Reproductive BioMedicine Online (2015) 30, 415–420

w w w. s c i e n c e d i r e c t . c o m
w w w. r b m o n l i n e . c o m

ARTICLE

Analysis of WNT4 polymorphism in Chinese

Han women with endometriosis
Zhangying Wu, Ming Yuan, Yan Li, Fangfang Fu, Wenqinq Ma, Haixia Li,
Wenwen Wang *, Shixuan Wang *

Cancer Biology Research Center, Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Jiefang Street 1095#, Wuhan, Hubei 430030, PR China
* Corresponding authors. E-mail addresses: wangwenwen1@gmail.com (W Wang); sxwang2012@hotmail.com (S Wang).

Shi-Xuan Wang, Professor of Gynaecology and Gynaecological Oncology, is Director of the Division of Gynecol-
ogy and Vice Director of the Cancer Biology Research Centre in Tongji hospital, Huazhong University of Science
and Technology. He received his PhD at Tongji Medical University, Wuhan, China in 1996. From 2002 to 2004,
he devoted himself to a tumour immunology programme as a postdoctoral researcher at UT Southwestern Medical
Centre, Dallas, Texas, USA. His research interests are gynaecology and gynaecological oncology.

Abstract Endometriosis is a complex disease that is influenced by genetic and environmental factors. In endometriosis, WNT4 plays
a likely role owing to its biological functions. In this study, the TaqMan allelic discrimination technique was used to investigate the
relationship between endometriosis and four single nucleotide polymorphisms in WNT4 (rs7521902 [A/C], rs16826658 [G/T], rs7515106
[C/T] and rs2235529 [A/G]) in Chinese Han women. A total of 646 patients with endometriosis and 766 normal controls were recurited.
Regression analyses revealed that rs2235529 was a risk locus for endometriosis (P = 1.80E-03, OR, 95% CI = 1.311, 1.129 to 1.522),
particularly in patients with stage III and IV disease. No significant association was found between endometriosis and rs7521902 (A/C),
rs16826658 (G/T), or rs7515106 (C/T). For each of the four single nucleotide polymorphisms, no association was found between patients
with endometriosis-related infertility or primary infertility and the controls. The results demonstrated that WNT4 rs2235529 is associated
with endometriosis in Chinese Han women, which may result in aberrant expression of WNT4, leading to the pathogenesis of
endometriosis.
© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

KEYWORDS: endometriosis, infertility, polymorphism, WNT4

http://dx.doi.org/10.1016/j.rbmo.2014.12.010
1472-6483/© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

