- Email: [email protected]
Association between XRCC1 single-nucleotide polymorphisms and infertility with idiopathic azoospermia in northern Chinese Han males
Reproductive BioMedicine Online (2012) 25, 402– 407
www.sciencedirect.com www.rbmonline.com
ARTICLE
Association between XRCC1 single-nucleotide polymorphisms and infertility with idiopathic azoospermia in northern Chinese Han males Lie-rui Zheng a,d, Xiao-fang Wang b, Dang-xia Zhou Yong-wei Huo c, Hong Tian c
a,d,*
, Jing Zhang a,
a Department of Pathology, Medical School, Xi’an Jiaotong University, Xi’an 710061, China; b Shaan’xi Blood Center, Xi’an 710061, China; c Reproductive Medical Center, Xi’an Jiaotong University, Xi’an 710061, China; d Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an 710061, China
* Corresponding author. E-mail address: [email protected] (D-x Zhou). Dang-xia Zhou, associate professor, works in the pathology department at the medical school of Xi’an Jiaotong University. She received her MSc in pathology in 2002 and PhD in histology and embryology in 2006. Her research interest is the genetic aetiology and pathogenesis of male infertility.
Abstract X-ray repair cross-complementing group 1 (XRCC1) is a scaffold protein that plays a critical role in DNA base excision
repair. To explore the association between XRCC1 single-nucleotide polymorphisms and infertility with idiopathic azoospermia in a northern Chinese Han population, PCR restriction fragment length polymorphism was used to genotype a SNP locus (rs25487) of XRCC1 in 112 patients with idiopathic azoospermia and 156 healthy controls. Furthermore, nucleotide sequences were sequenced. The results showed that, compared with GG genotype, the GA and GA + AA genotypes showed a significant association with an increased risk of idiopathic azoospermia (OR 2.119, 95% CI 1.245–3.606, P = 0.005), (OR 2.052, 95% CI 1.227–3.431, P = 0.006) respectively. Meanwhile, the A allele frequency was significantly higher in azoospermic patients than that in controls (OR 1.472, 95% CI 1.029–2.105, P = 0.034). The substitutions bring about an amino acid alteration: G fi A changes the arginine residue into glutamine. In conclusion, the SNP locus rs25487 of XRCC1 could be a marker for genetic susceptibility to idiopathic azoospermia and the A allele might be a risk gene of idiopathic azoospermia in the northern Chinese Han population. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: idiopathic azoospermia, restriction fragment length polymorphism, single-nucleotide polymorphism, XRCC1
1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2012.06.014
XRCC1 and idiopathic azoospermia
Introduction Infertility is a huge problem that affects 10–15% of couples world-wide, in approximately 50% of cases the problem being due to male factor infertility partner (Huynh et al., 2002). Although several causes have been proposed for aetiologies of male infertility, unfortunately, nearly 50% of male infertility is unexplained or idiopathic (Zhang et al., 2007). As one of important types of idiopathic male infertility (Zhang et al., 2007), idiopathic azoospermia is characterized by no spermatozoa in semen and its causes is unclear (Matsuzaka et al., 2002). Recent studies suggest that impaired spermatogenesis is an essential aetiology of male infertility and that genetic disorders affecting spermatogenesis might be responsible for many cases of idiopathic infertility (Yang et al., 2006). It is well known that spermatogenesis is a complex, multifactor, multistep physiological process, regulated by multiple genes. Other than Y-chromosome microdeletion, more recent studies have suggested that polymorphism of genes in autosomal chromosomes may also play an important role in male infertility (Poongothai et al., 2009; Zhang et al., 2011). All kinds of endogenous and exogenous environmental factors, such as physical (anatomical abnormality, hyperthermia), chemical (drugs, cigarette smoking) and biological damage, can give rise to human cellular DNA damage, and the germ cell is no exception. The absence or decrease of DNA repair ability could increase the risk of germ cell genetic mutation and cause failure of spermatogenesis, thus resulting in azoospermia and oligozoospermia (Hsia et al., 2003). Fortunately, an integrated DNA repair pathway (include various DNA repair genes) exists in the human genome, which can repair a series of DNA damage in somatic and germ cells. The DNA repair system can be categorized into: (i) nucleotide-excision repair (NER); (ii) base-excision repair (BER); (iii) mismatch repair (MMR); (iv) DNA strand-break repair (DSBR); and (v) direct repair (DR). Of all of these, NER and BER are the most common (Hartwig, 2010). X-ray repair cross-complementing group 1 (XRCC1) is a scaffold protein that interacts with several DNA repair proteins and plays a critical role in BER. XRCC1 contains 17 exons and is located on chromosome 19q13.2 (Langsenlehner et al., 2011). Large number of studies reported that the single-nucleotide polymorphisms (SNP) in XRCC1 may be associated with the change of DNA repair capacity, which may affect genetic susceptibility to complex disease and the sensitivity of individuals to treatment. It has been shown that polymorphisms in the XRCC1 SNP locus rs25487 (codon 399, exon 10, base G fi A, amino acid Arg fi Gln) may increase the risks of lung cancer, bladder cancer, breast cancer and other diseases (Saadat et al., 2008; Sreeja et al., 2008; Qu and Morimoto, 2005). Recent studies have suggested that XRCC1 is highly expressed in spermatogenic and Sertoli cells of mouse testis (Ahmed et al., 2010). XRCC1 is also expressed in early elongating spermatids and is involved in the extensive chromatin remodelling in these cells; meanwhile, it has significant DNA repair capability (Ahmed et al., 2010; El-Domyati et al., 2009). XRCC1 knockout in mice results in embryonic lethality, demonstrating it to be essential for development (Tebbs et al., 1999). It means that XRCC1 may play a key role in
403 spermatogenesis and DNA repair of germ cells. Another study described XRCC1 as being most abundant in pachytene spermatocytes as well as in round spermatids and suggested that it might maintain spermatogenesis by repairing DNA damage during meiosis in germ cells (Gu et al., 2007). However, as far as is known, there are few studies concerning the association of XRCC1 and idiopathic azoospermia. Therefore, the current study aimed to investigate the potential association between XRCC1 SNP and idiopathic azoospermia in a northern Chinese Han population using PCR restriction fragment length polymorphism (RFLP) method. These results will not only provide useful information for anthropological researches and population genetics but also a scientific basis for male reproduction studies (Zhou et al., 2011a).
