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Abstracts / Placenta 35 (2014) A1eA112 A57

B) ProtonMagnetic resonance spectroscopy was performed to evaluate thebiochemical/ metabolic profile in placentas. A significant decrease inphosphocholine was observed in APS-mice compared to Ctrl (PCh/Cr+PCr:APS(n¼7)¼0.50±0.78 vs control (n¼4)¼1.69±0.73, p¼0.0352). Decreasedglucose levels and increased lactate levels, indicative of hypoxia, weremeasured in placentas from APS-mice compared to Ctrl (Glc/Cr+PCr:APS(n¼6)¼ 0.03±0.0.05 vs control (n¼4)¼1.04±0.9, p¼0.0222; Lac/Cr+PCr:APS(n¼7): 0.47±0.0.80 vs control (n¼4): 4.4±4.1, p¼0.0310). Placentalinsufficiency in APS was also associated with abnormal fetal neuro-development characterised by cortical axonal cytoarchitecture dis-ruption(NF200 and MAP-2 expression) and increased neurodegeneration(FluoroJadeB). Increased anxietywas observed in the offspring of APS-mice.

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Conclusion: Using two novel non invasive in uterus MRI-based methodswe were able to determine complement activation and metabolism in theplacentas and predict fetal outcomes.

P1.149-N.PATHOLOGICAL MECHANISMS OF ANTIPHOSPHOLIPID ANTIBODIES INTROPHOBLASTIC CELL FUSION

Tess Marchetti a,b, Philippe de Moerloose b, Marie Cohen a a Laboratory ofHormonology, Geneva University Hospitals, Geneva, Switzerland;bHaemostasis Unit, Geneva University Hospitals, Geneva, Switzerland

Objectives: Obstetrical Antiphospholipid Syndrome (APS) associatespregnancy pathologies with antiphospholipid antibodies (aPL). It has beensuggested that aPL could affect trophoblastic cell fusion. However, theirpathogenicity is still poorly understood.

Toll-like Receptors (TLR) have been implicated in the pathological activa-tion of aPL on endothelial cells. Although inflammation has been incrim-inated in this process, endoplasmic reticulum (ER) stress activation seemsmore related to trophoblastic fusion impairment by aPL.The aim of our study was to evaluate the pathological mechanisms of aPLon trophoblastic cell fusion.Methods:BeWocell line is usedas amodel tomimic trophoblastic cell fusion,triggered by forskolin. Results were corroborated with primary cytotropho-blastic cells. Fusion index was determined by immunocytochemistry andbiochemical differentiation by ELISA measuring hCG secretion.Effects of TLR on BeWo cell fusionwere evaluated using blocker antibodiesfor TLR2 and 4, peptide binding for TLR2 and 4, and stable shRNA TLR4 inthe presence or not of aPL.

After determining the role of TLR4 in trophoblastic cell fusion impairmentby aPL, signalling cascade of inflammationwas studied by ELISAmeasuringTNFa and IL8 secretion. ER stress activation was assessed by qPCR and WBquantifying Glucose-regulated protein 78 (GRP78) and C/EBP-homologousprotein (CHOP) expressions.Result: Fusion index and hCG secretion are decreased by addition of aPL. Inthe presence of aPL and anti-TLR4 antibodies or in BeWo cells transfectedwith shRNA TLR4, trophoblastic cell fusion and hCG secretion wererestored.Neither TNFa nor IL8 secretion was observed in BeWo cells stimulated byaPL. Increase in GRP78 and CHOP expressions was observed in cellsstimulated by aPL, but not when blocked with anti-TLR4 antibodies, sug-gesting a role of ER stress in trophoblastic cell fusion impairment by aPL.Conclusion: aPL interfered with fusion process and biochemical differ-entiation of trophoblastic cells via TLR4 that triggers ER stress activation.

P1.150.EXAMINING THE EFFECT OF OBESITY-ASSOCIATED SYSTEMICINFLAMMATION ON THE UTERINE IMMUNE CELL NICHE IN EARLYPREGNANCY

Yoona Kim, Sofie Perdu, Mahroo Aghababaei, Alexander Beristain TheUniversity of British Columbia, Vancouver, Canada

Objectives: Low-grade inflammation associates with major obstetricaldisorders like preterm birth and preeclampsia and is thought to be causalin the development of these high-risk pregnancies. Control of immunecell-directed pro-inflammatory cytokine production and cytotoxicity iscrucial for utero-placental function; aberrant pro-inflammatory T helper(h) 1 cell activity within the uterine microenvironment associates withplacental dysfunction and poor pregnancy outcome. Immunological im-balances and low-grade inflammation are associatedwith excess adiposity.However, the cellular mechanism(s) linking obesity-associated inflam-mation to poor pregnancy outcome are poorly understood. As a first step inaddressing this knowledge gap, our study aims to examine multiple im-mune cell subsets within decidual tissues of non-obese and obese womenin early pregnancy.

