SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 118A

118A

Reproductive Sciences Vol. 25, Supplement 1, March 2018

T-023
Silk Hydrogels for Cervical Augmentation: An In Vitro Model of an
Injectable Cerclage. Stephanie L Bakaysa,1 Nicole Raia,2 David Kaplan,2
MIchael House*.1 1Tufts Medical Center, Boston, MA, United States; 2Tufts
University, Medford, MA, United States.
INTRODUCTION: Alternate treatments for cervical dysfunction are
needed. We previously proposed cervical augmentation with injectable
silk hydrogels as an alternative treatment to the traditional cerclage. Our
objective was to 1) develop an in vitro model to test cervical injection with
silk hyaluronan (HA) based hydrogels and 2) assess fibroblast responses
on the silk-HA hydrogels. We hypothesized that 1) injectable silk-HA
hydrogels increase tissue volume while maintaining tissue properties and
2) the silk-HA hydrogel surface supports cervical fibroblast proliferation
without excess of LPS-induced release of inflammatory cytokines.
METHODS: Cervical specimens (N=4) were obtained from non-pregnant
premenopausal women undergoing hysterectomy for benign indications.
Specimens of the cervical stroma were cut to a cylindrical shape (10 mm
diameter x 8 mm height). To mimic the properties of pregnant cervical
tissue, specimens were softened with Collagenase B for 2hr at 37OC.
Injection of 300 µL of a silk-HA hydrogel was performed for tissue
augmentation. To assess stiffness, cervical specimens were tested in
compression at 3 time points: baseline (Non-Pregnant, NP), after specimen
softening (Pregnant-Like, PL), and after injection of 300 μL of a silkHA hydrogel (Post-Injection, PI). The volume increase and modulus
was calculated. To assess cell responses, primary cervical fibroblasts
were cultured on three surfaces: silk hydrogel, silk-HA hydrogel and
tissue culture plastic (TCP) control. Cell responses were assessed for
viability (Live/Dead imaging), metabolic activity (AlamarBlue assay) and
proliferation (PicoGreen reagent). To measure inflammatory responses,
cells were exposed to LPS (0.01 µg/mL for 24hr) and IL-6 and IL-8
cytokine responses were measured with ELISA, normalized for DNA
content.
RESULTS: Collagenase digestion caused marked softening of cervical
specimens. The compressive modulus decreased 85% between NP and
PL specimens. The mean volume increase was 55% between PL and PI
specimens (p<0.05); the change in modulus between PL and PI specimens
was not significant. Regarding cell responses, fibroblasts remained
alive, metabolically active and proliferative over 5 days of culture as
demonstrated by LiveDead Imaging, and Alamar Blue and PicoGreen
assays. After LPS induction, IL-6 secretion was significantly decreased
on all hydrogels compared to TCP. No significant change in IL-8 secretion
was measured.
CONCLUSION: Injectable silk-HA hydrogel successfully augmented
human cervical tissue without excessive tissue stiffening. Silk-HA
hydrogels were non-toxic to cervical fibroblasts and a decreased
inflammatory cytokine release was observed. Injectable silk-based
hydrogels are a promising alternate treatment to prevent preterm birth in
cases where cervical dysfunction is the cause.

T-024
Progesterone-Driven Mesenchymal to Epithelial Transition (MET):
A Mechanism Maintaining Fetal Membrane Integrity. Lauren
Richardson†, Ramkumar Menon*. UTMB, Galveston, TX, United States.
INTRODUCTION: Fetal membrane cells undergo remodeling
throughout pregnancy by cell shedding and recycling shed amnion
epithelial cells (AEC) by transitioning them into mesenchymal cells
(EMT). However, it is unclear how transitioned AECs are recycled to
replenish gaps in membrane created by cell shedding. We report that
progesterone (P4) aids membrane remodeling through a reversal of EMT
where transitioned AECs or mesenchymal cells in the matrix are recycled
back by mesenchymal epithelial transition (MET) to epithelial cells to
strengthen membranes to support pregnancy.
METHODS: Human primary AEC and amnion extracellular matrix
mesenchymal cell (AMCs) cultures were established from term fetal
membranes. Cells were treated with either 30nM of progesterone (P4),
or P4+ 0.03um β-Estradiol (β-E) for 6 days. The following measures to
determine mesenchymal phenotypes were performed 1) cell viability
(crystal violet stain), 2) mRNA levels of epithelial transcription factors

