino acid transporter. Results. In vitro folate deficiency reduced proliferation compared to controls (p<0.05) in first trimester explants, but did not affect rates of apoptosis. Folate-free culture had no effect on cell turnover in term explants. Placental function was unaffected by culture in folate-free conditions at either gestation. Folate supplementation increased apoptosis in first trimester explants (p<0.05), with a similar trend in term explants. High dose folic acid had differing effects on placental function in early and late pregnancy. In first trimester, culture with 250 and 500nM folic acid stimulated system A activity (p<0.05), whilst hCG secretion was unaffected. hCG secretion (p<0.05) and system A activity were decreased by culture with 500nM folic acid (p<0.05). Conclusions. These data suggest that folate deficiency may be detrimental to early placental development. The lack of effect of folate deprivation in later pregnancy may be due to high levels of folate stored by the placenta. Treatment with high-dose folic acid impaired placental cell turnover and altered placental function, with gestation specific effects. Further research is needed into the potential detrimental effects of high dose folic acid during pregnancy.

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P2.64 A LONGITUDINAL STUDY OF MATERNAL WEIGHT GAIN AND SERUM LEPTIN IN PREGNANCY AND THEIR ASSOCIATIONS WITH FETAL WEIGHT GAIN, AMNIOTIC FLUID VOLUME, BIRTH WEIGHT, PLACENTAL WEIGHT AND UMBILICAL VEIN LEPTIN. Kari Flo1,2, Tom Wilsgaard1, Aase Vaartun1, Ganesh Acharya1,2 1 University of Tromsø, Tromsø, Norway, 2University Hospital of North Norway, Tromsø, Norway Objective: To explore the associations of maternal weight gain and serum leptin with estimated fetal weight, amniotic fluid volume, birth weight, and placental weight and umbilical vein leptin. Methods: Body weight was measured at booking and at 4-weekly intervals from 20 weeks of gestation to term in 85 women with uncomplicated pregnancies. Body mass index (BMI) was calculated as: weight (kg)/ height2 (m). Serum leptin was measured longitudinally in 53 women at each visit after 20 weeks. Estimated fetal weight (EFW) and amniotic fluid index (AFI) were calculated using ultrasonography. In 56 babies umbilical vein (UV) leptin was measured after delivery using leptin enzyme immunoassay kit (DRG, EIA-2395). Linear mixed models and linear regression models were used for statistical analysis. Results: Mean maternal weight gain was 12.983.72 Kg from first trimester to term. Maternal serum leptin increased from 18.2 to 22.0 ng/L during 22 to 39 weeks, but this was not associated with maternal or fetal weight gain assessed by EFW. Mean birth weight and placental weight were 3596.27459.01 g and 603.06 121.15 g, respectively. Mean UV leptin was 6.305.59 ng/L. We found a significant association of maternal weight gain with birth weight (p ¼ 0.034) and UV leptin (p ¼ 0.0012), but not with AFI and placental weight. There was a significant association between maternal leptin in late pregnancy (34 -39 weeks) and umbilical vein serum leptin (p ¼ 0.0006). Maternal leptin adjusted for gestational age (p ¼ 0.0073) and change in serum leptin concentration during pregnancy (P ¼ 0.028) were significantly associated with UV leptin. Conclusion: Maternal weight gain and serum leptin in normal pregnancy are associated with birth weight and UV leptin but not with amniotic fluid volume and placental size which emphasizes the importance of healthy maternal life style and nutrition during pregnancy.

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Abstracts / Placenta 32 (2011) A1–A149

P2.65 APOLIPOPROTEINS (APO) ACCOUNTS FOR THE ANTI-INFLAMMATORY FUNCTION OF FETAL HDL Ivana Sreckovic1, Ruth Birner-Gruenberger2, Sonia Philipose3, Michael Holzer3, Gunther Marsche3, Gernot Desoye1, Uwe Lang1, Christian Wadsack1 1 Clinic of Obstetrics and Gynecology, Medical University of Graz, Graz, Styria, Austria, 2Proteomics Core Facility, Center of Medical Research, Medical University of Graz, Graz, Styria, Austria, 3Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Graz, Styria, Austria

Average number of SNAs /1mm2 villous area

Objectives: High density lipoprotein (HDL) represents the major cholesterol carrying class of lipoprotein in cord blood while in adult blood that is low-density lipoprotein. To test the hypothesis whether fetal HDL carries proteins that might have anti-inflammatory novel functions in the ability to interact with the placental-fetal endothelium, the composition and enzymatic activities of maternal and fetal HDL was investigated. Methods: Maternal and fetal (from corresponding arterial and venous cord blood) plasma from normolipidemic (n¼11) pregnancies at term were studied. HDL was isolated by ultracentrifugation and liquid chromatography/electrospray mass spectrometry was used for identification of HDLassociated proteins. Global protein results were confirmed by immunoblot analysis. Paraoxonase (PON-1) activity, and lecithin:cholesterol acyltransferase (LCAT) mass and activity was quantified by commercially available kits. Results: On the basis of our analysis criteria we detected 33 distinct maternal and fetal HDL-associated proteins. Peptides derived from ApoAI and ApoE prevailed. Maternal HDL was statistically enriched in the antioxidative protein PON-1, and in proteins linked to cholesterol metabolism (apoCIV, apoF, apoL) compared to fetal HDL. The 7-fold higher PON-1 protein expression in maternal circulation was corroborated by higher PON-1 activity. In line with the anti-inflammatory properties of maternal HDL, LCAT mass was statistically increased in maternal plasma (p<0.01) compared to cord plasma. Since fetal HDL contains 3-fold higher amounts of apoE than adult HDL and since apoE is the major activator for LCAT, the similar LCAT-activity in both circulations appears comprehensible. Furthermore apoAIV, a1-antytripsin and vitamin D binding protein were more abundant in HDL which originates from arterial umbilical plasma while venous HDL had more IgG heavy chain. Conclusion: These findings suggest that maternal and fetal HDL are

250

SNA number in explants at 6% O 2

200

SNA number in explants at 20% O 2

150 100 50 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Day Ă

basically composed of distinct (apolipo)protein cargo. Alterations occurring in maternal HDL composition due to pathological states (i.e. chronic inflammation) may intimately associated with changes in fetal lipid metabolism.

P2.66 REDUCED EXPRESSION OF PLACENTAL SIALYLTRANSFERASE IN GESTATIONAL DIABETES MELLITUS AND IMPAIRED IMMUNO-PROTECTIVE EFFECTS OF PLACENTAL GLYCODELIN Man-Kin Chung1,2, Tat-San Lau1,2, Stephen S.C. Chim1,2, Wing S. Lee1,2, Terence T. Lao1,2 1 The Chinese University of Hong Kong, Hong Kong, Hong Kong, 2Department of Obstetrics and Gynaecology, Prince of Wales Hospital, Hong Kong Objectives: Recently, it was reported that in gestational diabetic (GD) pregnancies, the immuno-modulatory activities of placental glycodelin (placental protein 14) is affected by a reduction of a2-6 sialylation in the carbohydrate moieties through unknown mechanisms. We therefore examined gene expression levels of the placental sialyltransferase and oligosaccharyltransferase in GD pregnancies. Methods: Pregnant women of age <35 years, with GD or normal glucose tolerance (controls) and no other obstetric complications, were recruited before delivery at term (38 weeks) for this study. The placentas were collected freshly after delivery. A healthy-looking block (2 x 2cm2 diameters) of placental tissue was dissected, cleansed and then used for total RNA extraction using a commercial RNA extraction kit according to the manufacturer’s instruction. The gene expression levels of glycanprocessing enzymes STT3A, STT3B, ST6GALNAC6 and ST3GAL3 were investigated by real-time quantitative PCR. The expression levels were normalized to GAPDH for comparison between the GD and control groups, and comparison was performed with the Mann-Whitney U test. Results: Compared with the controls (n¼9), the GD group (n¼7) was associated with a significant reduction in the expression level of STT3A (p¼0.008) and ST6GALNAC6 (p¼0.002). The differences in the expression of the other enzymes showed a trend but failed to reach statistical significance. Conclusion: The ST6GALNAC6 gene encoding the sialyltransferase is responsible for a2-6 sialylation of the glycoprotein, and reduced expression of the ST6GalNac6 gene demonstrated in GD pregnancies in this study could explain reduced a2-6 sialylation in placental glycodelin.

Abstracts / Placenta 32 (2011) A1–A149

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P2.67 HYPERGLYCEMIA AND OXYGEN MODULATE HUMAN FIRST-TRIMSTER TROPOBLAST PROLIFERATION IN A ROS-INDEPENDENT MANNER

P2.68 L-METHIONINE TRANPORT IN HUMAN CYTOTROPHOBLASTS ISOLATED FROM DIABETIC PREGNANCIES.

Julia D. Fröhlich1,2, Gernot Desoye2, Peter M. Abuja3, Julia König1, Berthold Huppert1 1 Medical University Graz; Institute of Cell Biology, Histology and Embryology, Graz, Austria, 2Medical University Graz; Clinic of Obstetrics and Gynecology, Graz, Austria, 3Medical University Graz; Institute of Pathology, Graz, Austria

Elisa Keating1, João R. Araújo1, Ana Correia-Branco1, Carla Ramalho2, Fátima Martel1 1 Department of Biochemistry (U38-FCT). Faculty of Medicine. University of Porto, Porto, Portugal, 2Department of Gynaecology and Obstetrics. Hospital de São João, Porto, Portugal

Objectives: Pregestational diabetes retards embryonic growth in early pregnancy. Placental and fetal growth are tightly associated suggesting that also placental growth is impaired. During the first trimester of gestation, oxygen tension rises steeply leading to excessive production of reactive oxygen species (ROS), which is exacerbated in diabetes and may affect placental development. We hypothesize that oxygen modifies hyperglycemic effects on ROS formation resulting in decreased human first-trimester trophoblast growth. Methods: The first trimester trophoblast-derived cell line ACH-3P was cultured up to 3 days under normoglycemia (5.5mmol/l D-glucose) and hyperglycemia (25mmol/l D-glucose) at different (2.5%, 8% and 21%) oxygen tensions. Intracellular and mitochondrial ROS levels were measured by fluorescence assays (H2DCFDA; MitoSOX Red). Proliferation and cell cycle were determined with and without cytosolic and mitochondrial ROS inducers and inhibitors. Results: Proliferation was not altered under normoglycaemia at varying oxygen concentrations. Hyperglycemia reduced cell number by 65% and resulted in cell cycle (G1- and S-phase) changes but only at 21% oxygen. At 2.5% and 8% oxygen proliferation and cell cycle were unchanged. Intracellular ROS elevation under hyperglycemia was oxygen-independent, whereas mitochondrial superoxide levels were enhanced under hyperglycemia only at 21% oxygen. Intervention to modulate cytosolic and mitochondrial ROS did not alter cell growth under hyperglycemia at 21% oxygen. Conclusion: Hyperglycemia and high oxygen-levels (21%) reduce proliferation of human first-trimester trophoblasts in a ROS-independent manner. This may account for reduced placental growth and, therefore, also for embryonic growth during the first trimester of diabetic pregnancies when the oxygen tension increases.

Objectives: To characterize the uptake of 14C-L-Methionine (14C-Met) in primary cultured human cytotrophoblasts isolated from diabetic placentas (DTB cells). Methods: Human cytotrophoblasts were isolated from normal (NTB) or gestational or type 2 diabetic pregnancies (DTB) and 14C-Met uptake was quantified by liquid scintillometry. Results: Uptake of 14C-Met revealed overlapping characteristics in NTB and DTB cells, being: a) time-dependent (with similar Amax); b) saturable (with similar Vmax and Km), c) partially (23%) Naþ-dependent, c) strongly (50%) inhibited by BCH (2-aminobicyclo[2,2,1]heptane-2carboxylic acid) (1 mM), L-Phe and L-Trp (100 mM), d) less markedly (30%) inhibited by D-Leu and D-Phe (100 mM), and e) slightly (15%) inhibited by L-Ala (100 mM). However, distinct characteristics of 14C-Met uptake in NTB and DTB cells were also found. Namely, the process was unaffected neither by L-Ser, L-Lys and L-Arg (100 mM) nor by MeAiB (amethylaminoisobutyric acid) (1 mM) in NTB cells, but it was decreased (by 15-20%) by all these compounds in DTB cells. Conclusions: Uptake of 14C-Met in both NTB and DTB cells seems to involve mainly a Naþ-independent and BCH-, L-Phe-, and L-Trp-sensitive system L, more specifically its high-affinity, D-Leu- and D-Phe-sensitive isoform LAT1, which seems to be more active in DTB cells. LAT2, an isoform sensitive to L-Ser and L-Ala, may also contribute to 14C-Met uptake in DTB, but not in NTB cells. Additionally, in DTB cells, 14C-Met uptake may be mediated by the Naþ-independent and L-Lys- and L-Arg-sensitive system b0þ and by the Naþ-dependent and MeAiB-sensitive system A. In contrast, in NTB cells, system yþL, a Naþ-dependent, BCH-insensitive and L-Alasensitive system, may be present. In conclusion, these results suggest that DTB and NTB cells take up Met with the same capacity and affinity, albeit using different transport systems. Supported by FCT and COMPETE, QREN and FEDER (PTDC/SAU-OSM/ 102239/2008, SFRH/BD/63086/2009 and SFRH/BPD/40170/2007).

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Abstracts / Placenta 32 (2011) A1–A149

P2.69 UPTAKE OF L-METHIONINE BY BEWO CELLS: CHARACTERIZATION AND MODULATION BY DIABETES-ASSOCIATED CONDITIONS. João R. Araújo1, Ana Correia-Branco1, Carla Ramalho2, Elisa Keating1, Fátima Martel1 1 Department of Biochemistry (U38-FCT). Faculty of Medicine.University of Porto, Porto, Portugal, 2Department of Gynaegology and Obstetrics. Hospital São João, Porto, Portugal Objectives: We hypothesize that diabetes-associated conditions may interfere with the placental transport of L-methionine (Met). So, our aims were to characterize the placental transport of Met and to study the effect of hyperglycaemia, hyperinsulinemia, hyperleptinemia and proinflammatory mediators upon this process. Methods: Bewo cells, used as a model of human trophoblasts, where exposed for 1, 4, 24 or 48 h to high concentrations of glucose, insulin, leptin or tumour necrosis factor-a (TNF-a). 14C-Met uptake was quantified by liquid scintillometry. Results: 14C-Met uptake by BeWo cells was: a) time-dependent, b) slightly (7%) Naþ-dependent, c) strongly (46%) inhibited by BCH (2-aminobicyclo [2,2,1]heptane-2-carboxylic acid) (1 mM), d) less markedly (25-30%) inhibited by L-Phe and L-Trp (100 mM), e) slightly (11-13%) inhibited by DLeu, D-Phe and L-Ala (100 mM) but d) unaffected by L-Ser and by MeAIB (amethylaminoisobutyric acid). Additionally, 14C-Met uptake was: 1) unaffected by hyperleptinemia (100 ng/ml), 2) decreased (by 10%) after a 48h exposure to 10 mM D-glucose (hyperglycaemia) and after a 24h exposure to 100 ng/l TNF-a, and 3) increased (by 15%) after a 1h exposure to 50 nM insulin (hyperinsulinemia). Curiously, when uptake of 14C-Met was carried out in serum-free culture medium (instead of buffer, as above), a 4h exposure to 0.01 and 1 nM insulin (normoinsulinemia and hyperinsulinemia, respectively) increased 14C-Met uptake by 12% and 24%, respectively. Conclusions: 14C-Met uptake by Bewo cells mainly involves the Naþindependent and BCH-, L-Phe-, and L-Trp-sensitive system L, more specifically its high-affinity, D-Leu- and D-Phe-sensitive isoform LAT1. The Naþ-dependent and BCH-insensitive system yþL, most probably its L-Alasensitive isoform yþLAT2, may also contribute to 14C-Met uptake. We also conclude that short periods of hyperinsulinemia stimulate, but longer periods of hyperglycemia and inflammation inhibit Met uptake by BeWo cells. Supported by FCT, COMPETE, QREN and FEDER (PTDC/SAU-OSM/102239/ 2008, SFRH/BD/63086/2009 and SFRH/BPD/40170/2007).

P2.70 DESCRIPTION OF THE PLACENTAL MATERNAL CIRCULATION IN TYPE 1 DIABETES USING A NOVEL STANDARDIZED APPROACH TO 3D POWER DOPPLER. Fiona Parker, Ruta Deshpande, George Bugg, Igor Chernyavsky, Lopa Leach University of Nottingham, Nottingham, United Kingdom Increased chorionic villous branching is a feature of the human placenta in pregnancies complicated by maternal Type 1 diabetes. These villi lie bathed in maternal blood flowing from endometrial spiral arteries that pierce the basal plate. Altered flow dynamics here may contribute to adverse fetal outcome. Non-invasive imaging techniques to assess spiral arteries may be useful in describing the maternal circulation. Using 3D power Doppler, which allows computation of flow and vascular indices, we designed a novel standardized procedure which further manipulated the 3D renders to produce 2D cross-sections of spiral arteries suitable for robust and reproducible counting. Using the basal plate as a reference point, sampling position and volumes of renders were standardised; a final tilting of 900 produced the 2D cross-sections. Numbers and flow signal diameters of spiral arteries (SA) in normal (n¼9; N) and Type 1 diabetic (n¼9; T1D) women were measured at 20 weeks gestation. Normal placenta had an average of 0.6 spiral arteries/cm2. This distribution was similar in diabetic placenta. Flow signal diameters of SA however showed an increase whilst Doppler flow index revealed a statistically significant reduction in T1D (p<0.05). This is a first study showing that the number of maternal arteries supplying the placenta does not change in T1D, but the flow signal diameter does. Using a simple theoretical relation (describing the intervillous space as a porous medium at a fixed pressure drop across the maternal side) Q w D/R between the maternal flow rate (Q), average diameter of SA openings (D), and intervillous space resistance (R), we suggest that the decreased placental flow index may be a reflection of increased resistance to maternal flow by the chorionic villi, whilst the increase in flow signal diameter of SA may be a compensatory response to ensure the normal maternal placental perfusion.

Abstracts / Placenta 32 (2011) A1–A149

P2.71 INSULIN ACTIVATES EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 2 INVOLVING INSULIN RECEPTOR A IN HUMAN PLACENTA MICROVASCULAR ENDOTHELIAL CELLS FROM GESTATIONAL DIABETES Carlos Salomon, Francisco Westermeier, Enrique Guzmán-Gutierrez, Andrea Leiva, Paola Casanello, Luis Sobrevia Cellular and Molecular Physiology Laboratory (CMPL) & Perinatology Research Laboratory (PRL), Division of Obstetrics and Gynaecology, Medical Research Centre (CIM), School of Medicine, Faculty of Medi, SANTIAGO, Chile Objectives: Human equilibrative nucleoside transporter 2 (hENT2) expression and activity is modulated by insulin in macrovascular fetal endothelium from normal pregnancies; however, nothing is reported regarding the potential action of insulin on hENT2 expression and/or activity in human placental microvascular endothelial cells (hPMEC) from gestational diabetes (GD). Insulin preferentially actives p42/44mapk and Akt via insulin receptor isoforms A (IR-A) and B (IR-B), respectively. We studied insulin effect on hENT2 expression and activity, and the potential involvement of IR-A and IR-B in hPMEC from GD. Methods: hPMEC from full-term normal or GD pregnancies were cultured under standard conditions. hENT2-mediated adenosine transport (2 mCi/ ml [3H]adenosine, 20 seconds, 22oC), protein abundance and mRNA expression were determined by Western blot and Q-PCR. Total and phosphorylated p42/44mapk (Pwp42/44mapk) and Akt (PwAkt), and IR-A and IRB mRNA expression in response to insulin were assayed. An adenoviralbased siRNA delivering system was used to knockdown IR-A (siIR-A) and IR-B (siIR-B) expression. Results: GD is associated with reduced (P>0.05) hENT2 expression (protein, mRNA) and adenosine transport (w80%), IR-A expression (w65%) and (Pwp42/44mapk/p42/44mapk)/(PwAkt/Akt) ratio (w50%). Insulin blocked these GD-associated alterations. In hPMEC from normal pregnancies, insulin increased IR-A (w1.9-fold), but not IR-B mRNA expression. However, in GD insulin increased IR-A (w1.4-fold) and recovered IR-B to values in cells from normal pregnancies. Insulin effect was paralleled by recovery of (Pwp42/44mapk/p42/44mapk)/(PwAkt/Akt) ratio to values in cells from normal pregnancies. Insulin effects were abolished in siIR-A, but not in siIR-B cells. Conclusion: Insulin could acts as a selective agonist for insulin receptor isoforms A and B in the human placental microvascular endothelium in states of insulin resistance such as gestational diabetes. Support: CONICYT (ACT-73 PIA, AT-24100210), FONDECYT (1110977, 1080534), Faculty of Medicine, Pontificia Universidad Católica de Chile (FM-PUC) (PMD 03/10). CS, FW and EG-G hold CONICYT-PhD fellowships. CS holds a FM-PUC PhD fellowship.

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P2.72 TYPE 1 DIABETES IMPAIRES PLACENTAL MMP EXPRESSION IN EARLY PREGNANCY Ursula Hiden1, Patrick Greimel1, Nassim Ghaffari-Tabrizi2, Martina Dieber-Rotheneder1, Marina Ivanisevic3, Josip Djelmis3, Gernot Desoye1 1 Department of Obstetrics and Gynecology, Medical University of Graz, Graz, Austria, 2Institute of Pathophysiology, Medical University of Graz, Graz, Austria, 3Department of Obstetrics and Gyecology, University Hospital Petrova, Zagreb, Croatia Objectives: Trophoblast invasion is a crucial process in pregnancy in order to anchor the placenta and to establish adequate utero-placental blood flow. Therefore, trophoblasts are well equipped with matrix degrading enzymes such as matrix metalloproteinases (MMPs). Maternal Type 1 diabetes (T1D) is associated with a higher risk for early pregnancy loss, pre-eclampsia and fetal growth restriction. As underlying reason we hypothesise impaired trophoblast invasion. Because placental changes in early gestation are difficult to determine, indirect evidence is needed to demonstrate changes in trophoblast invasion. The aim of the present study was to identify changes in MMP expression in first trimester placentas from healthy vs. T1D pregnancies. Methods: Placentas from normal (n¼15) vs. T1D (n¼12) pregnancies between gestational week 6-12 were obtained after pregnancy interruption for psycho-social reasons and immediately frozen in liquid nitrogen. Total RNA was isolated and mRNA expression of all MMPs measured using RT-PCR. Expression signals were normalized to the trophoblast marker cytokeratin 7. Activity of MMP2 and 9 was determined by gelatine gel zymography. First trimester trophoblast model cells (ACH-3P) were cultured under various oxygen concentrations (2.5, 5, 12, 21) for seven days and expression of MMPs was measured. Results: 14 MMPs were detected in first trimester placenta. T1D was associated with significantly reduced expression of 11 MMPs in weeks 810. In parallel lower amount of MMP2 (-32%, p¼0.02) and MMP9 (-39%, p¼0.05) activity was measured. In ACH-3P cells only 6 MMPs were detected of which 4 were reduced under hypoxia. Conclusion: This is the first study to demonstrate lower placental MMP expression during week 8-10 resulting from maternal T1D which hence may impair trophoblast invasion. Diabetes associated lower uterine oxygen concentrations may be involved in the reduced MMP expression. These results may explain the higher incidence of spontaneous abortions in T1D women.

