Microscopic features of the placenta of Mirounga leonina (carnivora, pinnipedia, phocidae)

Placenta 36 (2015) 469e521

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Abstracts for the VI Latin American Symposium on Maternal-Fetal Interaction and Placenta and V Latin American Symposium on Reproductive Immunology Meeting 2015

Abstract Outline e SLIMP and LASRI 2015

Opening / Closing Lectures SLIMP Poster Award SLIMP Oral Award Mini Course A Mini Course B Keynote Lectures Symposium Presentations Hot topic Presentations Oral Presentations A Oral Presentations B Young Investigator Session Poster Session A Poster Session B

http://dx.doi.org/10.1016/j.placenta.2015.01.381

(L1 e L6) (SPA) (SOA) (MCA 1 e MCA 3) (MCB) (K1 e K12) (S1 e S17) (HT 1 e HT 6) (OA 1 e OA 7) (OB 1 e OB 6) (YI 1 e YI 4) (PA.1 e PA.61) (PB.1 e PB.51)

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“OPENING AND CLOSING LECTURES”. L1. PERICONCEPTIONAL IMPAIRMENTS AND FUTURE HEALTH

implantation or impair placental morphogenesis. Failure to successfully establish Treg cell-mediated immune tolerance is emerging as an important etiological pathway leading to poor fertility or imparting compromised placental function and associated pregnancy disorders, leading to adverse consequences for the fetus and offspring.

Tom P. Fleming. University of Southampton, Southampton, UK Across mammalian species and including the human the periconceptional (PC) period has been identified as vulnerable to environmental influences which may change the programme of development and have lasting effects on disease risk into adulthood. We have studied the effect of maternal undernutrition during the PC period in mice (low protein diet, LPD). Even restricting LPD to just the preimplantation period with normal nutrition thereafter and in postnatal life (Emb-LPD) is sufficient to induce increased cardiovascular, metabolic and behavioral disease in adult offspring. We have found the timeline of programming initiates through Emb-LPD dietinduced reduction in insulin and branched-chain amino acid (BCAA) concentrations within maternal serum and/or uterine fluid. These changes are sensed by blastocysts via the mTOR signal pathway. This sensing mechanism associates with altered programming even if Emb-LPD blastocysts are transferred to control mothers. Similarly, in vitro cultured embryos with reduced insulin and BCAAs and subsequent transfer lead to similar adult disease phenotype to maternal Emb-LPD treatment. Programmed embryos undergo compensatory responses within the extra-embryonic cell lineages (trophectoderm; primitive endoderm) to promote nutrient retrieval during gestation to promote fetal growth. These responses include increased proliferation, endocytosis and motility of affected tissues. However, embryonic lineages show evidence of increased apoptosis and reduced survival signalling. Derivation of embryonic stem cell lines from blastocysts from diettreated mothers retain programming characteristics over several passages and permit mechanistic analyses and reduced use of animals. These lines have also been effective in identifying epigenetic mechanisms underlying lineage-specific programming responses. Lastly, Emb-LPD compensatory responses induced within extra-embryonic lineages cause increased perinatal growth. However, perinatal weight in programmed offspring correlates positively with disease risk in later life. These data therefore show a continuum of biological processes from maternal PC diet to adverse adult phenotype. Funded through BBSRC, EU FP7 EpiHealth and EpiHealthNet.

L2. IMMUNOMODULATION IN IMPLANTATION Sarah A. Robertson. Robinson Research Institute, University of Adelaide, SA 5005, Australia In mammalian pregnancy, there is attenuated expression of paternalderived alloantigens by gestational tissues. Despite this, the fetus and placenta are recognized as non-self by the maternal immune system, and are vulnerable to immunological attack. An active system to suppress inflammation and prevent rejection must exist from when conceptus and maternal tissues first come into contact at implantation. Crucial mediators of immune protection are inducible regulatory T cells (Treg cells). Unless sufficient Treg cells are present in the endometrium, successful implantation and progression to pregnancy cannot ensue. This key role of Treg cells confers to the female immune system substantial capability to influence reproductive events, particularly around the time of conception, embryo implantation and early placental development. While on the one hand this risks susceptibility to immune-based reproductive disorders, the potential evolutionary trade-off is the benefit of quality control to avoid poor reproductive outcomes. Several factors are required to establish a robust Treg cell response and an immune environment conducive to successful implantation and pregnancy. These include (a) appropriate cytokine balance; (b) correct phenotype of endometrial macrophages and dendritic cells to enable Treg cell activation; (c) sufficient estrogen and progesterone to stabilize and strengthen Treg cell phenotype, and (d) adequate male partner seminal fluid composition, including MHC antigens and immune-deviating agents, to promote priming of Treg cell populations after coitus. Compromises in the quality of this immune adaptation at conception can influence the early embryo and either prevent

