The Possible Role of XRCC1 Gene Polymorphisms with Idiopathic Non-obstructive Azoospermia in Southeast Turkey Halit Akbas1, Mahmut Balkan2*, Mahir Binici2, Abdullah Gedik3 Purpose: X-ray repair cross-complementing group 1 (XRCC1) plays a role in repairing DNA damage during sper- matogenesis. We examined the effects the possible role of two single nucleotide polymorphisms of XRCC1 Arg- 194Trp and Arg399Gln in DNA repair gene XRCC1 with risk of idiopathic non-obstructive azoospermia (INOA) in a south-east Turkey population. Materials and Methods: The genotype and allele frequencies of two observed polymorphisms of XRCC1 Arg- 194Trp and Arg399Gln were examined by polymerase chain reaction-restriction fragment length polymorphism in 102 infertile men with INOA and 102 fertile controls. Result: In our study, all the observed genotype frequencies were in agreement with Hardy-Weinberg equilibrium. The genotype frequencies of the XRCC Arg194Trp were 84% (CC), 16% (CT) and 2% (TT) among the men with INOA, while the frequencies of those genotypes in the controls were found to be 88% (CC), 12% (CT) and 2% (TT) (P < .05). Similarly, the genotypes frequencies of GG, GA, and AA of the XRCC1 Arg399Gln were 44%, 39%, and 19% in the group of men with INOA, whereas these frequencies were 42%, 45%, and 15% in the control group, respectively. No significant difference between the control group and the men with INOA were found in the frequencies of genotypes and allele of XRCC1 Arg194Trp and Arg399Gln (P > 0.05). Conclusion: Neither Arg194Trp nor Arg399Gln polymorphisms in the XRCC1 gene influenced risk of INOA in our study. However, these findings may be helpful in improving the understanding of the etiology of male infer- tility. Keywords: DNA repair; idiopathic azoospermia; male infertility; single-nucleotide polymorphism; XRCC1. INTRODUCTION Male factor infertility is a multifactorial complex disorder that affects about 7% of male from the general population.(1,2) The most common cause of male infertility is impaired spermatogenesis, in which azoo- spermia is present in about 10%–15%.(3) Azoospermia is characterized by no spermatozoa in semen and can be caused by either a physical blockage in the genital track, known as obstructive azoospermia, or spermat- ogenic failure, known as non-obstructive azoospermia. (4) In about 50% of non-obstructive azoospermia, the causes of infertility are unknown and categorized as idiopathic.(5–8) In approximately 15% of idiopathic non-obstructive azoospermia cases (INOA), the eti- ology is related to known genetic disorders including chromosomal aberrations and single gene mutations, such as Y-chromosome microdeletions. However, ap- proximately half of INOA has some unidentified genet- ic basis, and this suggests that polymorphism of genes in autosomal chromosomes may also play an important role in the spermatogenesis.(5–8) Spermatogenesis is regulated by many infertility-relat- 1 Department of Medical Biology and Genetics, Faculty of Medicine, Harran University, Sanliurfa, Turkey. 2 Department of Medical Biology and Genetics, Faculty of Medicine, Dicle University, Diyarbakır, Turkey. 3 Department of Urology, Faculty of Medicine, Dicle University, Diyarbakır, Turkey. *Correspondence: Department of Medical Biology and Genetics, Dicle University, Faculty of Medicine, Diyarbakır 21280, Turkey. Tel: +90 0412 2488001-4638 Fax: +90 0412 2488523. E-mail: balkanmah@gmail.com. Received March 2018 & Accepted November 2018 ed genes which is about 10% in the genome.(9) Up to the present, approximately 150 DNA repair genes have been identified, and most of them are known to have ge- netic variations in humans.(6) Among them, X-ray repair cross-complementing group 1 (XRCC1) is a well-stud- ied DNA repair gene. It encodes a protein that interacts with several DNA repair proteins and plays a critical role in base excision repair (BER) pathway. XRCC1 is located on chromosome 19q13.2 and contains 17 ex- ons.(5, 8) Many studies have been reported that the sin- gle-nucleotide polymorphisms (SNPs) in XRCC1 may be associated with the change of the DNA damage-re- pair response, which may be risk factor for various complex diseases such as cancer.