Materials and methods Selection of subjects The protocol was fully approved by the Institutional Medical Ethics Committee (reference number 2008051) on 6 March 2008. All subjects were randomly selected from Chinese Han population in the Shaanxi Province, China. The risks and benefits of participating in the study were explained to all subjects. According to the methods of previous studies (Zhou et al., 2011a,b), the infertile patients were selected from couples attending the infertility clinic who had a history of infertility of 12 months. After obtaining the informed consent of patients, a detailed medical and reproductive history was obtained from all subjects, including reproductive history and infertility evaluation of the female partner. World Health Organization criteria (2010) were used to define semen parameters for all subjects who were examined. Finally, 112 azoospermia patients (age 30.41 ± 3.58 years) were selected for infertility samples, excluding the following factors such as immune (antisperm antibody IgG, IgA), infection (mycoplasma, chlamydia 1), biochemical abnormality (a1–4 glucosidase, acid phosphatase, fructose) and anatomical abnormality. Patients with chromosomal abnormalities and microdelections in the AZF region on Y chromosome were also excluded. At the same time, 156 healthy Han, married, male volunteers who had at least one child and lived in the same place of residence were recruited as the control group (age 30.28 ± 3.16 years).
Genotype analysis by PCR-RFLP After informed consent, 2 ml noncoagulated EDTA blood samples were collected and stored at 20C before DNA extraction. Genomic DNA was extracted from peripheral white blood cells using a DNA extraction kit (A004-1; Dinguo, Beijing, China). The polymorphisms of the XRCC1 SNP locus rs25487 were determined using the PCR-RFLP method. The primers used were: forward, 50 -TCACACCTAACTGGCATC TTCACT-30 ; reverse, 50 -CTCCTTCCCTCATCTGGAGTACC-30 . PCR was carried out in 20-ll reaction volumes [containing 7 ll PCR mix (10 · Taq buffer with (NH4)2SO4, 0.2 mmol/l dNTP, 1.5 mmol/l MgCl2, 0.5 lmol/l of each
404 primer, 2.5 U Taq DNA polymerase (Fermentas, Lithuania)], 1 ll of each primer, 9 ll ddH2O, and 2 ll genomic DNA (approximately 50 ng DNA template). Thermal cycling was performed using a MyCycler Thermal Cycler (Bio-Rad) with the following program: 95C for 5 min and 35 cycles at 95C for 30 s, 64C for 45 s and 72C for 1 min, followed by final extension at 72C for 10 min. The products were detected on 1% agarose gel (Figure 1). The restriction enzyme MspI (Fermentas, Lithuania) was used to distinguish SNP. In brief, 3 ll PCR product was digested with 0.3 ll of MspI at 37C for 14 h. The fragments of PCR-RFLP were detected on a 2% agarose gel (Figure 2). At the SNP locus rs25487, the GG genotype produces two MspI restriction fragments of 177 and 153 bp; the GA genotype produces three fragments of 330, 177 and 153 bp and the AA genotype produces only one 330 bp fragment. Finally, genotypes of randomly selected subjects, as determined by PCR-RFLP, were confirmed by direct resequencing in an ABI PRISM 3730 autosequencer. All the oligonucleotides were synthesized and sequenced by Sangon Biotech (Shanghai, China).
Statistical analysis Hardy–Weinberg equilibrium of the XRCC1 locus was estimated using Modified Powerstates (Promega Corporation, USA). Allele frequencies and all analyses related to the case–control study were calculated using the Statistical Package for Social Sciences version 13.0 (SPSS, Chicago, USA). The frequencies of XRCC1 alleles were compared between controls and patients with idiopathic azoospermia using the Chi-squared test. Fisher’s Exact test was applied when one of expected numbers was less than 5. Relative risk was calculated as an odds ratio (OR) with 95% confidence interval (CI) using Cornfield’s approximation. P < 0.05 was regarded as statistically significant.
Results Subjects In total, 112 idiopathic azoospermic patients were diagnosed as male infertility after semen analysis and
Figure 1 PCR products of XRCC1. Fragments were analysed on a 1% agarose gel with ethidium bromide. Lengths of product fragments are approximately 330 bp. M = DNA size marker; lanes 1–7 = PCR products of seven azoospermic patients.
L-r Zheng et al.
Figure 2 Restriction fragment length polymorphism RFLP for XRCC1 in different azoospermic patients. Fragments were analysed on a 2% agarose gel with ethidium bromide. M = DNA size marker; lanes 1 and 2 = AA genotype, one fragment of 330 bp; lanes 3–5 = GG genotype, two fragments of 177 and 153 bp; lanes 6 and 7 = GA genotype, three fragments of 330, 177 and 153 bp (lanes 6 and 7).
examination of infertility factors. The mean ± SD ages of azoospermic patients and controls were 30.41 ± 3.58 and 30.28 ± 3.16 years, respectively, and there was no significant difference between two groups. All azoospermic patients (v2 = 3.268, df = 2) and controls (v 0.043, df = 2) were in compliance with Hardy–Weinberg equilibrium at XRCC1.
Mutation detection All genotypes and allele frequencies of SNP locus rs25487 of XRCC1 are summarized in Tables 1 and 2. The sequences from a patient and a control are shown in Figure 3. In SNP locus rs25487 of XRCC1, genotype changes (G/G fi G/A) were found in 67 (59.8%) out of 112 azoospermic patients and in 69 (44.2%) out of 156 controls, at the same time (G/G fi A/A) were found in 12 (10.7%) out of 112 azoospermic patients and in 15 (9.6%) out of 156 controls. The substitutions bring about an amino acid alteration: G fi A changes the arginine residue (Arg) into glutamine (Gln). As shown in Table 1, the GA genotype of XRCC1, leading to Arg399Gln, showed a significant association with a increased risk of idiopathic azoospermia compared with the GG genotype (OR 2.119, 95% CI 1.245–3.606, P = 0.005), and the GA + AA genotype also demonstrated a significant association with a increased risk of idiopathic azoospermia compared with the GG genotype (OR 2.052, 95% CI 1.227–3.431, P = 0.006). Meanwhile, the A allele frequency of Arg399Gln (G fi A) was significantly higher in infertile patients with azoospermia than that in controls (OR 1.472, 95% CI 1.029–2.105, P = 0.034; Table 2), which suggested that the A allele of Arg399Gln (G fi A) may increase the infertile risk.
