Activated Hippo/Yes-Associated Protein Pathway Promotes Cell Proliferation and Anti-apoptosis in Endometrial Stromal Cells of Endometriosis
Activated Hippo/Yes-Associated Protein Pathway Promotes Cell Proliferation and Anti-apoptosis in Endometrial Stromal Cells of Endometriosis
Yong Song 0
Jing Fu 0
Min Zhou 0
Li Xiao 0
Xue Feng 0
Hengxi Chen 0
Wei Huang 0
0 Department of Obstetrics and Gynecology, West China Second University Hospital of Sichuan University , Chengdu Sichuan 610041 , People's Republic of China
Context: The imbalance in cell proliferation and apoptosis is considered an important role in the pathogenesis of endometriosis, but the exact mechanisms remains unclear. A newly established signaling pathway-Hippo/Yes-associated protein (YAP) pathway plays a critical role in the proliferation and apoptosis processes. However, studies focusing on Hippo/YAP pathway and endometriosis are lacking. Objective: The objective was to explore the function of the Hippo/YAP pathway in endometriosis. Setting and Design: The expression of YAP was first investigated in endometrium of women with or without endometriosis. The role of YAP in cell proliferation and apoptosis is identified by transfection of endometrial stromal cells (ESCs) in vitro, subsequent Verteporfin treatments in eutopic ESCs in vitro, and endometriosis animal model of nude mice in vivo. Results: Our results revealed that increased expression of YAP and decreased expression of p-YAP in ectopic and eutopic endometrium compared with normal endometrium. YAP knockdown in eutopic ESCs decreased cell proliferation and enhanced cell apoptosis companied with decreased expression of TEAD1, CTGF, and B-cell lymphoma/leukemia (BCL)-2; whereas overexpression of YAP resulted in increased proliferation and decreased apoptosis of normal ESCs with increased expression of TEAD1, CTGF, and BCL-2. By chromatin immunoprecipitation qPCR CTGF and BCL-2 were identified as directly downstream target genes of YAP-TEAD1 active complex. Eutopic ESCs treated with Verteporfin revealed decreased proliferation and enhanced apoptosis whereas in endometriosis animal models of nude mice treated with Verteporfin, the size of endometriotic lesions was significantly reduced. Conclusions: Our study suggests that the Hippo/YAP-signaling pathway plays a critical role in the pathogenesis of endometriosis and should present a novel therapeutic method against endometriosis. (J Clin Endocrinol Metab 101: 1552-1561, 2016) Abbreviations: BCL, B-cell lymphoma/leukemia; BrdU, bromodeoxyuridine; ChIP, chromatin immunoprecipitation; CTGF, connective tissue growth factor; ESC, endometrial stromal cell; KD, knockdown; shRNA, short hairpin RNA; TEAD, TEA domain; YAP, Yes-associated protein.
E disease that affects 10 ?15% of women of
reproducndometriosis is a commonly encountered gynecologic
tive age. This condition arises when endometrial tissue
grows outside of the uterine cavity, which can result in
chronic pelvic pain, infertility, and an elevated risk of
ovarian cancer (
). Current treatments include using
medicine to alleviate chronic pelvic pain and surgical
removal of lesions, all of which provide a temporary but not
permanent cure (3). Until now, the etiology and
pathogenesis of endometriosis are still unclear. Understanding
the subtle course of its pathogenesis is important for
exploring effective, thorough therapy of disease.
As a kind of disease with a feature of growth, the
development of endometriosis includes a course of cell
proliferation and apoptosis. Cell proliferation and apoptosis
are essential yet opposing cellular processes. The
coordination and balance between cell proliferation and
apoptosis are crucial for normal development and tissue-size
). The disease will occur once the balance is
disrupted. Until now, the most widely accepted theory for
pathogenesis of endometriosis is Sampson theory, in
which the exfoliated menstrual endometrial cells attach to
the peritoneal membrane, and subsequent cell
proliferation and invasion into the underlying tissue result in
endometriotic lesions (
). The retrograde menstruation is a
physiologic process that takes place almost in all
menstruation cycles of reproductive-age women. But the
morbidity of endometriosis is only 10 ?15%, the answer to this
question lies in the defect of eutopic endometrial cells. The
menstrual endometrial cells usually do not survive even
expelled inside the peritoneal cavity because of
programmed cell death. Accumulated evidence suggests that
endometrial cells from both eutopic endometrium and
ectopic endometrium of endometriosis exhibit impaired
apoptosis and excessive proliferation (
aberrant expression of proliferation-related and
apoptosisrelated molecules such as c-myc, B-cell lymphoma/
leukemia (BCL)-2, and connective tissue growth factor
(CTGF) should be responsible for viability of ectopic
endometrial cells. Therefore, imbalance of cell proliferation
and apoptosis is the foundation of progression of
). So far, it has been reported that
miRNAs, histone deacetylation, and several signaling
pathways play a role in regulation of endometrial cell
proliferation and apoptosis in disease (
mechanisms of endometrial cells imbalance are still unclear and
need for further study.