(10) XRCC1 knockout in mice has shown that XRCC1 is the most abundant gene in pachytene spermatocytes as well as in round spermatids, and it is suggested that this might main- tain spermatogenesis by repairing DNA damage during meiosis in germ cells. However, there have been only a few studies so far that examine the association between the XRCC1 polymorphisms and the risk of male infer- tility in human.(8) Therefore, in the current study, we aimed to investigate the possible association between SEXUAL DYSFUNCTION AND ANDROLOGY Urology Journal/Vol 16 No. 4/ July-August 2019/ pp. 380-385. [DOI: http://dx.doi.org/10.22037/uj.v0i0.4435] two known SNPs of Arg194Trp and Arg399Gln of the XRCC1 gene and INOA in a south-east Turkey pop- ulation. Understanding the molecular mechanism of abnormal spermatogenesis and the genes involved are important in developing both diagnostic tools and treat- ment strategies for male infertility.(9) PATIENTS AND METHODS Study population The total 102 infertile men aged between 22 and 39 were included in this study. All infertile men are diag- nosed with INOA, with at least one year of infertility. All men underwent at least two semen analyses. The semen analysis for sperm concentration, motility and morphology was performed according to the World Health Organization criteria.(11) Inclusion criteria for the INOA group were primary infertility; absence of any known causes of infertility; clinical eugonadism; azoo- spermia and normal karyotype. Individuals with known causes of infertility, including genetic factors (e.g. kar- yotyping, and Y-chromosome microdeletion screen- ing), lifestyle factors (e.g. alcoholism and occupation), clinical factors affecting the fertility (varicocele, cryp- torchidism and infections, etc.) and men whose partner had factors involved in infertility were excluded from this study. The control group was consisted of 102 fer- tile controls with their ages ranging from 24 to 41 years. The controls were selected from fertile men who had at least one child without assisted reproductive technolo- gies and had normal semen sperm parameters, and all the control cases had the normal karyotype. Both the in- fertile men and the fertile controls were recruited within the same geographical region in the Southeastern Ana- tolia Region of Turkey. All studied men were referred from the Urology Depart- ment to the Medical Biology and Genetics Department at Dicle University Hospital. The study was approved by the Ethics Review Board of Dicle University’s Fac- ulty of Medicine (reference number 87/26.02.2016). SNPs selection and genotyping of XRCC1 gene polymorphisms In the present study, for genotyping, we selected two known SNPs of the XRCC1 gene; Arg194Trp in exon 6 (rs1799782, NG_033799.1:g.27157C>T, NM_006297.2:c.580C>T ) and Arg399Gln in exon 10 (rs25487, NG_033799.1:g.29005A>G, NM_006297.2:c.1196A>G), which can alter DNA re- pair capacity. SNPs were selected from the HapMap project and PubMed (http://www.ncbi.nlm.nih.gov/ pubmed). The SNP ID number and detailed sequence information are available in the public SNP database(6). After informed consent from each subject, 2 mL hep- arinised peripheral venous blood was collected using a vacuum tube containing ethylenediaminetetra ace- tic (EDTA) to prevent coagulation. All samples were stored in tubes at -20°C until the DNA extraction. Genomic DNA was extracted from whole blood using whole blood genomic DNA purification kit (Thermo Scientific, St. Leon-Rot, Germany) explained in our previous study,(12) then was stored at −80°C until using Table 1. Primer sequences, annealing temperature, restriction enzyme and allele sizes used for Arg194Trp and Arg399Gln polymor- phisms of XRCC1 gene. NCBI Primer sequences Annealing temperature (°C) Restriction enzyme Allele size SNP* rs 1799782; Arg194Trp (580C>T) F: 5´- GCCAGGGCCCCTCCTTCAA-3´ R: 5´- TACCCTCAGACCCACGAGT-3´ 57 PvuII C:485 T:396+89 rs25487; Arg399Gln (1196G>A) F: 5´-TTG TGC TTT CTC TGT GTC CA-3´ R: 5´-TCC TCC AGC CTT TTC TGA TA-3´ 68 MspI A: 615 G:374+221 *www.ncbi.nlm.nih.gov/gene Figure 1. PCR-RFLP products of XRCC1 gene Arg194Trp and Arg399Gln polymorphisms obtained by 3% agarose gel electrophoresis. (A) Arg194Trp polymorphism; lanes 1,2: homozygous CC alleles; lanes 3,4: heterozygous CT alleles; Lanes 5,6: homozygous TT alleles. (B) Arg399Gln polymorphisms; lanes 1,2: homozygous AA alleles; lanes 3,4: heterozygous GA alleles; Lanes 5,6: homozygous AA alleles. XRCC1 Gene Polymorphisms and Azoospermia-Akbas et al. Sexual Dysfunction and Andrology 381 Vol 16 No 04 July-August 2019 382 it for genotyping. