SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 228A

228A

Reproductive Sciences Vol. 25, Supplement 1, March 2018

F-140
Changes in Rat Placental Transport Phenotype in Advanced Maternal
Age. Tina Napso†,3 Alison S Care†,1,2 Sandra T Davidge*,2 Amanda N
Sferruzzi-Perri*.3 1University of Adelaide, Adelaide, Australia; 2University
of Alberta, Edmonton, AB, Canada; 3University of Cambridge, Cambridge,
United Kingdom.
INTRODUCTION: Advanced maternal age in women is associated with
an increased risk of pregnancy complications including preeclampsia
and abnormal birth weight. Similarly in rats, advanced maternal age is
associated with elevated blood pressure in the mother during pregnancy,
and reduced litter size and fetal growth (Care et al., 2015, Hypertension,
(65) 1324). The placenta is the interface between the mother and her fetus
that controls nutrient and oxygen transfer. It responds to nutritional and
metabolic signals in the mother by altering its structure and function, and
thereby appears to link maternal perturbations to changes in fetal growth
(Sferruzzi-Perri and Camm, 2016, Front Physiol, (7) 33). However, little
is known about the effect of advanced maternal age on placental formation
and function. We aimed to study the effect of advanced maternal age on
placental structure and transport capacity in relation to fetal outcomes.
METHODS: Young (3-4 months old) and aged (9.5-10 months old)
female rats were mated with males (3-5 months old). On day 20 of
gestation (term 22 days), litter size and fetal and placental weights were
recorded. Placentas were then snap frozen for later qPCR gene expression
analysis of glucose (Slc2a1, Slc2a3) and amino acid transporters (Slc38a1,
Slc38a2, Slc38a4) and the insulin-like growth factor-2 (Igf2), which is
important for placental growth and transport function. Placentas were also
fixed, exhaustively sectioned, stained and morphology assessed using
stereology. Data were analysed by t test and significant when P<0.05.
RESULTS: Delayed pregnancy in rats significantly reduced litter size
by 40% and fetal weight by 15%. Placental weight was increased and the
calculated placental efficiency was reduced by 23% in aged dams. There
was no difference in the proportion of the placental regions, although the
calculated volume of the transport labyrinth zone and endocrine junctional
zone were increased by 15% and 51% in aged relative to young dams.
There was no significant effect of maternal age on the expression nutrient
transporter and Igf2 genes by the placenta.
CONCLUSION: Advanced maternal aged reduces placental efficiency
and fetal growth but gross placental morphology and nutrient transporter
gene expression were unaltered. Work is underway to assess the impact
of advanced maternal age on labyrinth morphology, including fetal vessel
and maternal blood density, surface area and the thickness of the barrier
to diffusion, which are determinants of placental nutrient and oxygen
supply to the fetus.

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A Role for Abnormal Early Implantation in Preterm Birth: Using
ART as a Model. Sneha Mani†, 2 Jayashri Ghosh, 1 Yemin Lan, 2
Suneeta Senapati,2 Teri Ord,2 Carmen Sapienza,1 Christos Coutifaris,2
Monica Mainigi*.2 1Temple University, Philadelphia, PA, United States;
2
University of Pennsylvania, Philadelphia, PA, United States.
INTRODUCTION: Preterm birth (PTB) is the leading cause of perinatal
morbidity and mortality. However, little is known about the etiology of
preterm birth, which in part may be due to the heterogeneity among cases.
Assisted reproductive technologies (ART) such as in vitro fertilization
(IVF) may increase the risk of PTB. IVF also causes genome-wide
epigenetic changes, which are hypothesized to underlie disorders of
placentation. Therefore, we utilized our IVF population as a unique
platform to study the etiology of PTB.
Objective: Identify genes and pathways that may play a role in the etiology
of PTB using a homogenous at-risk population.
METHODS: We used the Illumina 850K-Methylation Array to compare
genome-wide methylation pattern in 71 placental samples (24 term control,
12 preterm controls, 24 term IVF and 11 preterm IVF). Analysis was
limited to CpG sites with a mean methylation difference of at least 5%
and genes with at least 2 differentially methylated CpG sites. Ingenuity
pathway analysis was used to examine molecular networks associated