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416
Introduction
Endometriosis is defined by the presence of endometrial tissue
outside the uterus, and it affects 5–10% of women of repro-
ductive age and up to 50% of infertile women (Eskenazi and
Warner, 1997; Giudice and Kao, 2004; Meuleman et al., 2009).
It often results in dysmenorrhoea, chronic pelvic pain, dys-
pareunia and infertility (Giudice and Kao, 2004).
Epidemiological studies have determined that the risk of en-
dometriosis in women with a family history of the
diseaseisincreased by approximately five-fold (Stefansson
et al., 2002; Treloar et al., 1999). Although the pathogen-
esis of endometriosis is largely unclear, it is considered to be
a multifactorial disease involving environmental and genetic
factors and interactions between these factors (Montgomery
et al., 2008).
The wingless-type MMTV integration site family member
4 (WNT4) is a protein-coding gene that plays a crucial role
in the development of the female reproductive tract, human
endometrial stromal cell differentiation and embryonic
implantation (Kobayashi and Behringer, 2003; Li et al., 2013;
Tulac et al., 2003; Vainio et al., 1999). A previous study ob-
served abnormal expression of the WNT4 gene in eutopic en-
dometrium from women with endometriosis (Pabona et al.,
2012). Recently, several genome-wide association studies
(GWAS) of endometriosis have detected some endometriosis-
related genetic markers in the region close to or within an
intron of WNT4 (Nyholt et al., 2012; Painter et al., 2011; Uno
et al., 2010). A Japanese GWAS reported an association
between endometriosis and rs7521902(A/C) and rs16826658(G/
T) (Nyholt et al., 2012; Uno et al., 2010). In addition, a sig-
nificant association between rs7521902(A/C) and endometriosis
was confirmed in the study by Painter et al. (2011), as well
as an association between rs7515106(C/T) and endometrio-
sis. Some studies have repeatedly investigated associations
between these single nucleotide polymorphisms (SNPs) and
endometriosis in women of different races (Albertsen et al.,
2013; Rahmioglu et al., 2014; Sundqvist et al., 2013). These
results, however, are inconsistent. In a recent study by
Albertsen et al. (2013), an association between one novel SNP
(rs2235529[A/G]) and endometriosis was observed in Ameri-
can women. In the present study, therefore, three contro-
versial SNPs were studied (rs7521902[A/C], rs16826658[G/
T], and rs7515106[C/T]) and one novel SNP (rs2235529[A/
G]) in Chinese Han women with endometriosis.
Materials and methods
Patients
In this study, 1412 women were enrolled (646 endometriosis
patients and 766 controls). All women had undergone surgery
between 2007 and 2013 at Tongji Hospital, Tongji Medical
College, Huazhong University of Science and Technology. The
following inclusion criteria were applied: members of the
Chinese Han population, aged 25–45 years, preoperative 6
months without hormonal therapy, regular menstruation, lapa-
roscopy or laparotomy and with a histologically confirmed pres-
ence of endometriosis or absence of endometriosis. Patients
with ovarian malignant or benign cysts (except ovarian
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Z Wu et al.
endometriosis), genital tract anomalies and histories of ma-
lignant disease were excluded. The control patients underwent
surgery for uterine leiomyomas (231/766 [30.2%]) or infer-
tility (535/766 [69.8%]). The postoperative diagnosis of in-
fertility was hydrosalpinx. According to the revised American
Fertility Society classification (Anon., 1985), 102 (15.8%) stage
I-II patients and 544 (84.2%) patients had moderate to severe
endometriosis (stage III-IV). In our sample, 39.8% (257/646)
women had endometriosis-associated infertility, and 22.5%
(172/766) controls had primary infertility.
Peripheral blood samples were preoperatively collected
into tubes treated with ethylenediaminetetraacetic acid.
All patients provided signed informed consent. The study
was approved by Tongji Medical College, Huazhong Univer-
sity of Science and Technology on 26 July 2011 (reference
number S462).
DNA extraction
Genomic DNA was extracted from leukocytes with the
Quickgene DNA whole blood kit (Fuji Film, Japan), according
to the manufacturer’s protocol.
WNT4 genotyping
Four SNPs were selected for genotyping: rs7521902(A/C),
rs16826658(G/T), rs7515106(C/T) and rs2235529(A/G). De-
tailed information is presented in Table 1. TaqMan allelic
discrimination analysis was carried out using the Applied
Biosystems protocol. A 384-well plate was prepared with a
mixture of 1 × TaqMan SNP Genotyping assay (Applied
Biosystems, USA), 1 × TaqMan Universal Master Mix (Applied
Biosystems, USA) and 20 ng DNA per well. Polymerase chain
reaction (PCR) was carried out using a 7900 HT Fast-Real-
Time PCR system (Applied Biosystems, USA). The PCR
conditions were denaturation at 95°C for 10 min, followed
by 40 cycles of 95°C denaturation for 15 s and a 60°C anneal
for 1 min. After PCR, the plates were read, and the data
analysed using SDS 2.4 (Applied Biosystems, USA). For quality
control, a random 10% of the samples were repeatedly
genotyped; the reproducibility rate was 100%. It was deter-
mined that a call rate of at least 97% met the standard
criteria.
Statistical analyses
The Statistical Package for Social Sciences (SPSS) version 18.0
for Windows (SPSS, Chicago, IL, USA) was used to conduct
statistical analyses. Certain differences (i.e. current age, age
at menarche, menstrual cycle and menstrual period) between
the patients and controls were evaluated using the non-
parametric Kruskal–Wallis test. The chi-squared test was used
to test the infertility rate, Hardy–Weinberg equilibrium, and
allele and genotype frequencies. Statistically significant values
were corrected for multiple tests using the Bonferroni
correction, and P < 0.05 was considered to be statistically
significant. The odds ratio (OR) and 95% confidence intervals
(CI) were calculated by binary logistic regression. A post-hoc
WNT4 polymorphism in Chinese Han women with endometriosis 417

Table 1 WNT4 single nucleotide polymorphisms in previous genome-wide association studies of endometriosis.
Location Allele Distance to WNT4 P-value OR (95% CI) Ethnicity Reference
SNP

rs7521902 Chr1:22164231 A/C Upstream 21 kb 4.23E-05 1.25 (1.12 to1.39) Japanese Uno et al. (2010)
8.90E-05 1.16 (1.08 to 1.25) White Painter et al. (2011)
rs16826658 Chr1:22159278 G/T Upstream 16 kb 3.46E-05 1.25 (1.12 to 1.39) White Painter et al. (2011)
rs2235529 Chr1:22123994 A/G Intron 1 1.36E-07 1.28 (1.16 to 1.41) White Albertsen et al. (2013)
rs7515106 Chr1:22146917 C/T Upstream 3.9 kb 3.60E-05 1.13 (1.07 to 1.05) White Painter et al. (2011)

CI = confidence interval; OR = odds ratio; SNP = single nucleotide polymorphism.

