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Analysis of WNT4 polymorphism in Chinese Han women with endometriosis

  • Zhangying Wu
    Affiliations
    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
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  • Ming Yuan
    Affiliations
    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
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  • Yan Li
    Affiliations
    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
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  • Fangfang Fu
    Affiliations
    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
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  • Wenqinq Ma
    Affiliations
    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
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  • Haixia Li
    Affiliations
    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
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  • Wenwen Wang
    Correspondence
    Corresponding authors.
    Affiliations
    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
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  • Shixuan Wang
    Correspondence
    Corresponding authors.
    Affiliations
    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
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Published:January 07, 2015DOI:https://doi.org/10.1016/j.rbmo.2014.12.010

      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.

      Keywords

      Introduction

      Endometriosis is defined by the presence of endometrial tissue outside the uterus, and it affects 5–10% of women of reproductive age and up to 50% of infertile women (
      • Eskenazi B.
      • Warner M.L.
      Epidemiology of endometriosis.
      ,
      • Giudice L.C.
      • Kao L.C.
      Endometriosis.
      ,
      • Meuleman C.
      • Vandenabeele B.
      • Fieuws S.
      • Spiessens C.
      • Timmerman D.
      • D'Hooghe T.
      High prevalence of endometriosis in infertile women with normal ovulation and normospermic partners.
      ). It often results in dysmenorrhoea, chronic pelvic pain, dyspareunia and infertility (
      • Giudice L.C.
      • Kao L.C.
      Endometriosis.
      ). Epidemiological studies have determined that the risk of endometriosis in women with a family history of the diseaseisincreased by approximately five-fold (
      • Stefansson H.
      • Geirsson R.T.
      • Steinthorsdottir V.
      • Jonsson H.
      • Manolescu A.
      • Kong A.
      • Ingadottir G.
      • Gulcher J.
      • Stefansson K.
      Genetic factors contribute to the risk of developing endometriosis.
      ,
      • Treloar S.A.
      • O'Connor D.T.
      • O'Connor V.M.
      • Martin N.G.
      Genetic influences on endometriosis in an Australian twin sample.
      ). Although the pathogenesis of endometriosis is largely unclear, it is considered to be a multifactorial disease involving environmental and genetic factors and interactions between these factors (
      • Montgomery G.W.
      • Nyholt D.R.
      • Zhao Z.Z.
      • Treloar S.A.
      • Painter J.N.
      • Missmer S.A.
      • Kennedy S.H.
      • Zondervan K.T.
      The search for genes contributing to endometriosis risk.
      ).
      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 A.
      • Behringer R.R.
      Developmental genetics of the female reproductive tract in mammals.
      ,
      • Li Q.
      • Kannan A.
      • Das A.
      • Demayo F.J.
      • Hornsby P.J.
      • Young S.L.
      • Taylor R.N.
      • Bagchi M.K.
      • Bagchi I.C.
      WNT4 acts downstream of BMP2 and functions via beta-catenin signaling pathway to regulate human endometrial stromal cell differentiation.
      ,
      • Tulac S.
      • Nayak N.R.
      • Kao L.C.
      • Van Waes M.
      • Huang J.
      • Lobo S.
      • Germeyer A.
      • Lessey B.A.
      • Taylor R.N.
      • Suchanek E.
      • Giudice L.C.
      Identification, characterization, and regulation of the canonical Wnt signaling pathway in human endometrium.
      ,
      • Vainio S.
      • Heikkila M.
      • Kispert A.
      • Chin N.
      • McMahon A.P.
