SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 117A

Scientific Abstracts

Influence of Placental Microbiome on Trophoblast Survival, Immune
Response and Invasion. Martha Heusler†, Damián O Muzzio†, Rebekka
Einenkel†, Jens Ehrhardt, Marek Zygmunt*. University of Greifswald,
Greifswald, Germany.
INTRODUCTION: Recently, an organ specific physiologic microbiome
has been described in the placenta. The question how these bacteria
influence pregnancy has to be addressed. Commensal bacteria might
trigger a proinflammatory response in the fetomaternal unit. Due to
the expression of toll like receptors, trophoblast cells are able to sense
and respond to bacteria by increase of inflammatory cytokine release
or apoptosis. Fusobacterium nucleatum (Fus n) has been described as
a part of the placental microbiome. Additionally it interacts through
E-cadherin with colon carcinoma cells and promotes their proliferation.
Here, we postulate that low concentrations of Fus n can affect trophoblast
survival, immune response and invasion without engaging significant
LPS-mediated apoptosis.
METHODS: Three different cell lines were used: HTR8/SVneo (low
E-cadherin expression, functional TLR4), JEG-3 (high E-cadherin,
functional TLR4) and BeWo (high E-cadherin expression, non-functional
TLR4 pathway). Cells were stimulated for 2 to 48 h with different
concentrations of oxygen inactivated Fus n to simulate the presence of
non-infective bacteria. Appropriate controls including E. coli were used.
At least five independent experiments each in triplicate were performed.
Viability, apoptosis rate, cell cycle, invasion, expression and secretion
of various cytokines were analyzed by Annexin V Apoptosis Detection
Kit, Nicoletti Cell Cycle Analysis, Cell Titer Blue Cell Viability Assay,
transwell invasion assay, qPCR and ELISA. Data were analyzed by
Student's T-test. p value <0.05 was considered statistically significant.
RESULTS:
Proliferation: The number of viable HTR8/SVneo cells decreased
significantly after 2 d exposure with high Fus n concentration (ratio
Fus n:trophoblast 1 and 10). BeWo numbers increased in the first 2 h of
stimulation (ratio 0.1; 1; 10) but decreased after 48 h.
Apoptosis: Only for HTR8/SVneo apoptosis was heightened with high
Fus n concentrations.
IL-6: Secretion by HTR8/SVneo declined with low concentrations of
Fus n (ratio 0.01) but was induced by higher concentrations (ratio 0.1; 1).
MMP-2: Secretion was decreased in BeWo by Fus n treatment but
increased in HTR8/SVneo, also it was not increased by E. coli.
Invasion: Fus n influenced invasion in HTR8/SVneo.
CONCLUSION: We showed that low concentrations of non-infective
Fus n can influence trophoblast invasive phenotype without affecting cell
viability. This phenomenon seems to be dependent on the balance between
E-cadherin and TLR4 expression.

T-021
Alpha Lipoic Acid, in an In-Vitro Model, Inhibits GM-CSF Induced
Human Fetal Membrane Weakening. D Kumar, A Sharma†, R Moore, B
Mercer, J J Moore*. CWRU, MetroHealth, Cleveland, OH, United States.
INTRODUCTION: We have developed an in-vitro model for study
of the fetal membrane (FM) weakening process that leads to rupture in
pPROM and resultant preterm birth. In the model system we utilize TNFα
as a surrogate for inflammation/infection and thrombin as a surrogate
for decidual bleeding/abruption, both leading causes of pPROM. Both
agents induce a concentration dependent weakening of human FM in
vitro and concomitantly reproduce the biochemical signature seen in
the natural weak zone of fresh FM. Choriodecidua (CD) rather than
amnion has been shown to be the primary target for TNFα and thrombin
induced weakening in this model system. Recently we have also shown
that GM-CSF is a critical intermediate for both TNFα and thrombin
induced FM weakening. That is: GM-CSF increases in a concentration
dependent manner, concomitant with weakening with both agents; GMCSF neutralizing antibody blocks weakening; and GM-CSF itself weakens
intact FM. Previously we have shown that α-lipoic acid (LA) blocks
TNFα and thrombin induced FM weakening. In these experiments, we

