SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 73A

Scientific Abstracts

O-049
Loss of the Grb2 Associated Binding Protein 3 is Associated with
Poor Uterine NK Cell Proliferation and Placental Accreta in Mice.
Anna Sliz†,3,4 Kristin Lampe,2 Alzbeta Godarova,2 Helen Jones*,1
Kasper Hoebe*.2 1Division of General and Thoracic Surgery, Cincinnati
Children's Hospital Research Foundation, Cincinnati, OH, United States;
2
Division of Immunobiology, Cincinnati Children's Hospital Research
Foundation, Cincinnati, OH, United States; 3Immunology Graduate
Program, Cincinnati Children's Hospital Medical Complex, Cincinnati,
OH, United States; 4University of Cincinnati, Cincinnati, OH, United
States.
INTRODUCTION: Placental accreta represents a significant cause of
maternal mortality, however the pathogenesis is unknown. Interestingly,
reduced frequency of uterine natural (uNK) cells was recently shown
to be associated with accreta. uNK cells play a key role in placental
development, however, the molecular mechanisms that control their
proliferation and function remain poorly defined. We have identified
the Grb2-associated binding protein 3 (Gab3) as a required component
for IL-2/IL-15 signaling and successful pregnancies. Mice that carry
Gab3 loss of function mutations have a significantly increased risk of
developing accreta.
METHODS: Our lab generated a hypomorph gremlin mouse carrying a
missense mutation in Gab3, resulting in an Arg27→Cys amino acid change
(Gab3R27C) by ENU mutagenesis. Gab3 deficient animals (Gab3KO)
were generated by CRISPR/Cas9 genome editing.
RESULTS: Pregnant Gab3R27C and Gab3KO mice have significantly
increased risk of developing accreta (up 9% & 16% respectively).
The complications during pregnancy were associated with increased
trophoblast invasion and impaired arterial remodeling at gd12.5 and
gd18.5. Importantly, the frequency of uNK cells at gd8.5 were significantly
reduced (down 25% vs 50%, respectively) in pregnant Gab3R27C and
Gab3KO females. At the molecular level, we identified a critical role
for Gab3 in IL-15 induced NK cell expansion with both Gab3R27C
and Gab3KO NK cells showing impaired proliferation. Further studies
identified a selective requirement for Gab3 to mediated MAPK (but not
STAT5 or PI3K) signaling downstream of the IL-2/IL-15 receptor.
CONCLUSION: Our studies identify Gab3 as a key mediator of IL-2/
IL-15 signaling required for peripheral NK cell priming and expansion.
We show that loss of functional Gab3 is associated with poor uNK cell
expansion during pregnancy. Failure of uNK cells to expand during
pregnancy was associated with poor decidualization and over-invasion
of the labyrinth by trophoblasts, resulting in placental accreta. We here
identify unique mouse models to study uNK cell function and provide new
insights into the cellular and molecular mechanisms that lead to accreta.