      Female development in mammals is regulated by Wnt-4 signalling.
      ). A previous study observed abnormal expression of the WNT4 gene in eutopic endometrium from women with endometriosis (
      • Pabona J.M.
      • Simmen F.A.
      • Nikiforov M.A.
      • Zhuang D.
      • Shankar K.
      • Velarde M.C.
      • Zelenko Z.
      • Giudice L.C.
      • Simmen R.C.
      Kruppel-like factor 9 and progesterone receptor coregulation of decidualizing endometrial stromal cells: implications for the pathogenesis of endometriosis.
      ). 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 D.R.
      • Low S.K.
      • Anderson C.A.
      • Painter J.N.
      • Uno S.
      • Morris A.P.
      • Macgregor S.
      • Gordon S.D.
      • Henders A.K.
      • Martin N.G.
      • Attia J.
      • Holliday E.G.
      • McEvoy M.
      • Scott R.J.
      • Kennedy S.H.
      • Treloar S.A.
      • Missmer S.A.
      • Adachi S.
      • Tanaka K.
      • Nakamura Y.
      • Zondervan K.T.
      • Zembutsu H.
      • Montgomery G.W.
      Genome-wide association meta-analysis identifies new endometriosis risk loci.
      ,
      • Painter J.N.
      • Anderson C.A.
      • Nyholt D.R.
      • Macgregor S.
      • Lin J.
      • Lee S.H.
      • Lambert A.
      • Zhao Z.Z.
      • Roseman F.
      • Guo Q.
      • Gordon S.D.
      • Wallace L.
      • Henders A.K.
      • Visscher P.M.
      • Kraft P.
      • Martin N.G.
      • Morris A.P.
      • Treloar S.A.
      • Kennedy S.H.
      • Missmer S.A.
      • Montgomery G.W.
      • Zondervan K.T.
      Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
      ,
      • Uno S.
      • Zembutsu H.
      • Hirasawa A.
      • Takahashi A.
      • Kubo M.
      • Akahane T.
      • Aoki D.
      • Kamatani N.
      • Hirata K.
      • Nakamura Y.
      A genome-wide association study identifies genetic variants in the CDKN2BAS locus associated with endometriosis in Japanese.
      ). A Japanese GWAS reported an association between endometriosis and rs7521902(A/C) and rs16826658(G/T) (
      • Nyholt D.R.
      • Low S.K.
      • Anderson C.A.
      • Painter J.N.
      • Uno S.
      • Morris A.P.
      • Macgregor S.
      • Gordon S.D.
      • Henders A.K.
      • Martin N.G.
      • Attia J.
      • Holliday E.G.
      • McEvoy M.
      • Scott R.J.
      • Kennedy S.H.
      • Treloar S.A.
      • Missmer S.A.
      • Adachi S.
      • Tanaka K.
      • Nakamura Y.
      • Zondervan K.T.
      • Zembutsu H.
      • Montgomery G.W.
      Genome-wide association meta-analysis identifies new endometriosis risk loci.
      ,
      • Uno S.
      • Zembutsu H.
      • Hirasawa A.
      • Takahashi A.
      • Kubo M.
      • Akahane T.
      • Aoki D.
      • Kamatani N.
      • Hirata K.
      • Nakamura Y.
      A genome-wide association study identifies genetic variants in the CDKN2BAS locus associated with endometriosis in Japanese.
      ). In addition, a significant association between rs7521902(A/C) and endometriosis was confirmed in the study by
      • Painter J.N.
      • Anderson C.A.
      • Nyholt D.R.
      • Macgregor S.
      • Lin J.
      • Lee S.H.
      • Lambert A.
      • Zhao Z.Z.
      • Roseman F.
      • Guo Q.
      • Gordon S.D.
      • Wallace L.
      • Henders A.K.
      • Visscher P.M.
      • Kraft P.
      • Martin N.G.
      • Morris A.P.
      • Treloar S.A.
      • Kennedy S.H.
      • Missmer S.A.
      • Montgomery G.W.
      • Zondervan K.T.
      Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
      , as well as an association between rs7515106(C/T) and endometriosis. Some studies have repeatedly investigated associations between these single nucleotide polymorphisms (SNPs) and endometriosis in women of different races (
      • Albertsen H.M.
      • Chettier R.
      • Farrington P.
      • Ward K.
      Genome-wide association study link novel loci to endometriosis.
      ,
      • Rahmioglu N.
      • Nyholt D.R.
      • Morris A.P.
      • Missmer S.A.
      • Montgomery G.W.
      • Zondervan K.T.
      Genetic variants underlying risk of endometriosis: insights from meta-analysis of eight genome-wide association and replication datasets.
      ,