L3. ROLE OF THE ENDOMETRIUM IN SUPPORTING EARLY DEVELOPMENT OF THE HUMAN PLACENTA Graham J. Burton. Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, UK In all mammals the initial nutritional support for the co Cambridge nceptus is provided by histiotrophe secreted from oviductal cells and endometrial glands. In the human, it was previously thought that the duration of histiotrophic nutrition is short due to our uniquely invasive form of implantation that removes the conceptus from the uterine lumen. However, it is now realized that secretions from the endometrial glands are delivered into the intervillous space during the first trimester through openings in the developing basal plate. These secretions, rich in glycogen, lipid droplets and large maternal glycoproteins such as MUC-1, are phagocytosed by the syncytiotrophoblast, where they co-localize with the lysosomal pathway suggesting their breakdown. Immunohistochemistry also indicates that these secretions contain a range of powerful mitogenic growth factors, including epidermal growth factor (EGF) and vascular endothelial growth factor. Application of EGF to first trimester explants stimulates proliferation of cytotrophoblast cells, and so the secretions may play an important role in promoting early placental development. Importantly, the pattern of glycosylation of the secretions changes between the late secretory phase and early pregnancy, with the loss of sialic acid endcaps. Consequently, any of the growth factors that reach the maternal circulation through the uterine veins will be rapidly cleared by asialoglycoprotein receptors in her liver. This arrangement allows a highly proliferative microenvironment to be created within the placenta without putting the mother at risk of excessive stimulation. Evidence from other species indicates that the conceptus signals to the glands to upregulate secretory activity and so promote its own development. In the human, the glandular epithelium undergoes characteristic morphological changes in early pregnancy indicative of hypersecretion, the Arias-Stella reaction. Experiments in vitro using an immortalized gland epithelial cell line indicate that chorionic gonadotropin and placental lactogen from the trophoblast and prolactin from the decidua may mediate this effect.

L4. SEXUAL DIMORPHISM IN THE EFFECT OF MATERNAL OBESITY ON PLACENTAL MITOCHONDRIAL FUNCTION L. Myatt, S. Muralimanoharan, A. Maloyan. Center for Pregnancy and Newborn Research, University of Texas Health Science Center San Antonio, San Antonio, TX 78229, USA Maternal obesity programs the offspring for adverse outcomes in later life. In adults obesity is associated with decreasing mitochondrial function. We hypothesized that maternal adiposity would affect placental mitochondrial respiration and function. Placentas were collected at term by C section in the absence of labor from women (n¼33) of pre-pregnancy BMI 18.5 e 40. Isolated villous cytotrophoblast cells were allowed to syncytialize over 72 hr. Mitochondrial respiration was measured in a Seahorse XF24. Basal, ATPstimulated and maximal respiration and spare capacity were all significantly reduced with increasing maternal adiposity. Increasing maternal adiposity was associated with significantly increased generation of reactive oxygen species but significantly decreased ATP generation, mitochondrial biogenesis and expression of mitochondrial complexes I-V. Culture of trophoblast from lean, but not overweight or obese women in galactose as glycolytic substrate increased respiration illustrating a lack of metabolic flexibility with increasing adiposity. Expression of the hypoxamir, miR-210 was increased with greater adiposity, but only in trophoblast from a female fetus, and accompanied by decreased expression of the mitochondrial

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target genes NDUFA4 and ISCU. Greater adiposity was associated with increasing expression of the transcription factor NFkB p65 in both sexes but increased NFkb p50 (NFkB1) only in female trophoblast. Binding of NFkB1 to the mir210 promoter was significantly increased only in female trophoblast of overweight and obese groups. Addition of TNFa to mimic inflammatory stress increased expression of NFkb p65 in both male and female but increased NFkB1 only in female trophoblast accompanied by increased mir-210 expression and decreased NDUFA4 and ISCU. Inhibition of NFkB1 prevented the TNFa-induced decrease in mitochondrial respiration. There is a sexual dimorphism in NFkB-regulated placental miR-210 expression with increasing maternal adiposity perhaps representing an integral part of the adaptation by the female fetus to an adverse in utero environment and susceptibility to programming in utero.

