Human serum – An alternative growth supplement for cell culture?

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Abstracts / Journal of Reproductive Immunology 115 (2016) 71–90

P2-23 Glucocorticoid receptors are upregulated and histone proteins responsible for rapid gene activation are downregulated in IUGR placentas Stefan Hutter, Simone Hofmann, Christina Kuhn, Julia Messner, Ulrich Andergassen, Doris Mayr, Sven Mahner, Petra Arck, Udo Jeschke Department of Gynaecology and Obstetrics, Ludwig-Maximilians-University Hospital, Munich, Germany Study hypothesis: The purpose of this study was to analyze the expression of both glucocorticoid receptor (GR) subtypes in placentas of pregnancies complicated by Intrauterine Growth Restriction (IUGR) and investigate the impact of GR changes on histone protein modification. Materials and methods: We screened for the fetal gender specific expression of GR␣ and GR␤ in 14 IUGR and 15 normal control placentas, equally divided in both gender. The expression of the histone proteins H3K4me3 and H3K9ac was analyzed in the same panel and finally in trophoblast tumor cells BeWo after stimulation with prednisolone in vitro. Results: In female IUGR placentas, upregulation of GR␣ was found in the Extravillous Trophoblast (EVT) and GR␤ upregulation was found in both the syncytiotrophoblast and the EVT in male IUGR placentas. Upregulation of GRs is accompanied with gender specific and trophoblast subtype specific inhibition of histone protein modification. H3K4me3 is down regulated in female syncytiotrophoblast and H3K9ac is downregulated in male EVT. Conclusion: Elevated cortisol level and or reduced 11␤-HSD2 activity leads to a gender specific upregulation of GRs in IUGR placentas. These changes are accompanied with histone modifications that are specific for fetal gender and for the trophoblast subtype. Histone modifications may be responsible for general epigenetic changes found in the IUGR offspring. http://dx.doi.org/10.1016/j.jri.2016.04.242 P2-24 GnRH analogues induce pro-inflammatory changes in T cell immunity Nayoung Sung, Maria D. Salazar, Li Wu, Annie Skariah, Svetlana Dambaeva, Kenneth Beaman, Alice Gilman-Sachs, Joanne Kwak-Kim Chicago Medical School at Rosalind Franklin University of Medicine and Science, Chicago, OH, USA Introduction: The potential function of Gonadotropin-releasing hormone (GnRH) and GnRH analogues in the immune system has been studied. In this study, we aim to investigate the effect of GnRH analogues in human immunity, such as the alteration of T cell immunity, in-vitro. Methods: Peripheral blood mononuclear cells (PBMCs) were treated with different doses of GnRH agonist (Buserelin acetate) or antagonist (Cetrorelix acetate), 0.1, 1, 5 and 10 ␮M for 4 h. TH1, TH2 and TH17 cell levels and the intracellular NF-kB signaling pathway in T lymphocytes were analyzed by flow cytometry. Results: TNF-␣+/IL-10+ T helper (TH) cell ratios were increased in PBMCs treated with 1, 5 and 10 ␮M GnRH agonist for 4 h culture when compared to controls. IFN-␥+/IL-10+ TH cell ratios were significantly increased with 0.1, 1, 5 and 10 ␮M GnRH agonist as compared with controls. When GnRH-agonist and -antagonist effects were compared, TNF-␣+, IFN-␥+ and IL-10+ producing TH