Scientific Abstracts

with PTB. Validation of differentially methylated CpGs was performed by
bisulfite pyrosequencing. siRNA knockdown in an EVT cell line was used
to carry out functional studies of candidate genes identified from the array.
RESULTS: We found 1317 genes differentially methylated between term
and preterm IVF placentas. Ingenuity pathway analysis of these genes
revealed several members of the GALNT, COL and ADAM/ADAMTS
gene families that function in extra-cellular matrix breakdown and
trophoblast invasion, processes essential for early trophoblast invasion
and embryo implantation. DNA methylation changes in these genes were
validated by pyrosequencing. To understand the functional significance
of these genes, we carried out siRNA knockdown of ADAMTS2 and
GALNT2 and 7 in HTR8 cells. We found decreased expression of MMP9,
a marker of trophoblast invasion, with siRNA knockdown of these genes.
Addition of conditioned media from maternal endothelial cells, which
promotes trophoblast invasion, increased expression of ADAMTS genes
in extravillous trophoblasts.
CONCLUSION: Analysis of patients with PTB following ART suggests
epigenetic dysregulation in several genes critical for early implantation.
Further investigation into the regulation and function of these genes may
provide valuable insight into the etiology of this complex disease.

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Endotoxin Effects on 1st Trimester Human Placental Function Can Be
Rescued by PPARγ Activation. Leena Kadam†,3 Dora Baczyk,2 Brian
Kilburn,3 Hamid Reza Kohan-Ghadr,3 John Kingdom,2 Sascha Drewlo*.1
1
College Of Human Medicine, Michigan State University, Grand Rapids,
MI, United States; 2Lunenfeld-Tanenbaum Research Institute, University
of Toronto, Toronto, ON, Canada; 3Wayne State University, Detroit, MI,
United States.
INTRODUCTION: A wide variety of pregnancy-related disorders
are associated with abnormal placental function as well as elevated
inflammation. We previously reported that induction of inflammation in
1st trimester human placenta downregulated trophoblast differentiation
related marker expression, which was rescued by Rosiglitazone. In the
current study, we aimed to further study the effects inflammation on
trophoblast function and delineate the molecular mechanisms mediating
these effects. We hypothesized that endotoxin exposure dysregulates
trophoblast differentiation via the NF-κB pathway, which can be
reversed by induction of PPAR-γ activity via Rosiglitazone.
METHODS: First trimester placental explants and HTR-8/SV Neo cells
were exposed to 1μg/ml of bacterial endotoxin Lipopolysaccharide (LPS)
+/- 10μM Rosiglitazone, LPS+NF-κB inhibitor for 24hrs. The effects
on extravillous trophoblast invasion were assayed using the (i) matrigel
invasion assay - measuring outgrowth (for explants) and number of cells
in lower chamber (for HTR-8/SV Neo cells) and (ii) semi-quantitative
IHC for integrins α1, α6. The effects of LPS on villous trophoblast
differentiation were assayed by semi-quantitative IHC. The transcription
factor activities for NF-κB and PPARγ were assessed using the proximity
ligation assay for co-localization with active RNA polymerase II.
RESULTS: Exposure to LPS did not alter the expression of PSG1 (n=5,
p=0.7) in 1st trimester villous explants. However, LPS significantly
upregulated matrigel outgrowth in explants (n=3, p=0.0007) and the
number of invading HTR cells (n=6, p=0.01) when compared to PBS
treated controls. LPS treated HTR cells also had significantly higher
expression of invasive phenotype associated integrin α1 (n=6, p<0.0001).
LPS treatment also significantly increased the activity of NF-κB (n=6,
p<0.001) in HTR cells. Rosiglitazone treatment reduced invasion and
integrin α1 expression to control levels in both models. It also significantly
downregulated the activity of NF-κB and induced PPARγ activity (n=6,
p=0.005). The effects of LPS on invasion were abrogated in presence of
NF-κB inhibitor.
CONCLUSION: We report for the 1st time that endotoxin exposure alters
trophoblast function by impairing trophoblast invasion via activation of
the NF-κB pathway which can be rescued by PPARγ activation. Our
study provides a critical link between elevated inflammation and impaired
trophoblast function and highlights the crucial role of PPAR-γ in regulating
trophoblast differentiation and placental inflammatory response.
Table of Contents for the Digital Edition of SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018