Table 2 Characteristics of the study population.

Endometriosis Control P-value
Characteristic

Number of participants 646 766

Age (years) 33.25 ± 7.55 34.06 ± 7.46 NS
Age at menarche 13.24 ± 1.42 13.40 ± 1.50 NS
Menstrual cycle length 29.47 ± 4.23 29.42 ± 4.41 NS
Menstrual period 5.93 ± 1.32 5.59 ± 1.68 0.019
Infertility 257/646 (39.8) 172/766 (22.5) <0.001

Values are expressed as the mean ± SD or n (%); P-value: total number of endometriosis pa-
tients versus controls. NS = not statistically significant.

power calculation was obtained using Power version 3.0.0 for
Windows (Power software, National Cancer Institute, USA).
The analysis was based on a disease prevalence of 8% and a
relative risk of 1.25. According to these assumptions, the study
power for each variant single nucleotide polymorphism (SNP)
was as follows: 51.6% for rs7521902(A/C), 57.7% for
rs16826658(G/T), 80.0% for rs2235529(A/G) and 72.6% for
rs7515106(C/T). The analysis of linkage disequilibrium
structures among the four WNT4 SNPs was carried out using
Haploview, version 4.2 (http://www.hapmap.org).
difference was found between the endometriosis patients
and controls in age, age at menarche and length of men-
strual cycle. The length of the menstrual period in the patients
(5.93 ± 1.32 days), however, was longer than that in the
controls (5.59 ± 1.68 days P = 0.019). Moreover, infertility
in the endometriosis patients was notably higher than in the
controls (39.8% versus 22.5%; P < 0.001).
Association of WNT4 polymorphism and
endometriosis

Results The genotype frequencies of the four SNPs were in Hardy–

Clinical characteristics of study population
The characteristics of the 646 patients with endometriosis
and the 766 controls are presented in Table 2. No significant
Weinberg equilibrium (Table 3) in this study. The call rates
for the assays were as follows: 98.4% for rs7521902 (A/C),
98.4% for rs16826658 (G/T), 98.3% for rs7515106 (C/T) and
98.7% for rs2235529 (A/G). The genotypes and allele
frequencies of the rs7521902 (A/C), rs16826658 (G/T),

Table 3 Comparison of risk allele frequencies in Chinese Han women with endometriosis and controls.
Allele Population N HWE RAF P-value OR (95% CI)
WNT4 polymorphism
study (allele)

rs7521902 (A/C) Control 747 0.176 0.50 (A) NS 1.031 (0.888 to 1.199)
Endometriosis 643 0.108 0.49 (A)
rs16826658 (G/T) Control 748 0.242 0.50 (G) NS 1.044 (0.868 to 1.257)
Endometriosis 642 0.507 0.52 (G)
rs2235529 (A/G) Control 746 0.806 0.48 (A) 1.80E-03 1.311 (1.129 to 1.522)
Endometriosis 642 0.347 0.55 (A)
rs7515106 (C/T) Control 760 0.383 0.80 (C) NS 1.215 (1.000 to 1.475)
Endometriosis 634 0.516 0.83 (C)

CI = confidence intervals; HWE = Hardy-Weinberg equilibrium; NS = not statistically significant; OR = odds ratio; RAF = risk allele frequencies.

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418 Z Wu et al.

Table 4 Analyses of the single nucleotide polymorphisms by severity of endometriosis.

Stage I-II Stage III-IV
WNT4 polymorphism
OR (95% CI) OR (95% CI)

rs7521902 1.087 (0.811 to 1.457) 1.021 (0.872 to 1.196)

rs1682668 1.040 (0.776 to 1.394) 1.069 (0.914 to 1.250)
rs2235529 1.154 (0.859 to 1.550) 1.322 (1.130 to 1.547)a
rs7515106 1.498 (0.985 to 2.278) 1.172 (0.956 to 1.435)

CI = confidence intervals; OR = odds ratio.

