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Missing abstracts from the IFPA 2011 meeting in Geilo, Norway
Placenta 32 (2011) A173–A175
Contents lists available at SciVerse ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Missing abstracts from the IFPA 2011 meeting in Geilo, Norway P1.141 GLUCOSE TRANSPORTER (GLUT)-9 EXPRESSION IN PLACENTAL TISSUE M.U. Baumann1, M. Messerli1, R. Sager1, U. Reinhart1, C. Albrecht2, D.V. Surbek1 1 University Hospital of Berne, Department of Obstetrics and Gynecology 2 Institute for Biochemistry and Molecular Medicine, University of Berne, Switzerland Objectives: Maternofetal glucose transport is crucial for the fetal wellbeing since the fetus is not able to generate glucose de novo. Glucose transport (GLUT)-9 is a novel member of the GLUT family and exists as two isoforms. The aim of this study is to investigate the expression of GLUT-9 and GLUT-1 in different placental models. Methods: As placental model systems primary villous tissue of term placentae following normal pregnancies and the BeWo choriocarcinoma cell line were used. Protein expression was assessed by Western analysis of whole tissue- or cell lysate, or syncytial basal membrane-enriched (BM) and apical microvillous-membrane (MVM) fractions (obtained following differential ultra-centrifugation and magnesium precipitation). The mRNA level of transport proteins was analysed by real-time PCR. Results: GLUT-1 and GLUT-9 protein were detected in primary villous tissue. GLUT-1 protein concentration was lower in BM than in MVM. The gene expression of both GLUT-9 isoforms, namely GLUT-9a and GLUT-9b, were detected in placental villous tissue as well as in BeWo cells. The GLUT-9b mRNA level was higher than the one of GLUT-9a. Conclusions: GLUT-1 was asymmetrically expressed on syncytial basal and apical membranes. Further, in placental tissue and BeWo cells GLUT-9b mRNA levels were higher than those of GLUT-9a. Further studies are needed to investigate the role of GLUT-1 and GLUT -9 in the pathogenesis of pregnancy-specific diseases such as preeclampsia or gestational diabetes.
doi:10.1016/j.placenta.2011.10.002
P1.144 EFFECT OF HYPEROXYGENATION AND GROWTH BLASTOCYST-DERIVED TROPHOBLAST STEM CELLS
FACTORS
ON
Priyadarsini Kumar1, Twanda L. Thirkil1l, Catherine Vandevoort1,2, Gordon C. Douglas1 1 University of California, USA 2 California National Primate Research Center, USA Objectives: To study the effect of hyperoxygenation on rhesus monkey blastocyst-derived trophoblast stem cells in the absence and presence of growth factors. Methods: Blastocyst-derived rhesus monkey stem cells were cultured for 5 days in 2% O2 (normoxia for these cells). Growth factors (hCG, b-estradiol, HB-EGF, EGF, or FGF2) were added to media and the cells cultured for an additional 72 h either at 2% O2 or 4% O2 (hyperoxia). The cells were analyzed by immunofluorescence microscopy, Western blotting, and zymography to assess trophoblast marker expression and differentiation. Results: Trophoblast stem cells cultured at 2% O2 proliferated and maintained viability. When switched to 4% O2 the cells underwent apoptosis. Jar cells did not show this effect when switched from 2% to 4% O2. With the exception of HB-EGF, the addition of growth factors protected stem cells from the apoptotic effects of hyperoxygenation. In addition, preincubation of cells with N-acetylcysteine or caspase inhibitor protected the cells from apoptosis. At 2% O2 hCG, b-estradiol, EGF, and FGF2 increased cell proliferation. When cultured in 4% O2 in the presence of hCG or b-estradiol, large multinucleated cells were generated. In the presence of EGF or FGF2 the cells became multinucleated but were also migratory and invasive. This effect was more pronounced in the presence of EGF at 4% O2. Cells cultured at 4% O2 in the presence of growth factors expressed cytokeratin 7, b1 integrin, a5 integrin and secreted MMPs. They also expressed CD9 which was not seen with the other treatments. Conclusions: Exposing rhesus monkey trophoblast stem cells to 4% O2 after culture at 2% O2 caused apoptosis which was prevented by the addition of growth factors. The invasive multinucleated cells formed at 4% O2 in the presence of EGF could represent the first trophectoderm-derived cells that penetrate the endometrial epithelium during implantation.
