SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 247A

Scientific Abstracts

F-200
Cilia-Associated Genes Are Upregulated in the Endometrium of
Women with Ectopic Pregnancy. Jessica Lentscher†, Nicole Ortogero,
Richard Burney, Gregory Chow*. Madigan Army Medical Center, Tacoma,
WA, United States.
INTRODUCTION: Ectopic pregnancy is the leading cause of morbidity
and mortality among women in the first trimester. A reliable diagnostic test
for the detection of ectopic pregnancy in cases of non-viable pregnancy
of unknown location (NV-PUL) is needed. An accurate serum biomarker
for this purpose is not available. The objective of the current study
is to describe endometrial molecular signatures toward discovery of
biologically plausible ectopic classifier candidates.
METHODS: Endometrial biopsies were collected from 69 women with
non-viable pregnancies undergoing surgical or manual vacuum aspiration
intervention with estimated gestational ages ranging from 37 to 65 days.
From this biorepository, nine samples from women with AIUP and eight
from women with ECT were selected for microarray analysis using the
Affymetrix Human Gene 2.0 ST Array. Linear Models for Microarray
Data (limma) was used to determine differentially expressed genes
(DEG). Predictive modeling was performed on cilia-associated DEG using
the k-nearest neighbors (KNN) algorithm with repeated 10-fold crossvalidation. Orthogonal validation of gene array results was conducted
using quantitative real time PCR.
RESULTS: Comparison of ECT versus AIUP samples identified 28
endometrial DEG. Fisher exact test revealed a statistically significant (p
< 0.0001) enrichment of cilia-associated genes. All 12 cilia-associated
genes were upregulated in ECT samples and known to be highly expressed
in human Fallopian tube. qPCR validation confirmed gene expression
results with 7 of 7 genes tested having statistically significant (p < 0.05)
upregulation in ECT versus AIUP samples (>2 fold). Based on KNN
predictive classification, an average accuracy of 91.1% for the detection
of ectopic pregnancy was achieved using a classifier consisting of the 12
cilia-associated genes. To further substantiate the classifier, a separate
KNN analysis of the 12 DEG classifier was performed on AW Horne's
2011 data. Cross-validation analysis revealed 87.9% average accuracy in
detecting ectopic pregnancy with this external dataset.
CONCLUSION: The observed enrichment of fallopian tube ciliaassociated genes in the endometrium may uniquely represent the
pathophysiological changes in ectopic pregnancy. This research identifies
cilia-associated genes as a promising classifier for ectopic pregnancy
with potentially high accuracy for delineating ectopic from intrauterine
pregnancies.

F-201
Differential Effects of IVF Stimulation Protocols on Endometrial
Development. John Wu†, Bruce Carr, Samir Babayev, Ann Word, Orhan
Bukulmez*. UT Southwestern, Dallas, TX, United States.
INTRODUCTION: It is understood that controlled ovarian
hyperstimulation adversely affects endometrial receptivity secondary to
supraphysiologic levels of estrogen (E2) and progesterone (P) leading to
advanced maturation of the endometrium. Sparse information is available
regarding the effect of different stimulation protocols on endometrial
differentiation. Here, we sought to compare in vivo, histologic, and
molecular profiles of endometrium from women undergoing IVF using
conventional (C-IVF) versus minimal stimulation (MS-IVF) protocols.

METHODS: Human endometrial tissue was collected at time of
oocyte retrieval from 10 patients undergoing either C-IVF or MS-IVF
(clomiphene citrate (CC) + hMG) protocols. Total RNA libraries were
sequenced on the Illumina HiSeq2500. Differential expression analysis
was performed using EdgeR. P-values were corrected for multiple testing
using the false discovery rate (FDR) method. The threshold of significance
was set as FDR < 0.01 for significantly differentially expressed (SDE)
genes. Histologic assessment was conducted by H&E staining.
RESULTS: MS-IVF was characterized by decreased peak E2 and P
levels and thinning of the endometrium (Table 1). Histomorphology
revealed dramatic changes with small undifferentiated glands dispersed
in a sea of edematous polygonal stromal cells in MS-IVF compared with
large glands of advanced secretory epithelium in C-IVF. Sequencing and
pathway analysis revealed several mechanisms for these endometrial
phenotypes. 723 of 20764 (3.4%) genes expressed in the endometrium
were SDE (log2FC >= 1.5) between MS-IVF and C-IVF. SFRP4
and WIF1, stromal cell products that inhibit Wnt-induced glandular
proliferation, were increased 7- and 18-fold in MS-IVF (p = 1.8E-30,
SRFP4 and 1.4E-11, WIF-1). Further, the anti-proliferative gene RALDH2
was increased 4-fold and IGFBP-4 and -5 were increased 3-4 -fold. In
contrast, P-responsive genes DKK-1 and HSD17β2, were upregulated
11- and 8- fold respectively in C-IVF. Importantly, our data revealed
no significant change in co-regulators of ER signaling, transcriptional
repressors, or cell cycle inhibitors.
Patient Demographics and Characteristics
C-IVF (n=5)

