SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 315A

Scientific Abstracts

METHODS: The human first trimester trophoblast cell line (Sw.71)
was cultured in RPMI media with glucose concentrations of 5mM
(normoglycemic levels) and 10mM (hyperglycemic levels) in the presence
or absence of allopurinol (200μM) for 72 hours. Cell-free supernatants
were collected and measured for IL-1β, IL-8, sFlt-1, sEndoglin, and PlGF
by ELISA (n=3-5).
RESULTS: Compared to 5mM glucose, excess glucose (10mM) increased
trophoblast secretion of IL-1β by 4.3±2.3 fold (p=0.0004); IL-8 by
3.4±1.1 fold (p<0.0001); sFlt-1 by 3.9±0.5-fold (p <0.0001), sEndoglin
by 1.2±0.2 fold; and PlGF by 16.0±6.5 fold (p=0.0125). Under excess
glucose conditions, the presence of allopurinol significantly inhibited
the secretion of IL-1β by 45.6±13.0% (p=0.0007); IL-8 by 54.2±3.6%
(p<0.0001); sFlt-1 by 19.6±2.5% (p=0.0003); and PlGF by 69.6±11.4%
(p=0.0162); but had no effect on sEndoglin.
CONCLUSION: Our findings indicate that xanthine oxidase-mediated
uric acid/IL-1β and/or ROS regulates trophoblast inflammatory and
angiogenic responses to excess glucose. Furthermore, our findings suggest
that allopurinol may be a candidate medication to prevent placental
dysfunction and adverse pregnancy outcomes, such as preeclampsia, in
pregnant women with diabetes.

Effect of Blood Contamination on Amniotic Fluid Detection In Vitro
Using Immunoassays. Elisa T Bushman†,2,4 Lauren H Theilen†,2,4
Ibrahim Hammad†,2,4 M Sean Esplin*,3,4 Isaac Esplin.1 1Brigham Young
University, Provo, UT, United States; 2Intermountain Healthcare, Murray,
UT, United States; 3Intermountain Healthcare, Salt Lake City, UT, United
States; 4University of Utah Health, Salt Lake City, UT, United States.
INTRODUCTION: To determine the accuracy of commercially-available
immunoassays for detecting amniotic fluid proteins in the setting of blood
contamination.
METHODS: IGFBP-1, PAMG-1, and AFP are present in high
concentrations in amniotic fluid, and are detected by three commerciallyavailable immunoassays: ROM Plus®, Amnisure®, and Actim® PROM.
We diluted whole blood samples containing known concentrations of
these proteins (in the range expected in a woman with a term pregnancy)
with amniotic fluid (also containing known concentrations of proteins) to
blood levels of 50%, 20%, 10%, 5%, and 1%. ROM Plus®, Amnisure®,
and Actim® PROM tests were performed on each sample in duplicate
according to package instructions. Results were interpreted at 5, 10, 15,
and 20 minutes by 2 obstetricians blinded to the concentrations of blood
and amniotic fluid proteins in each sample. Physician-interpreted results
were determined to be true positive, false negative, false positive, or true
negative. Accuracy, intra-, and inter-observer concordance were calculated
(Table 1). Sensitivity, specificity, and predictive values were calculated
(Table 2). Fisher exact test was used to compare test characteristics.
RESULTS: Out of 120 tests, 2 of the ROM Plus® devices yielded invalid
results. There were no false positive results. Overall, ROM Plus® had
better accuracy (97.9%) than Amnisure® (80.7%) or Actim® PROM
(78.3%). Intra- and inter-observer concordance were similar for all 3
tests (98-100%). ROM Plus® had significantly higher sensitivity than
Amnisure® and Actim® PROM (p<0.0001). There was no significant
difference in sensitivity between Amnisure® and Actim® PROM
(p=0.51).
CONCLUSION: ROM Plus® maintains strong test characteristics for the
detection of amniotic fluid proteins in the setting of blood contamination,
and performs significantly better than Amnisure® and Actim® PROM
tests under this circumstance.
*Figure(s) will be available online.