Abstracts / Placenta 32 (2011) A173–A175
A174
P2.146 RISK ASSESSMENT IN EARLY PREGNANCY ALLOWS FOR PREDICTION OF PREECLAMPSIA
risk for late preeclampsia
late risk 100
Baumann Marc1, Wiedemann Ute2, Nydegger Urs2, Risch Martin2, Surbek Daniel1, Risch Lorenz2; for the predictive markers for the diagnosis of preeclampsia (PRADO) study group 1 University Hospital of Berne, Department of Obstetrics and Gynecology 2 Labormedizinisches Zentrum Dr. Risch, Liebefeld
early risk 100
Sensitivity
80 60 40 20 0 0
20
40
60
100-Specificity
Ă
80
100
Sensitivity
Objectives: Preeclampsia represents a complication during pregnancy and accounts for substantial fetal and maternal morbidity and mortality. It has been shown, that early intervention (before week 16) with aspirin is beneficial in preventing the occurrence of preeclampsia. Early recognition of pregnant women at risk for developing preeclampsia would allow for effective installation of pharmacological prevention. Aim: To evaluate an algorithm for identifying pregnant women at risk for development of preeclampsia. Methods: In a nested-case control study done in the ongoing prospective PRADO study, women developing preeclampsia were matched (for maternal age, gestational age, smoking status, and weight) to women with uneventful pregnancies. Blood drawn at the occasion of first trimester testing (gestational weeks 11-14) was analyzed for Placental Growth Factor (PLGF) and PAPP-A by an immunoanalytical method (Perkin Elmer, Turku, Finland). By using the “Screen info” software developed by Cuckle and colleagues, risks for developing early and late preeclampsia were calculated. Results: 11 cases with early preeclampsia occurring <34 weeks (early onset) were matched to 50 controls. ROC-analysis with an area under the curve (AUC) of 0.83 (95%CI 0.71-0.91) revealed good characteristics in predicting the occurrence of early onset preeclampsia. At a risk cut-off 1:134, the specificity in predicting development of early onset preeclampsia was 82% (95%CI 6991%), whereas the sensitivity was 91% (95%CI 59-99%).13 cases with late onset preeclampsia were matched to 61 controls. The AUC for prediction of late onset preeclampsia was somewhat lower at 0.72 (95%CI 0.61-0.82). At a risk cut-off of 1:84, the sensitivity for prediction of late onset preeclampsia was 62% (95% CI 32-86%), whereas the specificity amounted 80% (68-89%). Conclusions: Measurements of PLGF and PAPP-A, done at the occasion of first trimester screening, and used together with the “Screen info” software possess predictive power in identifying pregnant women developing early and late onset preeclampsia. The employed algorithm might provide grounds for the decision to install early preventive measures with aspirin. Early onset:
80 60 40 20 0 0
20
40
60
80
100
100-Specificity Ă
P2.147 STUDY OF PREECLAMPSIA AND PREGNANCY INDUCED HYPERTENSION THROUGH HEART RATE VARIABILITY ANALYSIS Eduardo Tejera1, Maria José Areias2, José Manuel Nieto-Villar3, Irene Rebelo1 1 Faculty of Pharmacy/IBMC, Porto, Portugal 2 Maternity Hospital CHP, Porto, Portugal 3 Chemistry Faculty, Havana University, Havana, Cuba Objectives: To study the maternal autonomic balance during normal, hypertensive and preeclamptic pregnancy through the analysis of maternal heart rate variability (HRV) in different gestational ages. Methods: In the present work we performed a maternal heart rate variability comparative analysis during pregnancy between normal, hypertensive, and preeclamptic women. A total of 563 short ECG (10 min) records were obtained from 217 pregnant women (137 normal (N), 53 hypertensive (HT) and 27 preeclamptic (PRE) during several gestational ages in the sitting position. We studied conventional spectral (LF and HF) and complexity (SE, ApEn) indexes as well as a multifractal spectrum comparison. Results: The obtained results revealed that during pregnancy HF significantly decreased with lower values in the PRE group and was significantly correlated with maternal age. Similarly, complexity indexes also decreased during pregnancy either normal or pathological, but the values were significantly lower in hypertension and preeclampsia (ApEnN>ApEnHT>ApEnPRE). On the other hand the LF decreased from normal to HT group but increased with respect to PRE group. A significant negative correlation was found between complexity indexes and LF/HF and significant differences were also found with respect to parity and to influence of methyldopa treatment. Conclusion: The autonomic response during pregnancy is highly affected by hypertension or preeclampsia conditions leading to an adaptation capability reduction. The consequent reduction of complexity and HF indexes reveals an associated reduction of the parasympathetic activity while the sympathetic activity presented a more complex variation. On the other hand, the autonomic balance tends to be more favourable in twoparous women and is related with other factor like maternal age and effect of the methyldopa treatment.