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Abstracts / Placenta 32 (2011) A1–A149

P2.73 ULTRASTRUCTURE CHANGES IN PLACENTAS IN PREGNANCIES COMPLICATED WITH TYPE 2 DIABETES

P2.74 ASPIRIN AND HEPARIN THERAPY FOR CASES SUFFERING FROM OBSTETRICAL COMPLICATIONS DUE TO ANTIPHOSPHOLIPID ANTIBODIES OR CLOTTING FACTORS DISORDERS

1

H Mirghani1, S Tariq2, N. Osman1, E. Adegate2, A. Malek3 Department of Obstetrics & Gynecology, Faculty of Medicine & Health Sciences, United Arab Emirates University, United Arab Emirates, 2 Department Anatomy & Gynecology, Faculty of Medicine & Health Sciences, United Arab Emirates University, United Arab Emirates, 3 Department of Obstetrics & Gynecology, Faculty of Medicine & Health Sciences, United Arab Emirates University, United Arab Emirates

Rie Kawaguchi1,2, Monika Siwetz1, Monika Sundl1, Yuki Nakada2, Michiko Suzuki2, Eri Takahashi2, Taizan Kamide2, Nagayoshi Umehara2, Tadao Tanaka2, Berthold Huppertz1 1 Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Graz, Austria, 2Department of Obstetrics and Gynecology, The Jikei University School of Medicine, Tokyo, Japan

Objective: To compare the ultrastructure of term placentas obtained from pregnant women with type 2 Diabetes to placentas of uncomplicated pregnancies. Materials Methods: A total of 10 placentas were studied. All pregnancies were delivered at term either vaginally or by Cesarean section. Pregnancies with confirmed multiple pregnancy or fetal anomalies were excluded from the study. Immediately after a placenta was delivered, tissue slices of about 3X3 mm and 3 mm thick were taken midway through the central cotyledon. First, they were fixed in 2.0% glutaraldehyde in phosphate buffer, then in 2.0% osmic acid. phosphate buffer wash muscular tissues was immersed immediately in the Karnovskys fixativerats. After rinsing with phosphate buffer the placenta was post fixed with 1% Osmium tetroxide for 1hour. The tissue was dehydrated in a series of graded ethanol and then finally in propylene oxide. Blocks were trimmed and semithin & utrathin sections were cut. Semithin sections(130 mm thickness) stained with1% aquous toluidine blue on glass slides and ultrathin sections(95nm thin) on 200mesh Cu grids then contrasted with Uranyl acetate and followed by lead citrate double stain. Then grids were studied/examined and photographed under a Philips CM10 Transmission Electron Microscope. Results: At total of 10 placentas were studied; five of pregnancies complicated with type 2 diabetes, and five of normal pregnancies. The mean maternal age was 33.6 (2.3) years, the mean parity was 2.7 ( 1.5), and the mean gestational age at delivery was 38.5 ( 0.5) weeks. Transmission electron microscope showed several changes in the ultrastructure of placentas of type 2 diabetes compared to placentas of normal pregnancies: The villi are reduced in number and much thinner, thickening of the trophoblastic basement membrane, reduced stromal, and increase in cytotrophoblast organelles. Additional results and photos will be presented

Objectives: Aspirin and heparin have used as therapy for cases suffering from obstetrical complications. Antiphospholipid antibodies (APLs) and clotting factor (CF) disorders are recognized as causes associating placental insufficiency with obstetrical complications. To investigate the effect of a combined aspirin and heparin therapy on pregnancy outcome, we compared pregnancy outcome and placental pathologies in two successive pregnancies in the same patients suffering from APLs or CF disorders. Methods: All 1st pregnancies were complicated by FGR. During 2nd pregnancy, all cases were treated with low dose aspirin and heparin. The APS group included five patients with lupus-anticoagulant and other autoantibodies, the CF group included four patients with clotting factor abnormalities. In each group the placentas of 3 cases were obtained from both pregnancies and double-immunostained for Ki-67 and cytokeratin 7 to selectively identify proliferating villous cytotrophoblasts. Villi were randomly selected, cells were counted and rates of proliferating cytotrophoblasts were calculated. Results: Therapies in the 2nd pregnancy were effective in terms delivery week (from 31.72.2 to 36.71.3 weeks) for all cases, and for placental weight in the APS group (34065g vs. 51787; p <0.05) but not in the CF group (359130g vs 40939; NS). Fetal weight significantly increased in APLs cases compared to CF cases (APLs vs CF, 2731222g vs 2020233; p <0.01). In the placentas, the ratio of Ki67 positive cytotrophoblasts and fibrin regions in the intravillous area significantly increased in the CF group but not in the APLs group. Conclusion: Heparin and aspirin therapy is effective to improve pregnancy outcome especially in cases with APLs in combination with placental improvement. However, this is not the case in the CF group in terms of fetal growth and increased placental infarction. Hence, there is the need for a further assessment of APLs and CF disorder effects on placental development.

Abstracts / Placenta 32 (2011) A1–A149

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P2.75 PHENOTYPIC DEVIATIONS OCCUR IN LYMPHOCYTE LINEAGES DURING TYPE 1 DIABETIC PREGNANCY

P2.76 ISOLATION OF TERM PLACENTAL MACROPHAGES LEADS TO A POPULATION HIGHLY ENRICHED IN MATERNAL CELLS

Alexandra Seaward, Suzanne Burke, Graeme Smith, B. Anne Croy Queen’s University, Kingston, Ontario, Canada

Meghan Riddell1, Yanyan Jiang2, Bonnie Winkler-Lowen3, Larry Guilbert3, Sandra Davidge2,1 1 Department of Physiology, University of Alberta, Edmonton, AB, Canada, 2 Department of Obstetrics and Gynecology, University of Alberta, Edmonton, AB, Canada, 3Department of Medical Microbiology and Immunology, Edmonton, AB, Canada

Objectives: UNK initiate decidual spiral arterial remodelling in early gestation. Circulating CD56bright NK cells are thought to include uNK cell precursors that employ a unique pattern of homing molecules and chemokine receptors for decidual extravasation. We previously reported that diabetes alters CD56þ cells in a manner that restricts their ability to interact in vitro under shear forces with decidual endothelium. We hypothesized that the mechanisms for diabetes-induced alterations in leukocyte homing potential to decidua include changes in frequencies of cells expressing receptors used for interactions with decidual endothelial cells and chemokines. Methods: T1D (n¼8) and normal control (n¼8) women were recruited in their 1st trimester of pregnancy. Peripheral blood samples were collected once per trimester and once post partum. Lymphocytes were isolated using standard density gradient separation methods. Four-colour flow cytometry assessed stained lymphocytes as CD3 (T cells), CD56 (NK and NKT cells), IL-18Ra (pan Type 1 cytokine marker), ST2L (pan Type 2 cytokine marker), CXCR3, CXCR4 (chemokine receptors), CD49d (a4 integrin adhesion molecule) and CD62L (L-selectin homing molecule). Two-way ANOVA with Bonferroni’s post-test was used to determine statistical significance (P<0.05). Results: CD56bright ST2Lþ cells were transiently elevated during 2nd trimester in control patients (P<0.01) but not in T1D patients. CD56bright cells had the highest frequencies of CD49dþ and CD62Lþ cells. This abundant percentage was linked to a type 1 cytokine profile and was not affected by T1D. However, relative intensity of CD62L was decreased by T1D in the first trimester compared to controls (P<0.05). T1D T cell CXCR3 expression was greater at all four sampling times in T1D (P<0.05). Conclusions: Phenotypic deviations occur in T1D lymphocyte subsets during pregnancy that have the potential to alter homing to decidua. Supported by CIHR and the Canada Research Chairs Program

Objectives: Placental macrophages/Hofbauer cells engage in essential processes of immune surveillance, tissue remodelling and angiogenesis. Elucidation of the mechanisms through which Hofbauer cells function requires culturing; thus, we have developed a protocol for isolation and culture of term human placental macrophages. Methods: Placentas were obtained from normal pregnancies with nonlaboured caesarean deliveries and the decidua and fetal membranes removed. The tissue was extensively washed to remove blood and minced tissue was subjected to serial trypsin/DNase digestion. Single cell suspensions were incubated for 1hr in 37oC 20% O2. Non-adherent cells were removed by vigorous washing, adherent cells were again incubated for 24hrs and then CD45 FACS sorted. CD45-positive cells were cultured in IMDM 10% FCS plus the macrophage differentiation factor CSF-1 at 6% O2. The cells were then characterized for expression of the monocyte/ macrophage markers CSF-1R, CD14 and CD68 and the hematopoietic marker CD45. The maternal/fetal origin was determined by Y chromosome detection in male pregnancies. Results: Isolated cells comprised initially of stellate fibroblast-like cells and classical macrophages but by day 7 of culture >95% of the cells were classical macrophages. All were positive for CD14, CSF-1R and CD45 but variable expression of CD68 was observed. Importantly, only w15% of the population were of fetal origin. Conclusion: Isolation of monocyte/macrophage cells from extensively washed term placenta via trypsin/DNase digestion and CD45 selection, when cultured, yields a highly purified population of macrophages predominantly of maternal origin. Significance: Attention to the maternal/fetal origin of isolated placental macrophages has not previously been addressed by most investigators. Therefore the mixed origin of cultured placentally-derived macrophages must be considered when interpreting the results of in vitro experiments.

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Abstracts / Placenta 32 (2011) A1–A149

P2.77 FULLY DIFFERENTIATED UTERINE NATURAL KILLER CELLS BECOME CD127D IN A DECIDUAL MICROENVIRONMENT ENRICHED FOR THYMIC STROMAL CYTOKINES Jianhong Zhang1, Jorg Fritz2, Yrina Rochman3, Warren Leonard3, Jennifer Gommerman2, Zhilin Chen1, Adam Plumb4, Ninan Abraham4, B. Anne Croy1 1 Queen’s University, Kingston, Ontario, Canada, 2University of Toronto, Toronto, Ontario, Canada, 3Laboratory of Molecular Immunology, National Heart, Lung and Blood Institute, NIH, Bethesda MD, 4University of British Columbia, Vancouver, BC, Canada Objectives: Endometrial decidualization causes an influx of uterine Natural Killer (uNK) cells in humans and mice. Dolichos biflorus agglutinin (DBA) lectin divides mouse uNK cells into reactive cells that traffick from marrow and blood and non-reactive cells of distinct but undefined origins. We examined expression of CD127, the alpha chain in the dimeric receptors of interleukin 7 (Il7) and thymic stromal lymphopoietin (Tslp) and a marker of early NK progenitor and peripheral cells, as a potential aid to resolving relationships between DBA lectin reactive and non-reactive uNK cells. Methods: Timed implantation sites (gestation days (gd) 6-12) were collected from C57BL/6J (B6), Il7-/-, Il7ra-/- and Tslpr-/- mice for immunohistochemistry, supernatant production, western blotting and realtimePCR studies. Results: By immunohistochemistry, DBAþ and DBA- uNK cells were CD127at gd6.5. By gd10.5, mature, post-mitotic DBAþ and DBA- uNK cells of decidua basalis (DB) were strongly CD127 reactive. Less mature DBAþ and DBA- uNK cells of the gd10.5 mesometrial lymphoid aggregate of pregnancy (MLAp) had weak CD127 reactivity. Il7 mRNA and protein were detected in DB as were mRNA and biologically active Tslp. Additional lymphoid stromal factors (gp38/Podoplanin and ERTR7 proteins and mRNA for the transcription factor autoimmune regulator Aire) were expressed by mesometrial decidua. DBAþ and DBA- NK cells differentiated in Il7-/-, Il7ra-/-and Tslpr-/- mice and expressed CD127 at midgestation. However, interferon gamma (Ifng) production by Tslpr-/- uNK cells was severely impaired. Conclusion: Mature, midgestational DBAþ and DBA- uNK cells acquire CD127, a component of the Tslp receptor that appears to sustain peak, midgestational Ifng production. Tslp and other thymic stromal cell factors important for tolerance induction are products of decidual cells, suggesting new roles for decidua in immune protection of conceptuses. Supported by Awards from NSERC, CIHR, CRC Program and National Heart, Lung, and Blood Institute, NIH.

P2.78 INDOLEAMINE 2,3 DIOXIGENASE1 EXPRESSION IN VASCULAR ENDOTHELIA OF THE HUMAN PLACENTA Astrid Blaschitz1, Martin Gauster1, Dietmar Fuchs2, Ingrid Lang1, Petra Maschke1, Gottfried Dohr1, Peter Sedlmayr1 1 Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Graz, Austria, 2Division of Biological Chemistry, Innsbruck Medical University, Innsbruck, Austria Objectives: In the present study we investigated the expression of indoleamine 2,3 dioxigenase1 (IDO1) in placenta endothelial cells of the fetal and the maternal circulations. Methods: For immunolocalization we used paraffin-embedded tissues from early and term placentas. Furthermore we measured the kynurenine (kyn) and tryptophan (trypt) levels and calculated the kyn/trypt-ratio allowing an estimate of IDO enzyme activities in sera and tissues. We identified IDO1 mRNA in isolated endothelial cells from the placenta and other organs. Results: On the fetal side IDO1 positivity was restricted to vascular endothelium, which did not co-express HLA-DR. The expression extended from sub-trophoblastic capillaries in first-trimester to a nearly general presence on endothelia at term. These findings paralleled the detectability of IDO1 mRNA and a high increase in the kyn/trypt-ratio in villous tissues from first trimester to term. Umbilical cord vessels were generally negative for IDO1 throughout all stages of development. Endothelial cells isolated from the chorionic plate expressed IDO1 mRNA in contrast to such originating from umbilical vein, iliac vein, or aorta. On the maternal side we found decidua arteries expressing IDO1, which was complementary to the endothelial HLA-DR expression in veins there. IDO1 expression was higher in vessels located close to the feto maternal interface than in those at the periphery of the uterine wall. IDO1 activity in blood taken from chorionic plate and umbilical cord vessels indicated far higher values than those found in the peripheral blood of adults. Conclusions: Our data suggest that there exists an inclining gradient of IDO1 expression in vessel endothelia at both sides towards to the fetomaternal contact zone in the human placenta.

Abstracts / Placenta 32 (2011) A1–A149

P2.79 THE IMMUNOMODULATORY MICROVESICLES.

ROLE

Jennifer Southcombe, Ingrid Christopher Redman, Ian Sargent University of Oxford, Oxford, UK

OF

SYNCYTIOTROPHOBLAST

Granne,

Dionne

Tannetta,

Objective: Maternal immune adaptation is essential for a successful pregnancy. Typically pregnancy is associated with increased production of inflammatory cytokines but down-regulation of adaptive cell-mediated immune responses. We have investigated whether syncytiotrophoblast microvesicles (STBM), which are shed from the placenta into the maternal circulation, could be responsible for these changes by measuring cytokine production from maternal peripheral blood mononuclear cells (PBMC) in response to STBM. Methods: PBMC from non-pregnant women were incubated with STBM (produced from normal placentas by perfusion) and cytokine profiles detected using cytokine arrays. The production of cytokines which were highly up or down regulated was confirmed by ELISA. STBM were incubated with PBMC from third trimester normal pregnant (n¼10) and nonpregnant women (n¼10) and ELISAs performed to determine whether cytokine production was altered. Binding and uptake of STBM by PBMC was investigated by imaging flow cytometry and the cellular source of the cytokines determined by intracellular cytokine flow cytometry. Results: STBM triggered cytokine release from PBMC. Cytokine arrays revealed induction of MIP-1a, MIP-1b, IL-1a, IL-1b, IL-6 and G-CSF by more than 20-fold with smaller changes in TNFa, IL-10, I-309 and IL-5. Other cytokines, such as IP-10, were down-regulated. PBMC from normal pregnant women produced more TNF-a and IL-6 in response to STBM than non-pregnant women. STBM bound to monocytes and B cells and imaging flow cytometry confirmed that STBM were phagocytosed by these cells. Monocytes were responsible for most of the cytokine production. Conclusion: STBM are phagocytosed by monocytes and B cells. In response, monocytes produce proinflammatory cytokines which may contribute to the increased inflammation seen in normal pregnancy. PBMC from pregnant women produce greater amounts of cytokines, suggesting the cells are primed by pregnancy. Some cytokines, such as the interferon gamma related cytokine IP-10, are inhibited by STBM which could be beneficial for pregnancy.

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P2.80 SOLUBLE ST2 AND IL-33 IN PREGNANCY AND PRE-ECLAMPSIA Jennifer Southcombe, Ingrid Granne, Christopher Redman, Ian Sargent University of Oxford, Oxford, United Kingdom

Dionne

Tannetta,

Objective: Normal pregnancy is associated with a type 2 cytokine immune bias, whereas pre-eclampsia has a type 1 dominance. IL-33 has strong immunomodulatory functions and predominantly induces the production of Th2 cytokines in T cells and NK cells. Soluble ST2 (sST2) is thought to act as a ‘decoy receptor’, competing with the membrane bound receptor (ST2L) for IL-33 binding. Increasing levels of sST2 may therefore negatively regulate IL-33 signalling and diminish the cytokines overall bioactivity. We have investigated the levels of these proteins in the maternal circulation in pregnancy and pre-eclampsia and whether the placenta could be a potential source. Methods: Peripheral blood levels of IL-33 and sST2 were measured by ELISA throughout the three trimesters of pregnancy in normal pregnancy and in pre-eclamptic women. sST2 and IL-33 expression in the normal and pre-eclampsia placentas was analysed by western blotting and immunohistochemistry. Placenta perfusion and explants models were used to study sST2 and IL-33 secretion. Results: Although maternal blood levels of IL-33 levels were unchanged throughout normal or pre-eclamptic pregnancies, sST2 was elevated in the third trimester of normal pregnancy (p<0.05). sST2 levels were even higher in pre-eclamptic women (p<0.001) and this increase could be detected prior to the onset of clinical symptoms (p<0.05). Normal and preeclampsia placentas expressed both molecules, however only sST2 could be detected in the maternal effluent of perfused placenta lobes. Placenta explants treated with proinflammatory cytokines (IL-1b/ TNFa) and hypoxia reperfusion injury secreted higher levels of sST2. Conclusion: These results suggest that the placenta is a source of the increased levels of sST2 detected in the maternal circulation in preeclampsia. Raised levels of sST2 may inhibit the function of IL-33, preventing activation of the type 2 immune cytokine bias that is essential for successful pregnancy.

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Abstracts / Placenta 32 (2011) A1–A149

P2.81 PROFILING CYTOKINE AND ANGIOGENIC FACTOR SECRETION BY DECIDUAL NATURAL KILLER CELLS FROM FIRST TRIMESTER PREGNANCIES AT HIGH OR LOW RISK OF PRE-ECLAMPSIA. Rupsha Fraser, Guy Whitley, Alan Johnstone, Baskaran Thilaganathan, Judith Cartwright St. George’s, University fo London, London, United Kingdom Objectives: Decidual natural killer cells (dNK) are key players in controlling the maternal-fetal interface. dNK protein secretion will regulate both placentation and the uterine adaptations to pregnancy with altered cytokine production likely to be a factor in the pathogenesis of pre-eclampsia. Our objectives were to profile cytokine and angiogenic protein secretion from first trimester dNK isolated from pregnancies characterised as at the highest and lowest risk of developing pre-eclampsia. Methods: Uterine artery Doppler was used to identify groups of women undergoing termination of pregnancy who were most likely and least likely to have developed pre-eclampsia. dNK were isolated and culture supernatant collected over 24h. Secreted proteins in these supernatants from 19 high-risk and 21 low-risk pregnancies were characterised using multiplex assays (Fluorokine or Luminex). Results: First trimester dNK secreted numerous factors including angiogenic factors (angiogenin, endostatin, PlGF, VEGF), cytokines (IL-1b, IL-6, IL8, IL-10, IL-12, IFN-g, TNFa), soluble cytokine receptors and antagonists (IL2 receptor, IL-1 receptor antagonist), growth factors (HGF, basic FGF, G-CSF, GM-CSF) and chemokines (IP-10, MCP-1, MIG, MIP-1a, MIP-1b, RANTES). Two factors which can affect the angiogenic process, angiogenin (generally pro-angiogenic) and endostatin (generally anti-angiogenic), were at significantly higher levels in the culture supernatant from high-risk dNK. Similarly, the soluble IL-2R was secreted at higher levels by high-risk dNK. Conclusion: Analysis of secreted factors by dNK cells will be informative for understanding the role of dNK in normal pregnancy. The altered levels of angiogenin and endostatin may be important in how dNK affect vascular cells during spiral artery remodelling. If dNK from the high-risk group are more active (perhaps reflected by their increased IL-2R) this could result in them being more prone to attack neighbouring trophoblast. The functional significance of angiogenin, endostatin and IL-2R at the maternal-fetal interface will now be investigated.

P2.82 TOLL-LIKE RECEPTORS AND VIRAL INFECTION IN TROPHOBLASTS Guro Sannerud Stødle, Line Haugstad Tangerås, Guro Dalheim Olsen, Bjørg Steinkjer, Astrid Solberg Gundersen, Liv Ryan, Bente Skei, RigmorAustgulen, Ann-Charlotte Iversen Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway Objectives: Viral infections in pregnancy may cause complications such as spontaneous abortion and preterm delivery. Intrauterine infections may evoke local inflammatory responses by activation of the pathogen-sensing receptor system; the Toll-Like Receptors (TLRs), disturbing the delicate interaction between the maternal and fetal cells of the placenta. The viralresponsive TLRs include TLR2, TLR3, TLR7, TLR8 and TLR9, and trophoblast expression of these TLRs suggests that trophoblasts contribute to TLRmediated immune activation upon viral infections. Human Cytomegalovirus (CMV) infection may cause pregnancy complications and fetal sequelae, and CMV is shown to activate the viral-responsive TLRs. In this study, immune activation of primary first trimester trophoblasts through viral-responsive TLRs is elucidated, and the influence of CMV infection on cytokine response and TLR expression in trophoblasts is determined. Methods: Primary trophoblasts were isolated from first trimester placentas (5-12 gestational weeks) by enzymatic degradation and LSM gradient centrifugation. Trophoblast choriocarcinoma cell lines were included for comparison. Cells were infected with HCMV strain AD169 for 4-72 hours. Gene expression of TLR 1-10 and viral genes were analyzed by real-time quantitative RT-PCR, and pro-inflammatory cytokine response was determined by Multiplex. Results: Primary first trimester trophoblasts were actively infected by CMV and expressed all TLRs 1-10. The primary trophoblasts responded to activation of viral-responsive TLRs by potent release of pro-inflammatory cytokines, such as IL-6, IL-8 and IP-10. Both trophoblast TLR expression and cytokine response were influenced by CMV infection. Conclusion: Viral-responsive TLRs mediate potent trophoblast immune activation by release of pro-inflammatory cytokines, suggesting that TLRs may be involved in harmful consequences of viral infection in the placenta.

