SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 111A

Scientific Abstracts

T-002
Dexamethasone Cause Amnion Cell Senescence through p38MAPK
Independent but p21 Dependent Pathway. Laura Martin†,1 Lauren
Richardson†, 2 Ramkumar Menon*. 2 1São Paulo State University,
Botucatu, Brazil; 2UTMB, Galveston, TX, United States.
INTRODUCTION: Dexamethasone (Dex), a glucocorticoid, is used in
utero for fetal organ maturation to improve birth outcomes. However,
Dex induced accelerated proliferation of fetal cells can result in telomere
dependent cellular senescence and premature aging of organs can produce
long-term consequences. Here we test Dexamethasone's ability to induce
telomere dependent replicative senescence in amnion membrane cells
(AEC) leading to premature aging.
METHODS: Human primary AECs in culture were treated with 200nM
of Dex or Dex and 0.4µm RU486 for 48 hours. Semi-quantitative crystal
violet dye exclusion was used determined cell proliferation at 6, 24, and
48 hours. Using flow cytometry we examined progression of cell cycle,
telomere length, type of cell death (senescence, apoptosis, necrosis
and autophagy) in treated and untreated cells (controls). Western blots
(wb) determined senescence pathway (p38MAPK, p21, p16, and p19),
inflammation (P-Rel A), senescence (SA-β-Gal staining) and epithelial
to mesenchymal transition of cell vimentin, cytokeratin-18, E-cadherin,
N-cadherin, SLUG. Glucocorticoid receptor expression and inflammatory
markers (cytokines) were measured using qRT-PCR. ANOVA compared
statistical differences between groups.
RESULTS: Dex did not cause AEC proliferation or telomere depletion
regardless of duration of exposure compared to control (both p=NS). WB
analysis showed p21 dependent but p38MAPK independent senescence
(p<0.05) by Dex with no change in inflammatory markers (P-Rel-A, IL-6,
and IL-8) compared to control. Additionally, cells lacked signs of necrosis,
autophagy or apoptosis after Dex treatment. Conversely, co-treatment
of Dex with RU486 produced DNA damage (p=0.0249) and necrosis
(p=0.0004), with significant increase in inflammatory cytokines (IL-6;
p=0.0211; IL-8; p=0.0008) compared to Dex alone.
CONCLUSION: Dex does not induce AEC proliferation; however, it
induces senescence in human AECs through a p21 dependent (cyclin
dependent kinase inhibitor), telomere, and p38MAPK independent
pathway. These data warrant further discussions on risk/benefit effects
of Dex during pregnancy, as it is capable of growth arrest of specific
cell types.

T-003
Co-Culture of Human Fetal Membranes and Uterine Myocytes Induce
Synergistic Release of a Series of Pro-Inflammatory Cytokines and
Chemokines Promoting Transitioning of the Uterus for Parturition.
Kelycia B Leimert†, Angela Messer, Theora Gray†, Xin Fang, David M
Olson*. University of Alberta, Edmonton, AB, Canada.
INTRODUCTION: Transitioning of the pregnant uterus into the
uterus of delivery is essential for parturition and is marked by increases
in uterine activation proteins (UAPs: FP, OTR and COX-2) and proinflammatory mediators (IL-1Β, IL-6). IL-1Β has an influential role in this
transition, including the up-regulation of 98 genes in uterine myocytes.
We developed a co-culture method to explore maternal myometrial
and fetal membrane tissue interactions, involving term non-labouring
primary human myometrial smooth muscle cells (HMSMC) and human
fetal membrane explants (FME). We hypothesize that crosstalk between
tissues promotes pro-inflammatory expression and therefore uterine
transitioning for parturition.
METHODS: HMSMC are plated in 12-well plates alone or with 6mm
FME in transwell inserts. Via shared culture medium, these tissues can be
simultaneously stimulated with IL-1Β (1 ng/mL), followed by collection
of supernatant for multiplex assay and RNA extraction of FME/HMSMC
for RT-PCR. UAP and cytokine/chemokine relative expression levels are