      • Sundqvist J.
      • Xu H.
      • Vodolazkaia A.
      • Fassbender A.
      • Kyama C.
      • Bokor A.
      • Gemzell-Danielsson K.
      • D'Hooghe T.M.
      • Falconer H.
      Replication of endometriosis-associated single-nucleotide polymorphisms from genome-wide association studies in a Caucasian population.
      ). These results, however, are inconsistent. In a recent study by
      • Albertsen H.M.
      • Chettier R.
      • Farrington P.
      • Ward K.
      Genome-wide association study link novel loci to endometriosis.
      , an association between one novel SNP (rs2235529[A/G]) and endometriosis was observed in American women. In the present study, therefore, three controversial 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, laparoscopy or laparotomy and with a histologically confirmed presence of endometriosis or absence of endometriosis. Patients with ovarian malignant or benign cysts (except ovarian endometriosis), genital tract anomalies and histories of malignant disease were excluded. The control patients underwent surgery for uterine leiomyomas (231/766 [30.2%]) or infertility (535/766 [69.8%]). The postoperative diagnosis of infertility was hydrosalpinx. According to the revised American Fertility Society classification (
      • Anonymous
      Revised American Fertility Society classification of endometriosis: 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 University 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). Detailed 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 determined that a call rate of at least 97% met the standard criteria.
      Table 1WNT4 single nucleotide polymorphisms in previous genome-wide association studies of endometriosis.
      SNPLocationAlleleDistance to WNT4P-valueOR (95% CI)EthnicityReference
      rs7521902Chr1:22164231A/CUpstream 21 kb4.23E-051.25 (1.12 to1.39)Japanese
      • Uno S.
      • Zembutsu H.
      • Hirasawa A.
      • Takahashi A.
      • Kubo M.
      • Akahane T.
      • Aoki D.
      • Kamatani N.
      • Hirata K.
      • Nakamura Y.
      A genome-wide association study identifies genetic variants in the CDKN2BAS locus associated with endometriosis in Japanese.
      8.90E-051.16 (1.08 to 1.25)White
      • Painter J.N.
      • Anderson C.A.
      • Nyholt D.R.
      • Macgregor S.
      • Lin J.
      • Lee S.H.
      • Lambert A.
      • Zhao Z.Z.
      • Roseman F.
      • Guo Q.
      • Gordon S.D.
      • Wallace L.
      • Henders A.K.
      • Visscher P.M.
      • Kraft P.
      • Martin N.G.
      • Morris A.P.
      • Treloar S.A.
      • Kennedy S.H.
      • Missmer S.A.
      • Montgomery G.W.
      • Zondervan K.T.
      Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
      rs16826658Chr1:22159278G/TUpstream 16 kb3.46E-051.25 (1.12 to 1.39)White
      • Painter J.N.
      • Anderson C.A.
      • Nyholt D.R.
      • Macgregor S.
      • Lin J.
      • Lee S.H.
      • Lambert A.
      • Zhao Z.Z.
      • Roseman F.
      • Guo Q.
      • Gordon S.D.
      • Wallace L.
      • Henders A.K.
      • Visscher P.M.
      • Kraft P.
      • Martin N.G.
      • Morris A.P.
      • Treloar S.A.
      • Kennedy S.H.
      • Missmer S.A.
      • Montgomery G.W.
      • Zondervan K.T.
      Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
      rs2235529Chr1:22123994A/GIntron 11.36E-071.28 (1.16 to 1.41)White
      • Albertsen H.M.
      • Chettier R.
      • Farrington P.
      • Ward K.
      Genome-wide association study link novel loci to endometriosis.
      rs7515106Chr1:22146917C/TUpstream 3.9 kb3.60E-051.13 (1.07 to 1.05)White
      • Painter J.N.
      • Anderson C.A.
      • Nyholt D.R.
      • Macgregor S.
      • Lin J.
      • Lee S.H.
      • Lambert A.
      • Zhao Z.Z.
      • Roseman F.
      • Guo Q.
      • Gordon S.D.
      • Wallace L.
      • Henders A.K.
      • Visscher P.M.
      • Kraft P.
      • Martin N.G.
      • Morris A.P.
      • Treloar S.A.
      • Kennedy S.H.
      • Missmer S.A.
      • Montgomery G.W.
      • Zondervan K.T.
      Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
      CI = confidence interval; OR = odds ratio; SNP = single nucleotide polymorphism.