MS-IVF (n=5)

Age (y)

33.4 ± 1.2

41.6 ± 1.1

<0.01

BMI (kg/m2)

25.4 ± 2.5

26.3 ± 2.9

Gonadotropins (units)

3646 ± 599

615 ± 55

<0.01

Endometrial Stripe (mm)

11.4 ± 1.3

5.6 ± 0.8

<0.01

Estradiol (pg/ml)

2937 ± 402

837 ± 145

<0.01

Progesterone (ng/ml)

1.4 ± 0.2

0.4 ± 0.08

<0.01

RNA integrity number (RIN)

8.7 ± 0.3

8.9 ± 0.2

CONCLUSION: Taken together, although MS-IVF protocols do not
cause advanced maturation of the endometrium, the WNT pathway for
endometrial glandular growth and differentiation is dysregulated and
this effect is primarily mediated through stromal-epithelial interactions.
We hypothesize that endometrial receptivity in MS-IVF is hindered by
cell- and gene-specific effects of the anti-estrogen clomiphene citrate.

F-202
Impaired Endometrial Decidualization in Unexplained Infertility
Due to a Defect in Autophagy. Lauren Reschke†, Darcy Broughton†,
Arin Oestreich, Kelle Moley*. Washington University in St. Louis, St.
Louis, MO, United States.
INTRODUCTION: Up to 15% of infertile couples carry the diagnosis
of unexplained infertility (UI). Evaluation of the endometrium is not a
routine part of the infertility workup, and an endometrial defect may be
contributing to UI. Decidualization of the endometrium occurs in the
luteal phase and is necessary for implantation and establishment of a
viable pregnancy. Autophagy has an important role in decidualization. In
prior studies, decidualized endometrial cells demonstrate an increase in
autophagic flux. We hypothesized that patients with UI would demonstrate
impaired endometrial decidualization due to a defect in autophagy.
METHODS: Women ages 18-37 with a diagnosis of UI (N=7) or
fertile with regular cycles (N=9) underwent endometrial biopsy in the
luteal phase. Exclusion criteria included BMI ≥ 30, irregular cycles,
endometriosis, or recurrent miscarriage. Endometrial stromal cells
(ESCs) were cultured for ≥ 9 days to remove endogenous hormone effect.
ESCs were then treated with medroxyprogesterone acetate for nine days
to induce decidualization. Cells were collected and protein isolated.
Immunoblotting was performed for two proteins involved in autophagy,
p62 and LC3B. Studies have shown that with increased autophagic flux,
levels of p62 decrease, while LC3B increases. Data are expressed as
means ± SEM and differences between cohorts were analyzed using the
Mann Whitney U test.

Friday Posters

silencing. We observed a total of 44,698 cis-eQTLs across 441 probes
satisfying a stringent Bonferroni genome-wide significance threshold of
p< 3.3x10-9, including genes implicated in endometriosis. An additional
2,964 trans-eQTLs affecting 89 probes were detected using the Bonferroni
genome-wide correction (p< 5.5x10-13). We observed 36 cis-eSNPs that
are also trans-eSNPs in endometrium, two of which affect genes (ITGB1
and SPARC) previously associated with endometrial biology.
CONCLUSION: Taken together, these data extend our understanding
of the dynamic nature of gene expression in endometrium, identify
strong genetic effects on genes with diverse functions in endometrium,
and provide a platform for better understanding of genetic effects on
endometrial-related pathologies.

247A

Reproductive Sciences Vol. 25, Supplement 1, March 2018

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