Abstracts / Placenta 32 (2011) A1–A149

P2.83 HMGB1 AND RAGE EXPRESSION ON HUMAN FOETAL MEMBRANES AT TERM GESTATION AND IN PREECLAMPSIA Alessandra Zicari1, Massimo Realacci1, Emanuela Mari1, Tullia Todros2, Simona Cardaropoli2, Carlo Ticconi3, Nicoletta di Simone4 1 University La Sapienza;, Rome, Italy, 2Osp. S.Anna -Univ Torino, Turin, Italy, 3Univ Tor Vergata, Rome, Italy, 4Univ Cattolica S.Cuore, Rome, Italy Objectives:Pre-eclampsia, a widespread and dangerous pregnancy complication that affects 3-5% of all pregnancies, is characterized by intravascular inflammation and endothelial disfunction.High Mobility Group Box 1 (HMGB1) firstly considered as a nuclear protein has been recently demonstrated to play important extracellular cytokine-like functions, to trigger inflammation and to promote tissue regeneration and it is now considered as an alarmin.The Receptor for Advance Glycated Endoproducts (RAGE), the main HMGB1 receptor, it is expressed by a variety of immune and vascular cells, including T cells, monocytes, DCs , endothelial and vascular smooth muscle cells.In this study we analyzed HMGB1 and RAGE expression on human foetal membranes obtained from elective cesarean section at term and from women affected by pre-eclampsia ,we also analyzed the effects of “in vitro” incubation with HCG and heparin on their expression. Methods: Samples of reflected foetal membranes were collected immediately after the delivery of the placenta, processed in laboratory and incubated for 24 hrs in 95% O2 and 5% CO2. Some samples have been incubated with 10UI/100UI HCG or with 25-100UI heparin. Each culture was performed in duplicate.Western blot analysis was performed to verify HMGB1 and RAGE protein expression in human foetal membranes. Results: Human foetal membranes of both at term gestation and preeclamptic women express HMGB1 and its main receptor RAGE. Their expression is modulated by HCG and heparin: HCG induces an increase of HMGB1 and RAGE expression mainly on samples obtained at term gestation; the Heparin treatment induces significant increase of HMGB1 and RAGE on both at term and preeclamptic membranes. Conclusion: The presence of HMGB1and RAGE on human foetal membranes suggest a possible unknown role played by these molecules in pregnancy. The modulation of their expression could be an useful tool to improve the local immune response.

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P2.84 MATERNAL ALLOIMMUNE RESPONSE DURING PREGNANCY: BRAKE OF MATERNAL TOLERANCE TO FETAL HUMAN PLATELET ANTIGEN 1A ANTIGEN MAY BE ASSOCIATED WITH REDUCED PLACENTAL FUNCTION Anne Husebekk1,2, Maria Theres Ahlen1, Mette Kjær Killie1, Heidi Tiller1, Mariana Eksteen2, Ida Killie2, Gøril Heide2, Bjørn Skogen1,2, Jens Kjeldsen-Kragh3,4, Tor Stuge1,2 1 University Hospital North Norway, Tromsø, Norway, 2University of Tromsø, Tromsø, Norway, 3Oslo University Hospital, Oslo, Norway, 4 University of Oslo, Oslo, Norway Background: Fetal/neonatal alloimmune thrombocytopenia is caused by antibodies to fetal HPA1a in >80% of the cases. The antibodies cross placenta, sensitize fetal platelets which are phagocytosed, rendering the fetus thrombocytopenic and at risk of bleeding. Human platelet antigen (HPA) 1a is an epitope on the b3 integrin of the fibrinogen receptor on platelets. b3 integrin is also present on invading throphoblasts as part of the vitronectin receptor. Immunization may occur during the first trimester of the pregnancy and platelets are probably not the source of antigen in this situation. Recent data show an association between maternal anti-HPA 1a antibody level and reduced birth weight, indicating that placental function may be affected by these antibodies during pregnancy. Objectives: Exploring the mechanism of immunization with the HPA 1a antigen during pregnancy and the consequences of the antibodies for the fetus. Method: Clonal B- and T-cells were isolated from peripheral blood mononuclear cells from HPA 1b homozygous mothers who had given birth to children with severe thrombocytopenia. Results: The T cells could be activated by HPA 1a peptides or HPA 1a positive platelets in culture. The monoclonal B-cells were EBV transformed and produced anti-HPA 1a antibodies with high affinity. Conclusion: The immune response against HPA 1a antigen represents a brake of maternal tolerance to this antigen. One consequence of the antibodies is thrombocytopenia and risk of bleeding in the fetus and newborn. As the vitronectin receptor carrying the HPA 1a epitope also is present on activated endothelial cells and throphoblasts, it is likely that these cells may be influenced by the antibodies and the reduced birth weight may indicate placental dysfunction.

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P2.85 MATERNAL SYSTEMIC IMMUNE NETWORKS DURING MID-GESTATION Douglas Creedon, Wendy Nevala, Alex Leontovich, Svetomir Markovic, Shernan Holtan Mayo Clinic, Rochester, MN, USA Objective: Trophoblast invasion and angiogenesis during placentation induce not only a local maternal immune response but also a reprogramming of maternal immunity that is detectable systemically. Our objective is to understand these systemic immune networks active during mid gestation. Methods: Serum obtained from 16 primiparous women in the second trimester (15-26 weeks gestation, 89 samples total) were assayed using a Millipore 42-plex cytokine/growth factor array. We determined correlations between cytokines and growth factors in a pair-wise fashion and performed unsupervised hierarchical clustering to reveal inherent patterns within the data. Highly correlated factors (Spearman’s rho >0.7 for positive associations or <-0.6 for negative associations) were then assessed for pathway enrichment with MetaCoreÔ software. Results: Pair-wise analysis identified correlations with IL-1b, IL-1ra, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-13, IL-15, IFNg, FGF2, MDC (positive), and MCP-1 (negative). Using this list, MetaCore pathway enrichment identified seven active cytokine/growth factor networks:

1) 2) 3) 4) 5) 6) 7)

an an an an an an an

IL 4, IL 10, IL 6, IL 7 receptor, IL 7 network, IL 4, CCL2, IL 12 beta chain, IL 6 receptor, IFNGR1 network, IL 4, IL 10, IL 15, IL 4R type I, FGF23 network, IL 15, IL 15RA, IL1RN, TNF beta, IL 12 alpha chain network, IFNGR1, FGF23, FGF2, FGF21, FGF20 network, IFNGR1, IL 15RA, IL 7, IL 7 receptor, IL 15 network, and IL 4, IL 4R type II, IL 6, IL 4R type I, c Src network.

Conclusions: Using this approach, we have begun to characterize immune networks involved in maintenance of a tolerogenic milieu during pregnancy. We believe that this informatics-based approach may help identify previously unknown targets for therapeutic intervention in conditions of pregnancy that may have an immunologic basis, such as preterm labor and preeclampsia. P2.86 CD8A RECEPTOR EXPRESSION OF MOUSE UTERINE NATURAL KILLER CELL BEARING FUNCTIONAL SUBSETS DURING PREGNANCY Karina Degaki, Alessandro Farias, Aureo Yamada Institute of Biology, Campinas,SP, Brazil Objectives: In the aim to evaluate the unique down regulation of innate immune type response of uterine natural killer cell in spite of full intracellular cytolytic arsenal, we investigate the possibility of perforinþ/uNK cells share CD8þ receptor of cytotoxic T lymphocyte bearing to adaptive immune response. Metodologia: Uterine mesometrial region from pregnant mice on gestational day (gd) 9th were dissected for uNK cell isolation using Dolichos biflorus agglutinin (DBA) lectin coated magnetic beads. The isolated cells were stained with fluorochrome conjugated anti-NK1.1, anti-CD8a, anti-CD14, anti-CD3, anti-perforin and DBA lectin for single, double and triple labeling to be quantified using Fluorescence Activated Cell Sorting (FACS). In parallel uterine fragments were processed for histological sections and immunostained with anti-perforin and DBA lectin cytochemistry for morphometry. Results: Both morphometry (26%) and flow cytometry (31%) confirmed the similar incidences of DBAþ/perforinþ uNK cell in the mice uterus at gd 9th. Co-expression of NK1.1þ(87%), CD8aþ(57%), CD14þ(85%) and no CD3þ cells among DBA lectin-magnetic bead affinity isolated uNK cells and most of perforinþuNK cells being CD8aþ, suggest diversity uNK cell phenotype but no incidence of NKT cell in the mouse pregnant uterus. Conclusions: Unequivocally there are functional subsets of uNK cells in the pregnant mouse uterus with hallmarked expression of CD8a receptor responsible for well known activation of CD8-T lymphocyte perforin, granzymes, TNFa and IFNg production related to cytotoxic response but not usual to the blood circulating NK cells.

P2.87 RECOGNITION OF PATERNAL TROPHOBLAST HLA-C BY MATERNAL UTERINE NK CELL KIR DETERMINES THE OUTCOME OF PREGNANCY A. Moffett, S.E. Hiby, R. Apps, A.M. Sharkey, L.E. Farrell, L. Gardner, M. Carrington University of Cambridge, Cambridge, UK Background: Successful pregnancy requires that trophoblast invasion of the uterus be appropriately calibrated to ensure that neither fetus nor mother is compromised. We have investigated the regulation of placentation by interactions between maternal KIR on uterine Natural Killer cells (uNK) and HLA-C expressed by fetal trophoblast. Methods: We used immunostaining of human implantation site sections and FACS analysis of primary uNK and trophoblast with HLA-C or KIR specific antibodies and primary trophoblast with KIR fusion proteins to investigate the expression and interaction of the two protein families. We carried out KIR and HLA-C genetic analyses of normal (n¼592) and affected pregnancies complicated by pre-eclampsia (n¼742), fetal growth restriction (n¼118) or recurrent miscarriage (n¼115). Results: We show, in vivo, that uNK and trophoblast are closely apposed, and that trophoblast expresses both maternal and paternal allotypes of HLA-C. We find that KIR2DS1, the activating receptor for HLA-C2, is expressed by uNK and binds specifically to trophoblast HLAC2. Genetic analyses of normal and affected pregnancies complicated by pre-eclampsia, fetal growth restriction or recurrent miscarriage show that in the presence of fetal C2 (in particular, paternally-derived C2), the telomeric end of the KIR B haplotype, containing KIR2DS1, is significantly protective. In contrast, KIR A haplotypes are detrimental. Conclusion: We conclude that KIR/HLA-C interactions at the maternal/ fetal interface are significant determinants of reproductive outcome. P2.88 CHARACTERIZATION OF PLACENTAL AND CIRCULATING REGULATORY T-CELLS G.M. Johnsen1,3, J. Landskron2,3, K. Taskén2,3, A.C. Staff1,4 1 Department of Obstetrics and Department of Gynaecology, Oslo University Hospital, Ulleval, Norway, 2Centre for Molecular Medicine Norway, Nordic EMBL Partnership, Oslo, Norway, 3Biotechnology Centre, Oslo, Norway, 4Faculty of Medicine, University of Oslo, Oslo, Norway Objective: In pregnancy there is an active maternal immunologic tolerance toward the semi-allogeneic fetus. Regulatory T-cells (Tregs) have suppressive properties involved in antigen-specific tolerance of pregnancy by subduing effector T-cell mediated rejection of the fetus. We wanted to characterize different subpopulations of T-cells, particularly Tregs, in the circulation and placental tissue from healthy pregnancies as a basis for investigating possible pathogenic changes in preeclampsia. Methods: Blood and decidua (the placenta at the maternal/fetal interface) samples were obtained from women with healthy pregnancies undergoing elective cesarean section. Peripheral mononuclear cells were isolated from freshly collected blood using gradient centrifugation. Decidual tissue was harvested by suction from the placental bed and mononuclear cells isolated by enzymatic tissue digestion followed by gradient centrifugation. The cells were next fixed, permeabilized and stained with fluorochromecoupled antibodies to CD3, CD4, CD8, CD45RA, FoxP3, CD25, and CD127, and analyzed by flow cytometry with BD FACSCantoÔII. Results: FoxP3 and CD45RA dual staining was used to identify naive and memory T-cells, cytokine-secreting effector T-cells, resting and activated Tregs. The percentage of activated Tregs and cytokine-secreting T-cells were higher in decidua compared to blood (5.4% vs. 1.1%, p<0.01 and 6.5% vs. 2.4%, p<0.01), while resting Tregs conversely were more abundant in blood than in decidua (2.2% vs. 0.3%, p<0.01). The percentage of CD4þ and CD8þ memory cells were significantly increased (70% vs. 45% and 50% vs. 27% of CD4þ and CD8þ T-cells, respectively, both p<0.01) in decidua compared to blood, whereas both CD4þ and CD8þ naive T-cells were more abundant in the circulation. Conclusion: We found clear differences in the populations of activated Tregs and memory T-cells between blood and decidual tissue, and hence we propose that Treg activation status could impact maternal tolerance towards the fetus. We will next extend our Treg investigations to pregnancy complications such as preeclampsia.

Abstracts / Placenta 32 (2011) A1–A149

P2.89 NONCHORIO PLACENTAL MATERNAL FEVER

PATHOLOGIES

ARE

ASSOCIATED

Pradeepkumar Charlagorla, Beata Dygulska, Carolyn Pramod NaruLa New York Methodist Hospital, Brooklyn, NY 11215, USA

WITH

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P2.90 ASSOCIATION OF PLACENTAL INFLAMMATORY CHANGES WITH DEGREE OF MATERNAL FEVER AND ELEVATED CRP IN INFANTS: A GUIDE FOR DURATION OF TREATMENT

salafia,

Background: Only a minority of cases of maternal fever demonstrate histologic evidence of intraamniotic infection, but other placental pathologies (such as infarct and abruption or chronic villitis) may also in theory present as elevated maternal temperature. We tested this hypothesis in a consecutive group of mothers with fever for which placental pathology examination was universally performed. Objective: To determine the placental differences in mothers with diagnosed fever prior to delivery in the presence and absence of histologic chorioamnionitis. Design/Methods: Over a period of 6 months, 61 women were identified with fever, defined as 100.4 F and above, prior to birth. All placentas were sent to the pathology department for review by a single observer blinded to clinical data. Diagnoses of histologic chorioamnionitis, including both maternal and fetal inflammatory responses, infarcts, intervillous thrombi, chronic villitis, abruption and fetal vascular pathology were coded as present/absent. The fetal placental weight ratio was calculated from the trimmed placental weight. Birth weight and gestational age were abstracted from the maternal record. Contingency tables and MannWhitney U tests considered p<0.05 significant. Results: 19 of the 61 (31%) and 34 (56%) patients with fever had histologic fetal inflammatory responses (FIR) or maternal neutrophils beneath or within the chorionic plate, respectively. There were no differences in gestational age, birth weight, or placental weight between those with and without histologic acute inflammatory responses. Of 6 cases with subacute infarcts, 5 (83%) occurred in the cases without FIR. All 4 cases of histologic abruption occurred in the cases without FIR. 4 of the 5 cases with chronic nonspecific villitis and all 6 cases of stem vessel thrombus of fetal were found in cases without FIR. In total, of the 18 cases with infarct, abruption, villitis or fetal vessel thrombus, 16 occurred in cases of mothers with fever but no histologic FIR (p¼0.029). Conclusions: No single placental pathology accounts for maternal fever in the absence of histologic acute inflammation, but 37% of such cases have evidence of tissue injury (infarct, abruption, fetal vessel thrombus) or other inflammatory pathology (villitis) that may account for maternal fever. Placental pathology examination is indicated in all cases of maternal fever.

Pradeepkumar Charlagorla, Catherine Abban, Beata Danathan Hoang, Pramod Narula New York Methodist Hospital, Brooklyn, NY 11215, USA

Dygulska,

Background: Chorioamnionitis (CA) complicates 1 to 10% of all pregnancies, and presents a risk for transmission of pathogens to the neonate. The early diagnosis of infection continues to be a challenging task to neonatologists, as standard hematological tests, such as band count, immature to mature neutrophil ratio (I/T) , and C reactive protein (CRP), have limited predictive values. Maternal fever (MF) is often used as a clinical predictor of infection, but may be affected by other factors such as epidural (EPI) and spinal anesthesia (SE). Histologic CA, the inflammation (IF) of the chorion laeve and amnion, is the most common pathologic finding during placental examination, and may occur in the absence of risk factors or clinical symptoms. More specifically, the invasion of fetal umbilical vessels by inflammatory cells (funisitis), has been reported to have a tighter association with positive signs and symptoms, cultures, and other markers of IF. Objective: To determine whether antepartum MF has a higher association with histologic evidence of IF on maternal and/or fetal surface. To determine whether histologic umbilical vasculitis correlates with CRP and I/T ratio in infants. To determine whether SE/EPI can produce placental inflammatory changes similar to CA. Methods: Study subjects were full-term (FT) babies (n¼62) born to mothers with temperatures >/¼ 38oF. Control subjects (n¼62) were FT infants born to mothers without any fever, matched with gestational age and mode of delivery. Hematological tests were done on all subjects for CBC with differential, I/T ratio, CRP and cultures. Placental tissues were examined for acute inflammatory changes along maternal and fetal membranes in both groups. Maternal records were reviewed for type of anesthesia received (EPI/ SE). Results: There was a significant association of cases vs. controls (fever vs no fever) with maternal IF, chorionic plate IF, and umbilical cord IF (fetal) (p¼<.0001). There was no significant association between EPI/SE and placental IF. The presence of fetal IF was associated with significant increase in CRP above 1 mg/L (3.9 þ/- 2.5 vs. 0.59 þ/- 0.25, p¼.001). I/T ratio >0.2 does not predict placental IF. Conclusions: Acute inflammatory changes in the placenta strongly correlate with degree of antepartum MF and rise in CRP in infants. Maternal anesthesia (EPI/SE) may cause fever, but not placental IF. Finding of histologic funisitis is a reliable marker of congenital infection, and when found in conjunction with an elevated CRP, can be used as guide to initiate antimicrobial therapy in infants born to febrile mothers.

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Abstracts / Placenta 32 (2011) A1–A149

P2.91 PARTICIPATION OF PROTEASES IN THE INFECTION OF HUMAN CHORIONIC VILLI EXPLANTS BY TRYPANOSOMA CRUZI

P2.93 UP-REGULATION OF HYALURONAN SYNTASE 3 GENE EXPRESSION OF TROPHOBLAST BY TOXOPLASMA GONDII

Christian Castillo1, Juan Duaso1, Arturo Villarroel1, Gonzalo Cabrera1, Juan Diego Maya1, Norbel Galanti1, Ulrike Kemmerling1,2 1 Universidad de Chile, Santiago, Chile, 2Universidad de Talca, Talca, Chile

Tatiana Batanova1, Akihiro Unno1, Masahisa Watarai2, Katsuya Kitoh1, Yasuhiro Takashima1 1 Gifu University, Gifu, Japan, 2Yamagichi University, Yamaguchi, Japan

Chagas’ disease, one of the major public health concerns in Latin America, is caused by the haemophlagelated protozoan Trypanosoma cruzi (T. cruzi). In the past few years congenital transmission of T. cruzi has become more important, and partly responsible for the “globalization of Chagas’ disease”, constituting a public health problem of increasing relevance. Diverse pathogens, including T. cruzi, are able to cross the placental barrier and infect both the placenta and fetus. In order to determine the possible participation of proteases as part of tissue invasion mechanism, we analyzed the expression and activity of matrix metalloproteases (MMP-2 and MMP-9) in human chorionic villi as well as the participation of the T. cruzi protease cruzipain in collagen I degradation. T. cruzi trypomastigotes (Y strain) were harvested from infected VERO cell cultures and placentas were obtained from healthy woman. Chorionic villi were incubed in presence or absence of 105 and 106 parasites, with or without protease inhibitors (doxycycline for MMPs, E64 for cruzipain). Effective infection was tested by PCR. Expression of MMPs was determined by western blotting and immunohistochemistry while their activities were measured using the InnoZymeÔ Gelatinase Activity Assay kit (Merck). Effect of MMPs and cruzipain on collagen I organization in villous stroma of chorionic villi was analyzed histochemically with Picro Sirius red staining. T. cruzi induces expression and activity of MMPs. Inhibition of the proteases partially prevents collagen I destruction in the placenta but do not avoid infection of the tissue. We conclude that activation of endogenous and parasitic proteases play a role in the tissue invasion mechanism of human placenta by T. cruzi. Financed by FONDECYT Projects N 11080166 (UK), N 1090078 (JM) and N 1090124 (NG) and CONICYT-PBCT Anillo ACT 112, Chile.

Objectives: The expression of CD44, a hyaluronan receptor, of Toxoplasma gondii infected-leukocytes is higher than non-infected ones. We hypothesized that attachment of the infected maternal leukocytes via hyaluronan molecule in the placenta contributes to vertical transmission.of the parasite. We therefore examined whether maternal infected leukocytes accumulate in the placenta and whether serum of T. gondii infected mice and interferon-g (IFN-g) up-regulate the expression of hyaluronan synthases (Has 1, 2 and 3) genes of trophoblasts. Methods: To facilitate detection of maternal infected leukocytes in the placenta, we established GFPþ/– female mice were mated with GFP–/– male mice confirmed that only the maternally derived tissues showed green fluorescence in the placenta of GFP–/– genotype fetuses. The pregnant mice were inoculated with transgenic T. gondii expressing a red fluorescent protein, DsRED Express. To examine the effect of infected sera and IFN-g, mouse giant trophoblast was cultured in the presence of mouse serum or serially diluted IFN- g. And the expression level of Has 1, 2 and 3 genes was examined by RT-PCR. Result: Aggregated maternal infected cells, green fluorescent cells harboring red parasites, were observed in the non-fluorescent fetus derived tissue area of the placenta. Low concentration (25 ng/ml) of IFN-g and infected serum up-regulated the Has 3 expression of the trophoblast. Neither Has 2 nor Has 3 gene expression was changed. Conclusion: T. gondii infected-leukocytes in the maternal circulation attached to the fetus derived tissue of the placenta. Although it is still controversial whether hyaluronan molecule is synthesized at maternofetal border of healthy individuals, our results suggest that T. gondii infection induce hyaluronan synthesis on the border and facilitate the vertical transmission of the parasite.

P2.92 PLACENTAS FROM WOMEN WITH CHAGAS’ DISEASE PRESENT SIMILAR HISTOPATHOLOGICAL CHANGES AS EX VIVO HUMAN CHORIONIC VILLI EXPLANTS INFECTED WITH TRYPANOSOMA CRUZI Juan Duaso1, Christian Castillo1, Arturo Villarroel1, Gabriela Corral3, Cleo Bosco1, Juan Diego Maya1, Norbel Galanti1, Ulrike Kemmerling1,2 1 Universidad de Chile, Santiago, Chile, 2Universidad de Talca, Talca, Chile, 3 Hospital de Illapel, Illapel, Coquimbo, Chile Chagas’ disease is caused by the haemophlagelated protozoan Trypanosoma cruzi (T. cruzi). Diverse pathogens, including T. cruzi, are able to cross the placental barrier and infect both the placenta and fetus. In ex vivo infected human chorionic villi, the parasite induces detachment and disorganization of the syncythiotrophoblast as well as selective destruction of basal laminae and collagen I. These histopathological and molecular alterations have not been described in placentas from chagasic women. We performed histopathological analysis, immunohistochemical studies of basal lamina (collagen IV, heparansulphate and fibronectin) and histochemical studies of carbohydrate rich molecules and collagen I in the villous stroma of three placentas from chagasic women. Parasites in the placenta were detected by immunofluorescence (Ac anti-flagellar calcium binding protein) and by PCR. The diagnosis of Chagas’disease in women was performed by standard serological tests. “Chagasic” placentas showed similar tissue alterations, both at the histopathological and basal lamina levels, as ex vivo chorionic villi explants infected with T. cruzi. These results confirm the effect of this parasite on human placenta of infected women and validate the ex vivo infection model to study tissue invasion mechanisms. Financed by FONDECYT Projects N 11080166 (UK), N 1090078 (JM) and N 1090124 (NG). Support of CONICYT-PBCT Anillo ACT 112, Chile is also acknowledged.