      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 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).

      Results

      Clinical characteristics of study population

      The characteristics of the 646 patients with endometriosis and the 766 controls are presented in Table 2. No significant difference was found between the endometriosis patients and controls in age, age at menarche and length of menstrual 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).
      Table 2Characteristics of the study population.
      CharacteristicEndometriosisControlP-value
      Number of participants646766
      Age (years)33.25 ± 7.5534.06 ± 7.46NS
      Age at menarche13.24 ± 1.4213.40 ± 1.50NS
      Menstrual cycle length29.47 ± 4.2329.42 ± 4.41NS
      Menstrual period5.93 ± 1.325.59 ± 1.680.019
      Infertility257/646 (39.8)172/766 (22.5)<0.001
      Values are expressed as the mean ± SD or n (%); P-value: total number of endometriosis patients versus controls. NS = not statistically significant.

      Association of WNT4 polymorphism and endometriosis

      The genotype frequencies of the four SNPs were in Hardy–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), rs7515106 (C/T) and rs2235529 (A/G) WNT4 polymorphism in 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).
      Table 3Comparison of risk allele frequencies in Chinese Han women with endometriosis and controls.
      WNT4 polymorphismAllelePopulation studyNHWERAFP-valueOR (95% CI)
      (allele)
      rs7521902(A/C)Control7470.1760.50 (A)NS1.031 (0.888 to 1.199)
      Endometriosis6430.1080.49 (A)
      rs16826658(G/T)Control7480.2420.50 (G)NS1.044 (0.868 to 1.257)
      Endometriosis6420.5070.52 (G)
      rs2235529(A/G)Control7460.8060.48 (A)1.80E-031.311 (1.129 to 1.522)
      Endometriosis6420.3470.55 (A)
      rs7515106(C/T)Control7600.3830.80 (C)NS1.215 (1.000 to 1.475)
      Endometriosis6340.5160.83 (C)
      CI = confidence intervals; HWE = Hardy-Weinberg equilibrium; NS = not statistically significant; OR = odds ratio; RAF = risk allele frequencies.
      In the subgroup analyses, allele frequencies were compared between the patients with stage I-II or stage III-IV endometriosis 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).
      Table 4Analyses of the single nucleotide polymorphisms by severity of endometriosis.
      WNT4 polymorphismStage I-IIStage III-IV
      OR (95% CI)OR (95% CI)
      rs75219021.087 (0.811 to 1.457)1.021 (0.872 to 1.196)
      rs16826681.040 (0.776 to 1.394)1.069 (0.914 to 1.250)
      rs22355291.154 (0.859 to 1.550)1.322 (1.130 to 1.547)a
      rs75151061.498 (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).

      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.
      Table 5Distribution of genotype and allele frequencies in infertile women with and without endometriosis and controls.
      WNT4 polymorphism (1/2)Population studynRAF (allele)OR (95% CI)
      rs7521902 (A/C)Fertile controls5820.50 (A)
      Infertile controls1650.49 (A)0.976 (0.764 to 1.246)
      Infertile endometriosis1270.50 (A)0.984 (0.750 to 1.291)
      rs1682668 (G/T)Fertile controls5820.50 (G)
      Infertile controls1660.50 (G)0.993 (0.778 to 1.267)
      Infertile endometriosis1260.50 (G)0.978 (0.744 to 1.284)
      rs2235529 (A/G)Fertile controls5780.48 (A)
      Infertile controls1680.49 (A)1.068 (0.838 to 1.362)
      Infertile endometriosis1300.53 (A)1.257 (0.960 to 1.646)
      rs7515106 (C/T)Fertile controls5900.80 (C)
      Infertile controls1700.79 (C)0.916 (0.680 to 1.233)
      Infertile endometriosis1270.82 (C)1.143 (0.803 to 1.626)
      CI = confidence interval; OR = odds ratio; RAF = risk allele frequencies.
      No statistically significant differences were found.