The Hippo pathway was originally identified as the
signaling that controls organ size in Drosophila, with the
core architecture conserved in mammals. In the
mammalian Hippo pathway, the central components of this
pathway comprise a regulatory serine-threonine kinase
module and a transcriptional module. Mammalian Ste20-like
kinases (MST1/2) and large tumor suppressor kinases
(LATS1/2) as kinase module regulate transcriptional
coactivators, Yes-associated protein (YAP) and
Transcriptional coactivator with a PDZ-binding motif (TAZ) (
As a major downstream target of the Hippo pathway,
transcription cofactor YAP does not contain its own
DNA-binding motifs and initiates transcription by
interacting with the DNA-binding transcription factors TEA
domain (TEAD) family members; then activate expression
of target genes regulating cell proliferation,
differentiation, and apoptosis (
). YAP has been found to be
dysregulated in a variety of diseases, such as hepatocellular
carcinoma, neurological diseases, and malignant
). These findings highlight that dysregulation
by genetic inactivation of core pathway components or
amplification of its downstream effector YAP results in
increased cell proliferation and decreased cell apoptosis.
Verteporfin (trade name, Visudyne by Novartis) is used
clinically as a photosensitizer in photodynamic therapy
for neovascular macular degeneration. Recently, it has
been found that verteporfin repealed liver overgrowth
induced by YAP overexpression (OE) in vivo (
Verteporfin inhibits esophageal cancer development in vivo by
down-regulating SOX9 (another YAP-TEAD target gene)
in another report (
). These studies predict the
therapeutic potential of targeting YAP-TEAD interaction (
However, the expression and function of the Hippo/
YAP pathway in endometriosis have not been investigated
yet. Thus, the goal of this study is to gain further insight of
endometriosis pathogenesis by identifying the expression
and spatial distribution of YAP in endometriosis and
investigate the role of YAP in the regulation of endometrial
cell proliferation and apoptosis.
Materials and Methods
The study was approved by the Institutional Review Board of
West China Second University Hospital of Sichuan University,
and written informed consent was obtained from each patient.
Participants were age 20 ? 40 years and had regular menstrual
cycles. None had received steroid hormone treatment for at least
3 months before sampling. All were determined to be in the
proliferative phase according to their last menstrual period and
further confirmed by histological dating. Normal endometrium
was obtained from disease-free women laparoscopically as
controls, paired ectopic and eutopic endometrial tissues were
obtained from women with revised-American Fertility Society
(rAFS) stage III?IV ovarian endometriosis by laparoscopic and
histological diagnosis; the cyst wall of ovarian endometriosis
without adjacent ovarian tissue was obtained and the inner side
was used for further analysis.
Human endometrial samples and mice endometriotic lesions
were fixed in formalin, embedded in paraffin, cut to 4- m
sections, dried, and kept at 4?C. The hematoxylin-eosin and
immunohistochemistry staining were made. The primary
antibodies used were 1:400 monoclonal rabbit antihuman YAP
(ab52771, Abcam) and 1:100 monoclonal rabbit antihuman
Ki67 (ab15580, Abcam). The immunostaining of Ki67 was
quantified by normalizing the average number of positively
stained cells to the total number of cells from five different fields
of each image.
Flow cytometry analysis
Apoptosis was analyzed using cell surface expression of
Annexin V. Endometrial stromal cells (ESCs) were separately
isolated from normal endometrium of controls or eutopic
endometrium of endometriosis as described previously (
). ESCs were
transfected for 24 hours and then cultured for 72 hours before
determining the extent of apoptosis using the Annexin V-FITC
Apoptosis Assay Kit as described by the manufacturer (KeyGEN
BioTECH). Before being analyzed by flow cytometry, the cells
were trypsinized and incubated for 5 minutes at room
temperature with Annexin V-FITC and propidiumiodide.
