SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 62A

62A

Reproductive Sciences Vol. 25, Supplement 1, March 2018

be presented at the meeting. These will shed light on the underlying
genetic mechanisms of this debilitating condition and may point to novel
therapeutic interventions.

O-016
Ulipristal Acetate Reverses Impaired Autophagy in Human Uterine
Fibroids. Ihor Atabiekov†, 2 Ayman Al-Hendy*, 1 Nahed Ismail*, 3
Abdeljabar El Andaloussi*.1 1Augusta University, Augusta, GA, United
States; 2Augusta University, Evans, GA, United States; 3Universityof
Pittsburgh, Pittsburgh, PA, United States.
INTRODUCTION: Uterine fibroids (UF) is a highly prevalent pathology
in women of reproductive age all over the World. One of the important
functional constituents, essential for normal cell functioning, is autophagy.
We have shown that autophagy pathway is blocked in UF cells at the level
of fusion of autophagosome with lysosome, preventing degradation on
intracellular debris and microorganisms (El-Andaloussi et. al, 2017).
Progesterone is known to signal via its nuclear receptors (genomic
pathway) or via membrane progesterone receptors (mPR) (non-genomic
pathway) resulting in PI3K-AKT-mTOR pathway activation, which is
essential for growth and development of UF. Ulipristal acetate (UPA) is a
new effective oral selective progesterone receptor modulator for treatment
of UF. The exact anti-fibroid mechanism of action of UPA is not fully
understood. We hypothesized that UPA may enhance autophagy flux in
UF cells through inhibition of mPR-Akt3-mTOR pathway.
METHODS: The in vitro study was performed in human UF cells
(HuLM) and primary patient fibroid cells. We performed 48h treatment
with 100pM and 1nM of UPA. Transmission electronic microscopy (TEM)
was done to examine the effect of UPA on the formation/accumulation
of autophagosomes and autolysosomes in HuLM and patient primary
cells. In addition, we analyzed autophagy flux under UPA treatment (48h)
with and without: 1) serum starvation for 1h using Hank's balanced salt
solution, HBSS, to induce autophagy; and 2) chloroquine 1 and 10 µM
(1h) to inhibit autolysosomal enzymatic degradation. This experiment
was monitored by FACS and qPCR to quantify: LC3/P62 and autophagyrelated genes (ATGs); ULK1, ULK2, mTOR, Akt3, LAMP1, LAMP2,
Rab7b, Rab11 respectively. The results were supported by Western blot
technique.
RESULTS: We observed that UPA significantly decreases accumulation
of LC3I and P62 in HuLM cells and in primary cells (p<0.05). We also
observed the synergistic effect of UPA and serum starvation. We found
a significantly increased percentage of LC3I-negative cells in 100pM
UPA+ serum starvation samples compared to controls and serum starved
cells (p<0.05). UPA was able to significantly decrease accumulation of
LC3I in HuLM cells, antagonizing chloroquine inhibitory effect of the
autophagy flux. Interestingly, the mRNA obtained from both HuLM and
primary cells analyzed by qPCR, has shown a significant suppression of
Akt3 (by 50 % for 100pM and 1nM), suggesting the stimulatory effect
of UPA on autophagy initiation. We also observed a significant increase
in ATG12 by 2 folds (p<0.005), ULK2 by 2 folds (p<0.005) and LAMP1
by 4 folds in HuLM cells when treated with UPA (p<0.05).
CONCLUSION: These results suggest that UPA possesses additional
inhibitory effect on non-classical progesterone signaling, which enhanced
autophagy in UF.

O-017
Bone Marrow-Endometrial Stromal Cell Fusion in Experimental
Endometriosis. Aya Tal, Reshef Tal, Shafiq Shaikh, Ramanaiah
Mamillapalli, Hugh S Taylor. Yale University School of Medicine, New
Haven, CT, United States.
INTRODUCTION: Cell fusion, a well-established feature of bonemarrow derived cells (BMDCs), is involved in the development and
repair of various tissues, and evidence suggests it may give rise treatment
resistant cells that contribute to the pathogenesis of some cancers.
Endometriosis, though a benign disease, shares molecular aspects in
common with cancer. Further, mouse endometriosis-like lesions are
infiltrated by BMDCs. We therefore sought to explore the occurrence of
cell fusion in a mouse model of endometriosis.