L5. FETO-PLACENTAL ENDOTHELIUM IN PREGNANCY DISEASE

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Complex 1 and 2 in maternal tissues and in the placenta, decreased protein expression and activity of key amino acid transporters in the placental barrier and resulted in fetal growth restriction. Studies in cultured primary human trophoblast (PHT) cells demonstrated that folate sensing by mTOR involves both mTOR Complex 1 and 2 and requires the proton-coupled folate transporter (PCFT, SLC46A1). Moreover, the role of PCFT in mTOR folate sensing is not related to its function as a transporter mediating cellular folate uptake. In contrast to PHT cells, only mTORC1 functions as a folate sensor in HEK293 and MCF7 cells. Collectively, we have identified a novel specific molecular link between folate availability and cell growth and proliferation, which involves PCFT and mTOR. In proliferating cells, including cancer cells, PCFT and mTOR may modulate cell growth and proliferation in response to changes in folate availability. We report for the first time that maternal folate deficiency inhibits placental mTOR signaling and amino acid transporter expression and activity resulting in fetal growth restriction, providing a novel link between maternal folate availability and fetal growth.

Gernot Desoye 1, Ingrid Lang 2, Christian Wadsack 1, Monika Ute Panzenboeck 1, Martin Gauster 2, Luciana Scholler 1, 3, Lassance 1, Silvija Tokic 1,2, Jelena Loegl 1, 2, Berthold Huppertz 2, Erika Nussbaumer 1, Ursula Hiden 1. 1 Department of Obstetrics and Gynaecology, Medical University Graz, Austria; 2 Institute of Cell Biology, Histology and 3 Institute of Embryology, Medical University Graz, Austria; Pathoyphysiology, Medical University Graz, Austria

SLIMP POSTER AWARD. LYSOPHOSPHATIDIC ACID INCREASES THE PRODUCTION OF PIVOTAL MEDIATORS OF DECIDUALIZATION AND VASCULARIZATION

The feto-placental endothelium is exposed to metabolic and stress conditions (oxidative, nitrative, inflammatory changes) in the placenta and fetal circulation, which associate with pregnancy diseases. Little is known about molecular and cellular changes of the feto-placental endothelium in conditions such as pre-eclampsia and fetal growth restriction. Most studies have concentrated on maternal overnutrition found in diabetes mellitus and obesity. Prominent commonalities of both invariably include fetal hyperglycemia and hyperinsulinism. At the end of gestation insulin receptors prevail at the feto-placental endothelium making it a target for the effects of fetal hyperinsulinism. These encompass enhancing placental vascularization through synergistic effects on MMP14 activity (extracellular matrix degradation), NO production (leading to tube formation) and on the cytoskeleton (through Rac1). Placental hypervascularization in maternal diabetes is needed to compensate the elevated fetal needs for oxygen because of insulin-stimulated fetal aerobic metabolism. Insulin often cooperates with glucose in inducing endothelial changes. One example is the promotion of glycogen synthesis and glucose storage in the feto-placental endothelium, likely for local purposes. Glucose and insulin also upregulate phospholipid transfer protein, which will not only result in enhanced cholesterol release from the endothelium, but also in remodeling of fetal HDL, which, as a speculation, may enhance local atheroprotection in the feto-placental vasculature. Collectively, the placental endothelium responds to changes in the fetal circulation associated with metabolic derangements in the mother. Teleologically, these changes can be regarded as placental adaptation to protect the fetus from adverse effects of the environmental alterations.

