Vassilis Tsatsaris

Comparative Methylation of ERVWE1/Syncytin-1 and Other Human Endogenous Retrovirus LTRs in Placenta Tissues

Human endogenous retroviruses (HERVs) are globally silent in somatic cells. However, some HERVs display high transcription in physiological conditions. In particular, ERVWE1, ERVFRDE1 and ERV3, three proviruses of distinct families, are highly transcribed in placenta and produce envelope proteins associated with placenta development. As silencing of repeated elements is thought to occur mainly by DNA methylation, we compared the methylation of ERVWE1 and related HERVs to appreciate whether HERV methylation relies upon the family, the integration site, the tissue, the long terminal repeat (LTR) function or the associated gene function. CpG methylation of HERV-W LTRs in placenta-associated tissues was heterogeneous but a joint epigenetic control was found for ERVWE1 5′LTR and its juxtaposed enhancer, a mammalian apparent LTR retrotransposon. Additionally, ERVWE1, ERVFRDE1 and ERV3 5′LTRs were all essentially hypomethylated in cytotrophoblasts during pregnancy, but showed distinct and stage-dependent methylation profiles. In non-cytotrophoblastic cells, they also exhibited different methylation profiles, compatible with their respective transcriptional activities. Comparative analyses of transcriptional activity and LTR methylation in cell lines further sustained a role for methylation in the control of functional LTRs. These results suggest that HERV methylation might not be family related but copy-specific, and related to the LTR function and the tissue. In particular, ERVWE1 and ERV3 could be developmentally epigenetically regulated HERVs.

Human endogenous retrovirus-FRD envelope protein (syncytin 2) expression in normal and trisomy 21-affected placenta

Human trophoblast expresses two fusogenic retroviral envelope proteins, the widely studied syncytin 1, encoded by HERV-W and the recently characterized syncytin 2 encoded by HERV-FRD. Here we studied syncytin 2 in normal and Trisomy 21-affected placenta associated with abnormal trophoblast differentiation. Syncytin 2 immunolocalization was restricted throughout normal pregnancy to some villous cytotrophoblastic cells (CT). During the second trimester of pregnancy, syncytin 2 was immunolocalized in some cuboidal CT in T21 placentas, whereas in normal placentas it was observed in flat CT, extending into their cytoplasmic processes. In vitro, CT isolated from normal placenta fuse and differentiate into syncytiotrophoblast. At the same time, syncytin 2 transcript levels decreased significantly with syncytiotrophoblast formation. In contrast, CT isolated from T21-affected placentas fused and differentiated poorly and no variation in syncytin 2 transcript levels was observed. Syncytin 2 expression illustrates the abnormal trophoblast differentiation observed in placenta of fetal T21-affected pregnancies.

Expression and Regulation by PPARγ of hCG α- and β-subunits: Comparison between Villous and Invasive Extravillous Trophoblastic Cells

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.

Pattern of secretion of pregnancy-associated plasma protein-A (PAPP-A) during pregnancies complicated by fetal aneuploidy, in vivo and in vitro

BackgroundPregnancy-associated placental protein-A (PAPP-A) is a metalloprotease which circulates as an hetero-tetramer in maternal blood. Its maternal serum concentration in fetal trisomy 21 is decreased during the first trimester, so that PAPP-A is a useful screening biomarker. However, the regulation of PAPP-A placental secretion is unclear. We therefore investigated the secretion of PAPP-A in pregnancies complicated by fetal aneuploidies, both in vivo and in vitro.MethodsMaternal serum collected between 10 WG and 33 WG during 7014 normal pregnancies and 96 pregnancies complicated by fetal trisomy 21, 18, and 13 were assayed for PAPP-A using the Immulite 2000xpi system®. The pregnancies were monitored using ultrasound scanning, fetal karyotyping and placental analysis. Villous cytotrophoblasts were isolated from normal and trisomic placenta and cultured to investigate PAPP-A secretion in vitro (nu2009=u20096).ResultsAn increased nuchal translucency during the first trimester is a common feature of many chromosomal defect but each aneuploidy has its own syndromic pattern of abnormalities detectable at the prenatal ultrasound scanning and confirmed at the fetal examination thereafter. PAPP-A levels rise throughout normal pregnancy whereas in trisomy 21, PAPP-A levels were significantly decreased, but only during the first trimester. PAPP-A levels were decreased in trisomy 13 and sharply in trisomy 18, whatever the gestational age. In vitro, PAPP-A secretion was decreased in aneuploidy, and associated with decreased hCG secretion in Trisomy 21 and 18. These biochemical profiles did not appear to be linked to any specific histological lesions affecting the placenta.ConclusionsThese profiles may reflect different quantitative and qualitative placental dysfunctions in the context of these aneuploidies.