Abstract
TP53 is a tumor-suppressor gene which is involved in multiple pathways including apoptosis, transcriptional regulation, and cell cycle control. To analyze whether the TP53 gene Arg72Pro polymorphism (rs1042522) is responsible for susceptibility to idiopathic infertility in southeast Chinese Han males, we used the PCR restriction fragment length polymorphism technique to detect the genotype distribution of 361 infertile men (including 212 with non-obstructive azoospermia and 149 with severe oligozoospermia) in comparison with 384 fertile controls. Genotyping was confirmed by DNA sequencing from randomly selected samples. The frequency of rs1042522 indicated an association with risk of idiopathic male infertility under a dominant mode (GG + GC genotypes vs. CC genotype, P = 0.013; χ2 = 6.169; OR = 1.581; 95%CI = 1.1-2.272; df = 1). Comparison of the allele frequencies revealed a significantly higher incidence of Arg allele among azoospermia group compared with controls (P = 0.001; χ2 =10.864; OR = 1.502; 95%CI = 1.177-1.917; df = 1). Our data suggest that the Arg allele was related only to azoospermia, not to severe oligozoospermia (P = 0.133; χ2 = 2.261; OR = 1.23; 95%CI = 0.939-1.611; df = 1). This study indicated that the TP53 gene Arg72Pro polymorphism of the spermatogenic pathway may be associated with idiopathic infertility in southeast Chinese Han males.
Introduction
Human infertility is a worldwide health concern that affects approximately 10-15% of couples, among which about 50% of the cases are due to the male partner [Aston et al. Citation2010]. Several genetic and environmental causes have been associated with male infertility, such as, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, karyotype abnormalities, Y chromosome microdeletions [Dohle et al. Citation2002], working in high temperatures, and exposure to chemical substances [Kenkel et al. Citation2001], etc. However, its etiology remains unknown in most cases which are classified as idiopathic male infertility [Poongothai et al. Citation2009]. Semen quality is requisite for normal fertilization. Absence of sperm in semen and too few sperm cells, referred to as azoospermia and oligozoospermia, are common clinical manifestations of male infertility. Germ cell apoptosis, such as FAS/FASL mediated system, is the normal physiological mechanism during spermatogenesis. However, abnormal expression of apoptotic pathway will affect semen parameters and numbers of spermatogenic cells both in human and mice [Francavilla et al. Citation2000; Jee et al. Citation2010; Sakkas et al. Citation1999].
TP53 is a transcription factor gene located on chromosome 17p13, not only to maintain organismal development and homeostasis but also to suppress tumorigenesis. It can mediate multiple cellular processes including transcription, apoptosis, and cell cycle control. Previous studies have demonstrated essential roles for TP53 in the meiotic process of spermatogenesis [Almon et al. Citation1993]. Mice with reduced levels of TP53 expression have the testicular degenerative syndrome in the spermatogenic pathway [Rotter et al. Citation1993]. Moreover, Bornstein et al. [2011] provided evidence on the use of these mechanisms throughout spermatogenesis and fertility defects that are associated with TP53 deficiency.
The TP53 polymorphism at codon 72 in exon 4, with a transversion of G to C causes an amino acid substitution – Arg (CGC) by Pro (CCC). The Arg72 and Pro72 of TP53 have distinct functions in biochemical and biological protein pathways. Thomas et al. [1999] reported that TP53 gene Arg72Pro polymorphism causes irregular protein expression and impairment of function. Rs1042522 has been extensively found to be associated with cancer susceptibility such as breast cancer [Al-Qasem et al. Citation2012], acute leukemia [Dunna et al. Citation2012], and hepatocellular carcinoma [Yoon et al. Citation2008]. Although the possibility that rs1042522 might contribute to cancer susceptibility has been widely reported, few related research studies have investigated male infertility.
Recently, Mashayekhi and Hadiyan [2012] reported that rs1042522 might be associated with infertility development in Iranian men. Men carrying Arg/Arg genotype presented a higher risk factor of male infertility susceptibility compared with those carrying Arg/Pro or Pro/Pro. A Hospital-based case-control study was conducted to determine the possible association between TP53 gene Arg72Pro polymorphism and idiopathic male infertility in southeast Chinese Han individuals.