Abstracts / Placenta 32 (2011) A1–A149

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P2.94 PLACENTAL HEPATITIS B VIRUS INFECTION AND MATERNAL HBEAG STATUS IN HBSAG POSITIVE WOMEN

P2.95 PLACENTAL HEPATITIS B VIRUS INFECTION AND MATERNAL CIRCULATING HEPATITIS B DNA LOAD IN HBSAG POSITIVE WOMEN

Terence Lao1,2, Man-Kin Chung1,2, Stephen Chim1,2, Stephen Suen1,2, Tat-San Lau1,3, Vincent Wong1,3, Henry Chan1,3, Tak-Yueng Leung1,2 1 The Chinese University of Hong Kong, Shatin, Hong Kong, 2Department of Obstetrics & Gynaecology, Prince of Wales Hospital, Shatin, Hong Kong, 3 Department of Medicine & Therapeutics, Prince of Wales Hospital, Shatin, Hong Kong

Terence Lao1,2, Man-Kin Chung1,2, Stephen Chim1,2, Stephen Suen1,2, Tat-San Lau1,3, Vincent Wong1,3, Henry Chan1,3, Tak-Yeung Leung1,2 1 The Chinese University of Hong Kong, Shatin, Hong Kong, 2Department of Obstetrics & Gynaecology, Prince of Wales Hospital, Shatin, Hong Kong, 3 Department of Medicine & Therapeutics, Prince of Wales Hospital, Shatin, Hong Kong

Objective: To study the role of hepatitis B e-antigen (HBeAg) as risk factor for vertical transmission of hepatitis B virus (HBV), we correlated presence of placental HBV DNA with maternal HBeAg and anti-e (HBeAb) status in hepatitis B surface antigen (HBsAg) positive mothers. Methods: HBsAg positive mothers were recruited during antenatal visits or admission for blood sampling (HBeAg, HBeAb, alanine transaminase (ALT), and HBV DNA) and postpartum collection of placental tissue for DNA extraction (QIAamp DNA min-kit, Qiagen) and measurement by quantitative real-time PCR. Placental HBV DNA was expressed as per 200ng of total extracted DNA. Results: HBV DNA was isolated from 14 (38.9%) placentas of the 36 recruited cases. In this group, the mothers were older and delivered at a slightly earlier gestation (Table). Maternal HBV DNA, HBeAg and HBeAb results were available in 29, 33 and 28 cases respectively. Positive HBeAg was significantly associated with placental HBV DNA (OR 20.3, 95% CI 3.10132.25).

Objective: To determine if the risk of placental infection with the hepatitis B virus (HBV) is related to maternal circulating HBV DNA load, and whether there is a threshold of circulating DNA load for placental infection, we examined presence of placental HBV DNA with maternal HBV DNA load measured in the second half of pregnancy in hepatitis B surface antigen (HBsAg) positive mothers. Methods: HBsAg positive mothers, identified by antenatal screening, were recruited during antenatal visits for blood sampling for HBV DNA, and postpartum collection of placental tissue for DNA extraction (QIAamp DNA min-kit, Qiagen) and measurement by quantitative real-time PCR. HBV DNA was expressed as per 200ng of total extracted DNA. Results: The 29 recruited women were categorized by their circulating HBV DNA levels into three groups as follows: (1) <1,000 copies, n¼11 (37.9%), (2) 1,000-100,000 copies, n¼9 (31.0%), and (3) >100,000 copies, n¼9 (31.0%), while placental HBV DNA was found in 12 (41.4%). Incidence of positive placental HBV DNA was correlated with serum DNA load (18.2%, 11.1% and 100% from groups (1) to (3) respectively, chi square test p<0.001, Spearman’s correlation p<0.001). Compared with DNA copies 100,000, DNA copies >100,000 predicted placental infection (OR 6.67, 95% CI 2.35-18.92). Conclusion: HBV DNA was isolated in 41.4% of the placentas, and was strongly associated with maternal serum HBV DNA >100,000 copies. Nevertheless, 25% of the infected placentas were found in women with HBV DNA <100,000 copies. Therefore a low maternal serum HBV DNA load cannot rule out placental HBV infection.

Placental HBV DNA Maternal age (yrs) Delivery gestation (wks) ALT (mmol/l) Male infants (%) Obstetric complications (%) Vaginal delivery (%) Maternal HBV DNA þve (%) Maternal HBeAg þve (%) Maternal HBeAb þve (%)

Positive (n¼14) 32.3  4.0 38.9  1.7 14.8  6.7 64.3 28.6 28.6 100 (12/12) 69.2 (9/13) 88.9 (8/9)

Negative (n¼22) 31.1  6.0 39.2  0.8 17.0  6.5 36.4 22.7 22.7 82.4 (14/17) 10.0 (2/20) 78.9 (15/19)

Difference P¼0.028 P¼0.009 NS NS NS NS NS P<0.001 NS

Results are expressed in mean  SD or % as indicated.

Conclusion: HBV DNA was isolated in 39% of the placentas, and was strongly associated with positive HBeAg, but not HBeAb, status. Nevertheless, negative HBeAg status does not rule out such possibility as HBV DNA was isolated from 10% of the cases.

P2.96 PLACENTAL HEPATITIS B VIRUS INFECTION AND MATERNAL CIRCULATING HEPATITIS B VIRUS DNA LOAD IN HBSAG POSITIVE WOMEN IS INFLUENCED BY INFANT GENDER Man-Kin Chung1,2, Tat-San Lau1,2, Stephen SC Chim1,2, Stephen SH Suen1,2, Vincent WS Wong1,3, Henry LY Chan1,3, Tak-Yeung Leung1,2, Terence T Lao1,2 1 The Chinese University of Hong Kong, Hong Kong, Hong Kong, 2Department of Obstetrics and Gynaecology, Prince of Wales Hospital, Hong Kong, 3Department of Medicine and Therapeutics, Prince of Wales Hospital, Hong Kong Objective: To determine the influence of fetal gender on the risk of placental infection with hepatitis B virus (HBV) and maternal circulating HBV DNA load in singleton pregnancies carried by hepatitis B surface antigen (HBsAg) positive mothers. Methods: HBsAg positive mothers, identified by antenatal screening, were recruited during antenatal visits for blood sampling for hepatitis B eantigen (HBeAg) and anti-e (HBeAb), HBV DNA, and postpartum collection of placental tissue for DNA extraction (QIAamp DNA min-kit, Qiagen) and measurement by quantitative real-time PCR. HBV DNA was expressed as per 200ng of total extracted DNA. The results were related to fetal gender. Results: 14 (53.8%) of the 26 pregnancies with positive maternal circulating HBV DNA and placental HBV DNA measurement resulted in male infants. There was no significant difference between pregnancies with male and female infants in the incidence of positive HBeAg (50.0% vs 25.0%) or HBeAb (81.8% vs 90.0%). However, higher circulating HBV DNA load was associated with male infants (incidence of <100 copies, 100-100,000 copies, and >100,000 copies was 7.1%, 35.7% and 57.1% for males infants vs 0%, 91.7% and 8.3% respectively for female infants, p¼0.014). Pregnancies with male infants also tended to have higher incidence of placental HBV DNA isolation (64.3% vs 25.0%, Fisher’s test p¼0.062, OR 5.40, 95% CI 0.98-29.67). Conclusion: In singleton pregnancies carried by HBsAg positive mothers, those with a male fetus tended to have increased risk of placental HBV infection, which was probably related to the higher circulating HBV DNA load.

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Abstracts / Placenta 32 (2011) A1–A149

P2.97 INCREASED FIBROBLAST GROWTH FACTOR EXPRESSION IN PLACENTAS FROM CMV-INFECTED MICE Randi Gombos, Alexandria Kelly, Denise Hemmings University of Alberta, Edmonton, AB, Canada Introduction: Cytomegalovirus (CMV) is the main source of congenitally acquired infections and is associated with vascular diseases including preeclampsia. Maternal CMV infections are also associated with increased vascular endothelial growth factor expression in human placenta. In latepregnant (LP) C57Bl/6J mice, an active CMV infection resulted in increased endothelial-dependent vasodilation in mesenteric arteries. The uterine arteries from these infected LP mice showed decreased vasodilation and increased vasoconstriction. In CMV-infected LP mice placental weights were reduced with no differences in fetal weights leading to increased fetus-to-placenta ratios. This suggests that placental efficiency is increased to compensate for CMV- induced maternal vascular dysfunction to ensure adequate fetal growth. Objective: To determine if an active CMV infection during pregnancy leads to increased expression of angiogenic factors in the placenta in two mouse models differing in susceptibility to CMV infection. Methods: Balb/C and C57Bl/6J CMV-infected and uninfected mice were bred. Placentas from day 18 (late) pregnant mice were dissected from membranous tissue and wet weights were obtained. Fibroblast growth factor (FGF) localization and expression in placentas were measured with immunofluorescence. Hematoxylin and eosin stain measured placental pathology. Results: Immunofluorescence showed qualitative increases in expression of FGF in the placental vasculature from CMV-infected C57Bl/6J mice and decreased expression in uninfected Balb/C mice with no differences in pathology. CMV-infected Balb/C mice could not sustain their pregnancies at equal (106 PFU) and lesser (5x105 PFU) doses of CMV compared to that used in the C57Bl/6J strain. Conclusion: FGF expression increased in CMV-infected C57Bl/6J mice may compensate for the reduced placental size by increasing angiogenic capacity and the efficiency of the placenta. The more susceptible Balb/C strain was unable to maintain their pregnancy during an active CMV infection. Thus, a maternal CMV infection can affect both placental and fetal development; however, pregnancy outcome depends on susceptibility to the infection.

P2.98 YELLOW FEVER VACCINATION IN MOUSE PREGNANT FEMALES: GESTATIONAL PERFORMANCE AND VIRAL ANTIGEN DISTRIBUTION IN PLACENTA AND FETUSES Fernanda Carini da Silva1, Fernanda Milani Magaldi1, Helena Sato2, Estela Bevilacqua1 1 Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo – SP, Brazil, 2Immunization Program, Department of Health, University of São Paulo, São Paulo – SP, Brazil Background: The potential risk of placental transmission of the 17 DD viruses to the fetus associated with the viral neurotrophism led to the general recommendation that the vaccine against the Yellow Fever (YF) must not be administered during pregnancy, excepted when epidemiologically justified. In spite of that, vaccination can occur in periods the pregnancy has not been confirmed. Objective: This study aimed to evaluate the effect of YF vaccination in the gestational performance in mice correlating these data with the placental structure, immunolocalization of the viral antigen and the viral activity (viral RNA) at the maternal fetal interface and fetal organism. Results: Our results showed a decrease in the rate of fetal viability caused by the vaccine when administered during embryo implantation stage. Maternal liver exhibited discrete YF-reactive areas. The viral antigen has not been found at the maternal-fetal barrier, although reactivity was eventually seen in the fetal liver of normal fetuses, suggesting a transplacental passage. In stillborn fetuses this passage appears to be more intense; the viral antigen was immunolocalized in all placental zones and several fetal organs. Vaccine viral RNA analyzed by RT-PCR as an indicative of viral activity was detected in all fetuses even in those where the viral antigen was only discretely immunolocalized. Conclusions: These findings indicate that there are stages of pregnancy in which fetal viability is more susceptible to the viral vaccine infection. The presence of viral antigen and virus activity in fetal liver suggest the vaccine virus can be transmitted from mother to fetus in spite of the innate barrier represented by the placenta. Financial support: FAPESP, CNPq and CAPES.

Abstracts / Placenta 32 (2011) A1–A149

P2.99 ABNORMAL MATERNAL INFLAMATION IN RATS IS LINKED TO THE DEVELOPMENT OF COAGULOPATHIES ASSOCIATED WITH INTRAUTERINE GROWTH RESTRICTION Tiziana Cotechini, Shannyn K. Macdonald-Goodfellow, Maha Othman, Bani J. Falcon, Charles H. Graham Queen’s University, Kingston, Ontario, Canada Objectives: Inflammation-associated intrauterine growth restriction (IUGR) is characterised by the presence of maternal coagulopathies and placental infarcts. The latter may impede utero-placental blood flow, thereby restricting oxygen and nutrient delivery to the fetus. In the present study we determined whether maternal inflammation in rats during late gestation leads to the development of systemic coagulopathies. Since IUGR is associated with decreased placental nitric oxide (NO) bioavailability, a second objective of this study was to determine whether chronic administration of the NO mimetic nitroglycerin (GTN) prevents inflammation-induced maternal coagulopathies. Methods: To induce chronic inflammation during pregnancy, rats were injected with daily low doses of lipopolysaccharide (LPS) on gestational days (GD) 13.5-16.5 and euthanized on GD 17.5. Citrated blood collected by cardiac puncture at the time of euthanasia was analysed for haemostatic parameters (i.e. clotting time; percent clot lysis) using thromboelastography (TEG). To determine whether the development of systemic maternal coagulopathy is mediated by tumour necrosis factor a (TNFa), the TNFa inhibitor etanercept was injected to pregnant dams on GD 13.5 and 15.5. The effects of GTN on maternal haemostasis were assessed following continuous maternal delivery of GTN via daily application of a transdermal GTN patch (releasing 25 mg/h) on GD12.516.5. Results: Chronic inflammation resulted in various haemostatic alterations. Ninety percent of LPS-treated dams (vs. 33% of saline-treated controls) exhibited coagulopathy. These coagulopathies included hypercoagulability (decreased clotting time), hypocoagulability (increased clotting time), and hyperfibrinolysis. Etanercept administration reduced maternal coagulopathy by restoring clotting time to control levels and preventing hyperfibrinolysis. GTN treatment restored clotting time and decreased hyperfibrinolysis. Conclusion: This study links abnormal chronic inflammation with the development of systemic maternal coagulopathy. Our results provide a rationale for investigating a potential use of immunomodulation or GTN to prevent coagulopathies associated with inflammation-mediated IUGR.

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P2.100 FIRST TRIMESTER TROPHOBLASTS MEDIATE POTENT INFLAMMATORY RESPONSES THROUGH ACTIVATION OF TOLL-LIKE RECEPTORS Line Haugstad Tangerås, Guro Sannerud Stødle, Guro Dalheim Olsen, Astrid Solberg Gundersen, Bjørg Steinkjer, Liv Ryan, Bente Skei, Rigmor Austgulen, Ann-Charlotte Iversen NTNU, Department of Cancer Research and Molecular Medicine, Trondheim, Norway Objectives: Normal pregnancy is associated with a slightly elevated inflammatory state. Pathogenesis of preeclampsia is associated with a more harmful inflammation, and increased levels of several endogenous inflammatory mediators have been reported. However, their action at the molecular level is unclear. Some of these inflammatory mediators, such as HMGB1, heat shock protein 60 (HSP60) and HSP70, have recently been identified by their ability to activate Toll-like receptors (TLRs). The TLRs are critically important sensors for both pathogens and endogenous inflammatory inducers, and activation results in production of pro-inflammatory cytokines. The discovery that trophoblasts express TLRs suggests that trophoblasts play an active role in placental inflammation. The aim of this study was to define TLR-mediated immune activation in primary first trimester trophoblasts, and to determine whether endogenous inflammatory mediators activate trophoblasts through TLRs to produce proinflammatory cytokines. Methods: Primary trophoblasts were isolated from first trimester placentas (5-12 gestational weeks) by enzymatic digestion and LSM gradient centrifugation. The trophoblast choriocarcinoma cell line BeWo and the endothelial cell line HUVEC were included for comparison. Gene expression of TLRs was quantified by real-time quantitative RT-PCR. Cells were stimulated with specific TLR ligands, including endogenous inflammatory mediators (such as HMGB1, HSP60 and HSP70), and the proinflammatory cytokine response was analyzed by Multiplex. Results: Primary first trimester trophoblasts expressed all 10 TLR mRNAs and the expression level varied in trophoblasts isolated from different placentas. TLR activation by both specific TLR ligands and endogenous inflammatory mediators induced a potent pro-inflammatory cytokine response (such as up-regulation of IL-6, IP-10 and VEGF) in primary first trimester trophoblasts. Conclusion: The data show that primary first trimester trophoblasts express functionally active TLRs, and we therefore expect that trophoblasts contribute to placental inflammation by TLR activation.

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Abstracts / Placenta 32 (2011) A1–A149

P2.101 CULTURE NEGATIVE PREMATURITY IS ASSOCIATED WITH DECREASED PLACENTAL TAURINE INDEPENDENT OF THE CLINICAL INDICATION FOR PRETERM BIRTH. Mark Alexander1, Eunkyque Park1, Georgia Schuller-Levis1, Irina Buhimschi2, Catalin Buhimschi2, Carolyn Salafia1,3 1 Institute For Basic Research, Staten Island, NY, United States, 2Yale University School of Medicine, New Haven, CT, United States, 3Placental Analytics, LLC, Larchmont, NY, United States Objective: Taurine is an essential amino acid transported by the placenta which promotes healthy neurological development. The most common pathology underlying idiopathic prematurity, infection excluded, is maternal uteroplacental vascular pathology. We hypothesized that in culture negative preterm births, villous pathology caused by maternal uteroplacental vascular pathology results in altered placental taurine transport and lower placental taurine levels. Methods: Samples from 80 pregnancies were studied. 16 placentas from uncomplicated term pregnancies were compared to 64 preterm placentas obtained from cases of culture negative PTL/PPROM (GA range 22-34 w, n¼34), severe preeclampsia and/or idiopathic abruption (GA range 23-36 w, n¼ 29; abruption, n¼6, þFGR, n¼5). Within the PTL/PPROM group, intra-amniotic and fetal inflammation was assessed by proteomics analysis of amniotic fluid obtained at amniocentesis and cord blood IL-6, respectively. Taurine concentrations were determined in fresh frozen placental villous tissue by HPLC. Statistical tests performed included ANOVA, regression and bivariate and partial correlations, with p<0.05 considered significant. Results: Mean taurine concentrations were 2.70.1 mM, 3.30.1mM and 3.70.2mM in pregnancies delivered from 24-30 weeks, 30þ-36 weeks, and term, respectively (p<0.001). Linear regression showed a direct linear (r¼0.51, p<0.0001) relationship between placental taurine and GA which was independent of the clinical indication for preterm birth. (Figure 1) Among women with culture negative PTL/PPROM, taurine level was not independent of measures of intra-amniotic or fetal inflammation. Conclusions: Our data show that placentas delivered preterm and culture negative– regardless of clinical indication (PTL/PPROM, abruption, severe preeclampsia or fetal growth restriction)– have reduced placental taurine levels. Placental taurine levels have been reported to be identical in normal first and third trimester placentas (Placenta 1994;15:747-751); thus we speculate that our findings are not simply a maturational effect but reflect effects of the pathology(ies) causing culture negative preterm birth on placental taurine transport. Culture negative PTL/PPROM and abruption are each associated with maternal uteroplacental vascular pathology that is histologically indistinguishable from that seen in preterm PE and FGR. We are currently analyzing villous histology and capillary geometries to determine whether there is a structural villous correlate to decreased placental taurine levels, which could serve as a biomarker for neurodevelopmental risk.

P2.102 CORRELATION BETWEEN PLACENTAL BIOMETRY AND FETO-MATERNAL PARAMETERS IN NORMAL PREGNANCIES S. Calabrese, C. Mando’, E. Zaloshvili, M. Figus, L. Ferrante, M. Mazzocco, M.A. Marino, F. Parisi, I. Cetin Unit of Obstetrics and Gynecology and Center for Fetal Research Giorgio Pardi, Dept of Clinical Sciences L. Sacco, University of Milan, Milan, Italy Objectives: Low birth and placental weight has been associated with an increased risk of chronic diseases in adulthood. Barker et al have recently shown that a decrease in placental surface area is linked to higher risk of adult hypertension. We studied placental biometry and feto-maternal parameters in singleton pregnancies and their prediction on birthweight as a measure of fetal well-being. Methods: 348 placentas were collected from normal pregnancies, 13 from pregnancies complicated by gestational diabetes and 11 from pregnancies complicated by “placental insufficiency” (preeclampsia and intrauterine growth restriction). Maternal data (weight and height), fetal data (weight, cranial circumference and length) and gestational age were collected. After removing fetal membranes and umbilical cord, the larger and the smaller diameters were measured and placental weight obtained. With the hypothesis of an elliptical placental shape the surface area was calculated as: larger diameter X smaller diameter X p/4. Results: In the control group the bivariate analysis demonstrated that 5 variables (placental weight and surface area, gestational age, maternal height and weight) are statistically able to predict birthweight. In the multivatriate analysis 4 variables (placental weight and surface area, gestational age, maternal height) predicted birthweight independently. Mean placental weights were 421gr, 455 gr and 367 gr in the control, “diabetic” and “placental insufficiency” groups, respectively. Mean placental surface areas were 271 cm2, 274 cm2 and 243 cm2 in the control, “diabetic” and “placental insufficiency” groups, respectively. Conclusion: Placental surface area contributes significantly to birthweight independently from placental weight. Larger placental surface area means better fetal-maternal exchange. After adjusting for gestational age, a tendency of having larger placental surface area and greater placental weight was present in the “diabetic” group, while smaller placental surface area and lighter placental weight were present in the “placental insufficiency” group compared to controls.

Abstracts / Placenta 32 (2011) A1–A149

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P2.103 IMPACT OF A MATERNAL HYPERLIPIDIC HYPERCHOLESTEROLEMIC DIET ON PLACENTAL FUNCTION, IN A RABBIT MODEL.

P2.104 THE PLACENTAL RESPONSE TO EXCESS MATERNAL GLUCOCORTICOID EXPOSURE DIFFERS BETWEEN THE MALE AND FEMALE CONCEPTUS.

