SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 96A

96A

Reproductive Sciences Vol. 25, Supplement 1, March 2018

in current sources of ECFCs demonstrate the need for a supply that can
be obtained non-invasively and that has therapeutic potential. Animal
studies suggest that EPCs are enriched in the microvasculature of various
organs, and in humans, the number of ECFCs within the fetal circulation
gradually increases, suggesting that the placenta may be a reservoir
for these cells. Thus, our objective is to determine whether human
placental microvascular endothelial cells (HPMECs) contain ECFCs.
We hypothesize that ECs isolated from the placental microvasculature
will demonstrate an ECFC-like phenotype in comparison to mature,
fully-differentiated ECs.
METHODS: Placentas from uncomplicated, full-term pregnancies
undergoing scheduled C-section were obtained. Mature human placental
endothelial cells (PLECs) and HPMECs were isolated from the same
placenta. Cells were then cultured in 20% O2 as well as 6 - 8% O2,
mimicking physiologic fetoplacental O2 concentrations. PLECs and
HPMEC cell surface markers were assessed by flow cytometry. These
cells were then also subjected to Illumina HiSeq4000 system, MTT assays,
TrypanBlue cell counting assays, tube formation assays, and qPCR.
One-way ANOVA and Student's t-test were used for statistical analysis.
RESULTS: FACS analysis showed that both PLECs and HPMECs were
positive for CD31, CD105, and CD146 and negative for CD45 and CD14.
RNA-Seq data demonstrated HPMECs have significantly higer endothelial
progenitor markers and pro-angiogenic related genes compared with
PLECs from same placenta. HPMECs demonstrated higher metabolic
acitivity and proliferation rate via the MTT assay and TrypanBlue cell
counting assay than PLECs at both 20% O2 and 6% O2 (p<0.05). Both
HPMECs and PLECs formed capillary-like tube structures, but there
was significantly increased branch points and total tube length (p<0.01)
in HPMECs than PLECs at both 20% and 6% O2 tensions. qPCR results
showed significantly increased expression of key angiogenic genes
in HPMECs when they switched from 20% O2 to 8% O2 tension in
comparison to PLECs (p<0.01).
CONCLUSION: HPMECs demonstrate enhanced angiogenic potential
in comparison to matched, mature, PLECs. Further investigation to
determine whether thes cells are capable of enhanced angiogenesis and
neovasculogenesis in vivo are needed.

O-117
Molecular and Functional Memory in Human Uterine NK Cells
Following Pregnancy. Moriya Gamliel†,2 Debra S Goldman-Wohl,1
Ronit Haimov-Kochman,1 Avraham Nahum,1 Ofer Mandelboim,2 Simcha
Yagel*.1 1Hadassah-Hebrew University Medical Center, Jerusalem, Israel;
2
Hebrew University Faculty of Medicine, Jerusalem, Israel.
INTRODUCTION: Natural killer cells (NKs) are abundant in the human
decidua, and have been shown to support development of the placental bed.
Several of the Great Obstetrical Syndromes are associated with inefficient
placentation and are observed particulary in first pregnancies. We therefore
hypothesized that NK cells remember first pregnancy and respond better
by secreting increased growth factors in subsequent pregnancies.
METHODS: Characterization of decidual NKs (from elective pregnancy
terminations) from primigravid (n=85) vs multiparous (n=248) women.
The analysis included cell surface markers (flow cytometry), transcriptome
expression (RNA-seq), epigenetic signature (ATAC-seq and WGBS),
growth factors secretion (ELISA), and functional assays (in vitroaortic ring assay and in vivo- tumor growth mouse model). In addition,
endometrial NK cells as well as peripheral blood NK cells from women
following pregnancy, were tested.
RESULTS: We discovered NK cell population unique to all repeat
pregnancies included in our analysis. This finding was unique to NK
cells and found to be independent of the pregnancy week (6-14 weeks),
maternal age (range 17-47) or gravidity (in the multiparous). These NK
cells possess a novel transcriptome and epigenetic signature, along with
significantly high expression levels of the activating receptors NKG2C
and LILRB1. Triggering of these receptors by their ligands in the presence
of IL-15, leads to increased secretion of VEGFa and IFNγ. This secretion
was abolished when using appropriate blocking antibodies. These secreted
growth factors were shown to be functional, by supporting vascular
budding and trophoblast-tumor growth. Moreover, we show that higher