Results
The clinical characteristics and frequency distribution of 745 participants are shown in . The observed mean age was 29.8 years old (29.8 ± 3.205), ranging from 25 to 36 years. Infertile men presented significantly younger mean age values when compared with control (P < 0.001). There was a significantly higher percentage of smokers and radiation exposure among case and control groups (P < 0.001, P = 0.003). The frequency of men who had a drinking habit and exposure to high temperature were greater in the case group, but the difference did not reach statistical significance (P = 0.286, P = 0.284, respectively). In addition, there were no significant differences among the cases and controls when comparing their serum testosterone (T) and estradiol (E2 ) (P > 0.05). The follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) levels in infertile men were significantly higher than in controls (P < 0.05).
Table 1. Characteristics of 745 male participants.
The rs1042522 distribution and allele frequencies in 361 cases and 384 controls are displayed in . The rs1042522 was compatible with Hardy-Weinberg equilibrium (P = 0.812 for case group and P = 0.240 for control group, respectively). Frequency of rs1042522 indicated an association with increased risk of idiopathic male infertility under a dominant mode (GG + GC genotypes vs. CC genotype, P = 0.013; χ2 = 6.169; OR = 1.581; 95%CI = 1.1-2.272; df =1). However, the frequency of GC + CC genotypes was lower among cases than controls (62.33% in cases and 70.83% in controls). Comparison of the allele frequencies revealed a significantly higher incidence of the Arg allele among azoospermia group compared with controls (P = 0.001; χ2 = 10.864; OR = 1.502; 95% CI =1.177-1.917; df = 1).
Table 2. Genotype distribution and relative allele frequencies of the TP53 genotypes in 361 cases and 384 controls.
For subgroup analyses in cases, the 361 infertile men were classified as 212 non-obstructive azoospermia and 149 severe oligozoospermia. In the azoospermia group, the Arg allele was found more often than in Controls (P = 0.001; χ2 = 10.864; OR = 1.502; 95% CI = 1.177-1.917; df = 1). Similarly, the frequency of GG + GC genotypes vs. CC genotype in the azoospermia group compared with the controls also indicated a great difference (P = 0.003; χ2 = 8.92; OR = 1.988; 95% CI = 1.26-3.138; df = 1). However, in the severe oligozoospermia group the frequency of Arg allele was not significantly different between the patients and controls (P = 0.133; χ2 =2.261; OR = 1.23; 95%CI = 0.939-1.611; df = 1).
Discussion
TP53 plays key roles in the regulation of spermatogenesis and fertility defects may associate with TP53 deficiency [Bornstein et al. Citation2011]. The correlation between the TP53 gene Arg72Pro polymorphism and the risk for male infertility has not been well studied, and the conclusions of previous studies are contradictory. The present study indicates that rs1042522 may contribute to the pathogenesis of idiopathic infertility in southeast Chinese Han males and the Arg allele seems to be a risk factor.
Our results were in partial agreement with Mashayekhi and Hadiyan [2012], but different from Huang et al. [2012] and Lu et al. [2007], who found no significant difference between the rs1042522 and male infertility. Mashayekhi and Hadiyan [2012] found that men with idiopathic infertility showed an increased frequency of Arg/Arg homozygous genotype, suggesting that the Arg allele (56%) is correlated with possible increased risk of idiopathic infertility in Iranian men, which was similar to our data in a Chinese population (60.53% for Arg allele, P = 0.002). They also found a statistically significant difference between Arg/Pro + Pro/Pro genotypes and Arg/Arg genotype among infertile men compared with controls (P = 0.004; OR: 2.25) [Mashayekhi and Hadiyan Citation2012]. However, in our research the frequency of Arg/Arg homozygous + Arg/Pro genotypes had a significantly higher proportion than Pro/Pro genotype between cases and controls in southeast China (P = 0.013; OR: 1.581), in the azoospermia group, P = 0.003; OR: 1.988. From these two points of view, the discrepancy might be caused by the ethnic difference, moreover, the Arg allele may be a dominant effect in Chinese population, in contrast, to recessive in the Iranian population.