111A

measured in each tissue separately, as well as products released into shared
supernatant, in response to both monoculture and co-culture conditions.
N=5-7 subjects, two-way ANOVA, p<0.05.
RESULTS: After 24h in co-culture, HMSMC increased COX-2 and
IL-6 mRNA 34x and 523x, respectively, compared to HMSMC alone.
Co-incubated FME had 13x higher IL-6 and 17x higher COX-2 than FME
alone (all p<0.001). IL-1Β strongly up-regulates IL-6 and COX-2 in both
monocultures, but in co-cultures, IL-1Β incubation only induces a minimal
additional effect. Co-culture resulted in a 3.2x and 2.2x increase in FME
FP and OTR, and a 3x increase in HMSMC OTR, but this effect was
not significant (p>0.05). Supernatants from HMSMC alone, FME alone
and HMSMC/FME co-cultures were analyzed via multiplex; co-culture
induced the synergistic release of 18 different cytokines/chemokines
(including IL-6, IL-8, CCL2, CXCL1 and TNFα), producing outputs
much greater than the sum of each monoculture (p<0.001).
CONCLUSION: A better understanding of labour physiology, especially
the role of inflammatory mechanisms, is crucial. Our model studies in
vitro interactions between gestational layers using adjacent layers of
gestational tissues at term. After only 6h, indirect contact via shared
medium results in amplified production of a series of pro-inflammatory
cytokines/chemokines. After 24h co-culture, we see significantly increased
IL-6 and COX-2 (and increasing trends in FP and OTR) in both HMSMC
and FME. These data suggest that 'crosstalk' between the tissues initiate
a 'cytokine chain reaction' that results in up-regulation of IL-6 and COX2, promoting uterine transitions. Acknowledgements: WCHRI, CIHR.

T-004
Cytokine Changes in Maternal Peripheral Blood Correlate with
Time-to-Delivery in Pregnancies Complicated by Preterm Premature
Rupture of the Membranes. Stefania N Ronzoni*,1,3 Valerie Steckle†,2,4
Rohan D'Souza*, 1,2 Kellie Murphy*, 1,2 Stephen Lye*, 2,1,4 Oksana
Shynlova*.1,2 1Mount Sinai Hospital, University of Toronto, Toronto,
ON, Canada; 2Sinai Health System, Toronto, ON, Canada; 3Sunnybrook
Health Care Centre, Toronto, ON, Canada; 4University of Toronto,
Toronto, ON, Canada.
INTRODUCTION: Our study longitudinally assessed plasma cytokine
profiles of women whose pregnancies were complicated by preterm
premature rupture of the membranes (pPROM). We aimed to investigate
the value of cytokines as predictive biomarkers of the latency period
between pPROM and delivery. We hypothesized that women with higher
systemic inflammation at the time of pPROM admission would have
shorter latency periods until delivery.
METHODS: Peripheral blood samples were collected from 20
singleton pregnant women who presented with pPROM at Mount Sinai
Hospital, Toronto, between 24+1 and 35+6 weeks gestational age. The
first sample was drawn within 48 hours from admission, followed by
weekly blood draws until delivery. Pregnancies complicated with acute
chorioamnionitis, preeclampsia, intrauterine growth restriction, body
mass index >30, history of substance abuse and chronic maternal disease
were excluded. 20 uncomplicated, GA-matched pregnancies were used
as controls. The concentration of 39 cytokines in maternal plasma was
measured using Luminex assays (BioRad). Cytokine concentrations were
compared using Mann-Whitney U-test, and Wilcoxon P-test and p<0.05
was considered significant
RESULTS: At admission, women with pPROM exhibited significantly
lower plasma concentrations of pro-inflammatory mediators IL5, IP-10/
CXCL10, MIP1a/CCL3, PDGFbb and CTACK/CCL27 than controls.
In the pPROM group, IL1RA, IL4, IL8, IFNg, TNFa, MCP-1/CCL2
and MIP1a were significantly elevated in maternal plasma at delivery
compared to admission. Women with pPROM that subsequently delivered
within 7 days had significantly lower plasma concentration of antiinflammatory cytokine IL1RA than those with latency periods >7 days.
CONCLUSION: Higher levels of anti-inflammatory cytokines in women
with pPROM were associated with increased latency to delivery, probably
from counter-balancing pro-inflammatory load. When used in conjunction
with other predictive characteristics of time until delivery, cytokines may
further assist clinical decision-making and optimize pregnancy outcomes
in women with pPROM.

Thursday Posters

CONCLUSION: ADM2 is physiologically important for successful
completion of normal pregnancy, and its deficiency results in preterm
birth with increased pup mortality.

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