Anne Tarrade1,2, Marie-Christine Aubrière1,2, Olivier Morel1,2, Nicole Charpentier1,2, Pascale Chavatte-Palmer1,2 1 INRA, UMR 1198 Biologie du développement et reproduction, Jouy en Josas, France, 2Fondation PremUp, Paris, France

Hayley Dickinson1, Bree O’Connell1, Karen Moritz2, Claire Roberts3, David Walker1 1 Monash Institute of Medical Research, Melbourne, Australia, 2University of Queensland, Brisbane, Australia, 3University of Adelaide, Adelaide, Australia

Objectives: A hyperlipidic hypercholesterolemic diet in prepubertal rabbits has been shown to restrict fetal growth and increase offspring susceptibility to obesity. To better understand this IUGR phenotype, placental function has been explored. Methods: Female rabbits were fed with a control diet (C) or a high fat diet (HF) (6% of soybean oil and 0.2% cholesterol) from 10 weeks of age, throughout pregnancy. At D28 of gestation, they were anesthetized and a laparotomy was performed. Blood was drawn from fetuses for total cholesterol and triglycerides measurements. Placentas were collected for transmission electron microscopy and for gene expression including lipid and cholesterol metabolism using q-PCR. Results: At 28 days, fetal weight and the fetal/placenta ratio were significantly lower in the HF group compared to the C group. Interestingly, total cholesterol concentrations, in fetuses, were not statistically different in C and HF, whereas triglyceride concentrations were increased significantly in HF fetuses. The histological analysis of placentas revealed an abnormal accumulation of light vesicles localized in the trophoblast layer of the HF group, which were subsequently shown by ultrastructural analysis to be lipid droplets. Transcriptomic analysis of lipids and cholesterol metabolism indicated that the maternal HF diet lead to a significantly decrease in the expression of LDL-receptor, CD36 and LXR-alpha in placenta, while genes such as PPARgamma, FATP-4, adipophiline, HMG-coA reductase, SREBP-2 were not affected. Conclusion: Maternal HF diet induced a dyslipidemia in IUGR fetuses, associated with numerous lipid droplets and a modulation of genes implicated in lipid and cholesterol homeostasis in the placenta. It is interesting to note that cholesterol metabolism appears to be preferentially down regulated compared to fatty acids metabolism. Work is ongoing to confirm and validate these data.

Objectives: Excess maternal glucocorticoids are known to cause significant developmental challenges for the fetus and these can vary depending on the sex of the offspring. Here we investigate the effects of excess maternal glucocorticoids on the placenta with a specific focus on fetal sex, placental region, and time after glucocorticoid exposure. Methods: Dams were exposed to dexamethasone, (DEX; 125mg/kg) or saline (75ml) for 60h via osmotic pump from mid-gestation (d20; term is d39). Placentas were collected immediately after treatment (d23) and just before term (d37), and processed for histology or qPCR. Relative sizes of labyrinth and junctional zone (JZ) were determined and expression of Bmp4, Tgfb1, Wnt4, Gremlin, Bax, Bcl2, Vegfa, Vegfr2, Gcm1, and Map2k1 genes was quantified. Results: DEX exposure altered placental structure and mRNA expression of male and female fetuses both immediately (d23) and two-weeks post treatment (d37). The immediate consequences (d23) were similar between males and females with most changes observed within the labyrinth region, including decreases in the expression of Igf1, Igflr and Glut1 after DEX exposure. At day 37, the response of the placenta was dependant on the sex of the fetus and included transcriptional and structural changes. At d37, placentas of male fetuses had increased expression of Gcm1 and a decrease in Glut1 expression with an increase in maternal blood sinusoids, whereas in the female placenta increases in Glut1 and Map2k1 expression were observed with a decrease in maternal blood sinusoids. Conclusion: Maternal DEX exposure for 60h altered gene expression and structural development of the placenta, in a region, sex and time dependent manner. We propose that the different placental responses of males and females observed here, relates to the different strategies of the male and female fetus to maternal or intrauterine stressors.

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Abstracts / Placenta 32 (2011) A1–A149

P2.105 A RETROSPECTIVE PLACENTAL ANALYSIS OF NEWBORN INFANTS REFERRED FOR NEUROLOGICAL CONSULTATION: EARLY ROOTS OF NEURODEVELOPMENTAL RISK? Michael Presta1, Romaine Schubert1, Samantha VanHorn1,2, Pramod Narula1, Sanford Lederman1, Carolyn Salafia1,3 1 New York Methodist Hospital, Brooklyn, NY, United States, 2Institute For Basic Research, Staten Island, NY, United States, 3Placental Analytics, LLC, Larchmont, NY, United States Background: The correlation of placental pathology and poor neurological exam scores in newborns has not been studied. Specific lesions (e.g. fetal thrombotic vasculopathy, chorioamnionitis) have demonstrated associations with later poor outcomes. The objective of this study was to explore correlations of newborn neurologic exam score with placental shape and gross and histologic placental lesions. Methods: All newborn neurological consults at New York Methodist Hospital from 2007-2009 period (N¼59) were reviewed retrospectively. Three factors (gestational and early newborn medical history, general physical and neurological exam, and early CNS imaging) were scored as 1 (normal)- 3 (greatest likelihood of long-term neurodevelopmental impairment). The summed neurological score ranged from 3 to 9. Placental pathology reports were extracted for variables related to placental shape, infection, infarct and fetal vascular pathology (among others), by a single reviewer blinded to clinical data, and analyses were adjusted for birthweight. Regression considered p<0.05 significant. Results: As the neurological score increased, the umbilical cord insertion was more eccentric (p¼0.01), and both the number of infarcts in the placenta (p<0.0001) and the incidence of avascular villi (marking small fetoplacental vessels obliterated at least 5-7 days before birth, p¼0.02) increased. Placental thickness tended to decrease as the neurological exam score increased (p¼.05); chorionic surface dimensions showed no correlation (p>0.030). Chronic villitis and histological evidence of acute intraamniotic infection were not correlated with abnormal neurological score. Conclusion: Abnormal newborn neurological scores are associated with placental infarcts (a maternal vascular lesion) and avascular villi, the most common type of fetal vascular pathology in our data set, independent of birth weight. The lack of correlation of other types of fetal vascular pathology may have been due to their low incidence in this population. Eccentric cord insertion indicating early asymmetry in placental growth, and a trend toward reduced placental thickness, indicating reduced chorionic arborization, was also shown, suggesting that poor newborn neurologic function and future risk has its roots remote to delivery.

P2.106 THE LACK OF THE HDL RECEPTOR SR-BI AFFECTS EMBRYO CHOLESTEROL CONTENT AND NEURAL TUBE CLOSURE IN MICE Nicolás Santander, Andrea Leiva, Susana Contreras, Attilio Rigotti, Dolores Busso Pontificia Universidad Católica, Santiago, RM, Chile Objectives: The HDL receptor SR-BI participates in selective lipoprotein cholesterol uptake. Half of SR-BI-/- mice die before weaning, suggesting a role for SR-BI in embryo development. In this work, we studied: 1) the temporal morphology and demise of SR-BI-/- embryos during development; 2) the cholesterol content in SR-BI-/- embryos and 3) the localization of SR-BI in embryos and extraembryonic tissues. Methods: Embryos from C57BL6/129 SR-BIþ/- mice intercrosses were dissected at E9.5, E12.5 and E16.5 and individually genotyped by PCR. Embryos, yolk sacs and placentas were fixed, dehydrated and embedded in paraffin, or snap frozen and cryopreserved. Histological sections of SR-BIþ/ þ and SR-BI-/- embryos and placentas were stained with hematoxylin/ eosin and subjected to immunohistochemistry with anti-SR-BI. Cryopreserved embryos and tissues were lysed and immunoblotted with antiSR-BI or used to determine cholesterol content. Results: The proportions of genotypes at E9.5, E12.5 and E16.5 were similar to the Mendelian ratio. Interestingly, half of SR-BI-/- embryos showed neural tube closure defects (NTCD) at E9.5 and exencephaly at E12.5 and E16.5. Abnormal brain structure and neuroepithelium exposure to the amniotic fluid were demonstrated. Immunohistochemical studies showed strong and specific SR-BI staining in E9.5 giant trophoblast cells and in E12.5 placental labyrinth trophoblasts. Lysates from incipient placentas at E9.5 showed significant signal for SR-BI. SR-BI was not detected in embryos. In agreement with the localization of SR-BI and its role in cholesterol transport, E12.5 SR-BI-/- embryos showed significantly reduced body cholesterol contents (7.80.5 vs 11.50.8 mg/mg protein in SR-BI-/- vs SR-BIþ/þ, n¼5, p<0.05). Conclusion: The lack of SR-BI in extraembryonic tissues in mice reduces the cholesterol content of developing embryos and induces NTD/exencephaly with a 50% penetrance, what most likely explains the demise of SRBI-/- mice before weaning.

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P2.107 EXPRESSION PATTERN OF ANANDAMIDE-BINDING RECEPTORS IN RAT PLACENTA

P2.108 N-ACYLETHANOLAMINE LEVELS AND EXPRESSION OF RESPECTIVE METABOLIZING ENZYMES IN RAT PLACENTA

B.M. Fonseca1,2, G. Correia-da-Silva1,2, A.H. Taylor3, J.C. Konje3, N.A. Teixeira1,2 1 Departamento Ciências Biológicas, Laboratório de Bioquímica, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal, 2Instituto de Biologia Molecular e Celular da Universidade do Porto (IBMC), Porto, Portugal, 3 Endocannabinoid Research Group, Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, United Kingdom

B.M. Fonseca1,2, G. Correia-da-Silva1,2, A.H. Taylor3, P.M.W. Lam3, T.H. Marczylo3, J.C. Konje3, N.A. Teixeira1,2 1 Departamento Ciências Biológicas, Laboratório de Bioquímica, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal, 2Instituto de Biologia Molecular e Celular da Universidade do Porto (IBMC), Porto, Portugal, 3 Endocannabinoid Research Group, Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, United Kingdom

Objectives: In the last decade, the endocannabinoid system (ECS) has emerged as an endogenous signalling system, which mimetic the effects of Δ9-Tetrahidrocanabinol (THC), the main active component of Cannabis sativa plant. Although the detrimental effects of Cannabis sativa consumption during pregnancy are known for years, the underlying mechanisms have not been established. Endocannabinoids act through two G protein-coupled receptors, the cannabinoid receptors CB1 and CB2. However, anandamide (AEA), the main endocannabinoid, can also interact with other receptors, namely the transient receptor potential vanilloid 1 (TRPV1). Here, we examined the distribution of both cannabinoid and TRPV1 receptors in the rat placenta throughout pregnancy. Methods: Uterine horns were collected from days 14, 16 and 19 of rat pregnancy and prepared for routine histology. For quantitative RT-PCR and western blot, placentas were gently dissected. Results: Our data indicates that all the three receptors are expressed in rat placenta throughout pregnancy. However, protein analysis indicated that expression of both cannabinoid and TRPV1 receptors was stronger on day 14, compared to days 16 and 19. On the other hand, immunohistochemistry revealed that both cannabinoid receptors were found to be expressed in giant trophoblasts and spongiotrophoblasts on day 14, while TRPV1 was confined to giant trophoblast cells and syncytiotrophoblasts. Subsequently, and contrary to the cannabinoid receptors, TRPV1 also revealed positivity in the glycogenic trophoblast cells. Conclusion: The spatial and temporal pattern of expression for endocannabinoid receptors and TRPV1 suggest that placenta is a site of action for endocannabinoids supporting a role for endocannabinoid system during the period of placental development.

Objectives: Several works have described the detrimental effects of Cannabis consumption on pregnancy outcomes and the potential involvement of endocannabinoid signalling system in recurrent miscarriage and poor pregnancy outcomes. Anandamide (AEA) was the first endogenous cannabinoid described. Endocannabinoid system has been involved in a wide variety of actions, including the synchronization between the embryo and the uterus, acting as a regulator of the ‘‘implantation window’’. Consistently, high AEA systemic levels and low FAAH expression, the AEA-degrading enzyme, in circulating lymphocytes were associated with spontaneous miscarriage. Methods: In this work, the levels of AEA and two other N-acylethanolamines, N-oleoylethanolamine and N-palmitoylethanolamine, were measured in rat placenta between days 14 and 19 of pregnancy by ultraperformance liquid chromatography tandem mass spectrometry. The spatiotemporal expression of N-acylethanolamine metabolizing enzymes (NAPE-PLD , FAAH and Cox-2) was also determined by quantitative PCR, Western blot, and immunohistochemistry. Results: It was observed that placental levels of all three N-acylethanolamines fluctuate throughout pregnancy. While AEA levels increased and OEA levels decreased by the end of pregnancy, PEA levels were maintained unchanged. Q-PCR analysis revealed a decrease to the end of pregnancy only for cox-2 mRNA. On the other hand, FAAH expression was found unchanged, whereas on day 19 was observed a significant increase on NAPE-PLD expression, contrary to COX-2 expression. Immunohistochemistry showed NAPE-PLD expression on giant trophoblasts, spongiotrophoblast and syncytiotrophoblasts, while FAAH was also positive in the glycogenic trophoblast cells. COX-2 was only found to be expressed in giant trophoblasts and syncytiotrophoblast. Conclusion: Our results suggest that placental levels of endocannabinoids may be primarily regulated in situ,being NAPE-PLD the main contributor for AEA levels in rat placenta.

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Abstracts / Placenta 32 (2011) A1–A149

P2.109 EZRIN ORGANIZES A SUPRAMOLECULAR COMPLEX CONTROLLING COMMUNICATION THROUGH GAP JUNCTIONS DURING TROPHOBLAST CELL FUSION

P2.110 HEPARIN INTERFERES WITH THE TRANSCRIPTION-FACTOR NF-kB THEREBY MODULATING TNF-a-MEDIATED EFFECTS IN THE HUMAN ENDOMETRIUM

Guillaume Pidoux1,2, Pascale Gerbaud1,2, Jim Dompierre3, Birgitte Lygren4,5, Therese Solstad4,5, Danièle Evain-Brion1,2, Kjetil Tasken4,5 1 Inserm U767, Paris, France, 2PremUP, Paris, France, 3Inserm U895, Paris, France, 4The Biotechnology Centre of Oslo, Oslo, Norway, 5Centre for Molecular Medecine Norway, Oslo, Norway

Julia Spratte1, Henriette Meyer zu Schwabedissen2, Nicole Endlich3, Marek Zygmunt1, Herbert Fluhr1 1 University of Greifswald, Deaprtment of Obstetrics and Gynaecology, Greifswald, Germany, 2University of Greifswald, Institute of Pharmacology, Greifswald, Germany, 3University of Greifswald, Institute of Anatomy and Cell Biology, Greifswald, Germany

Objectives: The cell-cell fusion processes are complex biological phenomena. A limited number of human cell types can fuse to form a multinucleated syncytium. In the differentiation of human placenta, mononuclear cytotrophoblasts aggregate and fuse to form an endocrinologically active, non-proliferative, multinucleated syncytiotrophoblast, but the molecular events underlying this process remain poorly understood. Methods: Molecular biology and biochemistry experiments were performed on human trophoblastic cells purified from placentas obtained after caesarean sections. Cultured of human cytotrophoblastic cells were used to follow in vitro the fusion and the formation of a multinucleated syncytiotrophoblast. Results: We confirmed the involvement of the cAMP/PKA signaling pathway in syncytial formation and demonstrate the implication of AKAPs into this process by cellular delivery of PKA anchoring disruptor peptides. Moreover, we used a chemical proteomics approach to identify AKAPs expressed in trophoblastic cells. RNA interference demonstrated that ezrin was one of the AKAPs required for hCG regulation of the cell-cell fusion process. By a variety of immunoprecipitation and immunolocalization experiments, we showed that Ezrin organizes a supramolecular complex containing PKA, connexin 43 (Cx43) and ZO1, two proteins previously shown to be involved into cell fusion. Conclusion: By a combination of siRNA-mediated knockdown, reconstitution experiments using ezrin with or without the ability to bind PKA and PKA anchoring disruptor peptides, we demonstrated that ezrin-mediated coordination of PKA and Cx43 localization is necessary for discrete control of Cx43 phosphorylation by PKA which enhances GAP junctional communication leading to trophoblastic cell-cell fusion.

Objectives: Tumor necrosis factor (TNF)-a is a pro-inflammatory T helper (Th)-1 cytokine influencing the cytokine milieu in the human endometrium and playing a role in the pathophysiology of implantation disorders. Heparin and low molecular weight heparins (LMWHs) seem to be effective in the treatment of women suffering from recurrent miscarriages even though they are not afflicted with thrombophilic disorders. We investigated the impact of heparins on TNF-a-effects in human endometrial stromal cells (ESCs) beyond their classical anti-coagulatory function. Methods: ESCs were isolated from hysterectomy specimens and decidualized in vitro by incubating the cells with progesterone and 17b-estradiol for 9 days. After incubating the cells with TNF-a, heparin LMWHs, signalling- and transporter-inhibitors, interleukin (IL-) 6 and -8 were analysed by real-time RT-PCR and ELISA. The NF-kB signalling pathway was further investigated by flow cytometry, confocal fluorescence microscopy, nuclear transcription factor assays and luciferase-based reporter-assays. In addition, the cellular uptake of heparin was characterized. Results: Heparins inhibit the TNF-a-induced, NF-kB-mediated expression and secretion of IL-6 and -8 in ESCs. Thereby heparin has no influence on the activation of NF-kB in the cytoplasm, but diminishes the transcriptional activity of NF-kB in the nucleus by interfering with the binding of NF-kB to the DNA. OAT (organic anion transporters) like OAT6 and stabilin1 might play a role for the cellular uptake of heparin in ESCs. Conclusion: Heparins inhibit TNF-a-induced effects by interacting with the NF-kB signalling pathway on the transcriptional level. Therefore heparin has to be taken up into the cell by active transporters. These results underline the concept of heparin and LMWHs as modulating agents in ESCs beyond their classical anti-coagulatory properties. Based on these observations heparins might be interesting anti-inflammatory agents for the treatment of women suffering from implantation disorders.

Abstracts / Placenta 32 (2011) A1–A149

P2.111 THE UNDERLYING MECHANISMS INVOLVED IN AEA-DUAL EFFECT OBSERVED IN DECIDUAL CELL DEATH B.M. Fonseca1,2, G. Correia-da-Silva1,2, M. Almada1, B. Macedo2, N.A. Teixeira1,2 1 Departamento Ciências Biológicas, Laboratório de Bioquímica, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal, 2Instituto de Biologia Molecular e Celular da Universidade do Porto (IBMC), Porto, Portugal Objectives: Endocannabinoids (ECS) are a new family of lipid mediators involved in various physiological processes, including reproduction. Anandamide (AEA), the main endocannabinoid, has been reported to have pleiotropic effects on reproduction, but the mechanism by which it exerts these effects is unclear. We have previously shown that AEA induces cell death in a dose-dependent manner. While lower concentrations induced DNA fragmentation, nuclear condensation and upregulation of caspase-3/7 activities, with involvement of cannabinoid receptor 1 (CB1), the higher concentrations induced a dramatic effect in cell viability and morphology, independent of cannabinoid receptors. The aim of this study was to elucidate the pathways involved in the dual effect induced by AEA in vitro. Methods: In that way, we have quantified ceramide levels by HPLC-MS/MS after AEA treatment, using primary decidual cell cultures obtained from day 10 pregnant rats. Moreover, we also studied the production of reactive oxygen species (ROS) and the involvement of AEA-transporter by using NAcetyl-cysteine (NAC) and N-(4-hydroxyphenyl)-Arachidonoyl Amide (AM404), respectively. Results: We found that AEA in lower concentrations (10 mM) induced a significant increase in ceramide levels, effect inhibited by the pretreatment with the CB1 receptor antagonist. On the other hand, the effects induced by the higher concentrations (25 mM) were abolished by NAC pre-treatment. Interestingly, AM404 did not prevent the activation of the signalling pathways induced by AEA in decidual cells. Conclusion: The results suggest that AEA- induced apoptosis of decidual cells could be mediated by ceramide and in that way AEA may interfere with pregnancy progression, effect dependent on the activation of CB1 receptor. On the other hand, aberrant concentrations of AEA may also impair normal pregnancy. NAC pre-treatment can improve decidual cell viability suggesting that oxidative stress may play a causal role in the detrimental effects induced by Cannabis-derivatives during pregnancy.

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P2.112 INCREASED LEPTIN AND LEPTIN RECEPTOR EXPRESSION, SIGNAL TRANSDUCTION ACTIVATION AND PROTEIN SYNTHESIS IN PLACENTA FROM PREGNANT WOMEN WITH GESTATIONAL DIABETES MELLITUS Antonio Pérez-Pérez1, Flora Sánchez-Jiménez1, Julieta Maymó2, Yésica Gambino2, José Luis Dueñas1, Cecilia Varone2, Víctor Sánchez-Margalet1 1 Macarena University Hospital, Seville, Spain, 2Buenos Aires University, Buenos Aires, Argentina Gestational diabetes is the most frequent pathophysiological process associated with pregnancy, which increases the risk for perinatal morbidity and mortality. These patients have insulin resistance and high plasma levels of insulin and leptin. Placentas from gestational diabetes suffer from structural and functional changes including overgrowth. Objectives: Since we have recently found that leptin stimulates protein synthesis by activating protein signaling machinery, we aimed to study the expression of leptin and leptin receptor, as well as the leptin receptor activation that may correlate with protein synthesis in placenta from pregnant women with gestational diabetes in comparison with placenta from normal pregnancy. Methods: We have studied ten placentas from normal pregnancy and ten from patients with gestational diabetes. We measured leptin and leptin receptor expression by quantitative real time-PCR and western blot. We studied leptin receptor signaling by immunoblot using antibodies that recognizes activation of signaling proteins, ie. STAT-3, ERK, PKB, and the activation of different proteins of the initiation stage of translation (S6 Kinase, EIF4EBP1 and EIF4E), and the rate of protein synthesis was assessed by [3H]leucine incorporation experiments. Results: We have found that leptin and leptin receptor are upregulated, and the translation machinery is activated in placentas from gestational diabetes compared with normal pregnancies. Moreover, protein synthesis rate was also found to be increased in placentas from gestational diabetes. Conclusion: These results provide new data to understand the molecular mechanisms underlying the increased growth of placenta in gestational diabetes.

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Abstracts / Placenta 32 (2011) A1–A149

P2.113 ACTIVATION OF PLACENTAL MAMMALIAN TARGET OF RAPAMYCIN SIGNALING IN OBESE WOMEN

P2.114 PLACENTAL C-JUN N-TERMINAL KINASE SIGNALING IS INHIBITED IN MATERNAL OBESITY AND LABOR

Francesca Gaccioli, Thomas Jansson, Theresa Powell Center for Pregnancy and Newborn Research, Department of Obstetrics and Gynecology, University of Texas Health Science Center, San Antonio, TX

Susanne Lager, Thomas Jansson, Theresa L. Powell University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States

Objectives: Obese women have an increased risk to deliver a large-forgestational age baby and enhanced placental nutrient transport capacity may contribute to fetal overgrowth in these pregnancies. Insulin/IGF-I and mammalian target of rapamycin (mTOR) stimulate placental amino acid transport. We tested the hypothesis that placental insulin/IGF-I and mTOR signaling are activated by maternal obesity. Methods: Placentas were obtained from 25 normal pregnancies at term, 11 women with normal BMI (21.3  0.64; birth weight: 3404  91.4 g) and 14 women with high BMI (32.8  1.36; birth weight: 3583  135.9 g). The protein expression (phosphorylated and total) of key signaling molecules was determined using Western blot analysis. The expression of phosphoAkt (Thr308) was used as an indicator for insulin/IGF-I signaling. mTOR Complex 1 (mTORC1) activation was assessed by 4E-BP1 (Thr36/47 and Thr70) and rpS6 (Ser235/236) phosphorylation. Phosphorylation of Akt (Ser473) was used as functional readout for mTOR Complex 2 signaling. Furthermore, activation of AMPK, a negative regulator of mTORC1, was determined by the expression of phospho-AMPK (at Thr172). Group differences were analyzed by student’s T-test. Results: Phosphorylation of placental 4E-BP1 was increased in high BMI women compared to normal BMI both at Thr36/47 (2.17-fold increase, P¼0.004) and at Thr70 (3.07-fold increase, P¼0.003). The expression of phosphorylated rpS6, Akt and AMPK did not differ significantly between groups. Total protein expression 4E-BP1, rpS6, Akt and AMPK was not affected by maternal obesity. Conclusion: These results suggest that maternal obesity is associated with an activation of placental mTORC1, which appears not to be due to increased insulin/IGF-I signaling or inhibition of AMPK. Further studies are needed to establish if mTORC1 activation results in increased protein synthesis and up-regulation of amino acid transport in the placenta of obese women.