Discussion This study found an association of the SNP locus rs25487 of XRCC1 with idiopathic azoospermia, indicating that A allele
XRCC1 and idiopathic azoospermia
405
Table 1 The genotypes of SNP locus rs25487 of XRCC1 (codon 399, base G fi A, amino acid Arg fi Gln) for patients and controls. Genotype
Patients (n = 112)
Controls (n = 156)
Pvalue
OR (95% CI)
GG GA
33 (29.5) 67 (59.8)
72 (46.2) 69 (44.2)
0.020
AA
12 (10.7)
15 (9.6)
GG GA + AA
33 (29.5) 79 (70.5)
72 (46.2) 84 (53.8)
1.00 2.119 (1.245– 3.606), P = 0.005 1.745 (0.736– 4.140) 1.00 2.052 (1.227– 3.431)
0.006
Values are n (%). Two-sided v2 test. P < 0.05 was considered to be significant.
Table 2
The allele frequencies of SNP locus rs25487 of XRCC1.
Allele
Patients (n = 112)
Controls (n = 156)
Pvalue
OR (95% CI)
G A
133 (59.4) 91 (40.6)
213 (68.3) 99 (31.7)
0.034
1.00 1.472 (1.029– 2.105)
Values are n (%). Two-sided v2 test. P < 0.05 was considered to be significant.
might be a risk gene of idiopathic azoospermia in the northern Chinese Han population. Sperm DNA integrity is a prerequisite for normal sperm function. Sperm DNA must be correctly programmed and repaired to successfully pass on genetic and epigenetic information to the developing embryo (O’Flynn et al., 2010). Sperm DNA damage is easily incurred by the endogenous and exogenous factors, such as intrinsic testicular factors, protamine deficiency, apoptosis, drugs, chemotherapy, radiotherapy, testicular hyperthermia, cigarette smoking, varicoceles, genital tract infection and inflammation and environmental toxins (Zini et al., 2010). XRCC1 is involved in the BER pathway, which is essential in repairing DNA damage (Ahmed et al., 2010; El-Domyati et al., 2009;
Figure 3
Langsenlehner et al., 2011; Tebbs et al., 1999; Thompson and West, 2000). The processes of spermatogenesis require an estimated 2000 genes that function in numerous pathways controlling spermatogenesis as well as development and maintenance of the testis (Hargreave, 2000). It will inevitably need DNA repair genes to participate to exercise its normal function for spermatogenesis when these genes are damaged. As an important DNA repair system of human genome, including XRCC1 and its polymorphism is valuable in disease-association studies (Rubes et al., 2010). As one of the important members of DNA repair system, abundant studies have reported about the association between SNP locus rs25487 of XRCC1 with human disease (mostly about the cancer research). XRCC1 is highly conservatively expressed in spermatogenic cells of testis (Ahmed et al., 2010). It also plays an important role in maintaining normal spermatogenesis by repairing DNA damages during meiosis process (Gu et al., 2007). Owing to the valuable role for spermatogenesis, mutations or polymorphisms in XRCC1 may contribute to susceptibility to spermatogenesis impairment. The present study demonstrated that the SNP locus rs25487 of XRCC1 has variant genotypes (GA, GA + AA) associated with a higher risk of idiopathic azoospermia. The A allele in SNP locus rs25487 of XRCC1 seemed to be the risk allele for idiopathic zoospermia and it is speculated that G fi A in SNP locus rs25487 of XRCC1 may alter its DNA repair capacity and be associated with spermatogenesis failure. The present results are consistent with several previous cancer studies, such as the study of Sreeja et al. (2008), which indicated that the carriers of XRCC1 codon 399 AA genotypes were at higher risk of lung cancer (OR 2.1, 95% CI 1.224–3.669, P = 0.007) than carriers of GG genotype. The studies of Saadat and Ansari-Lari (2009) and Li et al. (2009) reported that the A allele of XRCC1 codon 399 might act as a recessive allele in its association with breast cancer risk in Asian countries (Li et al., 2009; Saadat and Ansari-Lari, 2009). However, Gu et al. (2007) reported a conflicting result, which indicated that the AA genotype of Arg399Gln showed a significant association with a decreased risk of idiopathic azoospermia compared with the GG genotype (OR 0.315, 95% CI 0.12–0.86) in the southern Han population of China. It is speculated that the conflicting result between the present study and Gu et al. (2007) might be attributed to
The sequences for SNP locus rs25487 of XRCC1 from a patient and a control.
406 differences in sample size, ethnic background and geographic variations. At first, sample size is a significant factor affecting the result of case–control association studies. In this study, all samples in idiopathic azoospermia group and control group were in Hardy–Weinberg equilibrium at SNP locus rs25487 of XRCC1. Moreover, in order to avoid the possible occurrence of sampling errors, subjects were selected from a uniform ethnic and geographic background from Shaanxi province. Secondly, ethnic background and geographic variations are play a vital and increasingly role in the study results. The conflicting result between Gu et al. (2007) and this study might be owing to different areas and different population in China. Now there are 56 officially identified ethnic groups in China (Yang et al., 2010), Han nationality constituting about 92% of population of China. The subjects in the study of Gu et al. mainly came from Jiangsu province, situated in the south of China, whereas the present subjects were selected from Shaanxi province, which located in the northwest of China. Shaanxi province is both the birthplace of the Chinese nation and the birthplace of human civilization in Asia and the cultural centre of prehistoric civilization (Zhou et al., 2011a). As a result of its unique geographic characteristics, men from Shaanxi province could therefore be the one of representatives of Chinese Han and may have population characteristics which are distinct from previous studies. In conclusion, this study investigated the association between the SNP locus rs25487 of XRCC1 and idiopathic azoospermia in a northern Chinese Han population using PCR-RFLP. The SNP locus rs25487 of XRCC1 could be a marker for genetic susceptibility to idiopathic azoospermia and the A allele might be a risk gene of idiopathic azoospermia in the northern Chinese Han population, which would provide useful information for anthropological and male reproduction studies. Predictably, with the development of molecular biology techniques and increasing studies with larger sample size and more various ethnic populations, the precise role of XRCC1 in the pathophysiology of idiopathic azoospermia will be clarified.
Acknowledgement The authors would like to thank all the subjects who participated in this study. In addition, they thank all the interviewers and all technologists for their co-operation and contribution for this study. This research was financially supported by National Natural Science Funding of China (30700654), Science Technical Development Project Funding of Shaanxi Province (2005K15-G2) and The Fundamental Research Funds for the Central University (XJJ 2011024).
References Ahmed, E.A., de Boer, P., Philippens, M.E., Kal, H.B., de Rooij, D.G., 2010. Parp1-XRCC1 and the repair of DNA double strand breaks in mouse round spermatids. Mutat. Res. 683, 84–90. El-Domyati, M.M., Al-Din, A.B., Barakat, M.T., El-Fakahany, H.M., Xu, J., Sakkas, D., 2009. Deoxyribonucleic acid repair and apoptosis in testicular germ cells of aging fertile men: the role of the poly(adenosine diphosphate-ribosyl)ation pathway. Fertil. Steril. 91, 2221–2229.