No statistically significant differences were found, except a difference between rs2235529 and
Stage III-IV(aP = 0.004).

rs7515106 (C/T) and rs2235529 (A/G) WNT4 polymorphism in Discussion

women with and without endometriosis are shown in Table 3.
The SNP rs2235529 (A/G) differed significantly between the
endometriosis patients and the controls (P = 1.80E-03, OR 95%
CI 1.311, 1.129 to 1.522). The other three SNPs did not differ
significantly between the patients and the controls. In
addition, no linkage disequilibrium was found between any
two SNPs (Supplementary Figure S1).
In the subgroup analyses, allele frequencies were compared
between the patients with stage I-II or stage III-IV endome-
triosis and the controls. The frequency of rs2235529(A/G) was
significantly different between the patients with stage III-IV
disease and the control group (P = 0.004, OR 95% CI = 1.322,
1.130 to 1.547), but no differences were observed between
the patients with stage I-II disease and the control group. No
association was detected between the other three SNPs and
the two disease stages (Table 4).
Analyses of WNT4 polymorphisms and
endometriosis-related infertility or primary
infertility
The relationship between four WNT4 SNPs and endometriosis-
related infertility or primary infertility is presented in Table 5.
No significant difference was observed.
Because of its biological functions, WNT4 plays a plausible
role in endometriosis, including promoting Müllerian-duct
differentiation during embryonic development, as well as
cell proliferation, migration and invasion in the endome-
trium (Biason-Lauber, 2012; Brosens et al., 2012; Matsuzaki
and Darcha, 2013; Tulac et al., 2003). The aberrant expres-
sion of WNT4 in eutopic endometrium with endometriosis
further indicates that WNT4 may be involved in the patho-
genesis of endometriosis (Pabona et al., 2012). Polymorphisms
of the WNT4 gene may change the function or quantity of
WNT4. To date, GWAS have identified four SNPs in WNT4
that are associated with the risk of endometriosis in Japanese
and white populations (Albertsen et al., 2013; Painter et al.,
2011; Uno et al., 2010). Surprisingly, no endometriosis-
related studies of the WNT4 polymorphism in the Chinese
Han population have been published.
A total of 646 endometriosis patients and 766 controls
were enrolled in the present study. No significant differ-
ences were found between the two groups in age, age at
menarche and menstrual cycle length, whereas a longer
menstrual period and a higher proportion of infertility were
found in patients with endometriosis. These observations
aligned with a clinical manifestation and epidemiological
survey (Giudice and Kao, 2004).

Table 5 Distribution of genotype and allele frequencies in infertile women with and without endometriosis and controls.
Population study n RAF (allele) OR (95% CI)
WNT4 polymorphism (1/2)

rs7521902 (A/C) Fertile controls 582 0.50 (A)

Infertile controls 165 0.49 (A) 0.976 (0.764 to 1.246)
Infertile endometriosis 127 0.50 (A) 0.984 (0.750 to 1.291)
rs1682668 (G/T) Fertile controls 582 0.50 (G)
Infertile controls 166 0.50 (G) 0.993 (0.778 to 1.267)
Infertile endometriosis 126 0.50 (G) 0.978 (0.744 to 1.284)
rs2235529 (A/G) Fertile controls 578 0.48 (A)
Infertile controls 168 0.49 (A) 1.068 (0.838 to 1.362)
Infertile endometriosis 130 0.53 (A) 1.257 (0.960 to 1.646)
rs7515106 (C/T) Fertile controls 590 0.80 (C)
Infertile controls 170 0.79 (C) 0.916 (0.680 to 1.233)
Infertile endometriosis 127 0.82 (C) 1.143 (0.803 to 1.626)

CI = confidence interval; OR = odds ratio; RAF = risk allele frequencies.

No statistically significant differences were found.