      Discussion

      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 endometrium (
      • Biason-Lauber A.
      WNT4, RSPO1, and FOXL2 in sex development.
      ,
      • Brosens I.
      • Brosens J.J.
      • Benagiano G.
      The eutopic endometrium in endometriosis: are the changes of clinical significance?.
      ,
      • Matsuzaki S.
      • Darcha C.
      In vitro effects of a small-molecule antagonist of the Tcf/ss-catenin complex on endometrial and endometriotic cells of patients with endometriosis.
      ,
      • Tulac S.
      • Nayak N.R.
      • Kao L.C.
      • Van Waes M.
      • Huang J.
      • Lobo S.
      • Germeyer A.
      • Lessey B.A.
      • Taylor R.N.
      • Suchanek E.
      • Giudice L.C.
      Identification, characterization, and regulation of the canonical Wnt signaling pathway in human endometrium.
      ). The aberrant expression of WNT4 in eutopic endometrium with endometriosis further indicates that WNT4 may be involved in the pathogenesis of endometriosis (
      • Pabona J.M.
      • Simmen F.A.
      • Nikiforov M.A.
      • Zhuang D.
      • Shankar K.
      • Velarde M.C.
      • Zelenko Z.
      • Giudice L.C.
      • Simmen R.C.
      Kruppel-like factor 9 and progesterone receptor coregulation of decidualizing endometrial stromal cells: implications for the pathogenesis of endometriosis.
      ). 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 H.M.
      • Chettier R.
      • Farrington P.
      • Ward K.
      Genome-wide association study link novel loci to endometriosis.
      ,
      • Painter J.N.
      • Anderson C.A.
      • Nyholt D.R.
      • Macgregor S.
      • Lin J.
      • Lee S.H.
      • Lambert A.
      • Zhao Z.Z.
      • Roseman F.
      • Guo Q.
      • Gordon S.D.
      • Wallace L.
      • Henders A.K.
      • Visscher P.M.
      • Kraft P.
      • Martin N.G.
      • Morris A.P.
      • Treloar S.A.
      • Kennedy S.H.
      • Missmer S.A.
      • Montgomery G.W.
      • Zondervan K.T.
      Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
      ,
      • Uno S.
      • Zembutsu H.
      • Hirasawa A.
      • Takahashi A.
      • Kubo M.
      • Akahane T.
      • Aoki D.
      • Kamatani N.
      • Hirata K.
      • Nakamura Y.
      A genome-wide association study identifies genetic variants in the CDKN2BAS locus associated with endometriosis in Japanese.
      ). 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 differences 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 L.C.
      • Kao L.C.
      Endometriosis.
      ).
      In a recent GWAS,
      • Albertsen H.M.
      • Chettier R.
      • Farrington P.
      • Ward K.
      Genome-wide association study link novel loci to endometriosis.
      reported a novel SNP, rs2235529(A/G), that was associated with endometriosis 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 hypersensitive sites (
      • ENCODE Project Consortium
      An integrated encyclopedia of DNA elements in the human genome.
      , http://genome.ucsc.edu/ENCODE/), which may contribute to transcription regulation and aberrant expression, eventually leading to the pathogenesis of endometriosis (
      • Chrysogelos S.A.
      Chromatin structure of the EGFR gene suggests a role for intron 1 sequences in its regulation in breast cancer cells.
      ,
      • Purugganan M.
      • Wessler S.
      The splicing of transposable elements and its role in intron evolution.
      ). Since rs2235529 (A/G) was found to be associated with endometriosis in the study by
      • Albertsen H.M.
      • Chettier R.
      • Farrington P.
      • Ward K.
      Genome-wide association study link novel loci to endometriosis.
      , the relationship between this SNP and endometriosis was first validated. Our results were consistent with those of
      • Albertsen H.M.
      • Chettier R.
      • Farrington P.
      • Ward K.
      Genome-wide association study link novel loci to endometriosis.
      , which increases the likelihood that this is a true causal association. Therefore, 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 composed of different entities (