Abstracts / Placenta 32 (2011) A173–A175
P2. 149 TGFb REGULATION OF TAZ DURING PLACENTAL DEVELOPMENT A. Tagliaferro1, T. Sivasubramaniyam1,3, I. Caniggia1,2 1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada 2 Department of Obstetrics & Gynaecology, University of Toronto, Toronto, Canada 3 Department of Physiology, University of Toronto, Toronto, Canada Objectives: TGFb signalling is vital in regulating trophoblast cell fate in pregnancy and its associated pathologies. Transcriptional co-activator with PDZ-binding motif (TAZ) has been recently demonstrated to contribute to TGFb signalling by interacting with Smads. In addition, evidence has shown that Partionioning defective protein 6 (Par6) is central to the TGFb Smad-independent signalling pathway during cancer progression. Herein we hypothesize that TAZ interacts with Par6 via its PDZ binding motif thereby regulating trophoblast cell differentiation via the Smadindependent TGFb pathway during placental development. Methods: Expression of TAZ and Par6 were examined in placental lysates throughout gestation (5-40wks) by immunoblotting and dual-labelled
A175
immunofluorescence staining in placental sections. Subsequently, TAZPar6 interaction was assessed by immunoprecipitation analysis across placental development and in JEG3 choriocarcinoma cells in the presence and absence of TGFb1/TGFb3. To establish a role for TAZ and Par6 in trophoblast migration, their expression was assessed following woundhealing assay in JEG3 cells. Results: During placental development TAZ and Par6 protein expression and interaction peaked at 10-14 wks of gestation. While at 5-9 weeks, TAZ and Par6 co-localized in the cytotrophoblasts and to a lesser extent in mesenchymal cells, with advancing gestation their spatial distribution shifted to the cytoplasm of syncytiotrophoblast cells and TAZ and Par6 staining were also detected in extravillous trophoblast cells within the distal part of the anchoring columns. During JEG3 cells migration, following wound-healing assays, TAZ and Par6 expression switched from the nucleus and cell-cell boundaries to the cytoplasm. Interestingly, while TAZ-Smad association increased upon TGFb1 exposure, TAZ-Par6 expression and interaction increased following TGFb3, but not TGFb1 treatments. Conclusion: Our data demonstrate a novel role for the Smad-independent signalling pathway during human placental development, whereby TAZ/ Par6 system may regulate trophoblast cells migration. (Supported by CIHR).
Contents lists available at SciVerse ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Missing abstracts from the IFPA 2011 meeting in Geilo, Norway P1.141 GLUCOSE TRANSPORTER (GLUT)-9 EXPRESSION IN PLACENTAL TISSUE M.U. Baumann1, M. Messerli1, R. Sager1, U. Reinhart1, C. Albrecht2, D.V. Surbek1 1 University Hospital of Berne, Department of Obstetrics and Gynecology 2 Institute for Biochemistry and Molecular Medicine, University of Berne, Switzerland Objectives: Maternofetal glucose transport is crucial for the fetal wellbeing since the fetus is not able to generate glucose de novo. Glucose transport (GLUT)-9 is a novel member of the GLUT family and exists as two isoforms. The aim of this study is to investigate the expression of GLUT-9 and GLUT-1 in different placental models. Methods: As placental model systems primary villous tissue of term placentae following normal pregnancies and the BeWo choriocarcinoma cell line were used. Protein expression was assessed by Western analysis of whole tissue- or cell lysate, or syncytial basal membrane-enriched (BM) and apical microvillous-membrane (MVM) fractions (obtained following differential ultra-centrifugation and magnesium precipitation). The mRNA level of transport proteins was analysed by real-time PCR. Results: GLUT-1 and GLUT-9 protein were detected in primary villous tissue. GLUT-1 protein concentration was lower in BM than in MVM. The gene expression of both GLUT-9 isoforms, namely GLUT-9a and GLUT-9b, were detected in placental villous tissue as well as in BeWo cells. The GLUT-9b mRNA level was higher than the one of GLUT-9a. Conclusions: GLUT-1 was asymmetrically expressed on syncytial basal and apical membranes. Further, in placental tissue and BeWo cells GLUT-9b mRNA levels were higher than those of GLUT-9a. Further studies are needed to investigate the role of GLUT-1 and GLUT -9 in the pathogenesis of pregnancy-specific diseases such as preeclampsia or gestational diabetes.