cell levels, and IFN-␥+/IL-10+ TH cell ratios were significantly different. The proportion of pNF-kB+ TH cells were decreased in PBMCs treated for 4 h with 0.1 and 1 ␮M of GnRH analogues but increased in PBMCs with 0.1 ␮M GnRH analogues for 48 h as compared to untreated controls. Conclusions: GnRH analogues led to pro-inflammatory changes in TH cells, the effect varied depending on GnRH analogues and doses. However, the changes were not explained with NF-␬B signaling pathway. http://dx.doi.org/10.1016/j.jri.2016.04.243 P2-25 Higher levels of peripheral natural killer cells in patients with primary recurrent miscarriage are associated with altered cytokine profile Franziska Müller, Ruben Kuon, Kilian Vomstein, Thomas Strowitzki, Volker Daniel, Bettina Toth Department of Gynecological Endocrinology and Fertility Disorders, Ruprecht-Karls University Heidelberg, Heidelberg, Germany Objectives: Natural killer cells (NK cells) are associated with recurrent miscarriage (RM). Elevated levels of NK cells as well as cytokine dysbalances have been described in recent studies. However, controversial debates are ongoing. The aim of this study was to identify factors regulating NK cell levels with focus on a high sensitive cytokine assay. Methods: In total n = 91 couples with ≥3RM were identified. RM subgroups consisted of n = 53 primary RM (pRM) and n = 38 secondary RM (sRM). Peripheral blood was drawn in non-pregnant patients and CD56+CD16+ NK cells were determined using fourcolor fluorescence flow cytometry. Cytokines were analyzed using LUMINEX technique and highly sensitive cytokine assays (R&D systems). Results: In our subset of RM patients, higher levels of CD56+CD16+ were observed in pRM vs sRM patients (/␮l and %, mean ± SD 290 ± 119, 15.2 ± 5.8 vs 219 ± 126, 11.6 ± 4.8, p = 0.002, p = 0.008). RANTES (CCL5) and TNF were higher in pRM patients (both p < 0.05). Ratios of cytokines reflecting TH1/TH2 profile showed no significant difference. Conclusion: NK cells as well as RANTES and TNF differed significantly between pRM and sRM indicating possible effects of previous live birth. However, no significant differences were present with regard to cytokine ratios reflecting TH1/TH2 profile. We hypothesis that NK cells as well as RANTES and TNF might reflect immmunological changes in pRM and sRM patients and help to identify patients which might profit from immunomodulatory treatment. http://dx.doi.org/10.1016/j.jri.2016.04.244 P2-26 Human serum – An alternative growth supplement for cell culture? Julia Heger, Jana Pastuschek, Karolin Fröhlich, Lisa M. Görke, Sven Peters, Udo R. Markert, André Schmidt University Hospital Jena, Department of Obstetrics, Placenta Lab, Jena, Germany Purpose: Most experimental setups include the use of fetal calf serum (FCS) as medium supplement for cell and tissue culture. Due to its unstable availability, biosafety, potential interaction of human

Abstracts / Journal of Reproductive Immunology 115 (2016) 71–90

cells with xenogeneic growth factors and ethical aspects limitations of FCS must be considered. Aim of this study was to compare the qualification of human serum (HS) and FCS. Methods: Proliferation, migration and invasion of the cervical cancer cell lines SiHa and SW756 and the granulosa cell lines KGN and COV434 cultured in FCS or HS supplemented medium were investigated. The effects of these two sera on spheroid formation via hanging drops were analyzed microscopically. Subsequently, the influence of HS and FCS on pre-formed spheroids was analyzed by pipetting and culturing them on poly-HEMA coated 96 well plates. Results: Both, FCS and HS, stimulate cell proliferation and migration similarly, whereas HS enhanced invasion. In FCS supplemented medium SiHa cells only formed loose aggregates, while HS induced regularly shaped spheroids. Transfer of SiHa cell spheroids pre-formed in HS to medium containing FCS led to a degradation. Conclusion: HS ensures cell viability as well as migration or invasion capacity and represents a good alternative for FCS in cell culture. We conclude that xenogeneic sera do not mimic the human situation properly. To evaluate additional effects on immune responses or toxic effects further investigations are necessary. http://dx.doi.org/10.1016/j.jri.2016.04.245 P2-27 IL-27 triggers IL-10 production in Th17 cell by c-Maf/ROR␥t/Blimp-1 signal to promote the progress of endometriosis Ming-Qing Li, Kai-Kai Chang, Li-Ping Jin, Xiao-Yong Zhu, Da-Jin Li Hospital & Institute of Obstetrics and Gynecology, Fudan University, Shanghai, China IL-10 production by Th17 cells is critical for limiting autoimmunity and inflammatory responses. However, induction of IL-10-producing Th17 cells is still large unknown. Here we report IL-10+Th17 cells are significantly increased in the peritoneal fluid of women with endometriosis, along with elevation of IL-27, IL6 and TGF-␤. Compared to peripheral CD4+ T, endometrial CD4+T highly expressed IL-27 receptors (WSX-1 and gp130), especially ectopic endometrium. Under external (2,3,7,8-tetrachlorodibenzop-dioxin, TCDD) and local (estrogen, IL-6 and TGF-␤) environmental regulation, IL-27 from macrophage and endometrial stromal cells (ESC) induces IL-10 produce in Th17 cells in vitro and in vivo. This process is mediated through the interaction between c-musculoaponeurotic fibrosarconma (c-Maf) and retinoic acidrelated orphan receptor gamma t (ROR␥t), and associated with the up-regulation of downstream B lymphocyte-induced maturation protein-1 (Blimp-1). Such IL-10+Th17 cells in turn stimulate the proliferation and implantation of ectopic lesion and accelerate the progress of endometriosis. These results suggest that IL-27 is a pivotal regulator in endometriotic immune tolerance by triggering Th17 to produce IL-10 and promote rapid growth and implantation of ectopic lesion. This provides a scientific basis on which potential therapeutic strategies targeted to prevent the development of endometriosis, especially for patient with high level of IL-27 and IL-10+Th17 cells. http://dx.doi.org/10.1016/j.jri.2016.04.246