Although the human population is in continuous growth and will probably reach nine billion people by 2016, global statistics indicate that 15% of couples have infertility problems. This rate is compounded considering that only 50e60% of pregnancies exceed 20 weeks of gestation, being implantation failure the cause of pregnancy loss in 75% of cases. The decidual reaction and the formation of new vessels in the uterus are two crucial processes during embryo implantation. Based on published data, we postulate that lysophosphatidic acid (LPA) might be a lipid promoter of vascularization and decidualization. Previously, we observed that the pharmacological blockade of LPA3, an LPA receptor, provoked aberrant embryo spacing and increased embryo resorption with negative consequences in the vascularization and decidualization reactions at the sites of embryo implantation. Thus, we investigated LPA3 potential signaling pathways. Both cyclooxygenase (COX) and nitric oxide synthase (NOS) are known enzymes involved in implantation. Uterine slices on day 5 of gestation (day of implantation) were isolated and incubated with LPA, DGPP (a highly selective antagonist of LPA3) and COX and NOS selective and non-selective inhibitors. We observed that LPA, through the activation of LPA3 receptor, increased inducible NOS activity and the production of COX-2 derived prostaglandin E2. In addition, nitric oxide and prostaglandins presented a crosstalk under the effect of LPA. LPA also induced the expression of markers of vascularization (IL-10) and decidualization (IGFBP-1) through LPA3 receptor and these signaling pathways involved iNOS and COX-2. Taking into account our previous data, the in vitro results suggest that the LPA3 receptor and its ligand, LPA, are involved in the process of vascularization and decidualization that take place during embryo implantation.

L6. FOLATE SENSING BY PLACENTAL MTOR SIGNALING: A NOVEL MECHANISM LINKING MATERNAL FOLATE LEVELS, PLACENTAL FUNCTION AND FETAL DEVELOPMENT Irving Aye 1, Theresa Powell 2, Thomas Fredrick Rosario 1, 1 1 Jansson . Department of OB/GYN, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA; 2 Department of Pediatrics, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA Folate deficiency during pregnancy is associated with neural tube defects and fetal growth restriction, however the underlying mechanisms remain to be fully established. The mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway integrates growth factor signaling and information about nutrient availability to regulate cell growth, proliferation and metabolism. We tested the hypothesis that mTOR functions as a folate sensor. Folate deficiency in pregnant mice caused inhibition of mTOR

J.S. Beltrame, M.S. Sordelli, A. Szmelc, M. Cella, S. Perez Martinez, A.M. Franchi, M.L. Ribeiro. CEFyBO, CONICET, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina

SLIMP ORAL AWARD. IMPACT OF PLACENTAL GROWTH FACTOR ON BRAIN VASCULAR DEVELOPMENT, COGNITION AND BEHAVIOR €tsep 1, Angelina Paolozza 1, 3, Bruno Zavan 1, Vanessa R. Matthew T. Ra 1 Brandon Maser 1, Andrew Hickman 1, Patrick W. Kay , Stroman 1, 3, Graeme N. Smith 2, James N. Reynolds 1, 3, Michael A. Adams 1, B. Anne Croy 1. 1 Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario, Canada; 2 Department of Obstetrics and Gynecology, Queen’s University, Kingston, Ontario, Canada; 3 Centre for Neuroscience Studies, Queen’s University, Kingston, Ontario, Canada In many pre-eclamptic (PE) pregnancies, maternal plasma is low in the placentally-produced angiokine “placental growth factor” (PGF). Offspring of PE compared to uncomplicated pregnancies are at greater risk for hypertension, cognitive impairment and stroke. However, mechanisms

