Molecular Regulation Of Trophoblast Survival During Placentation And Pathologies Of Placental Insufficiency

HBEGF, is present in the uterus at the time of embryo implantation and protects first trimester TB cells from apoptosis and promotes their invasion. The hypertensive disease, PE, in which TB invasion of the uterine arteries is reduced and TB apoptosis is elevated, is characterized by a reduction in HBEGF expression. In this study using a first trimester cell line and villous explant culture key components involved in HBEGF survival signaling pathway were identified. Specific MMP inhibitors established the requirement for MMP2 in HBEGF shedding and upregulation. NGS identified a HIF regulated gene, HSPA6 (HSP70B’) and using specific inhibitors it was established that HSP70 regulates MMP2 mediated shedding of HBEGF at low O2 and is functional upstream of MAPKs signaling cascade. To further investigate HBEGF upregulation at low O2 using NGS and siRNA knockdown of DGCR8 it was demonstrated that other components of RISC may be involved in regulation of HBEGF mRNA translation. These findings suggest that trophoblast survival during early pregnancy requires this signaling pathway and disruption of any component could lead to placental insufficiency. However, a global platform is needed to study the pathophysiology of trophoblast cells and their role in placental insufficiency. TRIC is an innovative platform to noninvasively acquire fetal cells. DNA was extracted from fetal cells isolated from 20 specimens with gestational age as early at 5 weeks to 19 weeks. This was followed by targeted sequencing using the Forenseq (Illumina) platform to identify informative SNPs. Using the Forenseq software 89% ± 12% of the 94 SNPs were called correctly in the fetal samples. Using the STR analysis out of the 20 fetal samples, 9 males were confirmed. Therefore, TRIC not only provides fetal cells but also opens new venues of perinatal testing. TRIC provides ample RNA from isolated fetal cells for extensive transcriptomic analysis. Using qPCR it was demonstrated that the fetal cells have higher expression for EVT specific markers such as HLA-G and KRT7 and markers for invasion/migration such as CDH5 and MMP9, whereas the maternal cells have a higher expression for epithelial markers such as CDH1 and ITGA6. This suggests that not only are the fetal cells EVT like but may also undergo an epithelial mesenchymal transition. Comparison of fetal and maternal cells from normal group using NGS identified 409 genes, of which top 5 upregulated and downregulated genes were validated by qPCR. Pathway analysis of the differentially regulated identified pathways related to invasion/migration, proliferation and differentiation, further suggesting the EVT like expression of the fetal cells. NGS revealed 348 differential expressed genes on comparison of the EVT like fetal cells from the EPL group to those from the normal group that were further validated by qPCR. Bioinformatic analysis identified pathways related to apoptosis, inflammation and placental disease. The exact origin and biology of cervical EVT cells, and their relationship to the human placenta and pregnancy outcomes, are widely unknown. Based on our study so far it can be hypothesized that as the placenta grows, EVT cells are naturally shed into the cervical canal, and their overall phenotype is comparable to the EVT cells residing in the placenta. These studies will provide a clearer view of the utility of EVT cells obtained by TRIC from ongoing pregnancies for investigating early placentation and mechanisms of pathology, and their potential as a platform for prenatal tests to predict maternal risk of placenta-based disease