Cell proliferation assays
The CCK8 proliferation assay was performed according to
the manufacturer?s instructions (Dojindo Laboratory). Briefly,
96-well plates were seeded with 1000 cells per well. Every 24
hours, cell counting kit-8 reagent was added to the wells of the
plate. After 3 hours? incubation, the plate was measured as
spectrophotometric absorbance at wave length of 450 nm.
Cell culture and transfection
Construction and production of Lentiviral vectors were made
by Shanghai Genechem Co., Ltd., the YAP1-specific
short-hairpin RNA (shRNA)-targeting coding sequence
(GGTGATACTATCAACCAAA) was cloned into GV118 vector to produce
YAP-knockdown (KD) vector. Human YAP cDNA (BC038235)
was amplified by PCR as a template, then cloned into GV358
vector to generate YAP expression vector. The ESCs were
suspended in DMEM/F12 (1:1) supplemented with 10% fetal
bovine serum (life technologies) and 50 U/mL
penicillin/streptomycin at 37?C under a 5% CO2 condition, plated at 1 106 cells
per six-well plate and cultured to 40% confluency, then
transfected with optional virus-containing supernatant supplemented
with polybrene. The cells were transfected with YAP expression
Lentiviral vector to establish the YAP-overexpressing normal
ESCs line; 72 hours after transfection, they were selected with 4
g/mL puromycinin culture medium for more than 10 days.
Transfection efficiency of YAP was measured by RT-qPCR and
Western blotting. All experiments were performed in triplicate
and repeated three times.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assays were
conducted following the manufacturer?s protocol (EMD Millipore).
Briefly, to crosslink proteins to DNA, fresh formaldehyde (final
concentration, 1%) was added to the culture medium and
incubated for 10 minutes at room temperature. Cells were scraped
and collected and then lysed successively with cell lysis buffer and
nuclear lysis buffer containing protease inhibitors. Aliquots of
cell lysates were sonicated to shear DNA into 0.2?1.0-kb
fragments, and the cellular debris was removed. Chromatin aliquots
were incubated with fully suspended protein A magnetic beads
and 2.5 g specific YAP antibody (ab52771, Abcam), 5 g
TEAD1 antibody (ab133533, Abcam), 5 g Histone H3
antibody (positive control; ab4729), or 5 g normal rabbit IgG
(negative control; #12?370, EMD Millipore) overnight at 4?C with
rotation. Beads were collected with the magnetic separator and
then washed. Protein/DNA complexes were decrosslinked with
proteinase K for 2 hours at 62?C, 10 minutes at 95?C. After that,
DNA was purified using spin columns and resuspended. The
purified DNA was then subjected to quantitative PCR (qPCR)
with indicated ChIP primers in Supplemental Table 1.
Total RNA was extracted from all tissue samples or primary
cultured ESCs using TRIzol according to the manufacturer?s
protocol (Life Technologies) and then followed the protocol
(supporting information). Relative gene expression was calculated
using the 2 CT method, normalizing with
glyceraldehyde-3phosphate dehydrogenase (GADPH) levels.
Total proteins were collected with immunoprecipitation lysis
buffer supplemented with protease inhibitors and phosphatase
inhibitors. Protein concentration was determined using a
bicinchoninicacid assay kit (ThermoFisher Scientific) and then
followed the protocol (supporting information). Protein bands was
analyzed with Quantity One (Bio-Rad Laboratories). Protein
levels were normalized to that of the internal control -actin.
Verteporfin (Selleckchem) was dissolved in dimethyl
sulfoxide (100 mg/mL), aliquoted, and stored at 80?C. In vitro, ESCs
were treated with dimethyl sulfoxide or verteporfin with a dose
of 1 M. In vivo, working solution of verteporfin was prepared
at 10 mg/mL in PBS freshly before use, and mice were
administered ip at a dose of 100 mg/kg every other day for 12 days after
day 2 of implantation whereas control mice were injected with
All animal experiments were conducted in accordance with
the National Institutes of Health Guide lines for the Care and Use
of Laboratory Animals. The protocol was approved by the Ethics
Committee of Animal Experiments of Sichuan University.
