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 firsttrimester TB cells from apoptosis and promotes their invasion. The hypertensive disease, PE, inwhich TB invasion of the uterine arteries is reduced and TB apoptosis is elevated, ischaracterized by a reduction in HBEGF expression. In this study using a first trimester cell lineand villous explant culture key components involved in HBEGF survival signaling pathway wereidentified. Specific MMP inhibitors established the requirement for MMP2 in HBEGF sheddingand upregulation. NGS identified a HIF regulated gene, HSPA6 (HSP70B’) and using specificinhibitors it was established that HSP70 regulates MMP2 mediated shedding of HBEGF at lowO2 and is functional upstream of MAPKs signaling cascade. To further investigate HBEGFupregulation at low O2 using NGS and siRNA knockdown of DGCR8 it was demonstrated thatother components of RISC may be involved in regulation of HBEGF mRNA translation. Thesefindings suggest that trophoblast survival during early pregnancy requires this signaling pathwayand disruption of any component could lead to placental insufficiency. However, a globalplatform is needed to study the pathophysiology of trophoblast cells and their role in placentalinsufficiency.TRIC is an innovative platform to noninvasively acquire fetal cells. DNA was extractedfrom 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 identifyinformative SNPs. Using the Forenseq software 89% ± 12% of the 94 SNPs were calledcorrectly in the fetal samples. Using the STR analysis out of the 20 fetal samples, 9 males wereconfirmed. Therefore, TRIC not only provides fetal cells but also opens new venues of perinataltesting.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 specificmarkers such as HLA-G and KRT7 and markers for invasion/migration such as CDH5 andMMP9, whereas the maternal cells have a higher expression for epithelial markers such as CDH1and ITGA6. This suggests that not only are the fetal cells EVT like but may also undergo anepithelial mesenchymal transition. Comparison of fetal and maternal cells from normal groupusing NGS identified 409 genes, of which top 5 upregulated and downregulated genes werevalidated by qPCR. Pathway analysis of the differentially regulated identified pathways relatedto invasion/migration, proliferation and differentiation, further suggesting the EVT likeexpression of the fetal cells. NGS revealed 348 differential expressed genes on comparison ofthe EVT like fetal cells from the EPL group to those from the normal group that were furthervalidated 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 humanplacenta and pregnancy outcomes, are widely unknown. Based on our study so far it can behypothesized that as the placenta grows, EVT cells are naturally shed into the cervical canal, andtheir overall phenotype is comparable to the EVT cells residing in the placenta. These studieswill provide a clearer view of the utility of EVT cells obtained by TRIC from ongoingpregnancies for investigating early placentation and mechanisms of pathology, and their potentialas a platform for prenatal tests to predict maternal risk of placenta-based disease.