Scientific Abstracts

METHODS: A Cre-Lox system was employed to detect cell fusion
occurring upon access of Cre-recombinase from one fusion donor cell
to the floxed-STOP cassette upstream of an enhanced green fluorescent
(EGFP) in the fusion cell partner. 5-Fluorouracil-based non-gonadotoxic
bone-marrow transplant (BMT) from floxed-STOP EGFP donors to WT
recipients was followed by endometriosis induction using Cre uterine
implants to investigate cell fusion between BMDCs and cells of the lesion.
Flow-cytometry, immunohistochemistry, and immunofluorescence were
used to assess the frequency of cell fusion in the endometriosis-like lesion
and characterize fused cells.
RESULTS: The overall frequency of cell fusion between transplanted
bone-marrow (BM) and endometrial cells in mouse endometriosis-like
lesions is approximately 1 in 3000. Fusion-derived cells were localized
to the stromal compartment of the mouse lesion and uniformly lacked
expression of hematopoietic (CD45), endothelial (CD31), epithelial
(CK8), stromal (VIM) or proliferation (PCNA) markers, however they
expressed the mesenchymal/stromal cell markers Sca-1 and CD29. This
data demonstrates that BM contributes to cell fusion events within the
endometriosis lesion.
CONCLUSION: This study is first to describe the phenomenon of
cell fusion in the context of endometriosis and points to a population of
BM-derived, non-proliferating mesenchymal cells in endometriosis-like
lesions resulting from fusion of BM with endometrial cells. Expression
profiling of fusion-derived cells is currently underway to assess stemness,
treatment-resistance and the pathophysiologic role of this population.

O-018
Role of miR-451 in Regulating Endometrial Cell Proliferation
Associated with Endometriosis. Niraj Joshi*, Samantha Bond, Asgerally
Fazleabas*. Michigan State University, Grand Rapids, MI, United States.
INTRODUCTION: Endometriosis is one of the most common
gynecological disorders, which is a cause of chronic pelvic pain and
infertility. The molecular mechanisms associated with the pathophysiology
of the endometriosis are not well understood. MicroRNA's which regulate
gene expression, have been shown to play a role in endometriosis. We
have previously shown that miR-451 expression is decreased in both
eutopic (EuE) and ectopic (EcE) endometrium of baboons and women
with endometriosis and our in vitro data suggest that decrease in miR451 leads to increased endometriotic epithelial cell (12Z) proliferation.
The objective of the present study is to identify the mechanism by which
miR-451 regulates cellular proliferation.
METHODS: Total RNA from the endometrium extracted from baboons
(n=3) before and after the induction of endometriosis for in vivo analysis
of cell cycle genes. 12Z cells were transfected with non-targeting negative
control or miR-451 mimics. The cell cycle genes (CDK4, CDK6, CCNA2,
CCND2, CDKN1A-p21, CCNB1, CCNE1) were evaluated using
qRT-PCR. Western blot analysis was performed for different cell cycle
checkpoint proteins (Cyclin D, CDK6, p21) to understand the mechanism
regulating the cell proliferation.
RESULTS: Induction of endometriosis leads to significantly altered
expression of cell cycle genes (CCNA2, CCNE1, CDK4, CCND2) in
both eutopic and ectopic endometrium of baboons. In vitro transfection
of 12Z with miR-451 mimic showed a significant decrease in CDK6,
CCNA2, CCNB1 and CCNE1 compared to the non-targeting negative
control transfected cells. Interestingly, the expression of CDKN1A (p21an inhibitor of CDK6) was significantly increased in 12Z cells transfected
with miR-451 mimic compared to non-targeting negative controls.
Western blot analysis confirmed the decrease expression of CDK6 and
Cyclin D1 and increased levels of p21 in miR-451 mimic transfected cells
compared to negative controls.
CONCLUSION: Endometriosis causes a decrease in miR-451 expression
leading to altered cell cycle genes in both eutopic and ectopic endometrium
and aberrant cell proliferation. Furthermore, overexpression of miR451 in 12Z cells leads to decrease in CDK6/Cyclin D1 expression by
increasing the p21 levels and thereby preventing G1/S transition. This
study suggest an important role of miR-451 in regulating the cell cycle
and cellular proliferation in endometriotic tissues and offers a novel target
for therapeutic intervention. (HD083273)

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