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It is now demonstrated that the sex-specific maternal-placental-fetal interaction plays an important role in placental functions and pathologies. Determination of fetal-sex may therefore be an important consideration in studies using placenta samples. In this present study, we describe a simple, fast, and cheap protocol, which allows the fetal-sex determination of placental tissues from various starting materials (villi or formalin-fixed, paraffin-embedded (FFPE) tissues, isolated cytotrophoblasts or cellular debris from whole cell lysates, and cDNA) by a single duplex PCR reaction followed by agarose gel electrophoresis.

Human chorionic gonadotropin (hCG) is the first hormonal message from the placenta to the mother. It is detectable in maternal blood two days after implantation and behaves like an agonist of LH stimulating progesterone secretion by the corpus luteum. hCG has also a role in quiescence of the myometrium and local immune tolerance. Specific to humans, hCG is a complex glycoprotein composed of two glycosylated subunits. The α-subunit is identical to the pituitary gonadotropin hormones (LH, FSH, TSH), contains two N-glycosylation sites, and is encoded by a single gene (CGA). By contrast the β-subunits are distinct in each of the hormones and confer receptor and biological specificity. The hCG β-subunit contains two sites of N-glycosylation and four sites of O-glycosylation and is encoded by a cluster of genes (CGB). In this review, we will stress the importance of hCG glycosylation state, which varies with the stage of pregnancy, its source of production and in the pathology. It is well established that hCG is mainly secreted by the syncytiotrophoblast into maternal blood where it peaks around 8-10 weeks of gestation (WG). The invasive extravillous trophoblast also secretes hCG, and in particular like choriocarcinoma cells, hyperglycosylated forms of hCG (hCG-H). In maternal blood hCG-H is high during early first trimester. In addition to its endocrine role, hCG has autocrine and paracrine roles. It promotes formation of the syncytiotrophoblast and angiogenesis through LHCG receptor. In contrast, hCG-H stimulates trophoblast invasion and angiogenesis by interacting with the TGFβ receptor 2. hCG is largely used in antenatal screening and hCG-H represents a serum marker of early trophoblast invasion. Other abnormally glycosylated hCG are described in aneuploidies. In conclusion, hCG is the major pregnancy glycoprotein hormone, whose maternal concentration and glycan structure change all along pregnancy. Depending on its source of production, glycoforms of hCG display different biological activities and functions that are essential for pregnancy outcome.

International audience; The syncytiotrophoblast layer plays a major role throughout pregnancy, since it is the site of numerous placental functions, including ion and nutrient exchange and the synthesis of steroid and peptide hormones required for fetal growth and development. Inadequate formation and regeneration of this tissue contributes to several pathologies of pregnancy such as intrauterine growth restriction and preeclampsia, which may lead to iatrogenic preterm delivery in order to prevent fetal death and maternal complications. Syncytiotrophoblast formation can be reproduced in vitro using different models. For the last ten years we have routinely purified villous cytotrophoblastic cells (CT) from normal first, second and third trimester placentas and from gestational age-matched Trisomy 21 placentas. We cultured villous CT on plastic dishes to follow the molecular and biochemical aspects of their morphological and functional differentiation. Taking advantage of this unique collection of samples, we here discuss the concept that trophoblast fusion and functional differentiation may be two differentially regulated processes, which are linked but quite distinct. We highlight the major role of mesenchymal-trophoblast cross talk in regulating trophoblast cell fusion. We suggest that the oxidative status of the trophoblast may regulate glycosylation of proteins, including hCG, and thereby modulate major trophoblast cell functions.

During pregnancy, the placenta ensures multiple functions, which are directly involved in the initiation, fetal growth and outcome of gestation. The placental tissue involved in maternal-fetal exchanges and in synthesis of pregnancy hormones is the mononucleated villous cytotrophoblast (VCT) which aggregates and fuses to form and renew the syncytiotrophoblast (ST). Knowledge of the gene expression pattern specific to this endocrine and exchanges tissue of human placenta is of major importance to understand functions of this heterogeneous and complex tissue. Therefore, we undertook a global analysis of the gene expression profiles of primary cultured-VCT (n = 6) and in vitro-differentiated-ST (n = 5) in comparison with whole term placental tissue from which mononucleated VCT were isolated. A total of 880 differentially expressed genes (DEG) were observed between VCT/ST compared to whole placenta, and a total of 37 and 137 genes were significantly up and down-regulated, respectively, in VCT compared to ST. The 37 VCT-genes were involved in cellular processes (assembly, organization, and maintenance), whereas the 137 ST-genes were associated with lipid metabolism and cell morphology. In silico, all networks were linked to 3 transcriptional regulators (PPARγ, RARα and NR2F1) which are known to be essential for trophoblast differentiation. A subset of six DEG was validated by RT-qPCR and four by immunohistochemistry. To conclude, recognition of these pathways is fundamental to increase our understanding of the molecular basis of human trophoblast differentiation. The present study provides for the first time a gene expression signature of the VCT and ST compared to their originated term human placental tissue.

Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 diverse topics were discussed in twelve themed workshops, six of which are summarized in this report. 1. The placental pathology workshop focused on clinical correlates of placenta accreta/percreta. 2. Mechanisms of regulation of trophoblast invasion and spiral artery remodeling were discussed in the trophoblast invasion workshop. 3. The fetal sex and intrauterine stress workshop explored recent work on placental sex differences and discussed them in the context of whether boys live dangerously in the womb. 4. The workshop on parasites addressed inflammatory responses as a sign of interaction between placental tissue and parasites. 5. The decidua and embryonic/fetal loss workshop focused on key regulatory mediators in the decidua, embryo and fetus and how alterations in expression may contribute to different diseases and adverse conditions of pregnancy. 6. The trophoblast differentiation and syncytialisation workshop addressed the regulation of villous cytotrophoblast differentiation and how variations may lead to placental dysfunction and pregnancy complications.

Fetal trisomy 21 is associated with elevated maternal serum hCG and its free beta-subunit (hCG-beta) in vivo, and abnormal placental hCG production and glycosylation in vitro. Other maternal serum markers may also be disrupted in major aneuploidies (T21, T18, T13). We evaluated our aneuploidy screening practices, focusing on hCG-beta and hCG glycoforms, and retrospectively analyzed 55 aneuploidy cases diagnosed over a 2 year period, determining maternal serum hCG glycoforms profiles using 2D-electrophoresis. Screening efficiency reached 96.7%. T21 was associated with elevated hCG-beta while T18 presented with diminished serum markers. hCG glycoforms tended to be basic in aneuploidy (mainly T13).

The discovery of the peroxisome proliferator-activated receptors (PPARs) in 1990s provided new insights in understanding the mechanisms involved in the control of energy homeostasis and in cell differentiation, proliferation, apoptosis and the inflammatory process. The PPARs became thus an exciting therapeutic target for diabetes, metabolic syndrome, atherosclerosis, and cancer. Unexpectedly, genetic studies performed in mice established that PPARγ are essential for placental development. After a brief description of structural and functional features of PPARs, we will summarize in this review the most recent results concerning expression and the role of PPARs in placenta and of PPARγ in human trophoblastic cells in particular.

Context The placenta plays an essential role in the fetomaternal exchanges of iodine and thyroid hormones. Propylthiouracil (PTU) is presently considered to be the treatment of choice for hyperthyroidism during the first trimester of pregnancy. Little is known on the expression of iodide transporters in invasive human trophoblast and the possible effect of PTU on this early phase of human placental development. Objective To analyze during early pregnancy expression of sodium/iodide symporter (NIS) and pendrin at the feto–maternal interface in situ in first trimester placentas, in vitro during human trophoblastic cell differentiation in presence or not of PTU. Design NIS and pendrin immunodetection were performed on 8–10 WG placental tissue sections and in primary cultures of first trimester placenta trophoblastic cells, which differentiate in vitro into syncytiotrophoblast or invasive extravillous cytotrophoblasts (EVCT). The effect of PTU (1 mM) was tested in EVCT on iodide transporters expression, cell invasion, and hCG secretion. Results NIS and pendrin were present in early human trophoblast at the maternofetal interface. Their expression was modulated with in vitro trophoblast differentiation. Early invasive EVCT were characterized by higher expression of NIS than pendrin. In vitro PTU did modify significantly neither EVCT iodide transporters expression nor EVCT biological functions: i.e. invasive properties and hCG secretion. Conclusion This study reveals that NIS is highly expressed in early human trophoblast at the feto–maternal interface. PTU has no effect on early human trophoblast invasion.

The placenta provides critical transport functions between the maternal and fetal circulations during intrauterine development. Formation of this interface is controlled by nuclear transcription factors including homeobox genes. Here we summarize current knowledge regarding the expression and function of homeobox genes in the placenta. We also describe the identification of target transcription factors including PPARγ, biological pathways regulated by homeobox genes and their role in placental development. The role of the nuclear receptor PPARγ, ligands and target genes in human placental development is also discussed. A better understanding of these pathways will improve our knowledge of placental cell biology and has the potential to reveal new molecular targets for the early detection and diagnosis of pregnancy complications including human fetal growth restriction.