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P2-28 Immunohistochemical analysis of CD138-positive plasma cells in the endometrium Maja Weber, Bettina Toth, Isabel Santillan, Ruben Kuon, Christin Bär, Ekkehard Schleussner, Udo R. Markert University Hospital Jena, Department of Obstetrics, Placenta Lab, Jena, Germany Background: Chronic endometritis is often mild, characterized by the infiltration of plasma cells in the endometrium and usually caused by bacteria. It may be a reason for recurrent miscarriage (RM) and repeated implantation failure (RIF). The aim of this study was to quantify plasma cell concentration in the endometrium of RM and RIF patients. Methods: Endometrial biopsies from 450 patients (232 RIF, 70 RM, 151 unknown reason of infertility) were investigated via immunohistochemistry using DAB staining of CD138+ plasma cells. A second analysis was performed in 15 patients after antibiotictherapy. ≤3 plasma cells/mm2 were assumed as normal value based on previous reports. Results: Elevated plasma cell counts have been detected in 14.7% of RIF patients, 15.7% of RM patients and 13.2% of patients with unknown reason of infertility. After antibiotic therapy of chronic endometritis, in 11 of 15 (73.3%) patients the plasma cell count decreased to normal range indicating successful treatment. Conclusion: A subgroup of patients under reproductive medical treatment may have asymptomatic chronic endometritis which is only reflected by elevated endometrial plasma cells and may be treated with antibiotic therapy. The effect of this treatment on infertility needs to be further evaluated. http://dx.doi.org/10.1016/j.jri.2016.04.247 P2-29 Immunohistochemical analysis of CD56-positive natural killer cells in the human endometrium Maja Weber, Bettina Toth, Isabel Santillan, Christin Bär, Ekkehard Schleußner, Udo R. Markert University Hospital Jena, Department of Obstetrics, Placenta Lab, Jena, Germany Question: Recurrent miscarriage (RM) and repeated implantation failure (RIF) are highly frustrating reproductive disorders. Uterine natural killer (uNK) cells are major players during implantation and early pregnancy. However, the distribution during the menstrual cycle and the pathophysiological role in pregnancy is only partially understood. Previous data indicates that the concentration of uNK cells in women with RM and RIF is increased. The aim of our study was to quantify uNK cell concentration in the endometrium of RM and RIF patients. Methods: Endometrial biopsies from 254 patients (117 RIF and 137 RM), and 15 fertile women (controls) were investigated via immunohistochemistry using DAB staining of CD56+ uNK cells. Results: The endometrium of RIF and RM patients contained 275 + 202 and 233 + 185 uNK cells/cm2 , respectively. 67.5% of RIF and 65.7% of RM samples were in the range of 40–300 uNK cells/cm2 and 30.8% of RIF and 27.0% of RM samples contained more than 300 uNK cells/cm2 . In contrast, 158 + 70 uNK cells/cm2 were detected in the controls, of which 93% were within the