The results from our and Huang's research [Huang et al. Citation2012] were not consistent with each other, although both involved a Chinese sample. Nevertheless, the frequency of GG, GC, and CC genotypes (29.17%, 46.88%, and 23.96%, respectively) in our study was in accordance with the rates (29.3%, 47.1%, and 23.6%) previously reported by Huang et al. [2012] in two control groups, the frequency of each genotype in the case individuals were significantly different, indicating that the same ethnicity but different groups may also bring divergent conclusions.
Unlike the conclusions of Lu's group [Lu et al. Citation2007], we found that rs1042522 was relevant to the disease risk of idiopathic male infertility in cases (P = 0.011) and azoospermia group (P = 0.004). The majority of our cases are non-obstructive azoospermia, which accounts for about 58.73%. We also found there was no significant difference between the severe oligozoospermia group and controls in the rs1042522 frequency (P = 0.28). The proportion of cases of the azoospermia group and the severe oligozoospermia group were not mentioned in Lu et al. [2007] and Huang et al. [2012] studies. It is possible that different sample proportion compositions may have lead to the divergent observations.
The frequency distributions of genetic polymorphisms often vary across diverse ethnic backgrounds and geographic locations, yielding conflicting results. For rs1042522, the GG genotype is found in 17.7% of Iranians [Mashayekhi and Hadiyan Citation2012] and 29.3% of Chinese [Huang et al. Citation2012]. Similarly, the frequency of C allele in a population varies between racial groups. For example in African Americans it is approximately 60%, while only 30-35% in Caucasian Americans [Murphy Citation2006], which showed ethnic divergence in the genotypes distribution of SNP. These discrepancies might be caused by several factors. One possibility is the genetic background of the studied population; the other factors include different sample size and composition. The effect of gene-environment interactions should also be taken into consideration.
Our present work also revealed that cigarette smoking and exposure to radioactivity have substantial negative impact on male reproductive health (P < 0.001). El-Melegy and Ali [2011] suggested that infertile smokers have significantly lower semen parameters (such as sperm concentration, motility, and morphology) and significantly higher levels of apoptosis variables (DNA fragmentation and s-Fas) in comparison with infertile non-smokers and controls (P < 0.05 and P < 0.001, respectively). Cigarette smoking may have negative correlations with sperm quality and DNA integrity which decreased male fertility [Kunzle et al. Citation2003]. Radiation exposure is another environmental factor that may directly induce germ cell apoptosis by disrupting the intrinsic pathway which is largely TP53 dependent [Campion et al. Citation2010; Hasegawa et al. Citation1998]. Interactions between environmental factors and genetics have been implicated in the conditions of poor sperm quality and male infertility.
As a complex hormone-related disorder, male infertility is regulated by the hypothalamus-pituitary-gonad axis. In the present study, the levels of FSH, LH, and PRL in infertile men were higher than the controls, especially in the azoospermia group. Lakpour et al. [2012] reported that levels of FSH and LH were significantly higher in a male factor infertility group compared with a non-male factor infertility group (P < 0.001). Besides, the FSH level was negatively correlated with sperm concentration (P < 0.001) and motility (P < 0.01). Lu and Hsieh [2012] suggested that patients with hyperprolactinemia had a significantly lower infertility rate.
In summary, our study showed that the Arg allele frequency of TP53 is correlated with respect to other risk confounding factors which might play a role in spermatogensis. Also, the TP53 gene Arg72Pro polymorphism contributes significant association to male idiopathic infertility in the southeast Chinese Han population. Idiopathic male infertility is a heterogeneous disorder, with multiple environmental factors and numerous genetic factors contributing to impaired spermatogenesis, further study of the TP53 protein is necessary to gain insight into the biological mechanisms of apoptosis in infertility.