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explaining these risks are not well understood. Pgf/ mice have less decidual and placental vascular branching and connectivity than controls and most experience stroke after unilateral common carotid artery occlusion. Thus, we hypothesized that PGF deficiency, which manifests in PE, diminishes brain vascular development and leads to impaired cognition and elevated stroke risk. Pgf/ mice brain vasculature were found to be deficient compared to Pgf+/+ controls during gestation and as adults. The circle of Willis in Pgf/ mice brains was often incomplete, explaining the stroke phenomenon after carotid occlusion. Cognitive behavioral testing performed in Pgf/ mice revealed impairments in numerous cognitive domains, which also displayed sexually dimorphic differences. We have subsequently recruited children born to PE or uncomplicated pregnancies to determine if similar brain vascular and cognitive impairments are present in humans. Preliminary analyses have shown brain vascular anomalies to be common in PE children. Analyses of cognitive function in these children are currently ongoing. Our data produced to date are compelling in linking placental paracrine signaling to alterations in the development and proper functioning of the offspring. Although more data are needed to draw firm conclusions in this regard, our preliminary work has uncovered a previously unknown link between PGF expression and brain development. Currently ongoing studies will aim to further understand this process and potentially augment it in utero.Supported by NSERC, CIHR, the CRC program and Kingston General Hospital Foundation. MINI COURSE A. MCA-1. VASCULAR REGRESSION IN THE FETOPLACENTAL VASCULAR BED, AND ITS POSSIBLE IMPLICATIONS FOR FETAL GROWTH RESTRICTION John Aplin, Stefanie Swietlik, Jayne Charnock, Mahmoud Khalid, Melissa Westwood, Edward Johnstone. Maternal and Fetal Health Centre, University of Manchester, UK A consistent feature of placenta in fetal growth restriction (FGR) is reduced vascularisation (Chen et al Am J Obstet Gynecol. 187:764, 2002). This is not uniform in the villous tree; in late onset FGR, peripheral areas are less well vascularized than central areas (Junaid et al Placenta 35:808, 2014). Early in gestation, primitive extraembryonic mesenchymal cells differentiate into endothelium and form the first vascular cords. This process occurs throughout the villous placenta as well as in the chorionic plate (CP). However, as demonstrated by thin sectioning (including early implantation site specimens fixed in situ) and whole mount immunofluorescence, vascular regression occurs at 9-12 weeks in villi overlying the superficial aspect of the placenta as well as in the associated CP. These areas give rise to the CL. We used first trimester villous explants and primary mesenchymal cell cultures to examine potential mechanisms. Over 72 h, explants showed declining vessel integrity and altered endothelial morphology, while surrounding stroma and trophoblast remained intact. Vascular endothelial growth factor (VEGF) delayed this decline. CD31-positive endothelial cells were isolated from villous first trimester digests and cultured on collagen with or without VEGF. Within 24h, they acquired aSMA-positive stress fibres indicative of an endothelial-mesenchymal transition (EndMT). Thus VEGF extends the lifespan of vascular cells in tissue, but not in culture. In first trimester villi, double-positive cells were present in vessel walls, and at 9-12 weeks vascular regression was observed in areas far from the cord insertion. Thus endothelial cells in placental tissue abutting the capsular decidua undergo EndMT, express aSMA and produce the avascular CL. We suggest that exaggerated regression may occur in fetal growth restriction.

MCA-2. WHOLE MOUNT IMMUNOFLUORESCENCE OF THE HUMAN PLACENTA Shawn P. Murphy 1, 2, Meghan E. Bushway 2, Paula Zozzaro-Smith 1, Ian D. Perry 2, Scott A. Gerber 2, Richard K. Miller 1, Edith M. Lord 2. 1 Department of Obstetrics and Gynecology, University of Rochester School of Medicine, Rochester, NY, USA; 2 Department of Microbiology and Immunology, University of Rochester School of Medicine, Rochester, NY, USA

Objective: The human placenta plays multiple critical roles in successful pregnancy, and it is well established that defects in placental development and/or function are associated with severe complications of pregnancy. However, the molecular mechanisms underlying development of the human placenta are incompletely understood. Furthermore, the methods currently utilized to image the human placenta lack the capacity to simultaneously examine cellular phenotype, morphology and spatial arrangement of cells within intact placental structures. We therefore developed a novel whole mount immunofluorescence (WMIF) method to examine the placental microenvironment over the course of human gestation. Methods: Intact explants were dissected from 1st trimester (7e12 weeks), 2nd trimester (16e22 weeks) and term (39 weeks) placentas, fixed, and stained overnight with fluorescently-conjugated antibodies against specific cell surface or intracellular proteins. Samples were subsequently prepared as whole mounts and visualized by either conventional or confocal microscopy. Results: All of the major cell populations within the human placenta can be distinguished by WMIF, including immune cells, blood vessel endothelial cells, fibroblasts and several subpopulations of trophoblast cells. Significant differences in the morphology of placental blood vessels were observed over the course of pregnancy. Furthermore, both qualitative and quantitative differences were readily detected in placental expression of a number of markers over the course of gestation by WMIF. Our laboratory is currently successfully utilizing WMIF to compare marker expression in normotensive versus preeclamptic placentas, signal transduction in response to proinflammatory cytokines through the JAK-STAT pathway in intact placental explants, and infection of placental explants by pathogenic bacteria. Conclusions: Our collective studies demonstrate that WMIF can be successfully used to study the placental architecture over the course of gestation, and in normal versus pathological pregnancies. Moreover, this method provides a powerful approach for examining the effects of various factors on placental signal transduction and morphometry. MCA-3. THE IMMUNOMODULATORY PROPERTIES OF HUMAN PLACENTA: IMPLICATIONS FOR ITS USE IN REGENERATIVE MEDICINE O. Parolini, M. Magatti, S. Pianta, A. Silini, A. Cargnoni. Centro di Ricerca E. Menni, Fondazione Poliambulanza, Brescia, Italy Objectives: We have shown that mesenchymal (hAMSC) and epithelial (hAEC) cells can be isolated from the amniotic membrane of human term placenta and we have contributed to their characterization [1]. Our research is aimed at studying the immunomodulatory capabilities of these cells, and specifically on their derivatives, such as conditioned medium. Methods: Cell isolation from human term placenta, cell characterization, and generation of conditioned media were performed as previously described [2]. In vitro immunomodulation tests and in vivo models [3,4] were performed as described elsewhere. Results: We have previously demonstrated that hAMSC reduce T cell proliferation and recent findings indicate that hAMSC alter T helper cell subsets by specifically inducing polarization towards T regulatory cells [5]. Additionally, we have demonstrated that hAMSC can inhibit monocyte maturation towards dendritic cells [6], and promote macrophage polarization towards an M2 phenotype [6]. Importantly, we have recently shown that the conditioned medium derived from hAMSC and hAEC culture is also able to modulate immune cells, thus supporting the hypothesis that hAMSC and hAEC produce and secrete paracrine-acting factors which act on/ communicate with immune cells [7]. Furthermore, we have shown that the application of cells or their conditioned medium in animal models of disease can significantly attenuate progression of several inflammatoryassociated diseases, such as in animals with ischemic hearts [8] and lung and liver fibrosis [3,9]. Very recently, we have shown that hAMSC can ameliorate clinical progression of autoimmune diseases in experimental mouse models, such as multiple sclerosis and rheumatoid arthritis [4]. Importantly, we have also shown that hAMSC can suppress the inflammatory responses in cells isolated from patients with rheumatoid arthritis [4].