DLX3, a member of the large homeobox gene family of transcription factors, is necessary for normal placentation. Targeted deletion of dlx3 in mouse resulted in embryonic death due to placental failure. This study demonstrates the presence of DLX3 mRNA expression in human first trimester and term placental tissue, cultured trophoblast-like cell lines and in isolated primary villous and extravillous trophoblast cells. Using an ovine polyclonal antibody, the spatial distribution was identified for DLX3 in human placental tissues, trophoblast cell lines and in freshly isolated primary trophoblast cells. A 50 kDa immunoreactive DLX3 protein was detected in the human placenta, in trophoblast cell lines and in primary trophoblast cells. Nuclear expression for DLX3 was observed in villous cytotrophoblasts, syncytiotrophoblast and extravillous cytotrophoblast in the proximal regions of the cytotrophoblast cell columns in first trimester placental tissues. Immunoreactivity was also detected in few stromal cells and microvascular endothelial cells surrounding the fetal capillaries. In the first trimester placental bed, DLX3 expression was predominantly observed in the cytoplasm of the endovascular and interstitial trophoblasts. We conclude that the cellular expression of DLX3 was extensive in the human placenta and propose that DLX3 may play an important role in normal placental development.

During human placental development trophoblast follows two differentiation pathways: the extravillous (EVCT) and the villous cytotrophoblasts (VCT) that display different phenotypes and functions. It is well established that human chorionic gonadotropin hormone (hCG) is mainly secreted by the endocrine VCT (syncytiotrophoblast) into the maternal compartment and stimulates the formation of the syncytiotrophoblast (ST) in an autocrine manner. We recently reported that the invasive EVCT also produces hCG that promotes trophoblast invasion in vitro. Herein, we compared hCG gene expression in primary culture of villous and extravillous trophoblasts obtained from the same first trimester human chorionic villi and differentiated in vitro into ST and invasive EVCT, respectively. Total hCG, free alpha and free beta subunits were quantified in cell supernatants by immunometric assays and normalized to DNA content. alpha and beta transcript levels were quantified by Q-PCR and normalized to cytokeratin 7. We show that free alpha-, free beta-subunits and total hCG are differently expressed and secreted by the two trophoblast subtypes during their differentiation in vitro. We found an alpha/beta ratio 100 times lower in invasive EVCT in comparison to the ST suggesting that beta subunit may not be step limiting for hCG production in EVCT. Finally we investigated the regulation of hCG gene expression by PPARgamma, a nuclear receptor that controls trophoblast differentiation and invasion. Interestingly, activation of PPARgamma by the agonist rosiglitazone gave opposite results in the endocrine VCT and invasive EVCT: alpha and beta subunit transcript levels and protein secretions were up regulated in VCT, whereas they were down regulated in EVCT. Our results demonstrated that hCG gene expression is differentially regulated in the two trophoblast lineages during their in vitro differentiation and modulated in an opposite way by PPARgamma.

Human trophoblast differentiates into two pathways: extravillous cytotrophoblasts (EVCT) that invade the uterus wall and villous cytotrophoblasts (VCT) that fuse to form the syncytiotrophoblast (ST) involved in placental exchanges and endocrine function. It is established that hCG is produced and secreted by the ST into the maternal compartment where it plays a key endocrine role and stimulates ST formation in an autocrine manner. Herein, we investigated hCG expression in early placentas by immunohistochemistry using different antibodies. We then compared hCG secretion by primary cultures of VCT and EVCT isolated from the same first trimester human chorionic villi. In situ hCG was immunodetected in EVCT all along their invasive differentiating pathway except in cells near the stromal core of the proximal column. hCG expression was confirmed in vitro by immunocytochemistry and hCG secretion quantified in cell supernatants. Interestingly, whereas hCG secretion increased during VCT differentiation into ST (from 60 to 350UI/L/microg DNA), EVCT secretion remained constant and at a high level during the same culture period (160UI/L/microg DNA). Our data demonstrated that in addition to the ST, invasive EVCT also expressed and secreted high levels of hCG, suggesting a specific paracrine and/or autocrine role for hCG from EVCT origin.