Objectives: The effects of maternal obesity on the placenta are largely unknown. Obese women have elevated levels of pro-inflammatory cytokine and lipids, which are well-established stimulators of the inflammatory c-Jun N-terminal kinase (JNK) signaling pathway. We hypothesized that (1) fatty acids stimulate trophoblast JNK signaling in vitro and (2) placental JNK is activated in maternal obesity. Methods: Human, term cytotrophoblast cells were cultured in presence or absence of 400 mM oleic acid between 66 and 90 hours after plating. Term placentas were collected from lean (pre-pregnancy body mass index (BMI) 21.50.6 kg/m2, n¼12) and obese (BMI 32.71.3 kg/m2, n¼15) women. Protein expression of total and phosphorylated JNK (Thr183/Tyr185) in total cell lysates and placental homogenates was determined by western blot. Results: Oleic acid increased JNK phosphorylation in cultured trophoblast cells (p<0.05, paired t-test, n¼5) while total JNK expression was unaffected. Placental JNK phosphorylation did not differ between lean and obese women (n¼27). However, when examining the effect of labor we found that labor decreased placental JNK phosphorylation in lean women (p<0.05, t-test, n¼12). Furthermore, placental JNK phosphorylation and maternal pre-pregnancy BMI were inversely correlated in non-laboring women (r¼ -0.521, p<0.05, n¼18). Total JNK expression was unaffected by labor and maternal BMI. Conclusion: In agreement with our hypothesis, oleic acid increased trophoblast JNK phosphorylation in vitro. Unexpectedly, both maternal obesity and labor inhibited placental JNK signaling. The underlying mechanisms remain to be established but may be related to low maternal adiponectin levels in obesity as adiponectin has been reported to activate JNK. Furthermore, hyperglycemia attenuates JNK signaling in muscle cells and we speculate that increased glucose levels, often observed in obesity and in labor, could contribute to placental JNK inhibition in these conditions.

Abstracts / Placenta 32 (2011) A1–A149

P2.115 ONTOGENY OF HUMAN AQUAPORINS THROUGHOUT PREGNANCY

IN

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MEMBRANES

Cécile Prat1, Damien Bouvier1, Alain Herbet1, Denis Gallot1,2, Geoffroy Marceau1, Vincent Sapin1, Loïc Blanchon1 1 GReD UMR 6247 CNRS INSERM U931, Clermont-Ferrand, France, 2 Obstetric and gynecology, Hopital Estaing, Clermont-Ferrand, France Objectives: As placenta, human amniotic membranes are transitory but essential fetal partners during pregnancy. Among the numerous functions of these membranes (such as fetal protection or hormone production), the control of the amniotic liquid homeostasis is one of the most important. The aquaporins (trans-membrane water channels) were already described to be strongly implicated on this phenomenon. To better understand their implications throughout all the pregnancy, the purpose of our study was to determine the ontogeny of these different family protein members. Methods: Fetal membranes were provided by the Maternity of Estaing Hospital (Clermont-Ferrand, France) after informed consent of the patients. Eleven different fetal membranes from the first, second and third trimester of pregnancy were included in our experiments. Total mRNAs and proteins were extracted from total membranes and isolated amnion and chorion. QPCR and Western-blot experiments were used to determine the presence of AQPs and to quantify theirs spatio-temporal expression patterns throughout pregnancy. All statistic analysis were conducted using ANOVA test with confidence intervals of p<0.05 to p<0.001. Results: At term and during all the pregnancy, we determined that five different AQPs were always expressed in total fetal membranes and isolated chorion and amnion. Furthermore, we demonstrated that the expression of each of these five aquaporins is spatially and timely specific throughout human gestation at the mRNA and protein levels. Conclusion: Our results are the first exhaustive demonstration of the aquaporins ontogenesis during the human pregnancy and establish that their levels of expression are location and trimester specific. Furthermore, our results confirmed that these water channel proteins could be sharply involved in the regulation of liquid amniotic homeostasis throughout pregnancy and suggested that abnormal expression could occur at anytime of the pregnancy to generate obstetrical pathologies such as polyhydramnios or oligohydramnios.

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P2.116 COMPARATIVE ANALYSIS OF DIFFERENT CANCER/TESTIS ANTIGEN EXPRESSIONS IN NORMOTENSIVE AND PRE-ECLAMPTIC PLACENTAE Anushuya Tamang, Mathieu Morgan, Shiva Das Sivasubramaniam Nottingham Trent University, Nottingham NG11 8NS, UK Objectives: This study aimed to compare the expressions of five novel cancer-testis (CT) antigens namely BUC11, NY-ESO-1, T21, T128 and HAGE. The process of placentation in humans has been found to resemble tumorigenesis in cell invasion, escape from immune system and high cell proliferation. However placenta still operates as a normal organ. Therefore understanding the similarities and the differences between placentation and tumorigenesis would help in forming different therapeutic modalities against cancer. CT antigens are proteins expressed specifically by cancer cells; but not usually expressed in normal cells. Some of these antigens have recently been shown to be expressed in human placentae. However there are no detailed studies to evaluate the relative expressions in normal (normotensive; NT) and in placental diseases such as pre-eclampsia (PE). Methods: The expressions of these CT antigens were investigated in 13 normotensive (NT) and 12 pre-eclamptic (PE) placental samples using quantitative real time PCR (qRT-PCR) techniques. Results: The data have shown that some of these CT antigens; such as BUC11 has low expression in placenta. On the other hand others like T128 and HAGE are highly expressed in both NT and PE placentae. Most importantly the expression of HAGE in PE placentae was found to be significantly higher [median expression relative to house keeping genes (MER) ¼ 0.096) than in NT placentae (MER ¼ 0.046) (p<0.05). Conclusions: These results suggest that most of the CT antigens are also expressed in both NT and PE placentae. Moreover the reduced expression of HAGE PE placentae may be linked to their reduced invasive properties.

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Abstracts / Placenta 32 (2011) A1–A149

P2.117 GLOBAL PLACENTAL GENE EXPRESSION PROFILING IN THE FIRST AND THIRD TRIMESTERS OF NORMAL HUMAN PREGNANCY

P2.118 FUNCTIONAL GENOMIC INVESTIGATION OF EUTHERIAN TROPHOBLAST EVOLUTION

Vasilis Sitras1,2, Chris Fenton3, Ruth Paulssen3, Åse Vårtun2, Ganesh Acharya2 1 Department of Obstetrics and Gynaecology, Akershus University Hospital, Lørenskog, Norway, 2Women’s Health and Perinatology Group, University of Tromsø and University Hospital of North Norway, Tromsø, Norway, 3 Microarray Resource Centre, University Hospital of North Norway, Tromsø, Norway

Edward Chuong1, Marilyn Renfree2, Julie Baker1 1 Stanford University School of Medicine, Stanford, CA, 2University of Melbourne Department of Zoology, Melbourne, VIC, Australia

Objectives: The human placenta is a rapidly developing organ that performs multiple functions until birth. Investigations into molecular mechanisms that control placental plasticity during its maturation might be useful in understanding pathophysiology of pregnancy-specific disorders. We hypothesized that molecular rearrangements and phenotypic adaptations that are necessary for normal placental development are reflected in its genotype. Our objective was to investigate global gene expression profile in the first and third trimester normal human placentas. Methods: We recruited 21 women with uncomplicated pregnancies that were delivered at term and 16 healthy women undergoing surgical abortion at 9-12 weeks gestation. We compared global placental gene expression profile by Human Genome Survey Microarray v.2.0 (Applied Biosystems). A total of 37 hybridisations were performed applying direct comparison design. Results: We found that 7519 genes were differentially expressed between first and third trimester placentas, representing almost 25% of the genes spotted on the array. Bioinformatic analysis demonstrated that the first trimester placenta undergoes intense cell proliferation and differentiation processes. Angiogenesis was shown to play a central role during early placental development. Pathway analysis showed that brain and placenta might share common developmental routes. This result was validated using Human Neurogenesis and Neural Stem Cell PCR Array. Principal component analysis based on the expression of 17 imprinted genes showed a clear separation of first and third trimester placentas, indicating that epigenetic modifications occur throughout pregnancy. We validated this result by RT-PCR and found that IGF2 and PHLDA2 were highly, but differentially expressed between the groups. Further analysis of placentas obtained from women smoking tobacco showed a set of genes encoding for oxidoreductases to be up-regulated in both trimesters. Conclusion: During normal human development in utero there is a profound alteration in placental gene expression profile which seems to be affected by genetic and environmental factors.

Objectives: The mammalian placenta emerged w147 million years ago, prior to the divergence of eutherians and marsupials. To gain insight into the evolutionary genetic processes that gave rise to this novel organ, it is necessary to elucidate the genes that were involved in early ancestral placentation. However, current molecular-level understanding of placental development has been largely restricted to eutherians. Thus, we set out to characterize the genes and regulatory elements that are conserved in both eutherian and marsupial placenta development, with the aim of elucidating the genetic processes that shaped early placenta evolution. Methods: We harvested trophoblast stem cells and placentas from mice (e11, e13) and a yolk sac placentas (d21, d23, d25) from the tammar wallaby. We utilized functional assays based on high-throughput sequencing, including ChIP-Seq against H3K4me1, H3K4me3, H3K27ac, H3K27me3, and H3K4me3 to generate genome-wide histone occupancy maps and RNA-Seq for transcriptome data. Results: Comparison of placental transcriptomes from both mouse and wallaby revealed 157 tissue-specific transcription factors with conserved placental expression. Notably these include CDX2, CITED2, PPARG, and KLF5, which are key factors regulating eutherian trophoblast development. Candidate gene expression was validated in situ in tammar wallaby placentas. From mouse histone ChIP-Seq we identified w15,000 predicted trophoblast enhancers, of which w5,000 exhibit conserved expression across mammals. Notably, we find evidence that a number of enhancers are derived from exapted transposable elements. We are utilizing a trophoblast-specific lentiviral assay to test the activity of predicted enhancers. Conclusion: We are now investigating the genetic mutations that may have facilitated the evolution of the ancestral trophoblast gene network in mammals. Ongoing work involves further genomic characterization of placentation in multiple eutherian species to better understand how trophoblast specialized and diversified throughout eutherian evolution. In conclusion, high-throughput sequencing genomic approaches to characterize placenta development in non-model species provide important insight into mammalian placenta evolution.

Abstracts / Placenta 32 (2011) A1–A149

P2.119 CIRCADIAN VARIATION IN COMPONENTS OF THE PLACENTAL GLUCOCORTICOID BARRIER CONTRIBUTE TO RHYTHMIC PLACENTAL AND FETAL GLUCOCORTICOID EXPOSURE IN THE RAT Peter Mark, Michaela Wharfe, Jessica Lewis, Brendan Waddell The University of Western Australia, Perth, Western Australia, Australia Objectives: Fetal and placental glucocorticoid exposure is determined by the placental glucocorticoid barrier (PGB) which consists of placental expression of 11b-hydroxysteroid dehydrogenase type 1&2 (11b-HSD1 & 2) and the P-glycoprotein efflux-pump homologues, ABCB1a & 1b, each of which exhibit circadian variation in expression in various tissues. We recently demonstrated the rat placenta displays circadian variation in clock gene expression late in gestation. The aim of this study was to determine whether the key components of the PGB also exhibit rhythmic expression. Methods: Pregnant rats (n¼6/time point) were sampled over days 21-22 of gestation (term¼day 23). Samples of junctional (JZ) and labyrinth zones (LZ) of the placenta were collected at 0800, 1400, 2000 and 0200 h, which equate to zeitgeber times ZT1, ZT7, ZT13 and ZT19 respectively. Expression of 11b-hsd1, 11b-hsd2, Abcb1a, Abcb1b and GR were measured in placental tissues by qPCR. Data were analysed by 1- or 2-way ANOVA, as appropriate. Results: LZ expression of 11b-hsd1, Abcb1b and GR varied with time-of-day. The most marked circadian variation in LZ expression occurred in 11b-hsd1 (P<0.001), which exhibited a peak at ZT13 that was 3.8-fold higher than the trough at ZT1. Abcb1b expression in the LZ was 1.7-fold higher (P<0.05) at ZT13 than at ZT7. Labyrinthine GR expression also peaked at ZT13 (P<0.05) coinciding with onset of the active phase. JZ expression of 11bhsd2 was 10-fold higher (P¼0.019) at ZT1 than at ZT19, while there was a trend (P¼0.068) for a rhythm in Abcb1a expression, with a peak at ZT7 and a trough at ZT19. No time-of-day variation was observed for 11b-hsd2 and Abcb1a in the LZ or 11b-hsd1 and Abcb1b in the JZ. Conclusion: The rat placenta exhibits surprising circadian variation in PGB components late in gestation. This is likely to alter exposure of the placenta and fetus to maternal glucocorticoids. P2.120 A CANDIDATE GENE FOR X-LINKED GENOMIC IMPRINTING IN HUMAN FULL-TERM PLACENTA Joana C. Moreira de Mello, Fernando G. Sábio, Lygia V. Pereira Instituto de Biociencias - USP, Sao Paulo - SP, Brazil Genomic imprinting is characterized by monoallelic gene expression dependent on parental origin. Imprinted genes on the X chromosome is a puzzle phenomena as this chromosome is subjected to transcriptional inactivation in mammals, a process called X chromosome inactivation (XCI). In normal female mammals, each somatic cell shows one active and one inactive X, chosen in a random fashion regarding its parental origin. In humans the existence of X-linked imprinting is only from indirect evidences, as differences regarding cognitive functions in Turner’s syndrome women, according to parental origin of their single X (Nature, 1997; 387:705-708). By allele-specific X-linked gene expression we demonstrated that in human placenta XCI occurs in a random fashion (PLoS ONE 5(6):e10947). However, one X-linked gene always showed expression from the same allele. To verify if this is a gene subjected to imprinting, we accessed its allele-specific expression pattern by direct sequencing coding regions containing SNPs in 16 placental samples. Parental origin of the expressed allele was determined in five cases, and it was always maternally expressed. In order to complement our findings, bisulfite sequencing was used to determine the methylation pattern of the promoter CpG island. The presence of a VNTR inside the island was useful to determine the parental origin of the preferentially methylated allele. Interestingly, the methylation pattern of the promoter region did not correlate with the parental origin of the expressed allele, being more associated to XCI allele status than to the expression status. Taken together, our results strongly indicate that this gene is an X-linked gene subjected to genomic imprinting at least in human full-term placenta.

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P2.121 IDENTIFICATION OF MICRORNAS THAT TARGET GENE NETWORKS INVOLVED IN TROPHOBLAST PROLIFERATION Farkhondeh Farrokhnia, Melissa Westwood, John Aplin, Karen Forbes University of Manchester, Manchester Objective: Placental development depends on coordinated cellular growth. Endogenous microRNAs (miRs) exert post-transcriptional regulatory influences on genes involved in growth signalling. We have shown that global miR suppression in placenta accelerates cytotrophoblast proliferation. This study aimed to identify microRNAs (miRs) involved in regulating trophoblast. Method: Human placental expansion is much more rapid in first trimester than at term. To identify candidate regulators of proliferation, miR expression in first trimester (n¼5) and term placental tissue (n¼5) was compared in an array assay, and selected data validated using QPCR. Target prediction databases (miRecords) and Ingenuity Pathway Analysis software were used to classify miRs. Cell types present in the placenta were screened for expression. Predicted miR target gene products in first and term placenta were investigated by immunohistochemistry. Results: 58 miRs were differentially expressed between first trimester and term (P<0.001). Bioinformatics analysis of the 41 with lower expression in first trimester identified 10 miRs with predicted targets in signalling cascades known to regulate trophoblast proliferation. All 10 were expressed throughout the placenta but expression was low in villous trophoblast, consistent with a role in growth suppression. Predicted targets of three of the candidates, miR-145, miR-377 and Let-7a, include IGF1R, EGFR, ERK, AKT and SHP-2 all of which localise to cytotrophoblast. Expression of genes in both the IGF and EGF signalling cascades was lower at term than in the first trimester; an inverse correlation with miR expression is consistent with a potential role for these miRs in regulating mitogenesis. Conclusion: Our study has identifed miRs that may be involved in regulating growth signalling in trophoblast; ongoing studies using miR mimetics will explore the possibility of manipulating this system as a means of modulating placental growth. P2.122 TRANSCRIPT ANALYSIS OF A NOVEL LONG NON-CODING RNA ASSOCIATED WITH THE PREGNANCY-RELATED HELLP-SYNDROME. Marie van Dijk, Hari Thulluru, Cees Oudejans VU University Medical Center, Amsterdam Objectives: Recently, by genome-wide linkage analysis in families with a familial history of the pregnancy-related HELLP-syndrome, the chromosomal region has been identified linked to this disease. Its first trimester origin, i.e. reduced fetal trophoblast invasion, indicates that the placental genotype dictates the maternal phenotype. Indeed, potential mutations have been identified in the 12q23 locus in children born from a HELLP-pregnancy. Although the linkage region is a so-called gene desert between PMCH and IGF1, initial experiments showed Watson strandspecific transcription products within this region. Methods: Reverse transcription was performed using random hexamers on total RNA from the invasive fetal trophoblast cell line SGHPL-5. By overlapping PCRs on cDNA the chromosomal region between PMCH and IGF1 was fully covered. Identification of the 5’ and 3’ ends of the transcript was performed by 5’ and 3’ RACE. Results: The 5’ end of the transcript identified on chromosome 12q23 overlaps with exon 1 of PMCH, which itself is transcribed from the Crick strand. The transcript subsequently covers the complete gene desert without introns. The 3’ end has not yet been located exactly but the transcript runs at least up to the 3’UTR of IGF1, which is also transcribed from the Crick strand. The transcript is therefore a minimum of 203kb in length. As no significant open reading frames are present no protein will be formed. More likely, this transcript belongs to the class of long non-coding RNAs. Conclusion: Long non-coding RNAs have so far been implicated in chromatin remodeling, transcriptional control and post-transcriptional processing. With almost the complete 12q23 HELLP-syndrome associated transcript known, the next step is to identify its precise function as a regulating non-coding RNA and its dysfunction in the HELLP-syndrome.

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Abstracts / Placenta 32 (2011) A1–A149

P2.123 TRANSCRIPTOME ANALYSIS OF PPARG-TARGET GENES IN HUMAN INVASIVE TROPHOBLAST FROM FIRST TRIMESTER HUMAN PLACENTA Nadine Segond1,2, Severine Degrelle1,2, Philippe Dessen3, Danièle Evain-Brion1,2, Thierry Fournier1,2 1 Inserm U767, Paris, France, 2Paris Descartes University, Paris, France, 3 CNRS FRE2939, Villejuif, France Objectives: Human placental development is characterized by a deep invasion of extravillous cytotrophoblasts (EVCT) in the uterin wall during the first trimester of pregnancy. We have previously shown that activation of PPARg with rosiglitazone reduces EVCT invasiveness in vitro. The research of PPARg-target genes allowed us to identify genes implicated in trophoblast invasion. Methods: RNAs were extracted from 5 different rosiglitazone-treated or not primary EVCT cultures isolated from 8-9 WG placentae. Microarray analysis was performed using Affimetrix technology. Gene expression in treated EVCT was compared with matched untreated controls using significance analysis of microarrays (SAM). A statistical comparison to determine genes with a change of at least two-fold (FDR<10%) was performed. Ingenuity analysis was used to compute the differences between treated and untreated EVCT in gene sets and to define cellular functions and metabolic pathways. To confirm the microarray results we performed in new EVCT cultures, a relative real time Q-PCR on the eight most regulated genes. Results: The analysis of 14,500 genes using 22,000 probe sets revealed a total of 175 probe sets corresponding to differentially regulated genes (149 up, 26 down). Ingenuity analysis showed fonctionnal areas with significant changes in gene expression and the most important genes in the first seven networks. Confirmation of this analysis by Q-PCR showed a significant increase of DLC1, DPP4, HMOX1, LOX, CX43 (GJA1), PAI-1 from 1.7 (CX43) to 5.2 fold (DLC1) in rosiglitazone-treated EVCT, and a decrease of ADAM12 and PPARg(0.2 to 0.4 fold). These results confirmed the gene chip analysis. Conclusion: PPARg-target genes in invasive EVCT were implicated in various placental developmental processes, fatty acid metabolism and signaling pathways. Some of them were involved in tumor and metastatic processes and/or in hypoxia-reoxygenation regulation. Some of these genes may be involved in PPARg-mediated inhibition of EVCT invasiveness.

P2.124 ASSOCIATION OF THE ACE A11860G SINGLE NUCLEOTIDE POLYMORPHISM (SNP) AND FETAL SEX IN SMALL FOR GESTATIONAL AGE (SGA) PREGNANCY Ang Zhou1, Gus Dekker1, Eugenie Lumbers2, Gary Heinemann1, Robyn North3, Lesley McCowan4, Claire Roberts1 1 University of Adelaide, Adelaide, SA, Australia, 2University of Newcastle, Newcastle, NSW, Australia, 3Kings College London, London, 4University of Auckland, Auckland, New Zealand Objectives: The activity of the circulating renin-angiotensin system (RAS) is increased in human pregnancy. The RAS is involved in maternal adaptation to pregnancy particularly in the cardiovascular system. We hypothesised that genetic factors associate with cardiovascular adaptations and with SGA. Methods: We recruited 3197 healthy nulliparous women to the Screening for Pregnancy Endpoints (SCOPE) study in Adelaide and Auckland and women had blood sampled at 15 weeks gestation. We extracted DNA from blood from both parents and babies and genotyped them for SNPs in RAS genes. Genotypes for 1056 Caucasian women with uncomplicated pregnancies and 203 women with SGA babies (defined as birthweight <10th customised centile) were studied. Maternal plasma ACE was measured by ELISA (R&D Systems). Results: The maternal ACE A11860G SNP was associated with SGA after adjustment for other risk factors for SGA (maternal birthweight, smoking, age, socioeconomic index, pre-pregnancy green vegetable intake, mean arterial pressure (MAP) at 15 weeks gestation and paternal height) (GG vs AAþAG; aOR 1.5,95%CI 1.062.1). After separating the data according to fetal sex, in pregnancies with a female fetus the odds of SGA increased with maternal ACE A11860G GG genotype (OR 1.75, 95%CI 1.08-2.82) but in pregnancies with a male the SNP was not associated with SGA. Maternal ACE A11860G GG genotype was associated with increased MAP and increased plasma ACE at 15 weeks gestation. Women with SGA pregnancies had higher plasma ACE at 15 weeks gestation (p¼0.034) which remained significant only in those bearing a female fetus (p¼0.008). Conclusion: The maternal ACE A11860G GG genotype appears to associate in a sex specific way with SGA in part by its association with circulating ACE and MAP. Sex specificity may be explained by sex differences in placental gene expression and distinct patterns of fetal growth and hence survival.