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Conclusions: Cells and derivatives from human term placenta could represent a valuable therapeutic tool in diseases based on inflammatory processes and also in those with altered immune reactions, such as autoimmune disorders. References [1]. Parolini O, et al. Stem Cells Dev 2010 [2]. Rossi D, et al. PLoS One 2012 [3]. Cargnoni A, et al. Cytotherapy 2014 [4]. Parolini O, et al. Arthritis Rheumatol 2014 [5]. Pianta S, et al. Stem Cells Rev and Reports 2014 [6]. Magatti M, et al. Cell Transplantation, 2009 [7]. Magatti M, et al. Cell Transplantation, 2014 [8]. Cargnoni A, et al. Cell Transplantation 2009 [9]. Sant'Ansssna LB, et al. Cell Transplantation 2011

MINI COURSE B. THE ANATOMY AND PHYSIOLOGY OF A MANUSCRIPT Yoel Sadovsky 1, L. Myatt 2, Graham Burton 3. 1 Magee-Womens Research Institute, Department of Obstetrics, Gynecology and Reproductive Sciences, and Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, USA; 2 Center for Pregnancy and Newborn Research, University of Texas Health Science Center San Antonio, San Antonio, TX 78229, USA; 3 Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, UK

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Placentation in Neotropical (platyrrhine) primates will be discussed in the context of evolution of invasive placentation in primates, which culminates in the deep penetration of the endometrium and its arteries in hominoids. In Old World monkeys and apes, columns of trophoblast cells advance to the base of the implantation site and spread out to form a cytotrophoblastic shell. In addition, cytotrophoblasts enter the spiral arteries and are responsible for remodeling them to form wide, low resistance conduits. Additionally, in human and great apes, there is invasion of the endometrium and its vessels by trophoblasts originating from the base of the anchoring villi. In platyrrhines, on the other hand, there is no cytotrophoblastic shell. Instead the fetal-maternal interface is formed by a thick pad of syncytiotrophoblast resting directly on the undecidualized endometrium. The conventional wisdom is that trophoblast does not invade the placental bed and its vessels. If this is the case, how is an adequate blood supply maintained? A distinctive feature of the platyrrhine placenta is the presence within it of large maternal capillaries with multiple openings into the intertrabecular space. It is known that they are derived from subepithelial uterine capillaries, but scant attention has been paid to the arteries supplying them. Our preliminary observations suggest there is transformation of the arterial wall consistent with widening of the lumen to reduce resistance to blood flow. We believe this occurs under the influence of invasive trophoblast and call for further studies using fresh material to test this hypothesis. The results would have intrinsic value in relation to the primate radiation in the Neotropics as well as broader implications for understanding the evolution of trophoblast invasion in higher primates, including the human species.