doi:10.1016/j.placenta.2011.10.002
P1.144 EFFECT OF HYPEROXYGENATION AND GROWTH BLASTOCYST-DERIVED TROPHOBLAST STEM CELLS
FACTORS
ON
Priyadarsini Kumar1, Twanda L. Thirkil1l, Catherine Vandevoort1,2, Gordon C. Douglas1 1 University of California, USA 2 California National Primate Research Center, USA Objectives: To study the effect of hyperoxygenation on rhesus monkey blastocyst-derived trophoblast stem cells in the absence and presence of growth factors. Methods: Blastocyst-derived rhesus monkey stem cells were cultured for 5 days in 2% O2 (normoxia for these cells). Growth factors (hCG, b-estradiol, HB-EGF, EGF, or FGF2) were added to media and the cells cultured for an additional 72 h either at 2% O2 or 4% O2 (hyperoxia). The cells were analyzed by immunofluorescence microscopy, Western blotting, and zymography to assess trophoblast marker expression and differentiation. Results: Trophoblast stem cells cultured at 2% O2 proliferated and maintained viability. When switched to 4% O2 the cells underwent apoptosis. Jar cells did not show this effect when switched from 2% to 4% O2. With the exception of HB-EGF, the addition of growth factors protected stem cells from the apoptotic effects of hyperoxygenation. In addition, preincubation of cells with N-acetylcysteine or caspase inhibitor protected the cells from apoptosis. At 2% O2 hCG, b-estradiol, EGF, and FGF2 increased cell proliferation. When cultured in 4% O2 in the presence of hCG or b-estradiol, large multinucleated cells were generated. In the presence of EGF or FGF2 the cells became multinucleated but were also migratory and invasive. This effect was more pronounced in the presence of EGF at 4% O2. Cells cultured at 4% O2 in the presence of growth factors expressed cytokeratin 7, b1 integrin, a5 integrin and secreted MMPs. They also expressed CD9 which was not seen with the other treatments. Conclusions: Exposing rhesus monkey trophoblast stem cells to 4% O2 after culture at 2% O2 caused apoptosis which was prevented by the addition of growth factors. The invasive multinucleated cells formed at 4% O2 in the presence of EGF could represent the first trophectoderm-derived cells that penetrate the endometrial epithelium during implantation.
Abstracts / Placenta 32 (2011) A173–A175
A174
P2.146 RISK ASSESSMENT IN EARLY PREGNANCY ALLOWS FOR PREDICTION OF PREECLAMPSIA
risk for late preeclampsia
late risk 100
Baumann Marc1, Wiedemann Ute2, Nydegger Urs2, Risch Martin2, Surbek Daniel1, Risch Lorenz2; for the predictive markers for the diagnosis of preeclampsia (PRADO) study group 1 University Hospital of Berne, Department of Obstetrics and Gynecology 2 Labormedizinisches Zentrum Dr. Risch, Liebefeld
early risk 100
Sensitivity
80 60 40 20 0 0
20
40
60
100-Specificity
Ă
80
100
Sensitivity
Objectives: Preeclampsia represents a complication during pregnancy and accounts for substantial fetal and maternal morbidity and mortality. It has been shown, that early intervention (before week 16) with aspirin is beneficial in preventing the occurrence of preeclampsia. Early recognition of pregnant women at risk for developing preeclampsia would allow for effective installation of pharmacological prevention. Aim: To evaluate an algorithm for identifying pregnant women at risk for development of preeclampsia. Methods: In a nested-case control study done in the ongoing prospective PRADO study, women developing preeclampsia were matched (for maternal age, gestational age, smoking status, and weight) to women with uneventful pregnancies. Blood drawn at the occasion of first trimester testing (gestational weeks 11-14) was analyzed for Placental Growth Factor (PLGF) and PAPP-A by an immunoanalytical method (Perkin Elmer, Turku, Finland). By using the “Screen info” software developed by Cuckle and colleagues, risks for developing early and late preeclampsia were calculated. Results: 11 cases with early preeclampsia occurring <34 weeks (early onset) were matched to 50 controls. ROC-analysis with an area under the curve (AUC) of 0.83 (95%CI 0.71-0.91) revealed good characteristics in predicting the occurrence of early onset preeclampsia. At a risk cut-off 1:134, the specificity in predicting development of early onset preeclampsia was 82% (95%CI 6991%), whereas the sensitivity was 91% (95%CI 59-99%).13 cases with late onset preeclampsia were matched to 61 controls. The AUC for prediction of late onset preeclampsia was somewhat lower at 0.72 (95%CI 0.61-0.82). At a risk cut-off of 1:84, the sensitivity for prediction of late onset preeclampsia was 62% (95% CI 32-86%), whereas the specificity amounted 80% (68-89%). Conclusions: Measurements of PLGF and PAPP-A, done at the occasion of first trimester screening, and used together with the “Screen info” software possess predictive power in identifying pregnant women developing early and late onset preeclampsia. The employed algorithm might provide grounds for the decision to install early preventive measures with aspirin. Early onset:
