SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 110A

110A

Reproductive Sciences Vol. 25, Supplement 1, March 2018

O-159
Human Embryo Secretions Regulate Human Endometrial Adhesion
via the Lnc RNA, PTENP1's, Sponge Effect on Endometrial
MicroRNA. Masashi Takamura†,1 Kate Rainczuk,1 Carly Cuman,1,2,3 Luk
Rombauts,2,3 Tiki Osianlis,3 Evdokia Dimitriadis*.1,3 1Hudson Institute
of Medical Research, Melbourne, Australia; 2Monash IVF, Melbourne,
Australia; 3Monash University, Melbourne, Australia.
INTRODUCTION: Embryo implantation failure is a major cause of
infertility. We have previously demonstrated that human blastocyst
secretions (BCM) influence endometrial epithelial cell adhesion, the
initiating event of implantation. The role of long non-coding (lnc)
RNA in embryo implantation is poorly defined, and there are no studies
investigating the role of human embryo sections on endometrial lncRNA
expression. We hypothesised that human embryos regulate embryo
implantation via altering lncRNA and aimed to determine the role of
human embryo secretions on endometrial lncRNA expression and
the functional and mechanistic effects in endometrial receptivity and
implantation.
METHODS: We used array analysis to determine the expression of
lncRNA in endometrial epithelial cells treated with human IVF embryo
secretions from embryos that implanted compared to embryos that did
not implant. lncRNA was localised in endometrium throughout the
menstrual cycle in fertile women and women with primary infertility by
in situ hybridization and expression levels determined by qRT-PCR. The
effect of endometrial lncRNA on sponging endometrial microRNA was
determined by array and confirmed by fluidigm analysis. To determine
the effect of lncRNA on endometrial epithelial cell adhesive capacity we
used a co-culture adhesion model of trophoblast cell line (HTR8 cells)
spheroids and primary human endometrial epithelial cells.
RESULTS: We identified that the lncRNA PTENP1 (PhosphataseAnd-Tensin-Homolog-Pseudogene-1) was reduced in primary human
endometrial epithelial cells (HEEC) treated with BCM from embryos that
did not implant after IVF. PTENP1 and PTEN expression in endometrial
tissue across the menstrual cycle (N = 8/gp) did change across the cycle or
with fertility status. PTENP1 localized in human endometrial epithelium
and maximally in luminal epithelium (site of blastocyst attachment) in the
receptive phase. siRNA knockdown of PTENP1 in HEEC blocked HTR8
spheroid adhesion to HEEC (reduced by 21±4.0%, p<0.001) and reduced
several microRNA. Specifically, miR-590-3p was augmented after
PTENP1 knockdown (2.28±0.23 fold, p<0.05) suggesting a 'sponging'
effect. miR-590-3p mimic reduced adhesion of HEEC (p<0.001) via LIF,
RHOA and ALCAM.
CONCLUSION: This data show for the first time that human embryos
regulate their destiny to implant via regulating endometrial lncRNAs and
have important implications in developing novel therapeutics to facilitate
implantation in women with implantation failure/infertility.

O-160
Plasma microRNAs Identified as Novel Markers of Embryo
Implantation. Shirin Khanjani†, Rumana Islam, Javairia Khalid, Vasso
Terzidou, Stuart Lavery, Kim Sung Hye, Phillip R Bennett*. Imperial
College, London, United Kingdom.
INTRODUCTION: MicroRNAs (miRNAs) are small non-coding RNAs
critical for regulating cellular functions. There is growing evidence
supporting a role for microRNAs in embryonic and endometrial cell
function. miRNAs are present within cells and are exported into plasma
where their stability makes them potential biomarkers for health and
disease. Our study aims to identify 1) plasma miRNAs expression profiles
associated with IVF treatment and 2) biomarkers for 'receptivity' to predict
the treatment outcome.
METHODS: After informed consent blood samples were collected
from women undergoing IVF at Hammersmith Hospital. Samples were
collected at two time points, first prior to the start of the treatment cycle
and secondly on the day of embryo transfer prior to the procedure.
Pregnancy outcomes were recorded. A cohort of 12 patients was used for
miRNA analysis selected to exclude confounding factors as far as posible.
Selected patients i) underwent a fresh cycle of IVF, ii) had AMH 5-25,
ii) had no endometrial disease and iv) had a single top quality blastocyst

