SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 249A

Scientific Abstracts

similar compared to age-matched Het controls and Wt pups from LPS
treated dams. However, at PN25, FoxP3 expression was significantly
decreased in HO-1 Het-LPS compared to Wt-LPS.
CONCLUSION: We have demonstrated that maternal exposure to
late gestational inflammation can result in a dysregulation of T cells in
offspring, and to a greater degree in those with a deficiency of HO-1. We
speculate that these immune alterations are the basis of adverse outcomes
in neonates from mothers exposed to low-grade (subclinical) infections.

F-206

F-207
B7-H4 is Preferentially Expressed at the Human Maternal-Fetal
Interface in Early Gestation and May Be Implicated in Both Invasion
and Tolerance. Jie Zhou†, Rowan Karvas, Megan Hollenbeck, Yuchen
Tian, Laura C Schulz, R Michael Roberts, Ezashi Toshihiko, Danny J
Schust. University of Missouri, Columbia, MO, United States.
INTRODUCTION: B7-H4, a B7 family molecule encoded by VTCN1,
negatively regulates cell-mediated immunity, promotes tolerance and
alters the progression of some tumors. We previously demonstrated that
VTCN mRNA expression increased during in vitro differentiation of

249A

human embryonic stem cells (hESCs) to trophoblast, a model for the
earliest events in human placental development. The expression pattern of
VTCN in human placenta across gestation and its potential role in placental
invasion or tolerance at the maternal-fetal interface had not been studied.
METHODS: Tissue was obtained from healthy women undergoing
legal therapeutic abortion of an intact pregnancy at 5-20 weeks of
gestation via suction curettage and from term placenta after routine
delivery (sections obtained from UC San Diego Perinatal Biorepository).
Immunohistochemistry was used to examine B7-H4 distribution across
gestation. Human ESCs (H1 cells) were treated with BMP4, A8301 and PD173074 (BAP treatment). Trophoblast resulting from this
protocol likely represents the invasive and syncytializing leading-edge
trophoblast formed during blastocyst implantation. Cells were transfected
with silencing VTCN1 SiRNA (10ng/ml) or control SiRNA on day 4
of differentiation. Proteins were collected on day 6 and expression of
B7-H4 and proteins involved in invasion pathways (ß-catenin, hCGß)
and immune regulation (hCGß and HLA-G, -C ,-E) compared to that
of a housekeepping protein (GADPH) using western immunoblotting.
RESULTS: B7-H4 was expressed in placental samples throughout the
first 20 weeks of gestation but was barely detectable in term placenta (Fig
1). The expression levels of proteins involved in invasion and in immune
tolerance were all increased in BAP treated hESCs upon suppression of
VTCN1. Expression of the housekeeping control was unchanged.
CONCLUSION: Supporting our previous results, we detected robust
B7-H4 protein expression in placentas from early but not term gestation.
The effects of silencing B7-H4 protein expression in an in vitro model
representing immediate post-implantation placental development
implicate B7-H4 in the regulation of placental invasion and feto-maternal
immune interactions. Future experiments will include cell invasion assays
following knockdown of VTCN1 and real-time PCR analyses of pathway
constituents known to be involved in invasion and tolerance.
*Figure(s) will be available online.