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to genotype two SNPs of XRCC1, Arg194Trp and Arg399Gln, with the use of appropriate primer sets and restriction enzyme as previously described.(13,14) The primer sets and enzymes were used in this study are shown in Table 1. The PCR reaction was performed in a 20 µL reaction volume containing 1xPCR buffer, 80 ng of DNA, 2 mmol/L MgCl2,0.2 mmol/L of each dNTP (Fermentas, St. Leon-Rot, Germany), 1 unit of Taq DNA Polymer- ase (Fermentas) and 0.2 mmol/L of primer for codon 194 or 0.8 µM primer for codon 399 (Bio Basic Inc., Markham, Canada). A thermal cycler (Senso-Quest labcycler, SensoQuest GmbH, Göttingen,Germany) was used with the follow- ing conditions: 4 min of initial denaturation at 94°C, followed by 30 amplification cycles. Each cycle was consisted of denaturation at 94°C for 30s, annealing at 57°C and 68°C (for codon 194 and codon399, respec- tively) for 30 sand extension at 72°C for 30 s, with a final extension step of incubation at 72°C for 5 min. For genotyping of the Arg194Trp and Arg399Gln SNPs of XRCC1 gene, RFLP analysis was carried out by using the restriction enzymes PvuII and MspI (New England Biolabs, Beverly, MA, USA). PCR products were digested by PvuII and MspI restriction enzymes, respectively, at 37°C overnight. The digested prod- ucts were then separated on a 3% agarose gels (FMC Bioproducts) along with a 100–1500 bp DNA ladder (BioBasic Inc., Markham, Canada) and stained with ethidium bromide. Ethidium bromide- stained gels were analysed using the AlphaImager Imaging System (Al- phaInnotech, San Leandro, CA, USA). The 485 bp fragment of codon 194 yielded a 396 + 89 bp band, acting as an indicator of complete digestion. XRCC1 Arg194Trp genotypes CC (Arg/Arg), CT (Arg/ Trp), and TT (Trp/Trp) generated 485 bp, 485 + 396 bp and 396 bp DNA bands, respectively (Figure 1.A). XRCC1 codon 399 Arg allele generated 2 DNA bands (221 and 374 bp), whereas the variant Gln allele has a single 615 bp uncut band, and the heterozygote (Arg/ Gln) displays all 3 bands (615, 374 and 221 bp) (Figure 1.B). Statistical analysis A goodness-of-fit Chi-square test was used to deter- mine the Hardy-Weinberg equilibrium of the observed genotype frequencies. Statistical significance was de- fined as P < .05 and all statistical tests were two-tailed. The results were expressed as means with standard de- viation (± SD) if the variables were continuous and as percentage if the variables were categorical. All statisti- cal data were obtained using SPSS software (SPSS 11.5 for Windows, SPSS Inc., Chicago, IL, USA). RESULTS In this study, we analyzed the distribution of XRCC1 Arg194Trp and Arg399Gln polymorphisms in a sam- ple of 102 men with INOA and 102 fertile controls in a Turkish population and investigated their possible asso- ciations with INOA. The genotype and allele frequencies of the XRCC Ar- g194Trp and Arg399Gln polymorphisms for the cas- es and controls and their associations with the risk of INOA are shown in Table 2. All observed SNPs were in agreement with HWE (χ2 test: P = .060 and .605, respectively). The genotype frequencies of the XRCC Arg194Trp were 84% (CC), 16% (CT) and 2% (TT) among the men with INOA, while the frequencies of those gen- otypes in the controls were found to be 88% (CC), 12% (CT) and 2% (TT) (χ2 test: P < .05). Similarly, the genotypes frequencies of GG, GA, and AA of the XRCC1 Arg399Gln were 44%, 39%, and 19% in the group of men with INOA, whereas these frequencies were 42%, 45%, and 15% in the control group, respec- tively. However, these differences were not statistically significant among the cases and controls using the P < .05 threshold (P = .611 for Arg194Trp, and P = .064 for Arg399Gln). Table 3 shows comparison of mean values (± SEM) of semen analysis parameters, such as ejaculated volume, sperm count, total motility and normal morphology be- tween fertile (control) and azoospermic group. Semen volume was significantly lower in azoospermic group (P < .001). XRCC1 Gene Polymorphisms and Azoospermia-Akbas et al. Table 2. Genotype distributions and allele frequencies of XRCC1 Arg194Trp (C>T) and Arg399Gln (G>A) polymorphisms in infertile men with idiopathic nonobstructive azoospermia (INOA) and fertile controls. Infertile men N = 102 (%) Controls N = 102 (%) OR 95% CI P-value XRCC1 580C>T (Arg194Trp) Genotype CC 84 (82%) 88 (86%) Reference CT 16 (16%) 12 (12%) 1.39 0.62-3.12 .41 TT 2 (2%) 2 (2%) 1.04 0.14-7.60 .96 Allele C 184 (90%) 188 (92%) Reference T 20 (10%) 16 (8%) 1.27 0.64-2.54 .48 XRCC1 1196G>A (Arg399Gln) Genotype GG 44 (43%) 42 (41%) Reference GA 39 (38%) 45 (44%) 0.82 0.45-1.51 .53 AA 19 (19%) 15 (15%) 1.20 0.54-2.68 .64 Allele G 127 (62%) 129 (63%) Reference A 77 (38%) 75 (37%) 1.04 0.69-1.55 .83 The distribution of the genotypes among the control subjects was in agreement with that predicted under the conditions of Hardy-Wein- berg equilibrium (χ2 test: P = .060 for the Arg194Trp polymorphism and P = .605 for the Arg399Gln polymorphism). DISCUSSION Several single nucleotide polymorphisms have previ- ously been identified as responsible for male infertil- ity. For example; in a case-control study, the possible association of SNPs in the follicle-stimulating hor- mone receptor (FSHR) gene and male infertility have been investigated in south-east Turkey, and the results showed that the FSHR haplotype is not associated with different serum FSH levels. However, it has been showed a different distribution between fertile and in- fertile men.(15) In another study, the association of the methylenetetrahydrofolate reductase (MTHFR), me- thionine synthase reductase (MTRR) and methylene- tetrahydrofolate dehydrogenase (MTHFD1) genes pol- ymorphisms have been investigated in INOA among a population in south-east Turkey. There has been found a synergistic interaction between some polymorphisms. Therefore, this suggested that there has been no individ- ual, but interactive association between four prominent folate metabolism pathway markers and male infertil- ity.(2) Furthermore, Balkan et al.(16) have investigated the association of the SNPs of FAS/FASLG genes in male infertility. Their results suggested that the AA-GG binary genotype for FAS-670A/G SNP might be a ge- netic predisposing factor of INOA among south-eastern Anatolian men. In a recent study, the possible associa- tion of the microRNA-related genes and male infertili- ty have been investigated in a population of south-east Turkey(17), and the results have showed a significant difference between patients and control groups for the individual AA genotype frequency of the GEMIN3 (rs197388) gene. It has indicated that the AA genotype may be considered as indicative of a high predisposition to INOA. Recently, the potential role of the ICAM-1 gene polymorphism has been investigated in male in- fertility with INOA in a Turkish population. It has been found that the E469K polymorphism of ICAM-1 is not posing a risk for INOA.(3) Various studies have shown that the single nucleotide polymorphisms in DNA repair genes affect DNA re- pair capacity, and the absence or decrease of DNA re- pair ability may increase the risk of several syndromes, such as renal disease, cancer, coronary artery disease and other diseases.(18,19) However, very few studies have reported the associations between these polymorphisms in male infertility. In our study, we investigate the as- sociations of two well-characterized polymorphisms (Arg194Trp and Arg399Gln) of XRCC1 gene with risk of INOA in a south-east Turkey population to reveal the possible role of genetic polymorphisms in XRCC1 gene during spermatogenesis. We did not identify any association between Arg194Trp and Arg399Gln pol- ymorphisms and the risk of INOA. Although the as- sociation of the XRCC1 Arg194Trp and Arg399Gln polymorphisms in male infertility has been shown pre- viously,(5–8) as yet, there has been no final conclusion about the association of those polymorphisms in male infertility. For example, Gu et al.(6) has explored the possible role of the XRCC1 Arg399Gln polymorphism in the susceptibility to risk of INOA in a Chinese pop- ulation and found that the AA genotype of Arg399Gln showed a significant association with a increased risk of INOA. These results are consistent with the study of Zheng et al.(8), which indicated that Arg399Gln SNP of XRCC1 gene could be a marker for genetic suscepti- bility to INOA and the A allele might be a risk gene of INOA in Northern Chinese Han population. However, Ghasemi et al.(20) has reported a conflicting result, which indicated that there has been no significant association between XRCC1 Arg399Gln polymorphism and risk of male infertility. In addition, another study investigated the associations of three polymorphisms (T-77C, Arg- 194Trp, and Arg399Gln) in XRCC1 gene with risk of INOA in a Chinese population. They do not have any evidence of involvement of XRCC1 T-77C and Arg- 194Trp polymorphisms in INOA.