Scientific Abstracts

transferred. Plasma miRNA was extracted using Norgen Biotek kit.
miRNA expression profile was obtained using the nCounterTM assay.
Analysis was performed separately on matching samples before and after
the start of treatment and data from women who went to be pregnant was
compared to those who did not. SIMCA-P and VIP scores were calculated
using a proprietary algorithm. Gene pathway analysis was performed
using the DIANA software.
RESULTS: 15 miRNAs were identified to have statistically significant
differential expression following IVF treatment compared with matching
samples before treatment. 3 distinct miRNAs were found to have a
statistically significant expression between the subsequently pregnant and
non-pregnant group (Fig 1). Pathway analysis strongly indicated that the
identified miRNAs are essential for immune and inflammatory processes.
*Figure(s) will be available online.
CONCLUSION: We have identified a set of plasma miRNAs which
appear to predict implantation success and are involved with immune
and inflammatory pathways regulating ovarian hypestimulation and
implantation. Once utility is confirmed in replication cohorts these
biomarkers have the potential to improve IVF success rates by identifying
the cycle with best receptivity status.

T-001
Global Ablation of Adrenomedullin 2 in Female Mice Causes
Impaired Pregnancy Outcome. Madhu Chauhan, Ancizar Betancourt,
Chandrasekhar Yallampalli. Baylor College of Medicine/Texas Children's
Hospital, Houston, TX, United States.
INTRODUCTION: Adrenomedullin2 (ADM2), a novel peptide
discovered in 2004 belongs to calcitonin gene related peptide (CGRP)
family of hypotensive peptides. ADM2 is widely expressed in several
tissues including reproductive organs such as placenta, uterus, ovary and
cervix. We have demonstrated earlier that ADM2 facilitates trophoblast
invasion in 1st trimester of human pregnancy and, that its plasma and
placental levels are decreased in spontaneous abortion and preeclampsia.
Therefore, this study was designed to assess the physiological importance
of ADM2 in pregnancy using Adm2-/- knockout (KO) mice generated by
CRISPR/Cas9 genome editing technology.
METHODS: This study was approved by animal care and use committee
(IACUC) of Baylor College of Medicine. To generate a null allele of Adm2,
two single guide RNAs (sgRNAs) were selected by the Baylor College of
Medicine Mouse Embryonic Stem Cell Core (BCM mES Core) positioned
to flank the genomic region containing the open reading frame of Adm2.
Microinjection of the sgRNA/Cas9/ss oligo mixture into the cytoplasm of
pronuclear stage zygotes from C57BL/6NJ female mice was performed
by BCM Genetically Engineered Mouse Core. Injected zygotes were
transferred into pseudo-pregnant ICR females. Mice (N0) were genotyped
by standard PCR using a three primer system, a single forward primer
shared between two different reverse primers. Two primers approximately
100-200 bases outside the two sgRNA sites were designed to amplify a
smaller deletion amplicon compared to the wild type amplicon. A separate
reaction using the second reverse primer, placed within the predicted
deleted interval, was designed to amplify a product in the endogenous
allele. All females mice used in this study were Adm2-/- KO (n=8) and
Adm2+/+ wild type littermates (WT) females (n=8) from N2 generation,
and males were wild type C57BL/6NJ (12 weeks old breeders) from
Jackson laboratories. At 8 weeks of age, Adm2-/- KO and Adm2+/+ WT
females (n=8) were crossed with wild type male (1:1). The mating pairs
were left in cages for 6 months. The gestational age at 1st delivery and
number of litters / mouse and number of total dead pups over a period of
six months were recorded.
RESULTS: 1) Adm2 -/- females are fertile with no significant difference
in the number of pups/litter in Adm2-/-KO compared to the Adm2+/+
WT, 2) Ablation of Adm2 gene in female mice causes preterm delivery
in Adm2-/- KO at gestational day 18 compared to the Adm2+/+ WT that
delivered on day 21 (P<0.05), 3) Total number of dead pups are higher
in Adm2-/- KO compared to the WT over the period of six months of
six consecutive pregnancies (50 in 48 liters in Adm2-/- KO and 11 in 48
litters in Adm2+/+ WT).

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