Female BALB/c nude mice age 6 ? 8 weeks were purchased from
Chengdu Dashuo Experimental Animals Limited Company.
Nude mice were housed in a barrier unit in a controlled
pathogen-free environment and regulated light/dark cycles (12 h/12 h).
All equipment and food entering the barrier were autoclaved.
Mice had free access to food and water.
Explanted eutopic endometrial tissue collected from women
with endometriosis was cut into 5-mm fragments under sterile
conditions and kept for 1 hour in culture medium (DMEM/F12;
1:1; Life Technologies) supplemented with Pen-Strep (MP
Biomedicals) at 37?C prior to transplantation. Fragments of human
endometrium were transplanted into nude mice when local
anesthesia by ip administration of chloralhydrate (KeLong
Chemical) at a dose of 10 mg/kg. All mice were implanted two pieces
of implants and fixed on both sides of the lateral abdominal wall
with surgical sutures as described before (
). The experimental
mice (n 5) and control mice (n 5) were treated as previously
described. Finally, mice were euthanized by cervical dislocation.
Implanted endometrial lesions were dissected, subsequently
measured in two perpendicular diameters (d D) with a caliper,
and their volume was calculated with the following formula: V
(3/4) r2R (r and R are the radii; r R) (
). Then, the lesions
were divided into two parts: one part was frozen immediately in
liquid nitrogen for RNA/protein extraction; and another one
fixed in 10% formalin for morphological and
For bromodeoxyuridine (BrdU) labeling, mice were injected
ip with BrdU (50 mg/kg; Sigma-Aldrich) for 24 hours before
euthanasia. The endometriotic lesions were collected, fixed, and
sectioned as described above, then detected with an anti-BrdU
antibody (R&D systems). The percentage of BrdU incorporation
was determined by counting BrdU nuclei among the total
number of cells in distinct fields.
Apoptosis was detected by In Situ Cell Death Detection Kit
(Roche) following the manufacturer?s protocol with some
modifications (supporting information).
Statistical analysis was performed using SPSS version 18.0
(SPSS). All data were expressed as mean SD. The Student t test
was used for comparisons between the two groups, and one-way
ANOVA with a post-hoc test least-significant difference was
used for multiple comparisons. P .05 was considered
statistically significant (two tailed).
Increased expression of YAP in eutopic and ectopic endometrium of endometriosis
To investigate the functional relevance of YAP
during endometriosis, we performed qRT-PCR to detect
mRNA level of YAP in ectopic and eutopic
endometrium from women with endometriosis and normal
endometrium from disease-free women. The YAP mRNA
expression was significantly higher in ectopic
endometrium than eutopic and normal endometrium (Figure
1A). Then we detected protein level of YAP and its
phosphorylated form phosphoS127-YAP (serine to alanine
at residue 127) by Western blotting, The result showed
that not only up-regulation of Yap in endometrium of
women with endometriosis, but an inhibition of
phospho-YAP in this disease (Figure 1, B and C). In
immunohistochemistry, YAP immune expression was
distributed strongly in the nucleus and cytoplasm of epithelial
and stromal cells of ectopic and eutopic endometrium
whereas YAP protein expression was weak in the
nucleus and cytoplasm of normal endometrium (Figure
1D). The Above results suggest up-regulated YAP in
KD of YAP in eutopic ESCs of endometriosis reduces proliferation and promotes apoptosis
Based on the abundant expression of YAP in
endometriosis and its well-established ability to regulate cell
proliferation and apoptosis, we hypothesized that YAP may
control proliferation and apoptosis of endometrial cells.
To test this hypothesis, we performed an shRNA-based
KD of YAP in eutopic ESCs of endometriosis. GFP-labeled
scrambled shRNA was used as controls. We obtained
greater than 70% KD efficiency at protein and mRNA
levels (Figure 2A). Then the transfected eutopic ESCs were
subjected to CCK8 assays. The data showed that YAP-KD
eutopic ESCs have a retarded proliferation rate when
compared with the control group (Figure 2B). The flow
cytometry revealed a statistically higher percentage of early
apoptotic cells in YAP-KD eutopic ESCs (Figure 2C).
These data demonstrated that loss of YAP expression
resulted in decreased proliferation and enhanced apoptosis
of eutopic ESCs.