s / Placenta 35 (2014) A1eA112 A4 hormones. The placenta, therefore, has an important role in integrating the various endocrine signals involved in regulating resource allocation to fetal growth. PL2.2. EVERYTHING YOU EVER WANTED TO KNOW ABOUT HCG Thierry Fournier a,b UMR-S 1139, INSERM, University of Paris Descartes, Paris, France; b PremUp Foundation, Paris, France Human chorionic gonadotrophin (hCG) is the first hormonal message from the placenta to themother. It is detectable inmaternal blood two days after implantation and behaves like a super agonist of LH stimulating progesterone secretion by the corpus luteum. hCG has also a role in quiescence of the myometrium and local immune tolerance. Specific to the primate superior, hCG is a complex glycoprotein composed of two glycosylated subunits. The a-subunit is identical to the pituitary gonadotrophin hormones (LH, FSH, TSH), contains two N-glycosylation sites, and is encoded by a single gene, whereas the b-subunits in each of the hormones are distinct and confer receptor and biological specificity. The hCG b-subunit contains two sites of N-glycosylation and 4 sites of O-glycosylation. It is encoded by a cluster of genes that have evolved by duplication from LHB. CGA and CGB expressions are controlled by transcription factors such as AP2 and SP1 that recognize specific response elements in their promoters. hCG expression is regulated by growth factors and cytokines such as EGF, TNFa, activators of the cAMP signalling pathways and by ligands of the nuclear receptor PPARg. In this talk we will stress the importance of hCG glycosylation state, which varies with the stage of pregnancy, its trophoblastic source of production and in the pathology (choriocarcinoma, foeto-placental trisomy 21), modifying its biological activity and clearance. It is well established that hCG is mainly secreted by the endocrine syncytiotrophoblast into the maternal serumwhere it peaks around 10WG. In addition, we have reported that invasive extravillous trophoblasts also secrete hCG, and like choriocarcinoma cells, hyperglycosylated forms of hCG (hCG-H). In addition to its endocrine role, hCG has autocrine and paracrine role. It promotes formation of the syncytiotrophoblast and angiogenesis through LHCG receptor and activation of cAMP and phospholipase C signalling. In contrast, hCG-H does not signal through LHCGR but stimulates trophoblast invasion and angiogenesis by interacting with the TGFs receptor 2. hCG is largely used in antenatal screening and hCG-H may present a good serum marker to screen pregnancy diseases from placental origin. In conclusion, hCG is the major pregnancy glycoprotein hormone, whose maternal concentration and glycan structure change all along pregnancy. Depending on its source of production, the different glycoforms of hCG display different biological activities and functions that are essential for pregnancy outcome. PL2.3. PROFILE OF PLACENTAL GENE EXPRESSION AND PREGNANCY OUTCOME Maris Laan University of Tartu, Tartu, Estonia Placenta is a mammalian pregnancy-specific temporary organ encoded by the fetal genome. Normal implantation process and placental function throughout pregnancy have critical contributions to guarantee the intrauterine development of the offspring and modulation of the maternal gestational metabolism. Also, the following temporal dynamics of placental transcriptional regulation across three trimesters of gestation is of high importance. Aberrant placental gene expression profile and its temporal dynamics may affect the normal pregnancy course. And vice versa, perturbed placental transcriptome may reflect a consequence of impaired implantation, placental development and function. There are numerous reports addressing expression levels of biological candidate genes in placenta in relation to pregnancy complications. Past 10 years have brought along an abundance of studies addressing differential expression of placental genes in pregnancy complications using microarray approach. These analyses have provided novel insights into placental biology and served as discovery studies to identify biomarkers in order to predict pregnancy complications. Next-generation sequencing approach enables to obtain the most detailed coverage of transcriptomes. In order to bring novel insights into the placental transcriptional landscape in normal gestation and study differential gene expression in complicated pregnancies we performed total RNA and small RNA sequencing in 40 samples from term placentas. Samples were selected from the REPROMETA sample collection (Tartu, Estonia) and represent normal pregnancies along with the most clearly defined cases of preeclampsia and gestational diabetes, as well as small (SGA) and large (LGA) for gestational age births. This dataset represents a high quality resource to detect differences in placental transcription over diverse pregnancy courses, to investigation of key molecular changes in placentas from complicated pregnancies and to suggest novel potential biomarkers for monitoring the risk to develop pregnancy complications. PL3.1. INTERCELLULAR COMMUNICATION BY EXOSOMES IN PLACENTA Michel Record INSERM UMR 1037 CRCT Toulouse-Oncopole, TOULOUSE

s / Placenta 34 (2013) A1–A99 A71 been associated with pregnancy disorders. We investigated if DSC isolated from pregnancies with normal or impaired spiral artery remodelling (screened by uterine artery Doppler for normal resistance index (RI) or high RI respectively) displayed different extents of decidualisation, and differentially affected trophoblast invasion or motility. Methods: Decidual stromal cells were isolated with ethical approval from first trimester terminations of pregnancy screened by uterine artery Doppler ultrasound for a high or normal RI. DSC were treated with vehicle control or cAMP and medroxyprogesterone acetate (MPA) for 72 hours to induce a decidualised phenotype before collection of conditioned media (CM). CM was assayed for the classic decidualisation markers prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP-1) by Q-RTPCR and ELISA. DSC CM was incubated with the trophoblast cell line SGHPL-4 to investigate trophoblast invasion. Results: DSC isolated from normal and high RI pregnancies showed a significant increase in RNA and protein expression of PRL and IGFBP-1 when treated with decidualising factors as compared to a vehicle control (p