KEYNOTE LECTURES. K1. FORMATION OF HUMAN EXTRAVILLOUS TROPHOBLAST: MECHANISMS CONTROLLING PROLIFERATION, DIFFERENTIATION AND MIGRATION € fler. Department of Obstetrics and Fetal-Maternal Medicine, Martin Kno Reproductive Biology Unit, Medical University of Vienna, Austria Signaling pathways regulating human extravillous trophoblast (EVT) migration have been intensively investigated. However, key regulatory mechanisms controlling proliferation of trophoblast progenitors of placental anchoring villi and their differentiation towards the EVT lineage remain largely elusive. Since failures in the intrinsic EVT differentiation program could contribute to gestational diseases with abnormal placentation, such as preeclampsia or intrauterine growth restriction, a better understanding of the underlying mechanisms could be beneficial for future diagnosis and treatment. Recent studies in the laboratory suggested that the developmental pathways Notch, Wnt, Hippo and EGF may interact in an integrated manner to regulate cell column proliferation and differentiation. Development of EVTs is associated with nuclear recruitment of Wnt-dependent b-catenin and the co-activators of Hippo signaling YAP and TAZ. Accordingly, canonical Wnt signaling and the Hippo-Off state were shown to promote trophoblast migration and EVT marker expression. In contrast, canonical Notch activity could be required for balanced rates of column proliferation and EVT formation involving the key regulatory transcription factor RBPJk. However, Notch receptors, such as Notch1 and Notch2, have distinct expression patterns in individual trophoblast subtypes and exhibit differential roles. Notch1 controls trophoblast survival, whereas Notch2 regulates EVT motility. Similar to Notch receptors, trophoblasts switch the expression of EGF receptor family members during EVT differentiation. Functional analyses showed that distinct combinations of EGF receptors promote cell column proliferation and suppress EVT apoptosis, respectively, in a ligand-dependent manner. Moreover, our recent analyses indicated that, like in breast cancer cells, formation of EVT is associated with DNA amplification of EGF receptor 2 (HER-2) suggesting a physiological role of the gene in EVT function or differentiation.

K2. WHAT CAN NEOTROPICAL PRIMATES TEACH EVOLUTION OF INVASIVE PLACENTATION?

US

ABOUT

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A.M. Carter. Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark

K3. ANTIAPOPTOTIC EFFECTS OF LEPTIN ON TROPHOBLASTS n Toro 1, Malena Schanton 1, Julieta Maymo  1, Antonio Pe rezAyele 2 3   Bernardo Maskin , Víctor Sanchez-Margalet 2, Cecilia Perez , gica, FCEN, UBA, IQUIBICEN Varone 1. 1 Departamento de Química Biolo edica y CONICET, Buenos Aires, Argentina; 2 Departamento de Bioquímica M Biología Molecular, Universidad de Sevilla, Sevilla, Spain; 3 Hospital Nacional Alejandro Posadas, Buenos Aires, Argentina Leptin produced by the placenta has many roles as an autocrine hormone. We have previously demonstrated that leptin promotes proliferation and survival of trophoblast cells. In the present work, we aimed to study the mechanisms that mediate the antiapoptotic effect of leptin in the placenta. Methods: BeWo and Swan cells, cultured under standard conditions, as well as human placental explants were used. Western blot, qRT-PCR and transfection assays with reporter constructs and expression vectors were carried out. All the procedures were approved by the ethical review committee at the Posadas National Hospital (Buenos Aires, Argentina). Results: Recombinant human leptin added to the BeWo cell line and human placental explants showed a decrease in Caspase-3 activation in a dose- dependent manner. Moreover, inhibition of endogenous leptin expression with 2 mM of an antisense oligonucleotide reversed Caspase-3 diminution. We also found that the cleavage of Poly [ADP-ribose] polymerase-1 (PARP-1) was diminished in the presence of leptin. Placental explants cultured in the absence of serum in the culture media increased the apoptotic cleavage of DNA and this effect was prevented by the addition of 100 ng leptin/ml. We found that under serum deprivation conditions, leptin increased the anti-apoptotic Bcl-2 protein expression and downregulated the pro-apoptotic Bax and Bid protein expression in Swan71 cells and placental explants. In both models, leptin augmented the Bcl2/Bax ratio. Moreover, we demonstrated that p53, one of the key cell cyclesignaling proteins, is downregulated in the presence of leptin under serum deprivation. Leptin also reduced the phosphorylation of Ser-46 p53, which plays a pivotal role in the apoptotic signaling by p53 and augments the levels of Mdm2 protein, a regulator of p53 half-life. Furthermore, leptin antiapoptotic effect and p53 regulation by leptin involved MAPK and PI3K signaling pathways. Conclusions: These results improve the understanding of leptin function during pregnancy and further support the importance of leptin in pregnancy.