Materials and Methods
Subjects
Three hundred and sixty-one patients diagnosed with idiopathic male infertility and 384 proven fertile individuals without a prior history of assisted reproductive technologies were enrolled. All of the participants in the study are of Chinese Han nationality. The subjects were recruited from the Institute of Reproductive Medicine between September 2009 and May 2012. Infertile men were identified by at least three semen analyses performed adhering to the 5th edition of the World Health Organization manual [WHO Citation2010] (non-obstructive azoospermia: absence of sperm in the ejaculated semen and except the obstructive factors; severe oligozoospermia: sperm count < 5 × 106 ml−1) and screened for karyotyping, Y chromosome microdeletion, and serum determination of LH, FSH, T, PRL, and E2. Cases with obstructive azoospermia, karyotype abnormalities, and Y chromosome microdeletions, varicocele, cryptorchidism, hypogonadotropic hypogonadism, and genital trauma were excluded. The control individuals fathered in the past three years and have normal semen clinical parameters defined by the World Health Organization [WHO Citation2010]. Semen specimens were obtained through masturbation and harvested in sterile containers after 3-7d of sexual abstinence. Venous blood (5ml) from patients as well as controls was collected in an EDTA anticoagulation tube and stored at -80°C until used. Epidemiological and clinical history such as occupation, family history, cigarette smoking, alcohol drinking, reproductive history, and radiation exposure were taken from the participants using a self-made questionnaire. Each subject signed an informed consent to participate in our study and to donate a blood sample for genomic DNA analysis. The study was conducted according to the Declaration of Helsinki principles and approved by the Ethics Committee of Wannan Medical College. The clinical characteristics of the 745 participants are shown in .
Genotyping
The 361 cases were further sub-grouped according to the sperm count as having non-obstructive azoospermia (n = 212) or severe oligozoospermia(n = 149, sperm count < 5 × 106 ml−1). Serum hormone levels were analyzed by magnetic-separation ELISA technology. Luteinizing hormone, 3.0–12.0 mIU ml−1, FSH, 1.67–11.98 mIU ml−1, T, 2.41–11.41 ng ml−1, prolactin, 5.0–17.0 ng ml−1, and E2, 41.42 pg ml−1 were considered as normal range. Genomic DNA was extracted from peripheral blood lymphocytes using a QIAamp DNA Blood Mini kit (QIAGEN, Alameda, CA, USA) according to the operating instruction. The Rs1042522 genotype was performed using PCR-RFLP analysis technique. The primer set used for the amplification was: 5'-TTGCCGTCCCAAGCAATGGATGA-3' (forward) and 5'-TCTGGGAAGGGACAGAAGATGAC–3' (reverse)[Ara et al. Citation1990]. The PCR conditions consisted of an initial denaturation step at 95°C for 3min, followed by 40cycles of 25 s at 95°C, 35 s of annealing at 57°C, 25 s of extension at 72°C, and final elongation at 72°C for 7min. For RFLP, the 199bp PCR product was digested with BstuI (New England Biolabs, MA, USA) at 60°C for 4 h, and the genotype discrimination were separated by electrophoresis on 4% TBE agarose gel, finally the results were visualized under UV illumination. The restriction fragments were found to identify the rs1042522, the Arg allele produced 113 and 86bp fragments and the Pro allele produced a single 199bp fragment. For quality control, PCR product samples with different genotypes were randomly selected and submitted to DNA sequencing to confirm the authenticity of genotype and the results were also consistent.
Statistical analysis
All the statistical analyses were performed using SPSS 10.0. The allele and genotype frequencies were calculated by direct gene counting. The Hardy-Weinberg equilibrium was tested using chi-square test to compare the obtained genotype frequencies to the expected frequencies within control group. The χ2 test was used to compare the difference of genotype frequencies among each group. The clinical data (age, serum hormones levels, and semen parameters) were compared using the independent-samples t-test for continuous variables and the χ2 test for categorical variables such as several risk factors that may affect fertility. Odds ratio and 95% confidence intervals (CI) were also calculated to evaluate the relative risk between TP53 codon72 polymorphism and idiopathic male infertility by SPSS 10.0. For all tests, two-sided P < 0.05 was considered statistically significant.
Abbreviations
| CFTR: | = | cystic fibrosis transmembrane conductance regulator |
| E2: | = | estradiol |
| FSH: | = | follicle-stimulating hormone |
| LH: | = | luteinizing hormone |
| PRL: | = | prolactin |
| SNPs: | = | single nucleotide polymorphisms |
| T: | = | testosterone |
| TP53: | = | tumor suppressor gene 53. |
Acknowledgments
We are grateful to the volunteer participants and all members of National Research Institute for Family Planning in Beijing for their close cooperation and technical support.