(7) In another study, the effects of the XRCC1 polymorphisms (T-77C, Arg194Trp, Arg280His, Arg399Gln) on male infertil- ity have been explored in a Chinese population. They do not have any evidence of involvement of XRCC1 T-77C, Arg194Trp, and Arg280His polymorphisms in INOA.(5) In another report, XRCC1 polymorphisms (Arg194Trp, Arg399Gln) and xerodermapigmentosum group D (XPD) polymorphism (Lys751Gln) are inves- tigated whether there was a risk of developing INOA in a Chinese population. They founded that the XPD 751Gln allele was seemed to be a risk allele for azoo- spermia. When combined the XPD 751 Lys/ Gln+Gln/ Gln genotype with the XRCC1 194 Arg/Arg or 399 Arg/Arg genotype, the risk of azoospermia increased. In conclusion, their study showed that the XPD and XRCC1 polymorphisms have contributed to the risk of Parameters Fertile (control) (n = 102) Azoospermic (n = 102) Volume (mL) 3.25 ± 1.37 2.15 ± 1.37 Sperm count (million/mL) 80.35 ± 44.23 0 Total motility (%) 71.16 ± 18.26 0 Normal morphology (%) 63.25 ± 5.49 0 Table 3. Semen analysis parameters of fertile (control) and infertile men with idiopathic nonobstructive azoospermia (INOA). All values are expressed as mean ± SEM. Population Arg194Trp Arg399Gln References Arg399 (%) 399Gln (%) Arg194 (%) 194Trp (%) Chinese 79 21 88 12 [10] Korean 75 25 67 33 [19] Thai 75 25 70 30 [26] Indian 78 22 87 13 [27] German 68 32 93 7 [24] Italian 72 28 92 8 [25] Turkish 66 34 91 9 [21] Turkish 69 31 89 11 [22] Turkish 65 35 94 6 [23] Turkish 63 37 92 8 This study Table 4. The XRCC1 Arg194Trp and Arg399Gln allele frequencies among control groups of various populations. XRCC1 Gene Polymorphisms and Azoospermia-Akbas et al. Sexual Dysfunction and Andrology 383 Vol 16 No 04 July-August 2019 384 developing INOA.(6,7) It is speculated that the results of these studies might be attributed to differences in sam- ple size, ethnic background and geographic variations. There is much evidence in the literature that the fre- quencies of genetic polymorphisms vary among differ- ent populations. In our study, 102 fertile controls were within the same geographical region in the Southeast- ern Anatolia Region of Turkey. The allele frequencies for the Arg399Gln and Arg194Trp variants of XRCC1 gene among various control populations are presented in Table 4. In the present study, the frequencies of these variant alleles were similar to the frequencies reported for other Turkish studies.(21–23) Besides, allele frequen- cies for these variants that found in the present study for Turkish population were quite similar to the frequencies reported for other Caucasian population (German and Italian).(24,25) CONCLUSIONS Our data suggests that the genotype of Arg399Gln and Arg194Trp polymorphisms are not associated with INOA in a Turkish population. Therefore, this does not appear to be responsible for spermatogenic failure in male infertility. Since sample size is a significant factor affecting the result of case–control association studies, more works with large sample size and more various populations are needed to further explore the pathophysiology of these functional SNPs in INOA. In addition, it may be far better to investigate the role of XRCC1 Arg194Trp and Arg399Gln SNPs and their re- lationship to the sperm DNA damage levels in the etio- pathogenesis of INOA. CONFLICT OF INTEREST The authors declare that they have no conflict of inter- est. REFERENCES 1. Yıldırım Y, Ouriachi T, Woehlbier U, et al. Linked homozygous BMPR1B and PDHA2 variants in a consanguineous family with complex digit malformation and male infertility. Eur J Hum Genet. 2018;26:876-85. 2. Balkan M, Atar M, Erdal ME, et al. The possible association of polymorphisms in MTHFR, MTRR, and MTHFD1 genes with male infertility. Int Med J. 2013;4:404–8. 3. Balkan M, Akbas H, Penbegül N, Rustemoğlu A, Yücel İ, Yıldız İ. A possible association between E469K polymorphism of ICAM- 1 gene and nonobstructive azoospermia in southern Turkey. Biotechnol Biotechnol Equip. 2017;31:143–7. 4. Ayhan O, Balkan M, Guven A, et al. Truncating mutations in TAF4B and ZMYND15 causing recessive azoospermia. 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