117A

investigated (a) if α-lipoic acid (LA) can inhibit FM weakening by the
critical intermediate GM-CSF, and (b) if GM-CSF can weaken isolated
amnion.
METHODS: Full thickness FM fragments from uncomplicated term
repeat cesarean section deliveries were mounted in 2.5cm Transwell
inserts and cultured with/without LA (1-1000 mM). After 24h, medium
was removed and replaced with/without addition of GM-CSF (200ng/
ml). After 48h culture, the FM fragments were ruptured-strength tested
and the medium (supernatant) from the maternal side compartment of
the model assayed for proteases. Separately isolated amnion fragments
(disc and reflected amnion) from fresh membranes were incubated with
GM-CSF (200ng/ml) for 48 hr and then strength tested.
RESULTS: GM-CSF, compared to controls, caused significant weakening
of full thickness FM (6.9±0.4 vs. 13.3±0.5 N; p<0.001) and preincubation
with all doses of LA [1 mM LA (11.7±1 N); 10 mM LA (13.3±1.5 N); 100
mM (12±1 N); 1000 mM (13.1±0.8 N); all <0.001) blocked this weakening
significantly. LA had no effect on FM strength. GM-CSF applied directly
to isolated amnion was unable to weaken isolated amnion. Medium from
all experiments is being assayed for proteases.
CONCLUSION: LA effectively blocks FM weakening by GM-CSF,
a critical intermediate in both inflammation and bleeding induced FM
weakening. The LA effect upon GM-CSF induced FM weakening occurs
in the CD as GM-CSF is unable to weaken isolated amnion. We speculate
that LA may be a viable potential agent to prevent pPROM.

T-022
Oxidative Stress and Senescence in Human Primary Decidual Cells.
Jin Jin*,1,3 Samantha Sheller-Miller†,3 Man Ling Luo*,1 Nanbert Zhong*,2
Ramkumar Menon*.3 1NanFang Medical University, Guangzhou, China;
2
New York City University, New York, NY, United States; 3University Texas
Medical Branch, Galveston, TX, United States.
INTRODUCTION: Dysregulated redox balance and inflammation
are major triggers for parturition at term and preterm. Oxidative stress
(OS) can induce reactive oxygen species (ROS) and activate stress
response through p38 mitogen activated protein kinase (p38 MAPK)
pathway, senescence and senescence associated inflammation in fetal
cells. Herein, we tested the hypothesis that OS can cause senescence in
maternal decidua either through the activation of either p38MAPK or
proapoptotic p53 pathway.
METHODS: Decidual cells isolated from normal term, not in labor,
fetal membranes were exposed to OS inducer cigarette smoke extract
(CSE) prepared in cell culture medium with or without progesterone and
estrogen for 3, 6 and 24 hours. Changes in ROS levels were detected
using 2'7'-dichlorodihydro-fluorescein. DNA base and strand damage
was determined using Fragment Length Analysis using Repair Enzymes
(FLARE) assay. Western blot determined activated p38 (P-p38 MAPK)
and p53 (P-p53) expression and flow cytometry using probes for
senescence-associated β-galactosidase (SA-β-gal) measured senescence.
Co-treatment of cells with antioxidant N-acetyl cysteine (NAC) and or
p38MAPK inhibitor SB203580 verified specificity of activation.
RESULTS: ROS levels in CSE-exposed decidual cells increased rapidly
(within 2 min) and significantly at all-time points (p<0.05). This effect was
reduced by NAC (p<0.05). In FLARE assays, CSE treated cells showed
comet like appearance indicating DNA damage. CSE induced p38MAPK
activation (p<0.05) while p53 activation was not seen. Senescence (SAβ-Gal stained cells) in decidual cells was increased after CSE treatment
compared to control. Senescence was reduced by both p38 inhibitor
SB203580 and SB+NAC (p<0.05) but not NAC alone.
CONCLUSION: The findings support the hypothesis that OS induce
decidual cell senescence by increasing ROS mediated DNA damage
and activation of p38MAPK. Based on our prior reports on fetal cells
and current data, we postulate that OS induce both feto-maternal tissue
senescence through p38MAPK pathway that can contribute to parturitionassociated change at term and preterm.

Thursday Posters

T-020

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