F-208
A Novel Strategy to Treat Fetal Myelomeningocele Using Human
Amniotic Fluid Stem Cells. Yushi Abe†, Daigo Ochiai*, Hirotaka
Masuda, Toshimitsu Otani†, Marie Fukutake†, Youhei Akiba*, Tadashi
Matsumoto, Kei Miyakoshi*, Mamoru Tanaka*. Keio University School
of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan.
INTRODUCTION: Although fetal myelomeningocele (MMC) results in
irreversible neurological impairments after birth, the options of prenatal
treatments are still limited. The aim of this study is to investigate the
effects of intraamniotic administration of human amniotic fluid stem cells
(hAFS) for MMC using the rat model.
METHODS: hAFS were isolated from human amniotic fluid as CD117
positive cells. Sprague- Dawley rat dams were orally administrated
retinoic acid (60 mg/kg) to induce fetal MMC. On E 17, these MMC model
rats were inoculated with 1.0 × 105 cells/fetus of hAFS suspended in PBS
(group I) or PBS only as controls (group II). The fetuses were harvested
by cesarean section on E 21 and their MMC lesions were investigated. The
area of the exposed spinal cord lesion (open area) were macroscopically
measured. The cross-sectional area of the spinal cord (spinal x-section)
were histologically analyzed at the level of maximum transverse diameter
of the exposed lesion. To evaluate the defective spinal cord, neuron and
astrogliosis were stained with Tubulin-III and GFAP immunostaining,
respectively. Additionally, hAFS migration into the lesion was visualized
by immunohistochemistry with human-specific antigen staining.
RESULTS: hAFS treatment significantly reduced the open area (group
I vs. group II; 37.4 ± 18.2 mm2 vs. 54.3 ± 26.4 mm2; p < 0.05) and
histological analysis demonstrated a significant increase of the spinal
x-section (group I vs. group II; 1.74 ± 1.07 mm2, 0.48 ± 0.50 mm2; p
< 0.05) and Tubulin-III positive area (group I vs. group II; 1.60 ± 0.75
mm2, 0.81 ± 0.37 mm2; p < 0.05) after hAFS treatment, indicating that
hAFS promoted skin coverage of the open area and protected neurons in
the exposed spinal cord. The decline of GFAP / Tubulin-III positive area
ratio of group I might show the ability of hAFS to reduce astrogliosis

Friday Posters

The Human Myometrial Stem Cell Compartment in Fibroid-Affected
Uteri is Significantly Expanded after Ovarian Steroid Hormone
Exposure. Lauren Prusinski Fernung†,1 Aymara Mas,2 Ayman AlHendy*.1 1Augusta University, Augusta, GA, United States; 2La Fe Health
Research Institute, Valencia, Spain.
INTRODUCTION: To date the unknown mechanisms of origin of uterine
fibroids (UFs) remain critical obstacles for optimal female reproductive
healthcare. Somatic mutations may convert myometrial stem cells (MSCs)
into UF-tumor initiating cells, leading to UF development under the
influence of ovarian steroids. Previous experiments in a murine model
have demonstrated estrogen (E2) and progesterone (P4)-responsive human
UF growth requires progenitor cells. These cells, however, lack E2 and
P4 receptors, suggesting paracrine signaling with nearby differentiated
cells. Although it has been shown that proliferation markers are highest in
the luteal phase (E2+P4 influence), implicating P4 as a major influence in
UF growth, this hormone response has not been widely studied in human
tissues. To assess if there was any significant difference at hormone and
cell level, we quantified the compartment of human Stro1+/CD44+ MSCs of
normal uteri versus from uteri with UF during the follicular (E2 dominant)
vs. luteal (E2+P4) phase of the menstrual cycle.
METHODS: Once patients signed informed consent, human
myometrial samples were obtained from 28 pre-menopausal women
with pathologically normal uteri (MyoN) and/or women undergoing
hysterectomy for UF (MyoF, where samples were collected >2 cm from
closest UF). Subjects used no hormonal treatment for >3 months presurgery, and menstrual phase designation was confirmed by pathological
examination of endometrial tissues: proliferative/follicular (Fol) or
secretory/luteal (Lut). Fresh tissue was digested, and Stro-1+/CD44+
MSCs were labeled in single-cell suspensions as we reported previously.
Percentage of MSCs (%MSC) in Fol vs. Lut-phase tissues (MyoF or
MyoN tissues) was determined using flow cytometry and compared using
two-sample, one-tailed Student's t-test. Differences in %MSC between
MyoF and MyoN for each Fol or Lut phase were also compared using
one-way ANOVA.
RESULTS: By flow cytometry, the mean (±SD) %MSC from Lut
myometrium [(MyoF: 0.60 ± 0.09%), (MyoN: 0.23 ± 0.09%)] were
significantly greater than those of matched Fol myometrium [(MyoF:
0.44 ± 0.10%), (MyoN: 0.14 ± 0.05%)], p<.05. Within each phase, MyoF
%MSC was significantly greater than MyoN (p<.0001).
CONCLUSION: Our data show that the paracrine effect described in
murine models is also operational in the human uterus. Higher numbers
of MSCs in pre-fibroid myometrium (MyoF) suggest that such tissues
are distinct at the cellular and molecular levels from normal myometrium
(MyoN). Further studies are needed to reveal early changes in MSCs
that may alter their response to otherwise-normal hormone regulation,
leading to UF development. Support: R01ES028615-01 (AAH) and
1F30HD089585-01A1 (LPF).

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