73A

O-050
LPS-Exposed Fetal Membranes Activate Neutrophils and Increase
Neutrophil Extracellular Trap Formation. Mancy Tong†, Julie A
Potter, Gil Mor, Vikki M Abrahams*. Yale University, New Haven, CT,
United States.
INTRODUCTION: Chorioamnionitis, a major risk factor for preterm
birth and poor fetal/neonatal outcomes, is thought to be initiated by
bacterial infection of the fetal membranes (FMs) and is characterized by
neutrophil infiltration. Neutrophil function at the FMs is unclear. This
study aimed to characterize the effect of FMs on neutrophil migration,
activation, and neutrophil extracellular trap (NET) formation; and whether
this is altered if FMs were exposed to bacterial lipopolysaccharide (LPS).
METHODS: Conditioned media (CM) were collected from term FM
explants treated with or without LPS (1ng/ml) for 48hr, and added to
peripheral blood neutrophils from healthy women for 1-3hr. Media or
LPS served as controls. Neutrophil migration was quantified using a
two chamber colorimetric assay (n=7). Cytokine/chemokine secretion
was measured by multiplex assay and ELISA (n=11). NET formation
was visualized by immunofluorescence staining for extracellular
DNA, citrullinated histone 3 and myeloperoxidase; and quantified by
fluorescence of extracellular DNA (n=7).
RESULTS: Compared to media alone, untreated FM-CM increased
neutrophil migration (1.6±0.1-fold), which was further augmented
1.8±0.1-fold by LPS-treated FM-CM (p<0.05). Compared to media alone,
untreated FM-CM induced neutrophil secretion of IL-6; IL-8; G-CSF;
IP-10 and MCP-1 (p<0.05). LPS-treated FM-CM further augmented
neutrophil secretion of IL-6 (44.2±11.6-fold); IL-8 (8.7±1.2-fold); G-CSF
(5.8±1.0-fold); IP-10 (9.0±1.4-fold); and MCP-1 (6.3±0.7-fold) (p<0.001).
Compared to media alone and untreated FM-CM, LPS-treated FM-CM
induced neutrophil secretion of GRO-α (12.0±2.1-fold); IFNγ (8.9±1.51fold); MIP-1α (146.4±16.9-fold); MIP-1β (24.3±4.0-fold); RANTES
(5.2±1.1-fold) and TNFα (306±135-fold) (p<0.002). Untreated FM-CM
also induced NET formation and this was further increased 1.5±0.3-fold
by LPS-treated FM-CM (p<0.05). LPS alone had little or no effect on
neutrophil function.
CONCLUSION: Our findings show that FMs actively recruit neutrophils
and induce their release of chemokines and NETs, suggesting that
under normal pregnancy conditions, neutrophils may have a protective
phenotype at the FMs. In contrast, FMs exposed to bacteria may recruit
more neutrophils, augment and induce their release of chemokines and
inflammatory cytokines, and increase NET formation. This potentially
leads to the influx of more leukocytes to the FMs, induction of tissue
injury, and poor pregnancy outcomes.

O-051
Peri-Implantation Progesterone Determines Regulatory T Cell
Competence to Impact Late Gestation Pregnancy Outcome in
Mice. Sarah A Robertson*, Ella S Green†, Shaun R McColl, Lachlan M
Moldenhauer. The University of Adelaide, Adelaide, Australia.
INTRODUCTION: Regulatory T cells (Treg cells) are essential for
embryo implantation and early placental development, and also protect
the fetus and placenta from inflammatory insults in later gestation.
Insufficient numbers or suppressive function of Treg cells are implicated
in contributing to preeclampsia in women. The factors controlling the
strength, quality and stability of the Treg cell response are not yet defined.
The Aim of this study was to investigate the role of progesterone (P4) in
early pregnancy as a determinant of Treg cell abundance and phenotype,
and the consequences for pregnancy success.
METHODS: Firstly, to investigate the effects of P4 on Treg cells and
their function in pregnancy, C57Bl/6 female mice were mated with Balb/c
males and administered various doses of the P4 antagonist RU486 (0.58.0 mg/kg) in the peri-implantation phase on day 1.5 and 3.5 post-coitus
(pc). Mice were killed on day 9.5 pc to measure implantation sites and
Treg cells were assessed in the uterus-draining para-aortic lymph nodes
by flow cytometry to quantify and phenotype CD4+CD25+Foxp3+ cells.
Pregnancy outcomes and susceptibility to late-gestation inflammationinduced fetal loss were evaluated in a second cohort of pregnant females
given low dose RU486 in the peri-implantation phase, followed by E.

Thursday Orals

RESULTS: Age predicted by WBC DNAm was consistent with patients'
chronological age (+/- 4 years). There was no trend toward accelerated
aging in the WBC DNAm patterns in DOR patients from either age
group. A decreased overall DNAm level was observed in CC DNA
compared to the WBC DNA from the same patient (p<0.001), and CC
DNA showed significantly younger DNAm age with an average of 9.3
years old (p<0.001). There was no difference between the 4 groups in the
age predicted by CC DNAm (p=0.19).
CONCLUSION: DNAm age calculated in WBC based on the Horvath
algorithm accurately predicts chronological age in women of reproductive
age. CC show a distinctly different pattern, resulting a significantly
younger cellular profile that is not altered based on female age or response
to ovarian stimulation. Further studies are required to delineate the
mechanism(s) responsible for altered DNAm age in CC, and to investigate
whether alternate sites can accurately predict chronologic age or response
to stimulation in these cells.

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