L-r Zheng et al. Gu, A.H., Liang, J., Lu, N.X., Wu, B., Xia, Y.K., Lu, C.C., Song, L., Wang, S.L., Wang, X.R., 2007. Association of XRCC1 gene polymorphisms with idiopathic azoospermia in a Chinese population. Asian J. Androl. 9, 781–786. Hargreave, T.B., 2000. Genetic basis of male fertility. Br. Med. Bull. 56, 650–671. Hartwig, A., 2010. The role of DNA repair in benzene-induced carcinogenesis. Chem. Biol. Interact. 184, 269–272. Hsia, K.T., Millar, M.R., King, S., Selfridge, J., Redhead, N.J., Melton, D.W., Saunders, P.T., 2003. DNA repair gene Ercc1 is essential for normal spermatogenesis and oogenesis and for functional integrity of germ cell DNA in the mouse. Development 130, 369–378. Huynh, T., Mollard, R., Trounson, A., 2002. Selected genetic factors associated with male infertility. Hum. Reprod. Update 8, 183–198. Langsenlehner, T., Renner, W., Gerger, A., Hofmann, G., Thurner, E.M., Kapp, K.S., Langsenlehner, U., 2011. Association between single nucleotide polymorphisms in the gene for XRCC1 and radiation-induced late toxicity in prostate cancer patients. Radiother. Oncol. 98, 387–393. Li, H., Ha, T.C., Tai, B.C., 2009. XRCC1 gene polymorphisms and breast cancer risk in different populations: a meta-analysis. Breast 18, 183–191. Matsuzaka, Y., Makino, S., Okamoto, K., Oka, A., Tsujimura, A., Matsumiya, K., Takahara, S., Okuyama, A., Sada, M., Gotoh, R., Nakatani, T., Ota, M., Katsuyama, Y., Tamiya, G., Inoko, H., 2002. Susceptibility locus for idiopathic azoospermia is localized within the HLA-DR/DQ subregion: primary role of DQB1*0604. Tissue Antigens 60, 53–63. O’Flynn O’Brien, K.L., Varghese, A.C., Agarwal, A., 2010. The genetic causes of male factor infertility: a review. Fertil. Steril. 93, 1–12. Poongothai, J., Gopenath, T.S., Manonayaki, S., 2009. Genetics of human male infertility. Singapore Med. J. 50, 336–347. Qu, T., Morimoto, K., 2005. X-ray repair cross-complementing group 1 polymorphisms and cancer risks in Asian populations: a mini review. Cancer Detect. Prev. 29, 215–220. Rubes, J., Rybar, R., Prinosilova, P., Veznik, Z., Chvatalova, I., Solansky, I., Sram, R.J., 2010. Genetic polymorphisms influence the susceptibility of men to sperm DNA damage associated with exposure to air pollution. Mutat. Res. 683, 9–15. Saadat, M., Ansari-Lari, M., 2009. Polymorphism of XRCC1 (at codon 399) and susceptibility to breast cancer, a meta-analysis of the literatures. Breast Cancer Res. Treat. 115, 137–144. Saadat, M., Kohan, L., Omidvari, S., 2008. Genetic polymorphisms of XRCC1 (codon 399) and susceptibility to breast cancer in Iranian women, a case-control study. Breast Cancer Res. Treat. 111, 549–553. Sreeja, L., Syamala, V.S., Syamala, V., Hariharan, S., Raveendran, P.B., Vijayalekshmi, R.V., Madhavan, J., Ankathil, R., 2008. Prognostic importance of DNA repair gene polymorphisms of XRCC1 Arg399Gln and XPD Lys751Gln in lung cancer patients from India. J. Cancer Res. Clin. Oncol. 134, 645–652. Thompson, L.H., West, M.G., 2000. XRCC1 keeps DNA from getting stranded. Mutat. Res. 459, 1–18. Tebbs, R.S., Flannery, M.L., Meneses, J.J., Hartmann, A., Tucker, J.D., Thompson, L.H., Cleaver, J.E., Pedersen, R.A., 1999. Requirement for the Xrcc1 DNA base excision repair gene during early mouse development. Dev. Biol. 208, 513–529, 15. Yang, G., Deng, Y.J., Qin, H., Zhu, B.F., Chen, F., Shen, C.M., Sun, Z.M., Chen, L.P., Wu, J., Mu, H.F., Lucas, R., 2010. HLA-B*15 subtypes distribution in Han population in Beijing, China, as compared with those of other populations. Int. J. Immunogenet. 37, 205–212. Yang, Y., Xiao, C.Y., A, Z.C., Zhang, S.Z., Li, X., Zhang, S.X., 2006. DAZ1/DAZ2 cluster deletion mediated by gr/gr recombination per se may not be sufficient for spermatogenesis impairment: a
XRCC1 and idiopathic azoospermia study of Chinese normozoospermic men. Asian J. Androl. 8, 183–187. Zhang, J., Qiu, S.D., Li, S.B., Zhou, D.X., Tian, H., Huo, Y.W., Ge, L., Zhang, Q.Y., 2007. Novel mutations in ubiquitin-specific protease 26 gene might cause spermatogenesis impairment and male infertility. Asian J. Androl. 9, 809–814. Zhang, J., Zhou, D.X., Wang, H.X., Tian, Z., 2011. An association study of SPO11 gene single nucleotide polymorphisms with idiopathic male infertility in Chinese Han population. J. Assist. Reprod. Genet. 28, 731–736. Zhou, D.X., Zhang, J., Wang, H.X., Wang, X.F., Tian, Z., Zhao, W.B., Han, S.P., Zhang, J., Huo, Y.W., Tian, H., 2011a. Association study of HLA-B alleles with idiopathic male infertility in Han population of China. J. Assist. Reprod. Genet. 28, 979–985.