Scientific Abstracts

levels of NKG2C are found in the endometrial NK cells of women who
had been pregnant previously, as compared to those who had not. This
difference was not detected when comparing peripheral blood NK cells.
CONCLUSION: We propose that NK cells in the decidua remember
pregnancy and react better in subsequent pregnancies by secreting
pro-angiogenetic factors. We propose that this population represents
NK memory of pregnancy, as we termed Pregnancy Trained decidual
NK cells (PTdNKs). PTdNKS are probably exist in the uterus between
pregnancies. PTdNKs may help in understanding the basis for, and may
have therapeutic potential in treating the "Great Obstetrical Syndromes".

O-118
IP-10 Supression by Histone Methylation is Mediated by cAMP and
the PRC2 Complex in Uterine Stromal Cells. Michelle Silasi, Yang
Yang-Hartwich, Yuan You, Paulomi Aldo, Gil Mor*. Yale University,
New Haven, CT, United States.
INTRODUCTION: Recent studies suggest that epigenetic modifications
and immune mechanisms contribute to recurrent pregnancy loss (RPL).
Human chorionic gonadotropin (whose secondary messenger is cAMP)
has been suggested as a therapy for RPL due to its function regarding
T-cell migration. IP-10 is a major T- cell chemoattractant, and elevated
levels due to infection or inflammation are associated with adverse
pregnancy outcomes. However, the epigenetic mechanisms that regulates
chemokine expression in the decidua is not fully known. The PRC2
complex (and its histone methyltransferase enzyme EZH2) is the main
protein complex regulating histone methylation, which silences target
genes by binding to DNA. We hypothesize that chemokine surpression in
the decidua occurs by histone methylation. In this study we demonstrate
that cAMP, through the PRC2 complex, enhances histone methylation
(resulting in H3K27me3) repressing IP-10 expression. Furthermore, LPS
(as in infection/inflammation) can modify histone methylation, decrease
histone binding, and induce IP-10 expression. Modifications to histone
methylation from inflammation may be a mechanism of RPL.
METHODS: In vitro studies were done using the human endometrial
stromal cell line (hESC). IP-10 and EZH2 expression were determined
by quantitative PCR. Expression of H3K27me3 was demonstrated by
western blot in cells treated with cAMP. Chromatin immunoprecipitation
was performed to determine the enrichment of the of H3K27me3 to the
IP-10 promoter. Organ cultures were prepared from freshly isolated human
decidua tissue (non labor) and treated with LPS. Expression and secretion
of IP-10 in decidua tissue was determined by quantitative PCR and ELISA.
RESULTS: cAMP-inhibits IP-10 expression by inducing H3K27me3
histone methylation, which binds to the IP-10 promoter, thereby
suppressing IP-10 expression. cAMP-induced histone methylation is
through EZH2, the major functional member of the PRC2 complex.
SUZ12 and EED (two other major components of the PRC2 complex) are
also induced by cAMP. LPS reverses the histone enrichment, increasing
IP-10 expression/secretion.
CONCLUSION: We describe the presence of epigenetic mechanisms
active at the maternal-fetal interface. Our data demonstrates inflammation
suppression by histone methylation in uterine stromal cells. Alterations
in immune regulatory functions such as during inflammation, will have
detrimental effects on the pregnancy and may lead to pregnancy loss.

O-119
Analysis of the Dynamic Changes in Chromatin Accessibility in
Decidualizing Human Endometrial Stromal Cells. Raffaella Lucciola†,1
Pavle Vrljicak,1 Emma S Lucas,1 Lauren Lansdowne,2 Nigel Dyer,2
Sascha Ott,1,2 Jan J Brosens.1 1Tommy's National Centre for Miscarriage
Research, Warwick Medical School, University of Warwick, Coventry,
United Kingdom; 2University of Warwick, Coventry, United Kingdom.
INTRODUCTION: Cyclic decidualization of the endometrium in
response to progesterone signaling is confined to menstruating species,
including humans and other higher primates. During this process,
endometrial stromal cells (EnSCs) differentiate into specialized decidual
cells that control embryo implantation. Although several transcription

Table of Contents for the Digital Edition of SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018