Scientific Abstracts

(c-MYC and Klf4), 3) MET indicators like intermediate filament
cytokeratin-18 /vimentin ratio (Immunocytochemistry), and E-cadherin/
N-cadherin ratio, c-MYC, snail, and slug (western blot).
RESULTS: AECs in culture maintains metastate with equal number of
vimentin+ (mesenchymal) and CK-18+ (epithelial) cells. P4 treatment
of both AECs and AMCs increased c-MYC expression coincided with
an increase in CK-18+ staining compared to control. Additionally,
mesenchymal transcription factors (snail and slug) decreased and
epithelial junction marker E-cadherin increased transitioning these cells
into epithelial phenotype compared to untreated cells. Co-treatment of
cells with P4 and β-E preserved mesenchymal phenotype with no change
in c-MYC or epithelial cell transcription factors.
CONCLUSION: We report a novel functional role for progesterone
during pregnancy where it maintains membrane homeostasis by recycling
transitioned amnion mesenchymal cells to epithelium. This transition is
mechanistically mediated by P4 induced activation of proto-oncogene
c-MYC and inhibition of mesenchymal transcription factors. At term
estrogen can override P4s signals and forces a proinflammatory EMT
state where recycling is inhibited to promote parturition.

T-025
Amnion Cell Epithelial to Mesenchymal Transition (EMT): A
Mechanism Associated with Parturition? Lauren Richardson†,1 Jie Yu,2
Robert Taylor,2 Ramkumar Menon,1 George Saade.1 1UTMB, Galveston,
TX, United States; 2Wake Forest School of Medicine, Winston-Salem,
NC, United States.
INTRODUCTION: Human amnion epithelial cells are senescent and
inflamed at term parturition. Based on histologic and molecular evidence
we determined that senescent amnion epithelial cells (AEC) establishes
a static state of epithelial to mesenchymal transition ( ) at term resulting
in inflammation associated with parturition.
METHODS: We tested three groups of samples 1) amnion membrane
from term not in labor (TNIL) and term labor (TL) (n= 5), 2) term not
in labor membranes in culture (control) and exposed to OS by cigarette
smoke extract (CSE) (n=4), and 3) membranes from pregnant CD9
mice on days 18day 19 (N=5). We performed transmission electron and
multiphoton microscopy (TEM) (for cellular and stromal anomalies),
immunohistochemistry (vimentin, cytokeritain-18, E-cadherin,
N-cadherin, and MMP9), trichrome stain (collagen degradation) and
western blots (transcription factors TWIST, ZEB1, and SLUG) to
determine /inflammation changes in both human and murine tissues.
Parametric student t-tests or One-Way ANOVAS were used for statistical
analysis between groups. P values greater than 0.05 was significant.
RESULTS: TEM showed swollen organelles, constricted desmosomes
(p=0.009), amnion puckering, and defects in the basement membrane
and extracellular matrix in TL membranes than TNIL. Cytokeratin and
E-cadherin were reduced, whereas vimentin (p=0.008), N-cadherin
(p=0.03), matrix metalloproteinase 9 (MMP9), and the transcription
factors Twist, Snail, and Slug were also increased (all p < 0.05) in TL. The
same findings were replicated in membranes cultured under conditions
of OS. Similarly, mouse membranes on D19 had increased vimentin
(p=0.005), N-cad/E-cad ratio (p=0.01), TWIST, SLUG, and SNAIL
(p=0.002) expressions than D18
CONCLUSION: Classic signs of are seen in senescent human and
mouse TL amnion. This was recapitulated in vitro in TNIL tissues in
response to OS, a condition that mimicked TL. These data suggest that
OS induced senescence at term forces a static state of and inflammation
causing collagen degradation predisposing the membranes to parturitionassociated changes.

T-026
Noninvasive Objective MRI Method to Evaluate Uterine
Hemodynamic Function and the Effects of Naproxen. Kevin M
Hellman*, Katharina Laus, Folabomi Oladosu, Ellen Garrison. NorthShore
University HealthSystem, Evanston, IL, United States.
INTRODUCTION: Although it is hypothesized that vascular dysfunction
contributes to menstrual pain, clinical diagnostic tests have not yet been
developed to direct treatment strategies. Therefore, we evaluated the

Table of Contents for the Digital Edition of SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018