407 Zhou, D.X., Wang, X.F., Zhang, J., Wang, H., Tian, Z., 2011b. Association study of human leucocyte antigen-A gene with idiopathic male infertility in Han population of China. Andrologia. http://dx.doi.org/10.1111/j.1439-0272.2011.01166.x. Zini, A., Phillips, S., Lefebvre, J., Baazeem, A., Bissonnette, F., Kadoch, I.J., Gabriel, M.S., 2010. Anti-sperm antibodies are not associated with sperm DNA damage: a prospective study of infertile men. J. Reprod. Immunol. 85, 205–208. Declaration: The authors report no financial or commercial conflicts of interest. Received 12 March 2012; refereed 20 June 2012; accepted 21 June 2012.
www.sciencedirect.com www.rbmonline.com
ARTICLE
Association between XRCC1 single-nucleotide polymorphisms and infertility with idiopathic azoospermia in northern Chinese Han males Lie-rui Zheng a,d, Xiao-fang Wang b, Dang-xia Zhou Yong-wei Huo c, Hong Tian c
a,d,*
, Jing Zhang a,
a Department of Pathology, Medical School, Xi’an Jiaotong University, Xi’an 710061, China; b Shaan’xi Blood Center, Xi’an 710061, China; c Reproductive Medical Center, Xi’an Jiaotong University, Xi’an 710061, China; d Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an 710061, China
* Corresponding author. E-mail address: [email protected] (D-x Zhou). Dang-xia Zhou, associate professor, works in the pathology department at the medical school of Xi’an Jiaotong University. She received her MSc in pathology in 2002 and PhD in histology and embryology in 2006. Her research interest is the genetic aetiology and pathogenesis of male infertility.
Abstract X-ray repair cross-complementing group 1 (XRCC1) is a scaffold protein that plays a critical role in DNA base excision
repair. To explore the association between XRCC1 single-nucleotide polymorphisms and infertility with idiopathic azoospermia in a northern Chinese Han population, PCR restriction fragment length polymorphism was used to genotype a SNP locus (rs25487) of XRCC1 in 112 patients with idiopathic azoospermia and 156 healthy controls. Furthermore, nucleotide sequences were sequenced. The results showed that, compared with GG genotype, the GA and GA + AA genotypes showed a significant association with an increased risk of idiopathic azoospermia (OR 2.119, 95% CI 1.245–3.606, P = 0.005), (OR 2.052, 95% CI 1.227–3.431, P = 0.006) respectively. Meanwhile, the A allele frequency was significantly higher in azoospermic patients than that in controls (OR 1.472, 95% CI 1.029–2.105, P = 0.034). The substitutions bring about an amino acid alteration: G fi A changes the arginine residue into glutamine. In conclusion, the SNP locus rs25487 of XRCC1 could be a marker for genetic susceptibility to idiopathic azoospermia and the A allele might be a risk gene of idiopathic azoospermia in the northern Chinese Han population. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: idiopathic azoospermia, restriction fragment length polymorphism, single-nucleotide polymorphism, XRCC1
1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2012.06.014
XRCC1 and idiopathic azoospermia
Introduction Infertility is a huge problem that affects 10–15% of couples world-wide, in approximately 50% of cases the problem being due to male factor infertility partner (Huynh et al., 2002). Although several causes have been proposed for aetiologies of male infertility, unfortunately, nearly 50% of male infertility is unexplained or idiopathic (Zhang et al., 2007). As one of important types of idiopathic male infertility (Zhang et al., 2007), idiopathic azoospermia is characterized by no spermatozoa in semen and its causes is unclear (Matsuzaka et al., 2002). Recent studies suggest that impaired spermatogenesis is an essential aetiology of male infertility and that genetic disorders affecting spermatogenesis might be responsible for many cases of idiopathic infertility (Yang et al., 2006). It is well known that spermatogenesis is a complex, multifactor, multistep physiological process, regulated by multiple genes. Other than Y-chromosome microdeletion, more recent studies have suggested that polymorphism of genes in autosomal chromosomes may also play an important role in male infertility (Poongothai et al., 2009; Zhang et al., 2011). All kinds of endogenous and exogenous environmental factors, such as physical (anatomical abnormality, hyperthermia), chemical (drugs, cigarette smoking) and biological damage, can give rise to human cellular DNA damage, and the germ cell is no exception. The absence or decrease of DNA repair ability could increase the risk of germ cell genetic mutation and cause failure of spermatogenesis, thus resulting in azoospermia and oligozoospermia (Hsia et al., 2003). Fortunately, an integrated DNA repair pathway (include various DNA repair genes) exists in the human genome, which can repair a series of DNA damage in somatic and germ cells. The DNA repair system can be categorized into: (i) nucleotide-excision repair (NER); (ii) base-excision repair (BER); (iii) mismatch repair (MMR); (iv) DNA strand-break repair (DSBR); and (v) direct repair (DR). Of all of these, NER and BER are the most common (Hartwig, 2010). X-ray repair cross-complementing group 1 (XRCC1) is a scaffold protein that interacts with several DNA repair proteins and plays a critical role in BER. XRCC1 contains 17 exons and is located on chromosome 19q13.2 (Langsenlehner et al., 2011). Large number of studies reported that the single-nucleotide polymorphisms (SNP) in XRCC1 may be associated with the change of DNA repair capacity, which may affect genetic susceptibility to complex disease and the sensitivity of individuals to treatment. It has been shown that polymorphisms in the XRCC1 SNP locus rs25487 (codon 399, exon 10, base G fi A, amino acid Arg fi Gln) may increase the risks of lung cancer, bladder cancer, breast cancer and other diseases (Saadat et al., 2008; Sreeja et al., 2008; Qu and Morimoto, 2005). Recent studies have suggested that XRCC1 is highly expressed in spermatogenic and Sertoli cells of mouse testis (Ahmed et al., 2010). XRCC1 is also expressed in early elongating spermatids and is involved in the extensive chromatin remodelling in these cells; meanwhile, it has significant DNA repair capability (Ahmed et al., 2010; El-Domyati et al., 2009). XRCC1 knockout in mice results in embryonic lethality, demonstrating it to be essential for development (Tebbs et al., 1999). It means that XRCC1 may play a key role in
403 spermatogenesis and DNA repair of germ cells. Another study described XRCC1 as being most abundant in pachytene spermatocytes as well as in round spermatids and suggested that it might maintain spermatogenesis by repairing DNA damage during meiosis in germ cells (Gu et al., 2007). However, as far as is known, there are few studies concerning the association of XRCC1 and idiopathic azoospermia. Therefore, the current study aimed to investigate the potential association between XRCC1 SNP and idiopathic azoospermia in a northern Chinese Han population using PCR restriction fragment length polymorphism (RFLP) method. These results will not only provide useful information for anthropological researches and population genetics but also a scientific basis for male reproduction studies (Zhou et al., 2011a).
