SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 289A

Scientific Abstracts

S-110

S-111
Multiplexed Analysis of Human Uterine Tissue Lysates Reveals
a Molecular Signature of Adenomyosis. Evan L Chiswick†,2 Manu
Kumar†,2 Keith Isaacson*,1 Douglas Lauffenburger*,2 Linda Griffith*.2
1
Harvard Medical School, Newton Wellesly Hospital, Newton, MA,
United States; 2Massachusetts Institute of Technology, Cambridge, MA,
United States.
INTRODUCTION: The human endometrium undergoes extensive
remodeling that is guided by proteolytic and inflammatory networks in a
spatially asynchronous yet temporally resolved manner, culminating in
implantation or menses. Aberrant proteolytic and inflammatory activity
is known to facilitate metastasis in cancer, and numerous studies have
detailed their aberrations in gynepathologies such as endometriosis.
Uterine pathologies such as adenomyosis have foci of disease often
surrounded by healthy tissue. It is unclear if molecular signatures exist
which distinguish healthy from diseased tissue. This preliminary study
uses a systems biology approach to delineate healthy vs diseased tissue,
using readily accessible high throughput multiplexed technologies.
METHODS: Fresh tissue was excised from several regions of the uterus
from patients post-hysterectomy and flash-frozen. 50mg of tissue was
sectioned per block, and lysed with RIPA buffer + protease inhibitors.
Total protein was normalized and assayed for 50+ inflammatory mediators
and MMPs via Luminex. Analyte concentrations were used for principal
component analysis (PCA) and visualized using donors and disease
presence as color coding schemes.

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RESULTS: Unsupervised PCA showed that the most variance is driven by
donor to donor differences in analyte profiles. Interestingly, PC2 captures
variance that separates adenomyosis tissues from normal endometrium
and myometrium. However, PCA does not show discrete clusters of
healthy tissue from affected donors vs. unaffected donors. Analysis of
PC2 loadings showed contributions of both MMPs and cytokines.
CONCLUSION: These data suggest a valid approach to study tissue
sections in a high throughput multiplexed manner. Normal tissue from
affected and unaffected donors overlapped in PC space, suggesting ectopic
events may result from an altered microenvironment (i.e. dysregulated
myometrium). These data also underscore the need to consider intrinsic
difference amongst donors to avoid misleading false variation.
*Figure(s) will be available online.

S-112
Micro-RNA Let-7b Treatment Suppresses Endometriosis in a Mouse
Model. Cagdas Sahin, Ramanaiah Mamillapalli, Yi Kyong, Hugh S
Taylor*. Yale School of Medicine, New Haven, CT, United States.
INTRODUCTION: Endometriosis is an estrogen dependent, chronic
inflammatory disorder characterized by the growth of endometrial-like
tissue outside the uterus causing pain and infertility in reproductive-aged
women. Current treatments are limited to hormonal therapies which
suppress the disease but are limited due to adverse side effects, loss of
fertility and recurrence of the disease after discontinuation of therapy.
Previously we have described a role for Let 7 micro RNA in this disease.
METHODS: Endometriosis was induced in 9 weeks old female C57BL/6J
mice by suturing donor mouse endometrium into the peritoneal cavity.
Sham surgeries were performed on control mice. After two weeks Let-7b5p mimic was injected into the peritoneal cavity by in vivo-jetPEI for two
weeks. Lesions size and volume were measured from sacrificed animals.
RNA was extracted from the lesions and used for qRT-PCR. Tissue from
lesions was fixed in 4% paraformaldehyde for immunohistochemical
staining.
RESULTS: The volume and histologic area of endometriotic lesions
were significantly reduced in Let-7b treated mice. Analysis of gene
expression by qRT-PCR and protein levels by immunohistochemistry
revealed significant reductions of endometriosis drivers including ER-α,
ER-ß, Cyp19A (aromatase), KRAS 4A, KRAS 4B and IL-6 in microRNA
Let-7b-5p treated mice compared to untreated mice with endometriosis.
CONCLUSION: Decreased lesion size, volume and expression of
genes involved in the pathophysiology of endometriosis was observed
in micro-RNA let-7b-5p treated mice. These data reveal that miR Let 7b
has a role in the pathophysiology of endometriosis. Moreover, treatment
of endometriosis with micro-RNA let 7b is a promising novel treatment
option for endometriosis.