      • Matsuzaki S.
      • Canis M.
      • Pouly J.L.
      • Botchorishvili R.
      • Dechelotte P.J.
      • Mage G.
      Differential expression of genes in eutopic and ectopic endometrium from patients with ovarian endometriosis.
      ,
      • Nisolle M.
      • Donnez J.
      Peritoneal endometriosis, ovarian endometriosis, and adenomyotic nodules of the rectovaginal septum are three different entities.
      ,
      • Painter J.N.
      • Anderson C.A.
      • Nyholt D.R.
      • Macgregor S.
      • Lin J.
      • Lee S.H.
      • Lambert A.
      • Zhao Z.Z.
      • Roseman F.
      • Guo Q.
      • Gordon S.D.
      • Wallace L.
      • Henders A.K.
      • Visscher P.M.
      • Kraft P.
      • Martin N.G.
      • Morris A.P.
      • Treloar S.A.
      • Kennedy S.H.
      • Missmer S.A.
      • Montgomery G.W.
      • Zondervan K.T.
      Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
      ,
      • Sundqvist J.
      • Falconer H.
      • Seddighzadeh M.
      • Vodolazkaia A.
      • Fassbender A.
      • Kyama C.
      • Bokor A.
      • Stephansson O.
      • Padyukov L.
      • Gemzell-Danielsson K.
      • D'Hooghe T.M.
      Endometriosis and autoimmune disease: association of susceptibility to moderate/severe endometriosis with CCL21 and HLA-DRB1.
      ). 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 P.R.
      • Oosterlynck D.
      • D'Hooghe T.
      • Meuleman C.
      Deeply infiltrating endometriosis is a disease whereas mild endometriosis could be considered a non-disease.
      demonstrated that endometrial stage I-II is an epiphenomenon rather than a disease, which may partially 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 endometriosis. 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 L.
      • Gentilini D.
      • Vigano P.
      • Panina-Bordignon P.
      • Busacca M.
      • Candiani M.
      • Di Blasio A.M.
      An Italian association study and meta-analysis with previous GWAS confirm WNT4, CDKN2BAS and FN1 as the first identified susceptibility loci for endometriosis.
      ,
      • Painter J.N.
      • Anderson C.A.
      • Nyholt D.R.
      • Macgregor S.
      • Lin J.
      • Lee S.H.
      • Lambert A.
      • Zhao Z.Z.
      • Roseman F.
      • Guo Q.
      • Gordon S.D.
      • Wallace L.
      • Henders A.K.
      • Visscher P.M.
      • Kraft P.
      • Martin N.G.
      • Morris A.P.
      • Treloar S.A.
      • Kennedy S.H.
      • Missmer S.A.
      • Montgomery G.W.
      • Zondervan K.T.
      Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
      ,
      • Uno S.
      • Zembutsu H.
      • Hirasawa A.
      • Takahashi A.
      • Kubo M.
      • Akahane T.
      • Aoki D.
      • Kamatani N.
      • Hirata K.
      • Nakamura Y.
      A genome-wide association study identifies genetic variants in the CDKN2BAS locus associated with endometriosis in Japanese.
      ), but not Belgian women (
      • Sundqvist J.
      • Xu H.
      • Vodolazkaia A.
      • Fassbender A.
      • Kyama C.
      • Bokor A.
      • Gemzell-Danielsson K.
      • D'Hooghe T.M.
      • Falconer H.
      Replication of endometriosis-associated single-nucleotide polymorphisms from genome-wide association studies in a Caucasian population.
      ). Similarly, a strong relationship between rs16826658 (G/T) and endometriosis was observed in Australian and British women (
      • Painter J.N.
      • Anderson C.A.
      • Nyholt D.R.
      • Macgregor S.
      • Lin J.
      • Lee S.H.
      • Lambert A.
      • Zhao Z.Z.
      • Roseman F.
      • Guo Q.
      • Gordon S.D.
      • Wallace L.
      • Henders A.K.
      • Visscher P.M.
      • Kraft P.
      • Martin N.G.
      • Morris A.P.
      • Treloar S.A.
      • Kennedy S.H.
      • Missmer S.A.
      • Montgomery G.W.
      • Zondervan K.T.
      Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.
      ), whereas a weak association with rs16826658 (G/T) was found in the USA (
      • Albertsen H.M.
      • Chettier R.
      • Farrington P.
      • Ward K.