Materials and methods Selection of subjects The protocol was fully approved by the Institutional Medical Ethics Committee (reference number 2008051) on 6 March 2008. All subjects were randomly selected from Chinese Han population in the Shaanxi Province, China. The risks and benefits of participating in the study were explained to all subjects. According to the methods of previous studies (Zhou et al., 2011a,b), the infertile patients were selected from couples attending the infertility clinic who had a history of infertility of 12 months. After obtaining the informed consent of patients, a detailed medical and reproductive history was obtained from all subjects, including reproductive history and infertility evaluation of the female partner. World Health Organization criteria (2010) were used to define semen parameters for all subjects who were examined. Finally, 112 azoospermia patients (age 30.41 ± 3.58 years) were selected for infertility samples, excluding the following factors such as immune (antisperm antibody IgG, IgA), infection (mycoplasma, chlamydia 1), biochemical abnormality (a1–4 glucosidase, acid phosphatase, fructose) and anatomical abnormality. Patients with chromosomal abnormalities and microdelections in the AZF region on Y chromosome were also excluded. At the same time, 156 healthy Han, married, male volunteers who had at least one child and lived in the same place of residence were recruited as the control group (age 30.28 ± 3.16 years).
Genotype analysis by PCR-RFLP After informed consent, 2 ml noncoagulated EDTA blood samples were collected and stored at 20C before DNA extraction. Genomic DNA was extracted from peripheral white blood cells using a DNA extraction kit (A004-1; Dinguo, Beijing, China). The polymorphisms of the XRCC1 SNP locus rs25487 were determined using the PCR-RFLP method. The primers used were: forward, 50 -TCACACCTAACTGGCATC TTCACT-30 ; reverse, 50 -CTCCTTCCCTCATCTGGAGTACC-30 . PCR was carried out in 20-ll reaction volumes [containing 7 ll PCR mix (10 · Taq buffer with (NH4)2SO4, 0.2 mmol/l dNTP, 1.5 mmol/l MgCl2, 0.5 lmol/l of each
404 primer, 2.5 U Taq DNA polymerase (Fermentas, Lithuania)], 1 ll of each primer, 9 ll ddH2O, and 2 ll genomic DNA (approximately 50 ng DNA template). Thermal cycling was performed using a MyCycler Thermal Cycler (Bio-Rad) with the following program: 95C for 5 min and 35 cycles at 95C for 30 s, 64C for 45 s and 72C for 1 min, followed by final extension at 72C for 10 min. The products were detected on 1% agarose gel (Figure 1). The restriction enzyme MspI (Fermentas, Lithuania) was used to distinguish SNP. In brief, 3 ll PCR product was digested with 0.3 ll of MspI at 37C for 14 h. The fragments of PCR-RFLP were detected on a 2% agarose gel (Figure 2). At the SNP locus rs25487, the GG genotype produces two MspI restriction fragments of 177 and 153 bp; the GA genotype produces three fragments of 330, 177 and 153 bp and the AA genotype produces only one 330 bp fragment. Finally, genotypes of randomly selected subjects, as determined by PCR-RFLP, were confirmed by direct resequencing in an ABI PRISM 3730 autosequencer. All the oligonucleotides were synthesized and sequenced by Sangon Biotech (Shanghai, China).
Statistical analysis Hardy–Weinberg equilibrium of the XRCC1 locus was estimated using Modified Powerstates (Promega Corporation, USA). Allele frequencies and all analyses related to the case–control study were calculated using the Statistical Package for Social Sciences version 13.0 (SPSS, Chicago, USA). The frequencies of XRCC1 alleles were compared between controls and patients with idiopathic azoospermia using the Chi-squared test. Fisher’s Exact test was applied when one of expected numbers was less than 5. Relative risk was calculated as an odds ratio (OR) with 95% confidence interval (CI) using Cornfield’s approximation. P < 0.05 was regarded as statistically significant.
Results Subjects In total, 112 idiopathic azoospermic patients were diagnosed as male infertility after semen analysis and
Figure 1 PCR products of XRCC1. Fragments were analysed on a 1% agarose gel with ethidium bromide. Lengths of product fragments are approximately 330 bp. M = DNA size marker; lanes 1–7 = PCR products of seven azoospermic patients.
L-r Zheng et al.
Figure 2 Restriction fragment length polymorphism RFLP for XRCC1 in different azoospermic patients. Fragments were analysed on a 2% agarose gel with ethidium bromide. M = DNA size marker; lanes 1 and 2 = AA genotype, one fragment of 330 bp; lanes 3–5 = GG genotype, two fragments of 177 and 153 bp; lanes 6 and 7 = GA genotype, three fragments of 330, 177 and 153 bp (lanes 6 and 7).
examination of infertility factors. The mean ± SD ages of azoospermic patients and controls were 30.41 ± 3.58 and 30.28 ± 3.16 years, respectively, and there was no significant difference between two groups. All azoospermic patients (v2 = 3.268, df = 2) and controls (v 0.043, df = 2) were in compliance with Hardy–Weinberg equilibrium at XRCC1.
Mutation detection All genotypes and allele frequencies of SNP locus rs25487 of XRCC1 are summarized in Tables 1 and 2. The sequences from a patient and a control are shown in Figure 3. In SNP locus rs25487 of XRCC1, genotype changes (G/G fi G/A) were found in 67 (59.8%) out of 112 azoospermic patients and in 69 (44.2%) out of 156 controls, at the same time (G/G fi A/A) were found in 12 (10.7%) out of 112 azoospermic patients and in 15 (9.6%) out of 156 controls. The substitutions bring about an amino acid alteration: G fi A changes the arginine residue (Arg) into glutamine (Gln). As shown in Table 1, the GA genotype of XRCC1, leading to Arg399Gln, showed a significant association with a increased risk of idiopathic azoospermia compared with the GG genotype (OR 2.119, 95% CI 1.245–3.606, P = 0.005), and the GA + AA genotype also demonstrated a significant association with a increased risk of idiopathic azoospermia compared with the GG genotype (OR 2.052, 95% CI 1.227–3.431, P = 0.006). Meanwhile, the A allele frequency of Arg399Gln (G fi A) was significantly higher in infertile patients with azoospermia than that in controls (OR 1.472, 95% CI 1.029–2.105, P = 0.034; Table 2), which suggested that the A allele of Arg399Gln (G fi A) may increase the infertile risk.
Discussion This study found an association of the SNP locus rs25487 of XRCC1 with idiopathic azoospermia, indicating that A allele
XRCC1 and idiopathic azoospermia
405
Table 1 The genotypes of SNP locus rs25487 of XRCC1 (codon 399, base G fi A, amino acid Arg fi Gln) for patients and controls. Genotype
Patients (n = 112)
Controls (n = 156)
Pvalue
OR (95% CI)
GG GA
33 (29.5) 67 (59.8)
72 (46.2) 69 (44.2)
0.020
AA
12 (10.7)
15 (9.6)
GG GA + AA
33 (29.5) 79 (70.5)
72 (46.2) 84 (53.8)
1.00 2.119 (1.245– 3.606), P = 0.005 1.745 (0.736– 4.140) 1.00 2.052 (1.227– 3.431)
0.006
Values are n (%). Two-sided v2 test. P < 0.05 was considered to be significant.