Abstracts / Placenta 32 (2011) A1–A149

P2.125 TARGETED GENE ARRAYS ACROSS THE SEVERELY-DISCORDANT GROWTH MONOCHORIONIC TWIN PLACENTA: IMPLICATIONS FOR ANGIOGENESIS AND METABOLIC PROGRAMMING Susanne Schrey1, Dora Baczyk2, Brendan Fitzgerald3, Sarah Keating3, Greg Ryan1, John Kingdom1, Sascha Drewlo2 1 University of Toronto, Division of Maternal Fetal Medicine, Toronto, Canada, 2Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada, 3University of Toronto, Department of Pathology and Laboratory Medicine, Toronto, Canada Objectives: To explore angiogenesis and metabolic gene expression pathways in severely growth discordant monochorionic twins that may be subject to epigenetic regulation. Further, to assess placental leptin DNA methylation ratios to test the hypothesis that placental leptin expression is epigenetically regulated. Methods: We studied 8 severely growth-discordant (birth weight difference  25%) and 5 growth concordant (birth weight difference <25%) MC twin pairs without twin-twin-transfusion syndrome (TTTS). Growth discordant twin placentas were phenotyped by histology, demonstrating distal villous hypoplasia or immaturity on the IUGR side. Two random and unbiased snap frozen placental samples were collected at time of delivery from each twin. Placental mRNA expression of 88 angiogenesis related genes were measured by PCR array. Enzyme-linked immuno-sorbent assay (ELISA) and immunohistochemistry are currently performed to confirm mRNA findings. EpiTYPTER for DNA methylation is used to determine methylation ratios for the known CpG-rich sites within the proximal leptin promoter region. Results: The PCR array analysis showed significant mRNA up-regulation in the IUGR side for several genes, such as leptin (24.6 fold, p¼0.045), Flt1 (fms-like tyrosine kinase 1, 2.4 fold, p¼0.004) and Eng (endoglin, 1.86 fold up-regulated, p¼ 0.049). None of the other 84 angiogenesis related genes showed significant differences using the ΔΔCt comparison method. Epigenetic regulation of leptin is currently assessed by targeted PCR for bisulphate-converted DNA. Conclusions: The IUGR MC twin placenta up-regulates Flt1 and Eng mRNA that may be associated with the villous pathology. The substantial upregulation of leptin mRNA may be epigenetically conferred and relevant to the post-natal risk of metabolic syndrome in IUGR offspring. MC discordant IUGR twins offer unique insights into the epigenetic basis of perinatal programming.

P2.126 PLACENTAL GESTATION

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SULFATE

TRANSPORTERS

IN ANIMAL AND HUMAN

David Simmons1, Joanna Rakoczy1, Francesca Grace1, David McIntyre2, Paul Dawson3 1 School of Biomedical Sciences, The University of Queensland, St. Lucia/ QLD, Australia, 2Mater Clinical School, The University of Queensland, South Brisbane/QLD, Australia, 3Mater Medical Research Institute, South Brisbane/QLD, Australia Objectives: Sulfate is a vital nutrient for normal fetal growth and development, and is supplied from maternal circulation to the fetus via placental sulfate transporters. To date, 10 sulfate transporter genes belonging to the SLC13 (13A1 and 13A4) and SLC26 (26A1, 26A2, 26A3, 26A6, 26A7, 26A8, 26A9 and 26A11) gene families have been identified in both human and mouse. To build a model of directional sulfate transport in the placenta, we determined the relative abundance and spatial expression of all 10 sulfate transporter mRNAs in human and mouse placentas. Methods: Human placentae (n¼ 10 male and 10 female) were obtained from uncomplicated healthy pregnancies 37 wk gestation at elective caesarean section. Mouse placentas were obtained at E6.5 to E18.5 gestational ages. Quantitative real time PCR was used to determine the relative abundance of sulfate transporter mRNAs, and the spatial expression of mRNAs was determined by in situ hybridisation with gene-specific DIGlabelled riboprobes. Results: Negligible SLC13A1, SLC26A3, SLC26A7, SLC26A8 and SLC26A9 mRNA levels were detected in human and mouse placentas, whereas we detected very strong mRNA expression of SLC13A4 and SLC26A2, and intermediate levels of SLC26A1, SLC26A6 and SLC26A11. SLC13A4 and SLC26A2 expression increased by approximately 10-fold from midgestation in mouse placenta, and were localised to the feto-maternal barrier within the labyrinth layer. In human placentae, SLC13A4 mRNA was localised specifically to syncytiotrophoblasts, whereas SLC26A2 was detected in the cytotrophoblast cells. Conclusions: These data show that SLC13A4 and SLC26A2 are the most abundant sulfate transporter mRNAs expressed in human and mouse placentae, and their expression levels peak in mid- to late-gestation in cells of the feto-maternal barrier, when high sulfate levels are required for fetal development.

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P2.127 IGF REGULATES MIR-483-3P EXPRESSION IN THE DEVELOPING HUMAN PLACENTA VIA AN IGF1R/ERK PATHWAY Susan Hibbert, Farkhondeh Farrokhnia, Melissa Westwood, John Aplin, Karen Forbes University of Manchester, Manchester Objectives: microRNAs (miRs) are highly abundant within human placenta and are known to influence trophoblast proliferation. The mechanism(s) involved in regulating placental miR expression is unknown though studies of other systems suggest a role for the intracellular signalling molecule, ERK. This study has used IGFs and specific signalling molecule inhibitors to assess if activation of signalling pathways known to control trophoblast kinetics also regulate placental miR expression. Methods: First trimester placental explants were cultured in serum-free conditions for 24 h and then exposed to IGF-I (10nM) or IGF-II (10nM) for a further 24 h (n¼6). In some experiments, explants were preincubated with IGF1R inhibitor (PPP; 5mM), PI3K pathway inhibitors (LY294002; 200nM or Wortmannin; 100nM) or MAPKK inhibitor (PD98059; 50mM) for 30 min prior to IGF exposure. miR expression was profiled using arrays and data were validated by QPCR. In-situ hybridisation was used to localise miRs within placenta. Results: IGF (-I and –II) significantly decreased expression of two miRs (P<0.001); one of these, miR-483-3p was expressed throughout the villous stroma and within cytotrophoblast in first trimester placenta. Interestingly, although miR-483-3p was absent from the syncytial layer in the first trimester, it was highly abundant in term syncytium. Pre-incubation of explants with pathway specific inhibitors revealed that the ability of IGF-I to regulate miR-483-3p expression was significantly reduced following inhibition of IGF1R (P<0.01) or ERK (P<0.05). However IGF regulation of miR-483-3p is not dependent on the PI3K pathway. Conclusions: To our knowledge this is the first report to demonstrate that expression of specific miRs can be regulated by an IGF1R/ERK cascade. This pathway also regulates trophoblast proliferation and differentiation; miR483-3p expression within the trophoblast suggests that it may function to regulate these events. Ongoing studies will determine the targets and thus potential roles of this miR in the placenta.

P2.128 DNA COPY NUMBER VARIATION IN RECURRENT MISCARRIAGE Liina Nagirnaja1, Laura Kasak1, Priit Palta2,6, Kristiina Rull1,3, Ole B. Christiansen4, Tõnu Esko5,6, Maido Remm2,6, Andres Metspalu5,6, Maris Laan1 1 Human Molecular Genetics Research Group, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia, 2Department of Bioinformatics, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia, 3Department of Obstetrics and Gynaecology, Tartu University Clinics, Tartu, Estonia, 4Rigshospitalet, Copenhagen, Denmark, 5Estonian Genome Centre, University of Tartu, Tartu, Estonia, 6Estonian Biocentre, Tartu, Estonia Objectives: Recurrent miscarriage (RM) (3 miscarriages before 22 gestational weeks or spontaneous abortion of fetus <500g) is a heterogeneous condition occurring in 1-2% of fertile couples and approximately half of the cases are defined as idiopathic. The current study aimed to explore whether DNA copy number variations (CNV) contribute to inheritable predisposition to RM. Methods: Altogether 73 Estonian samples (43 RM patients, 30 fertile controls, recruited at Tartu University Hospital, Estonia) were genotyped on Illumina Infinum platform. Twelve genomic loci with altered CNV frequencies in RM cases compared to controls were validated by TaqMan qPCR. Two CNV regions for which the results supported array data were selected for replication in additional Estonian (n¼119 RM patients, n¼128 controls) and Danish (n¼438 RM patients, n¼228 controls) cohorts. Comparative control frequency data was obtained on 2500 genotyped individuals from Estonian Genome Project (EGP) representing average Estonian population. Results: A 25.9kb deletion on Chr 2 that has been implicated in RM previously shows no frequency differences among Danish (3.1% controls, 2.7% patients) but is more prevalent in Estonian primary RM cases vs controls (8.7% and 4.8% respectively, p¼0.272 due to small sample size). A 52.4kb duplication (up to 5 copies) on Chr 5 is 4x more frequent among women with primary RM than controls and EGP samples (combined frequency 8.4% vs 2.0% and 1.5% respectively, p¼0.006). The region involves two genes associated with reproductive function for the first time. Conclusions: Two dosage sensitive genomic regions are probably found to affect reproductive success: (i) deletion on Chr 2 indicates association with RM only in Estonians, (ii) duplication of novel fertility-related genes on Chr 5 increases the risk of primary RM in females in both populations. One of these duplicated genes is linked to a pathway shown to be critical for placental growth, invasion and metabolism.

Abstracts / Placenta 32 (2011) A1–A149

P2.129 CLONAL PATTERNS OF PLACENTAL DEVELOPMENT EVALUATED BY XCHROMOSOME INACTIVATION PROFILING Maria Peñaherrera, Luana Avila, Ruby Jiang, Wendy Robinson Univ. of British Columbia, Vancouver, BC, Canada

Carolyn

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P2.130 GENE EXPRESSION PROFILING OF BOVINE PERIPARTAL PLACENTOMES: DETECTION OF MOLECULAR PATHWAYS POTENTIALLY INVOLVED IN RELEASE OF FOETAL MEMBRANES

Brown,

X-chromosome inactivation (XCI), the random inactivation of one of the two X chromosomes in females, occurs in the late blastocyst stage and is thereafter stably maintained through successive cell divisions. As a result, a patchiness will occur in terms of which X is inactivated if cells positioned near each other are clonally descended from a common precursor. Evaluating patch size and distribution can then give clues as to the developmental history of a particular tissue. Objectives: To clarify the clonal pattern of development of human placenta. Methods: XCI status was evaluated using the methylation-based AR assay in samples obtained at multiple sites and depths from term human placentas. This pattern was compared to patterns of methylation variation associated with the H19/IGF2 imprinting control region; promoter regions of KISS1, PTPN6, CASP8, and APC; and LINE-1 elements. The within-site correlation in XCI between amnion, chorion, trophoblast and mesenchyme was also evaluated. Results: While XCI is highly variable from site-to-site within a placenta, villus samples taken from different depths spanning the maternal to fetal side of the placenta are highly clonally related. Methylation variation at APC and LINE1 also showed a significant correlation by depth, though not as distinct as for XCI. XCI status in amnion and chorion was uncorrelated with any other tissue, reflecting their separate developmental origins. However, the degree of XCI-skewing within enzymatically separated mesenchyme and trophoblast samples from the same placental location was highly correlated. This may reflect imperfect separation of the two tissues resulting in contamination of mesenchyme with trophoblast. However, the data is also consistent with common developmental origins of these cell types within a single villous tree. Conclusion: Detailed evaluation of XCI within a placenta is consistent with the development of villous trees vertically upwards from a small number of precursor cells within the chorionic plate.

D. Streyl1, R. Kenngott2, N. Herbach3, R. Wanke3, H. Blum4, E. Wolf4,5, H. Zerbe1, S. Bauersachs4,5 1 Clinic for Ruminants with Ambulatory and Herd Health Services, LMU Munich, Oberschleißheim, Germany, 2Lehrstuhl für Tieranatomie II, Department of Veterinary Sciences, LMU Munich, Munich, Germany, 3 Institute of Veterinary Pathology, Centre for Clinical Veterinary Medicine, LMU Munich, Munich, Germany, 4Laboratory for Functional Genome Analysis (LAFUGA), Munich, Germany, 5Chair for Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany Objectives: The mechanisms underlying detachment of fetal membranes in cows are still unclear. To address this problem in a systematic manner, we performed the first holistic transcriptome study of bovine peripartal placentomes by hybridization of Affymetrix GeneChipÒ Bovine Genome Arrays Methods: Three placentomes were extracted from each of 8 cows (AP group: n¼4, 7-10 days ante partum; IP group: n¼4 intra partum). Tissue samples derived from the contact zones of the placentomes were recovered by systematic random sampling and processed for RNA extraction and for stereological quantification of cellular composition. The microarray data were analyzed by “statistic analysis of microarray data” (SAM, FDR 1%) The differentially expressed genes were classified according to biological processes and molecular functions using the Functional Annotation Clustering tool of the DAVID Bioinformatics Resources. Results: The SAM analysis revealed 759 mRNAs with at least two-fold higher levels in the samples of the AP group, whereas 514 mRNAs showed higher levels in the IP group. Genes with higher mRNA levels in the AP group were nearly completely related to mitotic cell cycle and tissue differentiation. In parturition a complete shift occurred, because the genes with higher mRNA levels i.p. are nearly all related to three different physiological complexes: 1) apoptosis, 2) degradation of extra cellular matrix and 3) innate immune response. Conclusion: These results demonstrate that within 15min after delivery of the calf, the local processes in placentomes switch physiologically from nutrition of the fetus to tissue degradation, cell apoptosis and activation of a local immune response to release the “foreign” fetal membranes. These results are an excellent basis for future studies to investigate the retention of the fetal membranes (RFM). Elucidating abnormal gene expression profiles in cows with RFM may provide an approach to identify underlying pathophysiological mechanisms.

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P2.131 A STUDY OF NUCLEAR CONDENSATION AND M30 STAINING WITHIN SYNCYTIAL NUCLEAR AGGREGATES. Sarah Coleman, Carolyn Jones, Colin Sibley, John Aplin, Alexander Heazell Maternal and Fetal Health Research Centre, University of Manchester, United Kingdom Background: Syncytial nuclear aggregates (SNAs) are more prevalent in post-mature placentas and placental pathologies including preeclampsia. The morphology of nuclei in SNAs suggests they are apoptotic. Contrary to this, previous studies have shown that most SNAs do not show staining for either the M30 neoepitope (caspase cleavage of cytokeratin 18) or TUNEL. Objectives: 1.To clarify whether different types of SNA show varied levels of M30 neoepitope. 2.To test whether the proportion of condensed nuclei localised in SNAs is greater than in the remaining syncytiotrophoblast layer (ST). Methods: Ten serial sections from normal placenta (n¼6) were stained for the M30 neoepitope. M30-positive SNAs were traced through sections to determine type of SNA (knots, bridges, sectioning artefact, other). To investigate nuclear morphology an archive of electron micrographs was reviewed for normal (n¼7), post-mature (n¼7) and pre-eclamptic (n¼7) placentas. The nuclear appearances were semi-quantitatively assessed on a scale of 0-5. 0 ¼ mostly euchromatic, 5 ¼ totally heterochromatic. Results: Knots were more likely to be M30-positive (50%, interquartile range (IQR) 44.6%-53.3%) than bridges (13.3%, IQR 5.8%-20.0%) (figure 1). Normal placentas had more condensed (grade 4/5) (nuclei in SNAs than in ST (22.58% IQR 0.00%-50.00% vs. 0.00% IQR 0.00%-3.03%; p<0.001). Postmature ST had more nuclei at both extremes of chromatin condensation. Pre-eclamptic SNA nuclei were less condensed than normal SNA nuclei (Grade 4/5 nuclei 5.882% IQR 0.00%-9.09% vs. 22.58% IQR 0.00%-50.00%; p<0.001). Conclusion: Apoptotic features are more pronounced in syncytial knots than in bridges, indicating these structures may have different origins and functions. More condensed nuclei accumulate in syncytiotrophoblast with advancing gestation. The occurrence of fewer condensed nuclei in SNAs in PE is consistent with reports of faster trophoblast turnover in this condition. The data suggest that SNAs are dynamic structures and indicate that nuclear condensation is not a prerequisite for incorporation therein.

P2.132 APOPTOSIS IN THE FETOPLACENTAL UNIT: COMPARATIVE PATTERNS OF EXPRESSION OF TNF-RELATED APOPTOSIS INDUCED LIGAND (TRAIL), DEATH RECEPTORS DR4, DR5 AND DECOY RECEPTORS DCR1, DCR2 IN NORMAL RAT PREGNANCY AND SPONTANEOUS FETAL RESORPTION M.A. Costa1,2, M. Almada1, A.R. Martins1, B.M. Fonseca1,2, N.A. Teixeira1,2, G. Correia-da-Silva1,2 1 Departamento Ciências Biológicas, Laboratório de Bioquímica, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal, 2Instituto de Biologia Molecular e Celular da Universidade do Porto (IBMC), Porto, Portugal Objectives: During pregnancy the fetal-placental unit undergoes very complex histological changes to allow fetal development. However, some fetal-placental units fail during early pregnancy and undergo complete “spontaneous resorption”. Studying the expression of TRAIL system, we intend to analyse the role of extrinsic pathway in decidual remodelling and analyse the possible involvement of this system in fetal resorption comparing the differences in spatial-temporal expression of the system in normal and resorpted fetal-placental units. Methods: Pairs of normal and resorpted fetal-placental units of days 14, 16 and 19 of gestation were collected from the same Nulliparous Wistar Rats and at least 3 pairs obtained from different animals were studied. To evaluate morphological differences, sections of normal and resorpted units were stained with haematoxylin and eosin. Differences in spatial-temporal expression pattern of TRAIL system members in normal pregnancy and fetal resorption were assessed by immunohistochemistry. Results: Comparing to the normal implantation units, the resorpted sites presented major alterations in tissue morphology and extensive areas of cell debris and amorphous material. We found out that TRAIL expression in arteries and decidual capillaries and DcR2 in the circular muscle was more intense in fetal resorption. The number of decidual cells expressing ligand and receptors was lower though the intensity of the signal increased for DR4 and decreased for TRAIL. The number of uNK cells expressing TRAIL system was higher and the signal for TRAIL (day 16) and DR4 (days 14 and 16) was more intense. Conclusion: These observations suggest the involvement of the TRAIL system in the reorganization of the implantation site and in pathophysiological mechanisms that lead to fetal resorption and also support a role for uNK cells in uterine remodelling and pregnancy loss.

Abstracts / Placenta 32 (2011) A1–A149

Anne Bingel, Uta Enke, Philip Schmidt, Tobias Poehlmann, Ekkehard Schleussner, Lydia Seyfarth Department of Obstretrics and Gynecology, Placentalabor, Jena University Hospital, Friedrich-Schiller-University Jena, Jena, Germany Objectives:Caspases, a family of cysteine dependent aspartate-specific proteases, are involved in several apoptotic and inflammatory signalling pathways, including processes relevant for successful embryo implantation. Nevertheless, the mechanisms underlying caspase action on implantation are still topic of ongoing investigations, e.g. the role of caspase-4 in invasion. To study cell signalling pathways, not only gene expression, but also enzyme activity of caspase is of high importance. Methods and Results: We present a new approach for assaying the activity of caspase-4 by RP-HPLC technique. After incubation of cells with the caspase-4 substrate Ac-LEVD-AFC the cleavage product AFC (7-amino4-trifluoromethyl coumarin) can be detected directly in cell culture supernatant. Therefore, this new method abandons the steps of cell lysis and lysate incubation with substrate prior to monitoring. Furthermore, applying the RP-HPLC-technique under optimized conditions, the sensitivity is highly increased to a limit of detection of about 500 pM, which is equivalent to 25 fmol per sample. In JEG-3 choriocarcinoma cells, we demonstrate the easy handling of samples and the reliability of the method. In view of commercially available kits for determination of caspase-4 activity and the recommended assay procedures, we discussed some problems associated with the use of fluorescence microplate reader or using a fluorescence photometer for the caspase assay. Conclusions: This RP-HPLC method should be easily applied with little or no modification to other caspase assays using the same fluorophore.

P2.134 PILOT INVESTIGATION: IMMUNOLOCALIZATION OF ACTIVATED CASPASE 8 IN CYTOTROPHOBLASTS OF NORMOTENSIVE AND PREECLAMPTIC PREGNANCIES Jennifer Ducray1, Thajasvarie Naicker2, Berthold Huppertz3, Jagidesa Moodley2 1 Durban University of Technology, Durban, South Africa, 2University of KwaZulu-Natal, Durban, South Africa, 3Medical University of Graz, Graz, Austria Objectives: To explore villous and extravillous cytotrophoblast numbers and the distribution of activated caspase 8 in these cells in placentas from normotensive versus pre-eclamptic pregnancies. Methods: Placental sections from 10 normotensive and 10 pre-eclamptic pregnancies (gestational ages 36-40 weeks) were immunostained using a cocktail of antibodies against cytokeratin 7 and activated caspase 8, and examined to determine cytotrophoblast locations, as well as the distribution of caspase 8 activity. Results: In the normotensive cohort almost no cytotrophoblasts were seen in the intermediate or terminal villi, although they persisted in greater numbers in stem villi where several demonstrated a positive caspase 8 reaction sometimes in association with a corresponding positive reaction in the overlying syncytiotrophoblast. Pre-eclamptic samples demonstrated dramatically higher numbers of villous cytotrophoblasts particularly in the intermediate villi. Positive caspase 8 reaction was observed in association with branching as well as clumping of the cytotrophoblasts of the stem villi in both groups supporting the theory of the apoptotic cascade being involved in trophoblast turnover. In the normotensive samples, small areas of the syncytiotrophoblast demonstrated a positive caspase 8 reaction which presented as a punctate staining. The latter presentation was highly extensive in the syncytiotrophoblast of the pre-eclamptic samples. Extravillous cytotrophoblasts persisted in large numbers in both groups. This was particularly noted where stem villous syncytiotrophoblast had been replaced by perivillous fibrin, as well as in the basal plates and septa where numerous cytotrophoblasts demonstrated positive caspase 8 reactions possibly associated with parturition. Conclusion: Increased cytotrophoblasts especially associated with branching could be contributing to density of the pre-eclamptic villi. Higher incidence of activated caspase 8 demonstrates more initiation of

A % of M30 stained SNAs composed of each subclassification

P2.133 CASPASE-4 ASSAY IN JEG-3 CHORIOCARCINOMA CELLS: A NEW APPROACH USING RP-HPLC

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Fig. 1. Composition of SNAs that stained positively for the M30 neoepitope.Ă

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apoptosis. Further investigations into downstream caspases are underway to explore whether this could be leading to increased cellular death, dysregulated trophoblast turnover, or increased syncytial shedding.