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Comparing the Levels of miRNA Expression in Plasma from Blood
Collected Using EDTA and Heparin Tubes, and Heparinase-Treated
Plasma. Sung Hye Kim†,1 David A MacIntyre,1 Lynne Sykes,1 Phillip R
Bennett,1 Vasso Terzidou.1,2 1Imperial College London, London, United
Kingdom; 2Imperial College School of Medicine, Chelsea and Westminster
Hospital, London, United Kingdom.
INTRODUCTION: There is an increasing interest in circulating
microRNAs (miRNAs) as biomarkers for various diseases including
malignancy and pregnancy. Commonly, real-time quantitative PCR (RTqPCR) is used to detect circulating plasma miRNA levels. However,
previous studies have demonstrated that anticoagulant agents such as
heparin used during collection can skew the miRNA quantification by
inhibiting DNA polymerase activity. Although heparinase treatments of
extracted RNA have been introduced to reduce this effect of heparin, it
is unclear whether this treatment enables plasma miRNAs collected from
heparin tubes to be comparable to that from EDTA tubes. In this study,
we assessed the effect of EDTA, heparin and heparinase-treatment on
the measurement of endogenous circulating miRNAs in plasma samples.
METHODS: Fresh blood samples were collected in Vacutainer tubes
containing heparin (sodium heparin, 143 USP units) or EDTA (EDTA,
7.2mg) from three healthy pregnant women. Plasma RNA was extracted
using Plasma/Serum circulating RNA Purification Kit (Norgen Biotek).
For cDNA synthesis of EDTA and heparin samples, we used the
miRCURY Universal cDNA Synthesis Kit II. In addition, 2ul of RNA
from heparin sample was subjected to heparinase treatment with 6U
Heparinase I and 10U RNase inhibitor during the reserve transcription
reaction. The cDNA was analysed by RT-qPCR using the miRCURY
LNA Serum/Plasma Focus miRNA PCR Panel. Spearman's correlation
coefficients for the expression of 179 miRNAs in EDTA, heparin, and
heparinase-treated samples were calculated using GraphPad Prism.
Following this, ClustVis was used to visualize clustering of multivariate
data using principal component analysis (PCA) and heatmap. Individual
primer efficiency was also calculated using LinRegPCR.
RESULTS: Our data demonstrated that heparin inhibits miRNA
quantification by interfering with the RT-qPCR reaction. Morover,
it revealed that the miRNA profiles of EDTA and heparinase-treated
samples were highly correlated (r = 0.9464). The heparinase treatment
of extracted RNA improved the RT-qPCR signals attenuated by heparin
and recovered the ability to differentiate patient-to-patient variability.
However, there were differences in the specific miRNA levels between
EDTA and heparinase-treated samples.
CONCLUSION: This study highlight the differences in the miRNA
profiles of plasma collected using different anticoagulants. There is a
need for caution when interpreting the PCR-based quantification of
circulating miRNAs from heparinized plasma following heparinase
treatment as heparin affected different miRNAs to varying extent among
patient samples.

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Architectural and Functional Adjustment of the Secretory Pathway
in Decidualized Endometrial Stromal Cells. Paola Panina-Bordignon*,1
Adele Ulisse†,1 Tiziana Anelli*,2 Roberto Sitia*.2 1San Raffaele Scientific
Institute, Milan, Italy; 2San Raffaele University, Milan, Italy.
INTRODUCTION: Endometrial stromal cells decidualization entails a
massive increase of secretory protein synthesis, transport and degradation.
Flaws in the decidualization process are associated with reproductive
failure, placental dysfunction and endometriosis.
METHODS: Endometrial biopsies were obtained from 6 pre-menopausal
women undergoing diagnostic histeroscopy. The human endometrial
stromal cell line T-HESC, was obtained from ATCC. Decidualization was
induced with 0.5 mM cAMP and 1 μM MPA for up to 6 days. Western
blot images were acquired with the Chemidoc-it Imaging System or
with FLA-900 Starion and quantified with Fiji. For immunofluorescence
(IF) cells were analyzed an Olympus inverted fluorescence microscope
(model IX70) with DeltaVision RT Deconvolution System. Images
were processed with Fiji. Electron microscopy (EM) was performed
on ultrathin sections (70 nm) and observed with a LEO 912AB Zeiss

Saturday Posters

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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