OE of YAP in ESCs of normal endometrium promotes proliferation and decreases apoptosis
To further explore the role of YAP in cell proliferation
and apoptosis of ESCs, we established a normal ESC line,
which expresses the wild type (WT) YAP protein (YAP
OE). The empty vector (GV358) was also transfected
into ESCs and served as controls. Results showed that
normal ESCs expressed more YAP mRNA and protein
than controls (Figure 3A). By CCK8 assays, YAP OE
ESCs revealed a higher proliferation rate compared
with controls (Figure 3B). In addition, the percentage of
early apoptotic cells significantly decreased after OE of
YAP in ESCs (Figure 3C).
BCL-2 and CTGF participate in cell proliferation
and apoptosis as downstream targets of
The TEAD/TEF family of transcription factors is
necessary for YAP?s biological activity through a stable,
transcriptionally active complex. In our experiments, we
found that KD YAP decreased TEAD1 mRNA and protein
expression in eutopic ESCs whereas OE YAP increased
TEAD1 mRNA and protein levels in normal ESCs (Figure
4, A and B). Furthermore, YAP KD in eutopic ESCs or OE
in normal ESCs significantly altered CTGF and BCL-2
mRNA and protein expression, both had been previously
characterized for their important roles in regulating cell
proliferation and cell apoptosis. Therefore, we considered
that YAP-TEAD1 complex regulated ESCs through
mediation of CTGF and BCL-2. Two TEAD1-binding sites
(GGGATTCCTGCG and GACATTTCTGTG) in the
promoter region of human BCL-2 gene were identified
according to prediction of Jaspar database (Figure 4C). The
target of YAP-TEAD1 on CTGF had already been
). Our ChIP experiments confirmed
YAPTEAD1 complex binding to the promoter region of CTGF
and BCL-2 (Figure 4D). Together, the results suggestd
that YAP involve in ESC cell proliferation and apoptosis
through transcriptionally regulated CTGF and BCL-2
Inhibition of YAP pharmacologically attenuates cell proliferation and induces cell apoptosis of eutopic ESCs in vitro and ectopic endometrial cells in vivo
After treatment with verteporfin (1 M) for 18 hours,
we found that a reduction in the expression of
antiapoptosis marker BCL-2 and proliferation marker CTGF
(Figure 5A). The data also showed that eutopic ESCs treated
by verteporfin have a significantly lower proliferation rate
after 1 day when compared with control group (Figure
5B). Moreover, the flow cytometry revealed a significantly
higher percentage of dying cells in verteporfin-treated
eutopic ESCs (Figure 5C). These data demonstrated that
inhibition of YAP-TEAD complex resulted in decreased
proliferation and enhanced apoptosis of eutopic ESCs.
In the nude mouse model of endometriosis, the
implantation of eutopic endometrial fragments collected from
women with endometriosis successfully led to the
development of endometrial-like lesions in gross and
histological findings (Figure 6A). After treated ip verteporfin (100
mg/kg), the size of the ectopic lesion in mice experienced
a marked reduction compared with those treated with PBS
(Figure 6B). In addition, these lesions shrank at least 50%
and even some lesions disappeared (Figure 6B). However,
the endometrial-like tissue and inflammatory adhesions
could be observed inside the peritoneal cavity of control
mice treated by PBS (Figure 6B). The cell proliferation of
the endometriotic lesions was severely affected by
verteporfin treatment, as suggested by Ki67 staining and BrdU
staining (Figure 6C). In addition, verteporfin led to higher
cells apoptosis ratio in the endometriotic lesions (Figure
6C). This effect was concomitant with a reduction in the
expression of antiapoptosis marker BCL-2 and
proliferation marker CTGF (Figure 6D).
In the present study, we found for the first time that YAP
mRNA and protein up-regulating and decreased
phosphoS127-YAP in ectopic and eutopic endometrium.
Immune staining intensity of YAP was higher in epithelial
and stromal cells in both eutopic and ectopic endometrium
compared with normal endometrium. This observation
suggested that YAP and related Hippo signals may play a
role in the development and progression of endometriosis.