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K4. GLYCOSYLATION PATTERNS OF PROTEINS IN TROPHOBLAST AND DECIDUA OF THE CAT PLACENTA

K6. IMPACT OF HIGH DENSITY LIPOPROTEIN METABOLISM IN MOUSE EMBRYO PHYSIOLOGY

 nica 1, Ortega Barbeito Claudio 1, Fern andez Patricia 1, Diessler Mo 2 1 1 1 Hugo , Gimeno Eduardo , Portiansky Enrique . School of Veterinary Science UNLP, CONICET. La Plata, Bs As, Argentina; 2 Faculty of Veterinary Sciences UNL, Esperanza, Santa F e, Argentina

Nicol as Santander 1, Attilio Rigotti 1, 2, Dolores Busso 1. 1 Departamento de n, Diabetes y Metabolismo, Escuela de Medicina, Pontificia Nutricio n Molecular y lica, Santiago, Chile; 2 Centro de Nutricio Universidad Cato nicas, Escuela de Medicina, Pontificia Universidad Enfermedades Cro lica, Santiago, Chile Cato

The aim of this study was to characterize glycoconjugates of syncytiotrophoblast, cytotrophoblast and decidual cells of mature feline placenta. Samples from 12 normal pregnant female cats, after 45±5 days of gestation were obtained by hysterectomy. Sections were processed for routine observation. Lectinhistochemistry was performed using biotinylated lectins to characterize the expression of saccharides. Trophoblast expressed a varied composition of glycans, highly exposing terminal N-Acetylglucosamine residues and non-sialylated galactose and N-Acetyl galactosamine oligomers. Phaseolus vulgaris erythroagglutinin lectin, which recognized highly branched N-linked residues, was restricted to the syncytiotrophoblast. Unlike results reported in humans, mice and rats on lectin affinity of decidual cells, sialic acids and complex N-linked oligosaccharides were not demonstrated in cats. Glycosylation of proteins determines many of their final properties, thus becoming essential for the embryo-maternal relation during implantation and placentation. Knowledge about the glycosylation profile of the normal cat placenta may lead to a better understanding of both normal and pathological reproductive events.

K5. ADIPONECTIN e CROSS TALK BETWEEN MATERNAL ADIPOSE TISSUE AND PLACENTA Theresa L. Powell, Irving Aye, Fredrick Rosario, Thomas Jansson. Departments of Pediatrics and Obstetrics and Gynecology, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA Adiponectin is a multimeric protein secreted from adipose tissue. Secretion decreases as adipose tissue expands during weight gain. Low circulating levels of adiponectin are associated with decreased insulin sensitivity in skeletal muscle and liver. In contrast, our studies demonstrate that trophoblast cells respond by increasing insulin sensitivity with low maternal adiponectin. This establishes adiponectin as an endocrine link between maternal adipose tissue and the placenta, which promotes nutrient flux to the fetus when supplies are high and limits nutrient delivery to the fetus and preserves maternal tissues when nutrients are in low abundance. With low nutrient status maternal fasting insulin is low and adiponectin levels are high. During the postprandial period, insulin stimulated nutrient transport in the placenta is blunted by the high maternal adiponectin, therefore limiting fetal growth when maternal supplies are reduced. In maternal obesity, low adiponectin and chronically high insulin (due to maternal insulin resistance) will lead to maximal insulin stimulation of placental nutrient transport, contributing to fetal overgrowth. We have demonstrated in cultured trophoblast cells that increasing adiponectin concentrations caused a loss of insulin sensitivity at the level of IRS-1 and blunts insulin stimulated nutrient uptake. We also studied responses to infused adiponectin in normal lean pregnant mice using mini-osmotic pumps to increase maternal adiponectin levels 4-fold (within the normal physiological range) between E14-18. This treatment caused a significant reduction (-18%, p