      Genome-wide association study link novel loci to endometriosis.
      ). The inconsistent results of these studies are largely attributed 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 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 association in this study because the analysis had a power of 51.6%. In addition, non-genetic factors might contribute to the conflicting results in various studies (
      • Klein C.
      • Lohmann K.
      • Ziegler A.
      The promise and limitations of genome-wide association studies.
      ,
      • Vercellini P.
      • Vigano P.
      • Somigliana E.
      • Fedele L.
      Endometriosis: pathogenesis and treatment.
      ). On the basis of the International HapMap Project, different linkage disequilibrium patterns of SNPs exist in different populations (
      • Consortium I.H.
      A haplotype map of the human genome.
      ,
      • Consortium I.H.
      • Altshuler D.M.
      • Gibbs R.A.
      • Peltonen L.
      • Dermitzakis E.
      • Schaffner S.F.
      • Yu F.
      • Bonnen P.E.
      • De Bakker P.I.
      • Deloukas P.
      • Gabriel S.B.
      • Gwilliam R.
      • Hunt S.
      • Inouye M.
      • Jia X.
      • Palotie A.
      • Parkin M.
      • Whittaker P.
      • Chang K.
      • Hawes A.
      • Lewis L.R.
      • Ren Y.
      • Wheeler D.
      • Muzny D.M.
      • Barnes C.
      • Darvishi K.
      • Hurles M.
      • Korn J.M.
      • Kristiansson K.
      • Lee C.
      • McCarrol S.A.
      • Nemesh J.
      • Keinan A.
      • Montgomery S.B.
      • Pollack S.
      • Price A.L.
      • Soranzo N.
      • Gonzaga-Jauregui C.
      • Anttila V.
      • Brodeur W.
      • Daly M.J.
      • Leslie S.
      • McVean G.
      • Moutsianas L.
      • Nguyen H.
      • Zhang Q.
      • Ghori M.J.
      • McGinnis R.
      • McLaren W.
      • Takeuchi F.
      • Grossman S.R.
      • Shlyakhter I.
      • Hostetter E.B.
      • Sabeti P.C.
      • Adebamowo C.A.
      • Foster M.W.
      • Gordon D.R.
      • Licinio J.
      • Manca M.C.
      • Marshall P.A.
      • Matsuda I.
      • Ngare D.
      • Wang V.O.
      • Reddy D.
      • Rotimi C.N.
      • Royal C.D.
      • Sharp R.R.
      • Zeng C.
      • Brooks L.D.
      • McEwen J.E.
      Integrating common and rare genetic variation in diverse human populations.
      ). 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 infertility in women with endometriosis (
      • Li Q.
      • Kannan A.
      • Das A.
      • Demayo F.J.
      • Hornsby P.J.
      • Young S.L.
      • Taylor R.N.
      • Bagchi M.K.
      • Bagchi I.C.
      WNT4 acts downstream of BMP2 and functions via beta-catenin signaling pathway to regulate human endometrial stromal cell differentiation.
      ,
      • Matsuzaki S.
      • Darcha C.
      • Maleysson E.
      • Canis M.
      • Mage G.
      Impaired down-regulation of E-cadherin and beta-catenin protein expression in endometrial epithelial cells in the mid-secretory endometrium of infertile patients with endometriosis.
      ,
      • Tulac S.
      • Nayak N.R.
      • Kao L.C.
      • Van Waes M.
      • Huang J.
      • Lobo S.
      • Germeyer A.
      • Lessey B.A.
      • Taylor R.N.
      • Suchanek E.
      • Giudice L.C.
      Identification, characterization, and regulation of the canonical Wnt signaling pathway in human endometrium.
      ), 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 infertility in our study, which suggests that WNT4 may not play a role in endometriosis-associated infertility and primary infertility. Meanwhile, it was observed that the small sample size in the sub-analysis may result in an absence of significant 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 findings 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

      The following is the supplementary data to this article:

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      Biography

      Shi-Xuan Wang, Professor of Gynaecology and Gynaecological Oncology, is Director of the Division of Gynecology 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.