SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 189A

Scientific Abstracts

F-020

F-021
Term Plasma Exosomes Promote Inflammatory Changes in
Prepartum Murine Cervix and Uterus. Samantha Sheller-Miller†,2
Jayshil Trivedi,2 George Saade,2 Steven Yellon,1 Ramkumar Menon*.2
1
Loma Linda University School of Medicine, Loma Linda, CA, United
States; 2University of Texas Medical Branch, Galveston, TX, United States.
INTRODUCTION: Exosomes function as communication vehicles
between tissues and are hypothesized to carry signals that promote
inflammatory processes for parturition. In this study, we determined that
late-gestation (gestation day [E] 18) exosomes injected into mice on E15
cause remodeling and labor-associated changes in the cervix and uterus.
METHODS: Exosomes were isolated and characterized from maternal
plasma of pregnant CD-1 mice on E9 and E18. On E15, mice were injected
intraperitoneally (3x at 6-h intervals) with either PBS, E9 exosomes
(3.33x1010), or E18 exosomes (9.16x1010) (n=4 per group). On E17,
cervix and uterine tissues were collected and analyzed by western blot
for NF-κB activation (Rel A phosphorylation), connexin (CX)-43, and
COX-2 expression or by immunoassay for IL-6 and TNF-α. The cervix
from other mice were fixed, sectioned and stained for resident F4/80
macrophages. Appropriate statistical analyses were performed and p <
0.05 was considered significant.
RESULTS: Maternal plasma exosomes from E9 and E18 had a size range
between 30-150 nm and expressed markers CD9 and CD81. Maternal
plasma E18 exosome concentration was higher than E9 exosomes
(p<0.0001) and carried markers of non-specific (acute phase) inflammation
(e.g. CRP, alpha-2 macroglobulin, apolipoprotein A2, F2, plasminogen).
Mice injected with E18 exosomes increased NF-κB activation, COX-2
expression, IL-6 and TNF-α concentration in the cervix compared to mice
injected with E9 exosomes or PBS (all p<0.05). F4/80 macrophages were
increased in the cervix of mice injected with E18 exosomes compared to
E9 exosome or PBS-injected mice. Inflammatory markers tested (COX2, NFκB, cytokines and macrophages) were not significantly different in

189A

the uterus of mice regardless of exosome or PBS injections. However, a
significant increase in CX-43 expression (p<0.05) was seen in E18 injected
mice uterus compared to E9 and PBS injected mice.
CONCLUSION: Pro-inflammatory proteins carried by E18 exosomes
induce remodeling and inflammatory changes in the cervix and uterus
within 2 days after treatment (E17), i.e., several days prior to the typical
day of birth in this mouse strain. These data support the hypothesis that
plasma exosomes have a functional role in preparing maternal tissues
for parturition.

F-022
The Role of the Amniotic (Pro)renin-Receptor in Mediating Fetal
Membrane Integrity. Sarah J Delforce†,2 Eugenie R Lumbers,2 Martha
Lappas,1 Tamas Zakar,2 Kirsty G Pringle*.2 1University of Melbourne,
Melbourne, Australia; 2University of Newcastle, New Lambton Heights,
Australia.
INTRODUCTION: Preterm birth (PTB) is the single largest cause
of death in infants and young children. 40-45% follow spontaneous
labour with intact membranes and 25-30% are associated with preterm
premature rupture of membranes (P-PROM). PTB as a result of P-PROM
is significantly higher in pregnancies carrying male babies. The primary
cause of membrane weakness is unknown. In kidneys, the prorenin/
(pro)renin receptor ((P)RR) interaction stimulates cell growth and the
production of pro-fibrotic factors (including fibronectin and collagens),
which maintain the extracellular matrix. However, the role of the prorenin/
(P)RR interaction in regulating the integrity of the amnion is unknown. We
postulate that prorenin, secreted by the decidua, acts upon amniotic (P)RR
to stimulate pro-fibrotic factors and cell proliferation in order to maintain
the amnion. Interestingly, at term, decidual REN mRNA is significantly
higher in the decidua of women carrying a female fetus compared with
those carrying male fetuses. Furthermore, amnion explant cultures from
women carrying female fetuses compared to those from male fetuses
have a significantly higher expression of (P)RR. We propose that 'female'
pregnancies may be more protected against insults that inflict preterm
birth than 'male' pregnancies.
METHODS: Combined fetal membranes (amnion, chorion and decidua)
from early trimester (<16 weeks), term and preterm non-labouring
deliveries were obtained and REN (prorenin) and mRNA expression was
determined by qRT-PCR. To validate the relationship between prorenin/
(P)RR and membrane integrity primary amnion cells were isolated and
transfected with 10 nM (P)RR siRNA and a negative control siRNA.
Following qRT-PCR validation for (P)RR knockdown, the effects of
knockdown on downstream targets of the prorenin/(P)RR interaction and
on measures of membrane integrity were determined.
RESULTS: REN mRNA abundance was significantly increased in early
trimester membranes compared with preterm and term membranes
(P=0.026 and 0.0001, respectively), which suggests that advancing
gestation is associated with lower levels of expression of REN to
stimulate amniotic (P)RR. Preliminary results suggest that early trimester
'male' fetal membranes may express lower levers of REN mRNA than
'female' fetal membranes.(P)RR mRNA expression was inhibited by
approximately 85% in primary amnion cells (P=0.001). Inhibition of (P)
RR was associated with decreased expression of Fibronectin, Collagen
IV, TIMP1 and TP53 mRNA (P=0.007, 0.048, 0.001, 0.020 respectively);
all of which play a major role in maintenance of the ECM.
CONCLUSION: Thus the prorenin/(P)RR interaction may be involved
in maintaining amnion integrity through gestation and preventing preterm
birth through stimulating cell growth and extracellular matrix stabilisation.

