The Role of Estrogen Metabolites in Endothelial Cell Dysfunction

Anastasia Lin, Sneha Ramesh Mani, Monica Mainigi

University of Pennsylvania, Philadelphia,PA

Introduction Experimental Design Gene Expression changes after 2ME2 addition

• Pregnancies conceived using assisted reproductive technology (ART) • The goal of this study is to examine how changing levels of 2-
are at increased risk for adverse outcomes like preeclampsia (PE), methoxyestradiol (2ME2) impact human endometrial endothelial
characterized by hypertension and proteinuria due to maternal vessel cell (HEMEC) function. *
abnormalities leading to poor utero-placental circulation.
Treat with Incubate 24
• ART relies on embryo transfer (ET); ET can be carried out in multiple low (10-9) and hours in
Plate HEEC high (10-6) Normoxic
different ways: fresh ET (supraphysiological hormonal environment, 1
dose 2ME2 conditions
corpus luteum (CL)); programmed frozen ET (pFET) (0 CL); natural
frozen ET (nFET) (1 CL).

• Modes of ET with too many or absent CL increase risk for PE suggesting Harvest cells Extract RNA RT-qPCR
that the CL and its products could impact PE pathogenesis.
40x **
• During early pregnancy, the CL is a rich source of estrogen and estrogen
metabolites (ExM).
RT-qPCR Probe Selection
• Alterations in serum ExM levels have been observed in patients with PE,
and in vitro studies have found effects of ExM on vascular endothelial
Probes
cells in other organ systems. • Ki67 is a marker of
Endothelial cell Ki67 proliferation
We hypothesize that ExM level imbalance after ART leads to uterine proliferation • VCAD is a marker of loss of
vascular endothelial cell dysfunction and increased risk for PE Vascular Integrity VE-Cadherin endothelial barrier function
(VCAD) • VCAM, ICAM, and SELE are
Immune cell VCAM-1 (VCAM), markers of adhesion and Conclusions
recruitment ICAM-1 (ICAM), E- transendothelial migration of
Preliminary Data selectin (SELE), TF leukocytes
• We saw a significant decrease in 2 genes associated with EC
Apoptosis BAX • BAX is a marker of cell
dysfunction: VCAM and SELE.
• ExM serum was collected from 5 patients from 3 different peri- death
Hypoxia HIF • HIF is a marker of low O2 • Significant effects were seen with the high dose (10-6) of 2ME2.
conception hormonal environments (pFET, nFET, and fresh ET).
• At 4-6 weeks gestation, ultrasensitive mass spectroscopy (MS) was levels
• The results indicate that ExM can impact EC function.
used to look at a panel of ExM.
• ExM labeled in red showed significant differences in ExM levels Cell culture:
based on mode of transfer.
Human Endometrial Endothelial Cells (HEEC) Next Steps
Low magnification High magnification • Repeat experiment using more biological replicates (5-6) to reduce
biological variation

• Explore more ExM that have shown variation between modes of

conception: 2OHE2, 2OHE1, 2MeE2, 4OHE2, 4OHE1, 4MeE2, and
16KE2.
• Use other assays to 1) confirm qPCR data and 2) explore EC function
such as flow cytometry (to assess apoptosis)
• Explore the role of O2 in ExM regulation of EC using hypoxic conditions
(5% vs. 20%)
Optimized seeding and growth at a concentration of 300,000 cells/well