Access Type

Open Access Dissertation

Date of Award

January 2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Physiology

First Advisor

D. Randall Armant

Abstract

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.

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