Methods: Patient information, blood serum and decidual tissue fromgestationally aged-matched (8-10 weeks) women undergoing electivepregnancy terminations were used to determine BMI, serum C-reactiveprotein (CRP) (Qg/mL) and immune cell composition. Women were cate-gorized as healthy BMI (20-24.9) not presenting with low-grade inflam-mation (CRP < 2 Qg/mL; ControlCRP-) or obese BMI (� 30) presenting withlow-grade inflammation (CRP � 4 Qg/mL; ObeseCRP+). Immune cells,including natural killer cells (NKs), T cells (Th1, Th2, Th17, Tregs and CD8+

cytotoxic cells), macrophages and dendritic cells, were quantitated bymulticolour flow cytometry analysis.Results:Wedid not detect differences in proportions of CD56bright decidualNKs, dendritic cells, macrophages or Th17 and CD8+ T cells betweendecidual tissues of ControlCRP- (n¼15) and ObeseCRP+ (n¼15) women.However, proportions of immunosuppressive CD4+/FoxP3+ Tregs, pro-in-flammatory IFN©+ Th1 cells, and cytotoxic CD16+ NK cells were elevated inObeseCRP+ decidua while IL-13+ Th2 proportions were decreased.Conclusion: This study suggests that obesity-linked inflammation inearly pregnancy does alter immune cell composition within thematernal-fetal interface. These new findings lay the groundwork forfuture studies aimed at dissecting the importance of these cellularchanges in pregnancy.

P1.151.PHAGOCYTIC ACTIVITY OF MACROPHAGES IS REGULATED BY FIRSTTRIMESTER DECIDUAL CELLS IN RESPONSE TO PRO-INFLAMMATORYSTIMULI VIA THE MODULATION OF “DON'T-EAT-ME” SIGNAL

Longzhu Piao a, Susheela Tridandapani b, S. Joseph Huang a aDeartment ofOb/Gyn, The Ohio State University, Columbus, OH, USA; b SBS-MicrobialInfection/Immunity, The Ohio State University, Columbus, OH, USA

Abstracts / Placenta 35 (2014) A1eA112A58

Objectives: Phagocytosis is crucial for tissue homeostasis and remodellingin addition to its role in defending against invading organisms and allo-geneic cells. A “Don’t-eat-me” signal generated by binding of signal reg-ulatory protein alpha (SIRPa) on the surface of macrophages and itsubiquitously expressed ligand, CD47, plays an important role in regulatingphagocytic activity. Our previous results show that pro-inflammatorystimulus-activated first trimester decidual cells (FTDCs) enhance the ac-tivity of macrophages in inducing apoptosis in first trimester extravilloustrophoblasts (EVTs). Seeking to enhance understanding of decidualremodeling during pregnancy, this proposal aims to study themechanismsof FTDC/macrophage interactions resulting in previously undisclosed ho-meostasis on placentation.

Methods: FTDCs were primed with estradiol (E) (10�8 M) + medrox-yprogesterone acetate (M) (10�7 M) for 7d followed by incubating with 1ng/ml of interleukin-1 beta (IL-1b) or tumor necrosis factor-alpha (TNF-a)for 24h. Peripheral monocyte-derived macrophages were incubated withconditioned media (CM) from these FTDC cultures for 2d. The phagocyticactivity of peripheral monocyte-derived macrophages was examined byco-culturing with apoptotic bodies derived from PKH67-labeled HTR-8/SVneo (HTR-8) cells, a first trimester EVT cell line. The phagocytosis of HTRapoptotic bodies by Mfs was evaluated by fluorescent microscopy andflow cytometric analysis. SIRPa expression on macrophages was examinedby flow cytometric analysis, qRT-PCR and Western blot analysis.Results: CM from IL-1b- or TNF-a-treated FTDCs consistently enhancedphagocytosis of HTR-8-derived apoptotic bodies by macrophages, whileSIRPa expression was suppressed.Conclusions: These results suggest that FTDCs may play a role in main-taining decidual homeostasis by promoting phagocytic activity of macro-phages upon remodelling in response to pro-inflammatory stimuli.However, the enhanced phagocytic activity can also lead to increasedkilling of invading semi-allogeneic EVTs.

P1.152.PREGNANCY RECALL IN DECIDUAL NK CELLS

Debra Goldman-Wohl a, Moriya Gamliel b, Ronit Gilad a, Tal Imbar a, RonitHaimov-Kochman a, Caryn Greenfield a, Iris Eisenberg-Loebl a, OferMandelboim b, Simcha Yagel a a The Magda and Richard Hoffman Centerfor Human Placenta Research, Department of Obstetrics and Gynecology,Hadassah-Hebrew University Medical Center, Jerusalem, Israel;b Lautenberg Center for Immunology, Hebrew University Hadassah MedicalSchool, Jerusalem, Israel

Objective: Natural killer (NK) cells, lymphocytes of the innate immunesystem, act to kill tumor and infected cells. However, recent studies sug-gest that NK cells possess some features of adaptive immunity includingimmune memory. Specifically, the expansion of the NKG2C+ NK cell pop-ulation is implicated in the “memory” response of NK cells to HCMVinfection. NK cells of the decidua (dNK) possess unique phenotypical andfunctional properties and are considered regulators of remodeling of thematernal fetal interface. Here we investigate if these dNK cells have theability to “remember” pregnancy.