The complexity of YAP regulation has expanded
considerably in recent years. Until now, it has been well
established that YAP is a primary downstream effector of the
Hippo pathway crucial for organ size control,
regeneration, and tumorigenesis by controlling proliferation,
apoptosis, and stemness in mammals (
). In 2014, Fu et al
) found that YAP KD not only resulted in reduction
in cell proliferation, but also decreased aromatase
(CYP19A1) protein expression and estrogen synthesis in
human ovarian granulosa cell tumors cell line. In our
study, we knocked down YAP in eutopic ESCs to
investigate whether YAP regulates ESCs viability in
endometriosis; the ESCs exhibited a lower proliferation and higher
apoptosis after the process. Meantime, YAP OE in normal
ESCs showed an increased proliferation and antiapoptosis
of ESCs. These data showed that YAP involve in the
viability of ESCs. Previous studies had suggested that eutopic
endometrium of endometriosis possesses many different
characteristics when compared with normal endometrium
of disease-free women, and most part of lies in ESCs
). In our study, up-regulated YAP adjusted the
viability of ESCs in endometriosis. It seems as though
activated Hippo/YAP signal pathway gives increase to
eutopic ESCs viability. These dynamic endometrial cells
attach to the peritoneal membrane through retrograde
menstruation, forward to continuous growth.
The mechanism by which YAP regulated ESC
proliferation and antiapoptosis needs to be evaluated further. The
expression of TEAD1, one of YAP DNA-binding
transcription factors, is inhibited by YAP KD in eutopic ESCs,
whereas elevated TEAD1 expression is caused by OE YAP
in normal ESCs. Our data suggest that YAP-TEAD1
complex may play a critical role in ESC proliferation and
apoptosis. Zhao et al (
) reported that YAP up-regulation
increased cell growth in MCF10A cell lines in a
CTGFdependent manner. In line with this study, we also found
that the YAP-TEAD1-signaling pathway regulating ESCs
proliferation to be dependent of regulator CTGF. BCL-2
is a novel downstream target of the YAP-TEAD1
transcriptional active complex to regulate cell apoptosis. In a
previous study, a BCL-2 family gene MCL1 was found to
be up-regulated in liver from an ApoE/rtTA-YAP
transgenic mouse (of which YAP was induced in the liver) and
suggests that YAP could partner with some unknown
DNA-binding protein(s) to regulate target genes
transcription, including MCL1 (
). Our finding demonstrated
that TEAD1 was the cofactor of YAP to regulate BCL-2.
In addition, variation of CTGF and BCL-2 expression was
consistent with TEAD1. Taken together, our study
confirmed that both of CTGF and BCL-2 were required for
YAP-controlled ESC proliferation and apoptosis.
In vitro, after treatment with verteporfin, eutopic ESCs
revealed decreased proliferation and enhanced apoptosis.
In vivo, we established a nude mouse model of
endometriosis and then treated ip injections of verteporfin. We
found that verteporfin could significantly shrink the
endometriotic lesion size, and even lead to disappearance of
some of them. The experiments in vitro and in vivo both
suggested that verteporfin or other specific compounds
can disrupt or weaken the YAP-TEAD complex, which
could be considered a latent choice in the pharmacological
study of endometriosis.
Our study has some limitations. The first is the nude
mouse model used. Endometriosis is widely considered
inflammatory disease, yet the use of
immunocompromised mice precludes assessment of the host immune
response in the disease model and to the treatment. The
other limitation is that only ESCs were used in in vitro
studies. Primary endometrial epithelial cells are very
difficult to grow and passage, which limit our study on this
kind cell. But the potential role of Hippo/YAP pathway in
endometrial epithelial cells is still necessary to be explored
in the following study.
In conclusion, up-regulation of YAP in endometriosis
has been presented. Elevated expression of YAP promoted
ESCs proliferation and antiapoptosis in vitro and in vivo.
As direct and functional targets of YAP-TEAD1
complexes, CTGF and BCL-2 involved in regulation of ESC?s
viability in endometriosis. Hippo/YAP-signaling pathway
may be involved in the pathogenesis of endometriosis and
should represent as a novel hypothesis for pathogenesis of
We thank Dr Wenming Xu, Huaqin Sun, and Ke Wang for their
help in the study; and Mr Ruibo Zhang for his help for
proofreading the manuscript.
Address all correspondence and requests for reprints to: Wei
Huang, MD, PhD, Department of Obstetrics and Gynecology,
West China Second University Hospital of Sichuan University, 3
Duan 20 Hao Renminnanlu, Chengdu Sichuan 610041, People?s
Republic of China. E-mail: .
This work was supported by a grant from the Science and
Technology Bureau of Sichuan (2012SZ0030).
Disclosure Summary: The authors have nothing to disclose.
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