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WNT4 polymorphism in Chinese Han women with endometriosis
In a recent GWAS, Albertsen et al. (2013) reported a novel
SNP, rs2235529(A/G), that was associated with endometrio-
sis in women in the USA. Our study of 646 patients and 766
controls found a significant association between this SNP and
endometriosis. Although no amino acids were changed by the
rs2235529(A/G) polymorphism owing to its location in intron
1, genetic mutations in this intron are located in DNase 1 hy-
persensitive sites (ENCODE Project Consortium, 2012,
http://genome.ucsc.edu/ENCODE/), which may contribute
to transcription regulation and aberrant expression, eventu-
ally leading to the pathogenesis of endometriosis (Chrysogelos,
1993; Purugganan and Wessler, 1992). Since rs2235529 (A/G)
was found to be associated with endometriosis in the study
by Albertsen et al. (2013), the relationship between this SNP
and endometriosis was first validated. Our results were con-
sistent with those of Albertsen et al. (2013), which increases
the likelihood that this is a true causal association. There-
fore, it would be worthwhile to validate an association between
rs2235529 (A/G) and endometriosis in different populations
in the future. Previous reports, including GWAS, and studies
of disease morphology and gene expression, have suggested
that endometriosis is a heterogeneous disease that is com-
posed of different entities (Matsuzaki et al., 2006; Nisolle and
Donnez, 1997; Painter et al., 2011; Sundqvist et al., 2011).
In the present study, 84.2% (544/646) of the patients were in
stage III/IV, and a significant association was observed between
rs2235529(A/G) and stage III/IV, suggesting that the failure
to replicate some aspects of Albertsen’s study resulted from
differences in the genetic backgrounds of the populations.
Koninckx et al. (1994) demonstrated that endometrial stage
I-II is an epiphenomenon rather than a disease, which may par-
tially explain why no association was observed between the
patients with stage I/II endometriosis and the controls in our
study. It is debateable, however, whether endometriosis stage
I-II is a minimal to mild disease or ‘normal’. Because of the
small sample size of patients with stage I/II endometriosis in
our study (i.e. the sub-analysis was not powerful enough to
detect an effect), no association was observed between the
patients with stage I-II endometriosis and the controls.
The other three SNP (rs7521902 [A/C], rs16826658 [G/T]
and rs7515106 [C/T]) were not associated with endometrio-
sis. This result contrasts with the results of other studies that
have reported a strong association of rs7521902 (A/C) with
susceptibility to endometriosis in Japanese, Australian, British
and Italian women (Pagliardini et al., 2013; Painter et al., 2011;
Uno et al., 2010), but not Belgian women (Sundqvist et al.,
2013). Similarly, a strong relationship between rs16826658 (G/
T) and endometriosis was observed in Australian and British
women (Painter et al., 2011), whereas a weak association with
rs16826658 (G/T) was found in the USA (Albertsen et al., 2013).
The inconsistent results of these studies are largely attrib-
uted to genetic variations in the different populations studied.
In the present study, the lack of an association between these
SNPs and endometriosis may also be caused by the different
genetic backgrounds of the patients. When comparing the SNPs
allele and genotype frequencies from our study with those from
the 1000Genomes Consortium (www.1000genomes.org), an
approximately coincident distribution of allele and genotype
frequencies was observed in a Chinese Han population, which
suggests that, although the control patients were recruited
from the hospital, the control group bias was small. This bias,
however, cannot be completely excluded even though a larger
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419
population was analysed in the study. In addition, because
the sample size was limited compared with previous GWAS,
type II errors may result in the absence of significant asso-
ciation in this study because the analysis had a power of 51.6%.
In addition, non-genetic factors might contribute to the con-
flicting results in various studies (Klein et al., 2012; Vercellini
et al., 2014). On the basis of the International HapMap Project,
different linkage disequilibrium patterns of SNPs exist in dif-
ferent populations (Consortium, 2005; Consortium et al., 2010).
The failure to find linkage disequilibrum between any two SNPs
in the present study may be attributed to population
differences.
Given a possible role for WNT4/WNT signalling in infertil-
ity in women with endometriosis (Li et al., 2013; Matsuzaki
et al., 2010; Tulac et al., 2003), an attempt was made to analyse
the association between WNT4 SNPs and endometriosis-
related infertility, which has previously been unreported. No
association was found, however, between the examined SNPs
and endometriosis-associated infertility and primary infer-
tility in our study, which suggests that WNT4 may not play a
role in endometriosis-associated infertility and primary in-
fertility. Meanwhile, it was observed that the small sample
size in the sub-analysis may result in an absence of signifi-
cant association.
In conclusion, of the four candidate polymorphisms assessed
herein, evidence is provided from Chinese women that the
WNT4 rs2235529 polymorphism is associated with the risk of
endometriosis, but no association was found between rs7521902
(A/C), rs16826658 or rs7515106 and endometriosis. These find-
ings may lead to a better understanding of mutations in the
aetiology of endometriosis in different populations. Our study,
however, was limited in sample size; therefore, the results
must be validated in an independent cohort. In addition, the
function of WNT4 during the pathogenesis of endometriosis
is unclear and requires further elucidation.
Acknowledgements
The authors would like to thank the patients for their
participation.
Appendix: Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.rbmo.2014.12.010.
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Declaration: The authors report no financial or commercial con-
flicts of interest.
Received 6 October 2014; refereed 12 December 2014; accepted 15
December 2014.