Methods: dNK cells from nulliparous and parous women were isolatedfrom first trimester elective pregnancy terminations .The dNK cell receptorrepertoire was analyzed by FACS. Transcriptome analysis was performedusing RNA-seq.Results: Significantly more NKG2C+ dNK cells (P¼0.0008 (2-tailed)) weredetected in the parous versus nulliparous women. This finding was specificfor the dNK population but not observed in T cells, and did not correlatewith CMV serology status. In parous samples, transcriptome analysisrevealed upregulation of genes related to angiogenesis, tissue remodeling,TH1 to TH2 shift, secretion of growth factors and NK cell function/recruitment. The most significant transcriptome results were validated byreal-time PCR; they include PAEP, IGFBP1 and SPP1.Conclusion: This observation suggests that dNK cells that play a role inremodeling the maternal fetal interface may additionally possess theability to “recall” pregnancy. This findingmay give insight to the etiology ofpreeclampsia with increased risk in first pregnancy.

P1.153-N.CYTOKINES PROLIFE IN MATERNAL AND CORD BLOOD OF DIABETICMOTHERS

Glilciane Morceli a, Cristiane Hara b, Adenilda Hon�orio-França b, EduardoFrança b, Danny Laura Fagundes a, Marilza Rudge a, D�eboraDamasceno a, Iracema Calderon a aBotucatu Medical School, Botucatu/S~aoPaulo, Brazil; b Institute of Biological and Health Science - FederalUniversity of Mato Grosso, Barra do Garças/Mato Grosso, Brazil

Background: Diabetes is an inflammatory condition and cytokines havebeen implicated in its physiopathology. Thus, the interaction betweencytokines pro-inflammatory and anti-inflammatory is essential for preg-nancy outcome.

Objective: Evaluated the cytokine profile in maternal and cord blood frompregnancies complicated by hyperglycemia.Methods: The women were classified into three groups, according to theirglycemic status: normoglycemic (N ¼ 15), mild hyperglycemic (N ¼ 15)and diabetic (N ¼ 15). The cytokines levels were determined by flowcytometry and the data were analyses by software FCAP Array 1.0.Results: No significant changes were detected in the IL2, IL4 and IFN-gconcentrations between maternal and cord blood, neither among threegroups. IL6 and IL10 concentrations were increased in cord blood, and IL17levels were higher in maternal blood of diabetic mothers. Maternal bloodIL10 and IL17 concentrations were found significantly correlated withlevels observed in the umbilical cord.Conclusions: These data suggest that diabetes modifies the cytokinesprofile in maternal and cord blood. These results corroborate the hy-pothesis that diabetes alters the role of cytokines, and thus may influencethe systemic and mucosal immune response.Key words: cord blood, maternal blood, cytokines, diabetes, flowcytometry.Acknowledgements - FAPESP (2012/18033-0)

P1.154.IMMUNE SYSTEM AND REPRODUCTIVE SUCCESS: THE GENETICS OF THEMATERNAL-FETAL INTERFACE

Olympe Chazara a, Annettee Nakimuli b, Lydia Farrell a, Susan Hiby a, HugoHilton c, Paul Norman c, Neda Nemat-Gorgani c, Alison Eliott d,e, PontianoKaleebu d, Florence Mirembe b, Peter Parham c, AshleyMoffett a aUniversity of Cambridge, Cambridge, UK; bMakerere University,Kampala, Uganda; c Stanford University School of Medicine, Stanford, USA;dMedical Research Council/Uganda Virus Research Institute, Entebbe,Uganda; e London School of Hygiene and Tropical Medicine, London, UK

Objectives: The success of human pregnancy depends on interactionsbetween maternal killer cell immunoglobulin-like receptors (KIR)and their fetal ligands, HLA-C1 and C2. Our previous genetic studiesof pregnancies show that in disorders of pregnancy due todefective placentation (pre-eclampsia, recurrent miscarriage and fetalgrowth restriction) there is an increased frequency of maternalKIRAA genotype in combination with a paternally derived fetal HLA-C2group. The telomeric KIRB region provides protection from thesedisorders.Methods: Firstly, the inhibitory KIR2DL1, located in the centromeric KIRregion, binds strongly to HLA-C2, whereas the activating receptor for HLA-C2, KIR2DS1, is found on the telomeric B region. We have typed for allelicvariation of KIR2DL1 and KIR2DS1 in our pregnancy cohorts (1300 preg-nancies comparing controls and pre-eclampsia or recurrent miscarriage).Secondly, we have now conducted a case-control study in Kampala,Uganda. Mother-baby pairs from 484 normal pregnancies and 254complicated with pre-eclampsia were genotyped for KIR and HLA-C1 or C2groups.Result: We show that KIR2DL1 is polymorphic, with 11 alleles observedwhereas KIR2DS1 is monomorphic. One of the common KIR2DL1 alleles,KIR2DL1*003, is associated with pre-eclampsia and recurrent miscarriage(P-value¼0.0016, OR 1.5 and P¼0.0125, OR 1.7 respectively).