P2.136 A SOCIOLOGY OF PLACENTA SCIENTISTS: TOWARDS TRANSDISCIPLINARY COLLABORATION

P2.135 IMMUNOMODULATORS AND EXOSOMES FROM THE PLACENTA: IMPLICATIONS FOR MATERNAL-FETAL IMMUNE TOLERANCE

Rebecca Scott Queen’s University, Kingston, Ontario, Canada

1

1

2

1

Sarika Kshirsagar , Khorshed Alam , Herbert Hodes , Margaret Petroff 1 University of Kansas Medical Center, Kansas City, KS, USA 2Center for Women’s Health, Leawood, KS, USA Objectives: The semiallogeneic fetus is tolerated by the maternal immune system through its control of non-specific and specific maternal immune responses. Trophoblast cells are known to secrete nanometer-scale membranous particles called exosomes, which have been implicated in maternal immunomodulation both locally and distally to the placenta. Here, we investigate whether exosomes secreted from first trimester and term placenta carry HLA-G and B7 family immunomodulators. Methods: In the first study, purified term cytotrophoblasts were cultured for 3-7 days in the presence or absence of epidermal growth factor, which promotes syncytialization. In the second study, first trimester placental explants were cultured for 24 hours. For both studies, culture supernatant was collected, cleared of cellular debris, and subjected to ultracentrifugation to purify small membrane fragments including exosomes. Pellets were subjected to electron microscopic and immunoblot analysis for B7 family members, HLA-G, and markers for endosomes and endoplasmic reticulum. Finally, first trimester and term placentas were subjected to dual immunofluorescence for CD63 and either B7-H1 or HLA-G5. Resutls: Ultracentrifugation of culture supernatants revealed the secretion of exosomes from both first trimester and term placental tissues. The immunomodulators B7-H1 (CD274), B7-H3 (CD276) and HLA-G5 were abundant in this pelleted fraction, as determined by immunoblot analysis. Immunofluourescence revealed the colocalization of B7-H1 with both CD63 and LAMP-1 in first trimester and term placentas. Although colocalization of HLA-G with CD63 and LAMP-1 was not observed in placental sections, these proteins were strongly associated in purified cytotrophoblast cells. Conclusion: The results suggest that the immunomodulatory proteins HLA-G5, B7-H1 and B7-H3, are secreted from early and term trophoblast, and raise the possibility that the proteins exit the placenta via exosomes. Further work is required to confirm their containment specifically within exosomes. However, the results have important implications in the mechanisms by which trophoblast immunomodulators modify the surrounding immunological environment.

Objectives: This research aims to forge transdisciplinary connections between scientists and sociologists, specifically by exploring how placenta scientists understand their scientific practice. It describes the opinions, ideas and, experiences of placenta scientists, and then relates them to larger social processes through the lenses of the philosophy and sociology of science. Methods: This research employs empirical sociological methods: namely, interviewing and participant observation. 25 in-depth, semi-structured interviews were conducted with placenta scientists, including senior researchers, graduate students, and laboratory technicians from around the world. Participant observation was conducted in 4 labs in two countries for several days, as well as during a number of conferences, symposia and talks. All transcripts and notes were coded to identify key emergent themes. Results: A number of important themes emerged from this study. First, placenta scientists view the placenta as absolutely central to human life, health, and disease; despite this, it is greatly under-studied and underappreciated due to various and competing interests and values. Second, ethical considerations, including both institutional and philosophical ones, colour every aspect of placenta science, but much of the system of institutional ethics does not reflect the realities of scientific practice nor desires of the public. Third, there are various structural barriers to conducting scientific research on the placenta, including religious, political, and social structural ones. Finally, the placenta is seen to be, to use the language of the sociology of science, a prolific ally-maker and ally-collector, bridging various individuals, groups, institutions and interests. These bridges are built at the level of a pregnancy, within the realm of science, and in the intersections of science and society. Conclusions: Placenta science is therefore not a neutral endeavor but a highly sociable process. In this way, the placenta emerges as a key site for transdisciplinary collaboration between scientists and sociologists.

Abstracts / Placenta 32 (2011) A1–A149

P2.137 THROMBOXANE AND ISOPROSTANE SHARE THE SAME PROSTANOÏD RECEPTORS TO REGULATE HUMAN PLACENTAL VASCULAR TONE Research centre, CHU Ste-Justine, and Departments of Obstetrics & Gynaecology and of Pharmacology, Faculty of Medicine, Université de Montréal, Canada L. Hausermann, J. St-Louis Introduction: Placenta being devoid of autonomic innervations, umbilical-placental vascular tone should be under the control of tissue and humoral factors. Among the numerous stimuli able to challenge the placental circulation, we propose that prostanoïds could be responsible for the regulation of placental vascular tone. Consequently, we have measured vasomotor responses of U-46619, a mimetic of thromboxane A2 (TXA2), and of the isoprostane 8-iso-prostaglandin E2 (8-isoPGE2) in human placental vasculature. Methods: Placental tissue were collected from normotensive women after delivery and cotyledons were set up in a perfusion system whereas chorionic arteries were prepared as rings and installed in glass-jacketed tissue baths. The effects of U-46619 and 8-isoPGE2 were measured in the absence and presence of blockers of TP (TXA2) receptors, SQ29,548 and ICI192,605, and of EP (PGE2) receptors, AH6809. The influence of nitric oxide (NO) was assessed using L-NAME. Results: U-46619 and 8-isoPGE2 markedly increased perfusion pressure in cotyledons and tension in chorionic arteries. Dose-response curves to both prostanoïds were competitively shifted to the right. L-NAME had no significant effect on dose-response curves to both stimulants. Discussion: Effects of U-46619 and 8-isoPGE2 which appeared to be mediated by TP and EP receptors support our postulate that prostanoïds have some important regulatory role on placental vascular tone. However, NO did not seem to be involved. This work was supported by the Québec Heart and Stroke Fdn

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P2.138 THE INCREASED LEVEL OF FERRITIN AND IRON ARE ASSOCIATED WITH EARLY CARDIOVASCULAR DISEASES DEVELOPMENT IN SEVERE PREECLAMPSIA 1

Xuelan 1Li, Danyan Feng, 2Qi Chen First Hospital of Medical School of Xi’an Jiaotong University. China, 2 Department of Obstetrics & Gynaecology, The University of Auckland, New Zealand 1

Introduction: Preeclampsia is characterised by hypertension and proteinuria. Recent evidence confirms that mothers with preeclampsia have higher risks of developing cardiovascular disease compared to other women of similar age. Studies have showed that the iron overload plays an important role in causing cardiovascular diseases. Other studies also suggested that serum ferritin level could be a risk factor of development of cardiovascular diseases. However how they act in developing cardiovascular disease in women with preeclampsia in their later life is unclear. The objective of the present study was to investigate the association of Iron level or serum ferritin with women with preeclampsia. Methods: 30 women with severe preeclampsia and 30 normal pregnant women were included in this study. The serum level of iron and ferritin in preeclamptic women were measured before treatment. In addition, all the patients were examined by echocardiogram (ECG) and ultrasonic cardiogram (UCG) for determining cardiovascular diseases. Results: The mean systolic blood pressure and diastolic blood pressure in severe preeclampsia group were 159.873.039mmHg and 108.772.230mmHg, respectively. The level of iron in severe preeclampsia group was significantly higher than control group (7.671.515 via 6.91.22). The serum level of ferritin in severe preeclampsia group was also significantly higher than control group (97.89117.7 via 21.0915.31). 14 of 30 preeclamptic women had cardiovascular diseases (abnormal ECG and UCG). The level of iron was no difference between severe preeclamptic women with cardiovascular diseases and that without cardiovascular diseases, whereas the serum level of ferritin in severe preeclamptic women with cardiovascular diseases was significantly higher than that in severe preeclamptic women without cardiovascular diseases. Conclusion: This study suggested that iron stores play an important role in the development of cardiovascular disease in women with preeclampsia, in particular in preeclamptic women with cardiovascular diseases.

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P2.139 THE LEVEL OF SURVIVIN, THE INHIBITOR OF APOPTOSIS PROTEIN WAS DECREASED IN PREECLAMPTIC PLACENTA

P2.140 THE EXPRESSION OF AQUAPORIN-4 DECREASES IN PREECLAMPTIC PLACENTAS

1

1

C. Li, 1W. Guo, 1X. Li, 1T. Yang, 2Q. Chen First Hospital of Medical School of Xi’an Jiaotong University. China, 2 Department of Obstetrics & Gynaecology, The University of Auckland, New Zealand 1

Introduction: Preeclampsia is a major complication of pregnancy affecting maternal and fetal heath. Although the pathogenesis of preeclampsia is unclear it is believed that trophoblasts apoptosis during the pregnancy plays an important role in the pathogenesis of preeclampsia. Survivin is a family member of the inhibitor of apoptosis protein (IAP) family, that uniquely also promotes trophoblasts proliferation. In this study we examined the protein expression and mRNA level of survivin in the preeclamptic placenta compare to normal pregnant placenta. Methods: 40 preeclamptic (including severe and mild preeclampsia) and 20 age-matched normal placentae were used in this study. The expression of survivin was measured by immunohistochemistry and the mRNA level of survivin was measured by Real time PCR. Results: Survivin was expressed on syncytiotrophoblasts and cytotrophoblasts. The expression of survivin was significantly decreased in preeclamptic placenta compared to normal placenta. Furthermore the expression of survivin in severe preeclamptic placenta was significantly lower than that in mild preeclamptic placenta. In addition, the mRNA level of survivin in preeclamptic placenta is significantly decreased compared to normal placenta. Similar, the mRNA level of survivin in severe preeclamptic placenta is significantly decreased compared to mild preeclamptic placenta. Conclusion: our current data suggest that lower expression of survivin in placenta is associated with preeclampsia development. Contact: Prof. Wenli Gou. E-mail: [email protected]

Valeria Dietrich, 2Natalia Szpilbarg, 3Cyntia Aban, 4Claudia Silberstein, Elsa Zotta, 6Alicia Damiano 1 Universidad de Buenos Aires - Facultad de Farmacia y Bioquímica - Lab. Biología de la Reproducción, Buenos Aires, Argentina , 2CEFyBO- CONICET Lab. de Fisiopatología Placentaria, Buenos Aires, Argentina , 3Universidad de Buenos Aires - Facultad de Medicina - Depto. de Fisiología - Lab. de Fisiopatogenia, Buenos Aires, Argentina 5

The syncytiotrophoblast of human term placenta (hST) plays an important role throughout pregnancy because it is the site of nutrient exchange. The transport of metabolites, ions and water from mother to fetus could take place primarily via transcellular routes. Water transport across hST may be facilitated by aquaporins (AQPs). In some pathological states such as preeclampsia, the trophoblast invasion of the spiral arteries leads to a failure to establish an adequate uteroplacental blood flow generating a relatively hypoxic trophoblast tissue attenuating considerably the transport functions of the hST. We have first reported that water transport across the preeclamptic placentas was dramatically decreased compared to normal ones and that the expression of AQP3 and AQP9 was altered. Recently, some authors have found that AQP4 expression decreased along gestation in hST and endothelium of normal human placentas. In addition, other authors have reported that brain AQP4 expression changed under hypoxic conditions. Objective: The aim of the present work was to study AQP4 protein expression in the hST from normal and preeclamptic placenta and in a model of ischemia-reperfusion [hypoxia-reoxygenation] type insult. Methods: AQP4 expression was evaluated by Western blot and immunohistochemistry in normal, preeclamptic placentas and normal placental explants cultured in normoxia, hypoxia, and in hypoxia/reoxygenation conditions. Results: We found that AQP4 was located in the apical membranes of hST and in the endothelium of normal placentas but it was almost undetectable in preeclamptic placentas. When explants were cultured under hypoxic conditions, AQP4 expression increased in both hST and endothelium but decreased after reoxygenation treatment. Conclusions: Alterations in AQP4 expression as a consequence of the different oxygenation conditions may explain the changes observed in water transport across preeclamptic placentas.

Abstracts / Placenta 32 (2011) A1–A149

P2.141 MICRORNA EXPRESSION PROFILES IN TROPHOBLASTIC CELLS 1 Diana M. Morales Prieto, 1Maja Weber, 1Stephanie Ospina Prieto, 1Justine S. Fitzgerald, 1 Ekkehard Schleussner, 2Bernd Gruhn, 1 Udo R. Markert 1 University Hospital Jena; Department of Obstetrics; Placenta Laboratories, 2 Germany University Hospital Jena; Department of Pediatrics, Germany

Background: MicroRNAs are small single-stranded RNA molecules playing an important role in the post-transcriptional regulation of gene expression. We analyzed miRNA expression in four trophoblastic cell lines (JEG-3, ACH-3P, AC1-M59, HTR8/SV neo) before and after LIF challenge. Methods: Cells were stimulated or not with LIF for 4h and total RNA containing microRNAs was isolated. Expression of 762 microRNAs was screened in biological duplicates. HTR-8 cells were karyotyped through Multiplex-Fluorescence In Situ Hybridisation (M-FISH). Furthermore, expression kinetics of miR-9, miR-141, miR-21, miR-93 and let-7g has been analyzed by real time PCR in JEG-3 after stimulation with LIF for 1 to 24h. As being the most affected, miR-141 has been silenced and over-expressed to test its role in proliferation of JEG-3 cells after 24h and 48h. Results: MicroRNAs expression profiles of JEG-3, ACH-3P and AC1-M59 were similar exhibing high expression of the C19MC miRNA cluster, known to be almost exclusively expressed by the placenta, as well as the three miRNAs recognized as specific markers for human embryonic stem cells. Conversely, HTR-8 exhibits almost no expression of the C19MC miRNAs but instead high expression of a miRNA cluster located in the chromosome 14. These results correlate with the observed in the M-FISH karyotype of HTR8 cells, where both C19 and C14 were found to be physically altered. MicroRNAs change differentially between cell lines under LIF stimulation, indeed only a small group of miRNAs were found simultaneously altered in all tested cells. Silencing of miR-141 completely inhibited proliferation of JEG-3 cells, while overexpression had no effect. Conclusions: LIF modulates microRNA expression in trophoblastic cells in a cell type-dependent manner. Karyotype and miRNA expression profile provide a tool for the identification of trophoblast cells and their malignant derivates. MiR-141 may have an essential role in regulation of functions in trophoblastic cells.

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P2.142 COMPARISON OF LEUKEMIA INHIBITORY FACTOR-INDUCED INTRACELLULAR SIGNALLING IN DIFFERENT TROPHOBLASTIC CELL LINES Wittaya Chaiwangyen, Francisco L. Pereira de Sousa, Diana M. Morales Prieto, Stephanie Ospina Prieto, Udo R. Markert University Hospital Jena, Department of Obstetrics, Placenta Laboratories, Germany Objectives: Well balanced functions and differentiation of trophoblast cells are essential for inception and maintenance of successful pregnancy. Trophoblast cells perform invasion similar to tumors, but in a well regulated physiological manner and dysregulation may lead to severe pathologies. Leukemia inhibitory factor (LIF) induces trophoblast invasiveness via signal transducers and activators of transcription 3(STAT3), but activation mechanisms seem to differ in different cell lines and are not yet completely investigated. Therefore, the aim of our study is to analyze and compare the role of Extracellular Regulated Kinase(ERK)/STAT in trophoblast and choriocarcinoma cells with different invasive capacities. Methods: The immortalized human trophoblast cell line HTR-8/svneo, the choriocarcinoma cell line JEG-3, and the hybrids of JEG-3 derivates and 1st and 3rd trimester trophoblast cells ACH-M59 and AC1-3P, were treated with or without LIF. The activation and expression of STAT3 and extracellular regulated kinase 1/2 (ERK1/2) were measured by gel electrophoresis and Western blotting and quantified by use of a chemiluminescence gel documentation system. The effect on cell proliferation and invasiveness were determined by a MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) colorimetric assay and and a Matrigel invasion assay. Results: LIF stimulated the phosphorylation of STAT3 and ERK1/2 in all analyzed cell lines, but with different intensities. LIF also enhanced invasiveness of AC1-3P cells and JEG-3 cells. In contrast, proliferation was not affected by LIF. Conclusion: These findings demonstrate that the LIF-ERK1/2-STAT3 axis exists in different trophoblastic cell lines, but its activation may lead to different functions, which may be due to specific properties of each cell line

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Abstracts / Placenta 32 (2011) A1–A149

P2.143 THE EFFECT OF CIRCULATING ANGIOGENETIC FACTORS ON TROPHOBLAST APOPTOSIS: AN IN VITRO STUDY

P2.144 INCREASING MATERNAL BODY MASS INDEX IS ASSOCIATED WITH IMPAIRED CHORIONIC PLATE ARTERIAL FUNCTION

Betsy Varughese, Rajesh Kalra, Kalpana Luthra, Neerja Bhatla, Rani Kumar, Renu Dhingra All India Institute of Medical Science, India

Lucy Higgins1, Tracey Mills1, Susan Greenwood1, Colin Sibley1, Mark Wareing1, Christina Hayward1 1 Maternal and Fetal Health Research Centre, University of Manchester, Manchester, UK Objectives: Maternal obesity is associated with increased risk of pregnancy complications. The placenta is the key regulator of fetal growth; altered function may underlie poor outcomes in obesity. Here we test the hypothesis that chorionic plate arterial (CPA) function is altered with increasing maternal body mass index (BMI). Methods: Term placentas were collected post-delivery (vaginal or caesarean section) following uncomplicated pregnancies in women whose first trimester BMI was classified as normal (18.5–24.9), overweight (25.0– 29.9) or obese (30). CPAs were mounted on a wire myograph, normalised to 0.9 of L5.1KPa(z20 mmHg) and equilibrated (37 oC; 20 min in 5%O2/5% CO2 z 7% O2). Constriction was assessed with U46619 (thromboxane mimetic: 1010–105.7 M) and relaxation of pre-constricted arteries (U46619 EC80) was determined using sodium nitroprusside (SNP) (nitric oxide donor: 1010–104 M). Maximum response and area under the curve (AUC) were compared (normal vs obese) using median and interquartile range (IQR) and correlated with BMI. Statistical significance (p < 0.05) was assessed using Mann–Whitney U Test and Spearman's Coefficient (SC). Results: 23 placentas were examined: 8 normal (BMI 22.9; IQR 21.8–24.1), 6 overweight (BMI 26.2; 25.5–26.7)) and 9 obese (BMI 33.2; 32.2–43.9). There were no differences in baseline characteristics or CPA diameter between normal and obese women. BMI did not affect constriction to U46619. In contrast, maximal SNP vasodilation was reduced in obese women (Figure 1) and inversely correlated with BMI; constriction remaining 45% (38–60) vs. 72% (54–88); p ¼ 0.046 [SC 0.47; p ¼ 0.025] and AUC 416 (373–468) vs. 499 (465–583); p ¼ 0.02 [SC 0.53; p ¼ 0.0087] for normal vs. obese respectively. Conclusion: Obesity was associated with reduced vasodilation of CPA in response to SNP. This suggests that the NO signaling pathway is dysfunctional in CPA smooth muscle cells in obesity. Such aberrant regulation in these vessels could place the fetus at risk.

SNP Dose Response Curve 100

Obese n=9

90 80 70

%U46619 EC

Introduction: Preeclampsia, the de novo occurrence of hypertension and proteinuria after the twentieth week of gestation, remains an enigma and even today continues to exert a significant burden on maternal and pediatric diseases globally. It is a pregnancy complication diagnosed by signs of widespread maternal endothelial dysfunction possibly secondary to abnormal placentation due to increased trophoblast cell apoptosis. Although the etiology of preeclampsia is still unclear, our previous studies suggest that its major phenotypes like high blood pressure and proteinuria, are due in part to excess circulating soluble fms-like tyrosine kinase-1 (sFlt-1) concentrations. sFlt-1 is an endogenous anti-angiogenic protein that neutralizes the pro-angiogenic protein Vascular Endothelial Growth Factor (VEGF) eventually causing systemic endothelial cell dysfunction. This may obstruct the vascular remodelling and the survival of cytotrophoblasts in the placenta. Objective: To determine whether the circulating angiogenetic factors like VEGF and sFlt-1 are involved in the placental apoptosis in preeclampsia. Methods: Trophoblast cell lines (JAR cells) were cultured with (i) preeclamptic sera, (ii) normotensive sera, (iii) preeclamptic sera with recombinant VEGF and (iv) normotensive sera with recombinant sFlt-1. Apoptosis of the trophoblast cells were studied by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) technique. Result: The apoptotic index was significantly higher in JAR cells treated with preeclamptic sera as compared to the JAR cells treated with control sera. This effect was reversed when JAR cells were treated with preeclamptic sera after the addition of recombinant VEGF and JAR cells treated with control sera after the addition of recombinant sFlt-1. Conclusion: Altered serum levels of VEGF and sFlt-1 may induce the apoptosis of trophoblast cell lines (JAR) suggesting a role for circulating angiogenetic factors in the pathogenesis of preeclampsia.

Normal n=8

60 50 40 30 20 10 0 -10

-9

-8

-7

-6

-5

-4

SNP (M) Figure 1. Obesity is associated with reduced CPA vasodilation to SNP.Ă

Abstracts / Placenta 32 (2011) A1–A149

P2.145 PERINATAL OUTCOMES AND MATERNAL CLINICAL CHARACTERISTICS IN IUGR WITH ABSENT OR REVERSED END-DIASTOLIC FLOW VELOCITY IN THE UMBILICAL ARTERY Byung-Joon Park1, Dong Gyu Jang2, Yun Sung Jo2, Sung Jong Lee2, Narinay Kim2, Gui Se Ra Lee2 1 Department of Obstetrics and Gynecology, The Catholic University of Korea, Incheon St. Mary’s Hospital, Bupyeong 6-dong, Bupyeong-gu, Incheon, Korea, Republic of, 2Department of Obstetrics and Gynecology, The Catholic University of Korea, St. Vincent’s Hospital, Ji-dong, Paldal-gu, Suwon, Kyeonggi, Korea, Republic of Korea. Objectives: The aim of this study was to evaluate the effect of absent or reversed end-diastolic umbilical artery Doppler flow on neonatal outcome independent of oligohydramnios, gestational age, and maternal factors. Methods: From January 2004 to March 2010 we reviewed 76 cases at our hospital, which were diagnosed with intrauterine growth restriction (IUGR). Among those cases, the existence of absent or reversed end-diastolic velocity of umbilical artery (AEDV) was considered abnormal. We set the group that had no abnormal signs as the control group (57 cases), and

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compared it with the AEDV group (19 cases). Logistic regression was used to control for oligohydramnios and gestational age. Results: The gestational age was lower in the AEDV group compared to that of the control group. Neonatal weight, platelet count were also lower in the AEDV group and serum SGOT level, the frequency of non-reassuring fetal heart beat pattern were higher in AEDV group compared to that of the control group independent of gestational age. Perinatal outcomes such as Apgar score at 1 min below 4, use of a ventilator, admission to the neonatal intensive care unit (NICU), respiratory disease, neurologic disease, neonatal sepsis, anemia, thrombocytopenia, and neonatal mortality were statistically less favorable in the AEDV group compared to those in the control group independent of gestational age and presence of oligohydramnios. There were more intrauterine fetal death histories and preeclampsia in the AEDV group compared the control group. Conclusion: The waveform of umbilical artery Doppler velocity is an informative parameter of perinatal outcomes independent of gestational age or the presence of oligohydramnios in IUGR patients. It is especially important to check the waveform of umbilical artery Doppler velocity in IUGR patients with preeclampsia and IUGR patients with FDIU history. Keywords: Intrauterine growth restriction, absent or reversed diastolic flow of umbilical artery, oligohydramnios, perinatal outcomes, Gestational age