F-023
Matrix Metalloproteinases-8 and 10 Are Upregulated in the Weak
Zone of Human Fetal Membranes. D Kumar, R Moore, B Mercer, A
Sharma†, JJ Moore*. CWRU, MetroHealth, Cleveland, OH, United States.
INTRODUCTION: Preterm premature rupture of the fetal membranes
(FM) (pPROM) accounts for 25-50% of all preterm births with subsequent
significant infant mortality and morbidity. We have shown that FM
rupture occurs in areas of biochemically pre-weakened FM; laboring
women cannot develop sufficient force to rupture full strength FM. These

Friday Posters

Porphyromonas gingivalis Induces Inflammation in Amniotic
Mesenchymal Cells. Haruhisa Konishi,2 Satoshi Urabe,2 Yuko Teraoka,2
Hisako Furusho,2 Mutsumi Miyauchi,2 Hiroshi Miyoshi,1 Takashi Takata,2
Yoshiki Kudo.2 1Hiroshima Prefectual Hospital, Hiroshima, Japan;
2
Hiroshima University, Hiroshima, Japan.
INTRODUCTION: Dental disease is recently reported to be associated
to preterm birth. We previously reported that mice with dental
Porphyromonas gingivalis (P.g; a common pathogen of dental disease)
infection could be used as an effective model of preterm delivery. In this
model, P.g colonies were observed in the fetal membrane, and COX-2 and
interleukin (IL)-1b levels are increased. However, whether P.g induces
inflammation in the fetal membrane remains unknown. We aimed to
investigate the association of P.g infection with the human amniotic
membrane in vitro.
METHODS: Amniotic membranes were obtained at normal delivery
under protocols approved by the Institutional Review Board at Hiroshima
University Hospital. The amniotic membrane was isolated to epithelial
cells (AEC) and amniotic mesenchymal cell (AMC) with trypsin and
collagenase, and primary cell cultures were carried out. Confluent cells
were stimulated with P.g lipopolysaccharide (LPS)(1ng,10ng,100ng,1μg/
ml,12hours) or P.g (multiplicity of infection(MOI):100, 12hours) . The
mRNA expression of IL-1b, IL-8, and COX-2 were investigated by
realtime RT-PCR.
RESULTS: The mRNA expression of IL-1b, IL-8 was observed only in
AMC, and the mRNA expression of TNF-a and COX-2 was observed in
both cells. Following stimulation with P.g-LPS, the expressions in AMC
were enhanced in a concentration-dependent manner, and those in IL1b, IL-8, and COX-2 were increased by 2.7, 3.7, and 3.6 fold with 1μg/
ml, respectively. Similarly, the expressions of IL-1b, IL-8, and COX-2
in AMC were increased by 3.5,3.2, and 3.5 fold after stimulation with
P.g, respectively. By contrast, there were no significant changes in AEC.
CONCLUSION: P.g and P.g-LPS induce inflammation in AMC, leading
to preterm birth.

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