80 60 40 20 0 0
20
40
60
80
100
100-Specificity Ă
P2.147 STUDY OF PREECLAMPSIA AND PREGNANCY INDUCED HYPERTENSION THROUGH HEART RATE VARIABILITY ANALYSIS Eduardo Tejera1, Maria José Areias2, José Manuel Nieto-Villar3, Irene Rebelo1 1 Faculty of Pharmacy/IBMC, Porto, Portugal 2 Maternity Hospital CHP, Porto, Portugal 3 Chemistry Faculty, Havana University, Havana, Cuba Objectives: To study the maternal autonomic balance during normal, hypertensive and preeclamptic pregnancy through the analysis of maternal heart rate variability (HRV) in different gestational ages. Methods: In the present work we performed a maternal heart rate variability comparative analysis during pregnancy between normal, hypertensive, and preeclamptic women. A total of 563 short ECG (10 min) records were obtained from 217 pregnant women (137 normal (N), 53 hypertensive (HT) and 27 preeclamptic (PRE) during several gestational ages in the sitting position. We studied conventional spectral (LF and HF) and complexity (SE, ApEn) indexes as well as a multifractal spectrum comparison. Results: The obtained results revealed that during pregnancy HF significantly decreased with lower values in the PRE group and was significantly correlated with maternal age. Similarly, complexity indexes also decreased during pregnancy either normal or pathological, but the values were significantly lower in hypertension and preeclampsia (ApEnN>ApEnHT>ApEnPRE). On the other hand the LF decreased from normal to HT group but increased with respect to PRE group. A significant negative correlation was found between complexity indexes and LF/HF and significant differences were also found with respect to parity and to influence of methyldopa treatment. Conclusion: The autonomic response during pregnancy is highly affected by hypertension or preeclampsia conditions leading to an adaptation capability reduction. The consequent reduction of complexity and HF indexes reveals an associated reduction of the parasympathetic activity while the sympathetic activity presented a more complex variation. On the other hand, the autonomic balance tends to be more favourable in twoparous women and is related with other factor like maternal age and effect of the methyldopa treatment.
Abstracts / Placenta 32 (2011) A173–A175
P2. 149 TGFb REGULATION OF TAZ DURING PLACENTAL DEVELOPMENT A. Tagliaferro1, T. Sivasubramaniyam1,3, I. Caniggia1,2 1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada 2 Department of Obstetrics & Gynaecology, University of Toronto, Toronto, Canada 3 Department of Physiology, University of Toronto, Toronto, Canada Objectives: TGFb signalling is vital in regulating trophoblast cell fate in pregnancy and its associated pathologies. Transcriptional co-activator with PDZ-binding motif (TAZ) has been recently demonstrated to contribute to TGFb signalling by interacting with Smads. In addition, evidence has shown that Partionioning defective protein 6 (Par6) is central to the TGFb Smad-independent signalling pathway during cancer progression. Herein we hypothesize that TAZ interacts with Par6 via its PDZ binding motif thereby regulating trophoblast cell differentiation via the Smadindependent TGFb pathway during placental development. Methods: Expression of TAZ and Par6 were examined in placental lysates throughout gestation (5-40wks) by immunoblotting and dual-labelled
A175
immunofluorescence staining in placental sections. Subsequently, TAZPar6 interaction was assessed by immunoprecipitation analysis across placental development and in JEG3 choriocarcinoma cells in the presence and absence of TGFb1/TGFb3. To establish a role for TAZ and Par6 in trophoblast migration, their expression was assessed following woundhealing assay in JEG3 cells. Results: During placental development TAZ and Par6 protein expression and interaction peaked at 10-14 wks of gestation. While at 5-9 weeks, TAZ and Par6 co-localized in the cytotrophoblasts and to a lesser extent in mesenchymal cells, with advancing gestation their spatial distribution shifted to the cytoplasm of syncytiotrophoblast cells and TAZ and Par6 staining were also detected in extravillous trophoblast cells within the distal part of the anchoring columns. During JEG3 cells migration, following wound-healing assays, TAZ and Par6 expression switched from the nucleus and cell-cell boundaries to the cytoplasm. Interestingly, while TAZ-Smad association increased upon TGFb1 exposure, TAZ-Par6 expression and interaction increased following TGFb3, but not TGFb1 treatments. Conclusion: Our data demonstrate a novel role for the Smad-independent signalling pathway during human placental development, whereby TAZ/ Par6 system may regulate trophoblast cells migration. (Supported by CIHR).