S-113
Proteomic and Transcriptomic Analysis of Oxidative Stress, Lipid
Peroxidation and Iron Uptake Signatures in Endometriotic Lesions.
Bernd Elger, 1 Marie L Thézénas, 2 Christian M Becker, 2 Krina T
Zondervan,2 Karl J Morton,2 Thomas M Zollner,1 Benedikt M Kessler,2
Udo Oppermann.2 1Bayer AG, Berlin, Germany; 2University of Oxford,
Oxford, United Kingdom.
INTRODUCTION: Endometriosis is characterized by chronic,
unresolved inflammation, in which oxidative stress is likely to play a
critical role. We set out for a systematic study to identify novel factors
defining oxidative stress in ectopic peritoneal lesions.
METHODS: Samples of eutopic endometrium (n=7), ectopic lesions
(n=6) and eutopic control (n=4) from patient subjects were used
for immunohistochemistry, label-free quantitative proteomic and
transcriptomic analysis. Samples were collected using WERF EPHect
criteria. Expressed transcripts were derived from RNAseq of peritoneal
macrophages.
RESULTS: We identified 270 proteins (1% FDR for confidence of
identification) showing significant differences between ectopic lesions,
eutopic endometrium and controls (ANOVA). Proteins involved in
detoxification of ROS, glutathione (GSH) conjugation, and phase II
conjugation were significantly enriched. Several metabolic proteins were

Saturday Posters

Menstrual Effluent: A Tool for Better Understanding Endometriosis.
Laura Warren†, 1,2 Brandon Blau, 2 Nicole Cacace, 2 Andrew Shih, 2
Xiangying Xue, 2 Susana Marquez, 2 Mushmoom Khan, 2 Margaret
Defranco,2 Maya Tevlin,2 Annette Lee,2 Peter Gregeresen*,2 Christine
Metz*.2 1Donald and Barbara Zucker School of Medicine, Hempstead,
NY, United States; 2The Feinstein Institute, Manhasset, NY, United States.
INTRODUCTION: Endometriosis, defined by the growth of endometrial
tissue outside of the uterus, affects up to 10% of females of reproductive
age and is commonly associated with pelvic pain and infertility. Although
the cause of endometriosis is unknown, the most commonly accepted
theory is Sampson's theory of retrograde menstruation which suggests
that menstrual effluent (ME) contains the source endometrial cells that
are proposed to form endometriosis lesions, making it a key material to
study endometriosis. We aim to harness ME as a tool for improving
the diagnosis and understanding of endometriosis through phenotypic
and functional cellular analyses.
METHODS: ME was collected from endometriosis and control subjects
on the 1st or 2nd day of menstruation using the DivaCup. ME was analyzed
by flow cytometry for CD45- and CD45+ cell populations or plated in T75
flasks to isolate stromal cells by adherence. ME-stromal cells isolated from
endometriosis and control subjects were grown until passage 2 and then
decidualized at confluence with 8-Br-cAMP (0.5mM). Cell-free media
and protein lysates were collected after 6, 24, and 48hrs; and RNA was
collected at 6hrs. Media was analyzed for IGFBP-1 by ELISA to assess
decidualization, and RNA was used for RNAseq and qPCR for markers
of decidualization and to identify genes differentially expressed between
endometriosis and control subjects.
RESULTS: Flow cytometric analyses of ME for CD45+ revealed that
the % NK cells were significantly decreased in endometriosis-ME vs.
control ME (p=0.01), while the CD45- cells revealed no significant
differences between endometriosis and control subjects. Additionally,
the decidualization assays revealed that control ME-stromal cells showed
higher decidualization capacity (IGFBP-1 levels) than endometriosis
ME-stromal cells at all 3 time points (p<0.05). RNAseq and qPCR of
samples decidualized for 6hrs showed that retinoic acid pathway genes
(ALDH1A1 & RBP1) were down-regulated in endometriosis stromal
cells under basal and cAMP conditions (p<0.05).
CONCLUSION: ME is a valuable source of endometrial cells for
studying decidualization defects associated with endometriosis and may
be a potential diagnostic/screening tool for endometriosis.

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