Table 2
The allele frequencies of SNP locus rs25487 of XRCC1.
Allele
Patients (n = 112)
Controls (n = 156)
Pvalue
OR (95% CI)
G A
133 (59.4) 91 (40.6)
213 (68.3) 99 (31.7)
0.034
1.00 1.472 (1.029– 2.105)
Values are n (%). Two-sided v2 test. P < 0.05 was considered to be significant.
might be a risk gene of idiopathic azoospermia in the northern Chinese Han population. Sperm DNA integrity is a prerequisite for normal sperm function. Sperm DNA must be correctly programmed and repaired to successfully pass on genetic and epigenetic information to the developing embryo (O’Flynn et al., 2010). Sperm DNA damage is easily incurred by the endogenous and exogenous factors, such as intrinsic testicular factors, protamine deficiency, apoptosis, drugs, chemotherapy, radiotherapy, testicular hyperthermia, cigarette smoking, varicoceles, genital tract infection and inflammation and environmental toxins (Zini et al., 2010). XRCC1 is involved in the BER pathway, which is essential in repairing DNA damage (Ahmed et al., 2010; El-Domyati et al., 2009;
Figure 3
Langsenlehner et al., 2011; Tebbs et al., 1999; Thompson and West, 2000). The processes of spermatogenesis require an estimated 2000 genes that function in numerous pathways controlling spermatogenesis as well as development and maintenance of the testis (Hargreave, 2000). It will inevitably need DNA repair genes to participate to exercise its normal function for spermatogenesis when these genes are damaged. As an important DNA repair system of human genome, including XRCC1 and its polymorphism is valuable in disease-association studies (Rubes et al., 2010). As one of the important members of DNA repair system, abundant studies have reported about the association between SNP locus rs25487 of XRCC1 with human disease (mostly about the cancer research). XRCC1 is highly conservatively expressed in spermatogenic cells of testis (Ahmed et al., 2010). It also plays an important role in maintaining normal spermatogenesis by repairing DNA damages during meiosis process (Gu et al., 2007). Owing to the valuable role for spermatogenesis, mutations or polymorphisms in XRCC1 may contribute to susceptibility to spermatogenesis impairment. The present study demonstrated that the SNP locus rs25487 of XRCC1 has variant genotypes (GA, GA + AA) associated with a higher risk of idiopathic azoospermia. The A allele in SNP locus rs25487 of XRCC1 seemed to be the risk allele for idiopathic zoospermia and it is speculated that G fi A in SNP locus rs25487 of XRCC1 may alter its DNA repair capacity and be associated with spermatogenesis failure. The present results are consistent with several previous cancer studies, such as the study of Sreeja et al. (2008), which indicated that the carriers of XRCC1 codon 399 AA genotypes were at higher risk of lung cancer (OR 2.1, 95% CI 1.224–3.669, P = 0.007) than carriers of GG genotype. The studies of Saadat and Ansari-Lari (2009) and Li et al. (2009) reported that the A allele of XRCC1 codon 399 might act as a recessive allele in its association with breast cancer risk in Asian countries (Li et al., 2009; Saadat and Ansari-Lari, 2009). However, Gu et al. (2007) reported a conflicting result, which indicated that the AA genotype of Arg399Gln showed a significant association with a decreased risk of idiopathic azoospermia compared with the GG genotype (OR 0.315, 95% CI 0.12–0.86) in the southern Han population of China. It is speculated that the conflicting result between the present study and Gu et al. (2007) might be attributed to
The sequences for SNP locus rs25487 of XRCC1 from a patient and a control.
406 differences in sample size, ethnic background and geographic variations. At first, sample size is a significant factor affecting the result of case–control association studies. In this study, all samples in idiopathic azoospermia group and control group were in Hardy–Weinberg equilibrium at SNP locus rs25487 of XRCC1. Moreover, in order to avoid the possible occurrence of sampling errors, subjects were selected from a uniform ethnic and geographic background from Shaanxi province. Secondly, ethnic background and geographic variations are play a vital and increasingly role in the study results. The conflicting result between Gu et al. (2007) and this study might be owing to different areas and different population in China. Now there are 56 officially identified ethnic groups in China (Yang et al., 2010), Han nationality constituting about 92% of population of China. The subjects in the study of Gu et al. mainly came from Jiangsu province, situated in the south of China, whereas the present subjects were selected from Shaanxi province, which located in the northwest of China. Shaanxi province is both the birthplace of the Chinese nation and the birthplace of human civilization in Asia and the cultural centre of prehistoric civilization (Zhou et al., 2011a). As a result of its unique geographic characteristics, men from Shaanxi province could therefore be the one of representatives of Chinese Han and may have population characteristics which are distinct from previous studies. In conclusion, this study investigated the association between the SNP locus rs25487 of XRCC1 and idiopathic azoospermia in a northern Chinese Han population using PCR-RFLP. The SNP locus rs25487 of XRCC1 could be a marker for genetic susceptibility to idiopathic azoospermia and the A allele might be a risk gene of idiopathic azoospermia in the northern Chinese Han population, which would provide useful information for anthropological and male reproduction studies. Predictably, with the development of molecular biology techniques and increasing studies with larger sample size and more various ethnic populations, the precise role of XRCC1 in the pathophysiology of idiopathic azoospermia will be clarified.
Acknowledgement The authors would like to thank all the subjects who participated in this study. In addition, they thank all the interviewers and all technologists for their co-operation and contribution for this study. This research was financially supported by National Natural Science Funding of China (30700654), Science Technical Development Project Funding of Shaanxi Province (2005K15-G2) and The Fundamental Research Funds for the Central University (XJJ 2011024).
References Ahmed, E.A., de Boer, P., Philippens, M.E., Kal, H.B., de Rooij, D.G., 2010. Parp1-XRCC1 and the repair of DNA double strand breaks in mouse round spermatids. Mutat. Res. 683, 84–90. El-Domyati, M.M., Al-Din, A.B., Barakat, M.T., El-Fakahany, H.M., Xu, J., Sakkas, D., 2009. Deoxyribonucleic acid repair and apoptosis in testicular germ cells of aging fertile men: the role of the poly(adenosine diphosphate-ribosyl)ation pathway. Fertil. Steril. 91, 2221–2229.
L-r Zheng et al. Gu, A.H., Liang, J., Lu, N.X., Wu, B., Xia, Y.K., Lu, C.C., Song, L., Wang, S.L., Wang, X.R., 2007. Association of XRCC1 gene polymorphisms with idiopathic azoospermia in a Chinese population. Asian J. Androl. 9, 781–786. Hargreave, T.B., 2000. Genetic basis of male fertility. Br. Med. Bull. 56, 650–671. Hartwig, A., 2010. The role of DNA repair in benzene-induced carcinogenesis. Chem. Biol. Interact. 184, 269–272. Hsia, K.T., Millar, M.R., King, S., Selfridge, J., Redhead, N.J., Melton, D.W., Saunders, P.T., 2003. DNA repair gene Ercc1 is essential for normal spermatogenesis and oogenesis and for functional integrity of germ cell DNA in the mouse. Development 130, 369–378. Huynh, T., Mollard, R., Trounson, A., 2002. Selected genetic factors associated with male infertility. Hum. Reprod. Update 8, 183–198. Langsenlehner, T., Renner, W., Gerger, A., Hofmann, G., Thurner, E.M., Kapp, K.S., Langsenlehner, U., 2011. Association between single nucleotide polymorphisms in the gene for XRCC1 and radiation-induced late toxicity in prostate cancer patients. Radiother. Oncol. 98, 387–393. Li, H., Ha, T.C., Tai, B.C., 2009. XRCC1 gene polymorphisms and breast cancer risk in different populations: a meta-analysis. Breast 18, 183–191. Matsuzaka, Y., Makino, S., Okamoto, K., Oka, A., Tsujimura, A., Matsumiya, K., Takahara, S., Okuyama, A., Sada, M., Gotoh, R., Nakatani, T., Ota, M., Katsuyama, Y., Tamiya, G., Inoko, H., 2002. Susceptibility locus for idiopathic azoospermia is localized within the HLA-DR/DQ subregion: primary role of DQB1*0604. Tissue Antigens 60, 53–63. O’Flynn O’Brien, K.L., Varghese, A.C., Agarwal, A., 2010. The genetic causes of male factor infertility: a review. Fertil. Steril. 93, 1–12. Poongothai, J., Gopenath, T.S., Manonayaki, S., 2009. Genetics of human male infertility. Singapore Med. J. 50, 336–347. Qu, T., Morimoto, K., 2005. X-ray repair cross-complementing group 1 polymorphisms and cancer risks in Asian populations: a mini review. Cancer Detect. Prev. 29, 215–220. Rubes, J., Rybar, R., Prinosilova, P., Veznik, Z., Chvatalova, I., Solansky, I., Sram, R.J., 2010. Genetic polymorphisms influence the susceptibility of men to sperm DNA damage associated with exposure to air pollution. Mutat. Res. 683, 9–15. Saadat, M., Ansari-Lari, M., 2009. Polymorphism of XRCC1 (at codon 399) and susceptibility to breast cancer, a meta-analysis of the literatures. Breast Cancer Res. Treat. 115, 137–144. Saadat, M., Kohan, L., Omidvari, S., 2008. Genetic polymorphisms of XRCC1 (codon 399) and susceptibility to breast cancer in Iranian women, a case-control study. Breast Cancer Res. Treat. 111, 549–553. Sreeja, L., Syamala, V.S., Syamala, V., Hariharan, S., Raveendran, P.B., Vijayalekshmi, R.V., Madhavan, J., Ankathil, R., 2008. Prognostic importance of DNA repair gene polymorphisms of XRCC1 Arg399Gln and XPD Lys751Gln in lung cancer patients from India. J. Cancer Res. Clin. Oncol. 134, 645–652. Thompson, L.H., West, M.G., 2000. XRCC1 keeps DNA from getting stranded. Mutat. Res. 459, 1–18. Tebbs, R.S., Flannery, M.L., Meneses, J.J., Hartmann, A., Tucker, J.D., Thompson, L.H., Cleaver, J.E., Pedersen, R.A., 1999. Requirement for the Xrcc1 DNA base excision repair gene during early mouse development. Dev. Biol. 208, 513–529, 15. Yang, G., Deng, Y.J., Qin, H., Zhu, B.F., Chen, F., Shen, C.M., Sun, Z.M., Chen, L.P., Wu, J., Mu, H.F., Lucas, R., 2010. HLA-B*15 subtypes distribution in Han population in Beijing, China, as compared with those of other populations. Int. J. Immunogenet. 37, 205–212. Yang, Y., Xiao, C.Y., A, Z.C., Zhang, S.Z., Li, X., Zhang, S.X., 2006. DAZ1/DAZ2 cluster deletion mediated by gr/gr recombination per se may not be sufficient for spermatogenesis impairment: a
XRCC1 and idiopathic azoospermia study of Chinese normozoospermic men. Asian J. Androl. 8, 183–187. Zhang, J., Qiu, S.D., Li, S.B., Zhou, D.X., Tian, H., Huo, Y.W., Ge, L., Zhang, Q.Y., 2007. Novel mutations in ubiquitin-specific protease 26 gene might cause spermatogenesis impairment and male infertility. Asian J. Androl. 9, 809–814. Zhang, J., Zhou, D.X., Wang, H.X., Tian, Z., 2011. An association study of SPO11 gene single nucleotide polymorphisms with idiopathic male infertility in Chinese Han population. J. Assist. Reprod. Genet. 28, 731–736. Zhou, D.X., Zhang, J., Wang, H.X., Wang, X.F., Tian, Z., Zhao, W.B., Han, S.P., Zhang, J., Huo, Y.W., Tian, H., 2011a. Association study of HLA-B alleles with idiopathic male infertility in Han population of China. J. Assist. Reprod. Genet. 28, 979–985.
407 Zhou, D.X., Wang, X.F., Zhang, J., Wang, H., Tian, Z., 2011b. Association study of human leucocyte antigen-A gene with idiopathic male infertility in Han population of China. Andrologia. http://dx.doi.org/10.1111/j.1439-0272.2011.01166.x. Zini, A., Phillips, S., Lefebvre, J., Baazeem, A., Bissonnette, F., Kadoch, I.J., Gabriel, M.S., 2010. Anti-sperm antibodies are not associated with sperm DNA damage: a prospective study of infertile men. J. Reprod. Immunol. 85, 205–208. Declaration: The authors report no financial or commercial conflicts of interest. Received 12 March 2012; refereed 20 June 2012; accepted 21 June 2012.