Declaration of interest: There is no conflict of interests. This work was supported by National Basic Research Program of China (No.2010CB945102), National Natural Science Foundation of China (No.30973197), National Science & Technology Pillar Program of China (No.2008BAH24B05), and National Infrastructure Program of Chinese Genetic Resources (No. 2006DKA21300).
Author contributions Main investigator, participated in the design of the study, carried out the molecular genetics experiment, and drafted the manuscript: QJ; Conceived and designed the experiments, analyzed the data, and revised the manuscript: BBW, JW; Collected all the samples: TL, XYY; Participated in the semen analysis: CJ and XF; Conceived the study and serve as corresponding authors of the article: YFP, XM.
References
- Al-Qasem, A., Toulimat, M., Tulbah, A., Elkum, N., Al-Tweigeri, T. and Aboussekhra, A. (2012) The 53 codon 72 polymorphism is associated with risk and early onset of breast cancer among Saudi women. Oncol Lett 3:875–878.
- Almon, E., Goldfinger, N., Kapon, A., Schwartz, D., Levine, A.J. and Rotter, V. (1993) Testicular tissue-specific expression of the 53 suppressor gene. Dev Biol 156:107–116.
- Ara, S., Lee, P.S., Hansen, M.F. and Saya, H. (1990) Codon 72 polymorphism of the TP53 gene. Nucleic Acids Res 18:4961.
- Aston, K.I., Krausz, C., Laface, I., Ruiz-Castane, E. and Carrell, D.T. (2010) Evaluation of 172 candidate polymorphisms for association with oligozoospermia or azoospermia in a large cohort of men of European descent. Hum Reprod 25:1383–1397.
- Bornstein, C., Brosh, R., Molchadsky, A., Madar, S., Kogan-Sakin, I., Goldstein, I. , (2011) SPATA18, a spermatogenesis-associated gene, is a novel transcriptional target of 53 and 63. Mol Cell Biol 31:1679–1689.
- Campion, S.N., Sandrof, M.A., Yamasaki, H. and Boekelheide, K. (2010) Suppression of radiation-induced testicular germ cell apoptosis by 2,5-hexanedione pretreatment. III. Candidate gene analysis identifies a role for fas in the attenuation of X-ray-induced apoptosis. Toxicol Sci 117:466–474.
- Dohle, G.R., Halley, D.J., Van Hemel, J.O., van den Ouwel, A.M., Pieters, M.H., Weber, R.F., (2002) Genetic risk factors in infertile men with severe oligozoospermia and azoospermia. Hum Reprod 17:13–16.
- Dunna, N.R., Vure, S., Sailaja, K., Surekha, D., Raghunadharao, D., Rajappa, S., (2012) TP53 codon 72 polymorphism and risk of acute leukemia. Asian Pac J Cancer Prev 13:347–350.
- El-Melegy, N.T. and Ali, M.E. (2011) Apoptotic markers in semen of infertile men: Association with cigarette smoking. Int Braz J Urol 37:495–506.
- Francavilla, S., D'Abrizio, P., Rucci, N., Silvano, G., Properzi, G., Straface, E., (2000) Fas and Fas ligand expression in fetal and adult human testis with normal or deranged spermatogenesis. J Clin Endocrinol Metab 85:2692–2700.
- Hasegawa, M., Zhang, Y., Niibe, H., Terry, N.H. and Meistrich, M.L. (1998) Resistance of differentiating spermatogonia to radiation-induced apoptosis and loss in 53–deficient mice. Radiat Res 149:263–270.
- Huang, C., Liu, W., Ji, G.X., Gu, A.H., Qu, J.H., Song, L., (2012) Genetic variants in TP53 and MDM2 associated with male infertility in Chinese population. Asian J Androl 14:691–694.
- Jee, Y., Noh, E.M., Cho, E.S. and Son, H.Y. (2010) Involvement of the Fas and Fas ligand in testicular germ cell apoptosis by zearalenone in rat. J Vet Sci 11:115–119.
- Kenkel, S., Rolf, C. and Nieschlag, E. (2001) Occupational risks for male fertility: an analysis of patients attending a tertiary referral centre. Int J Androl 24:318–326.
- Kunzle, R., Mueller, M.D., Hanggi, W., Birkhauser, M.H., Drescher, H. and Bersinger, N.A. (2003) Semen quality of male smokers and nonsmokers in infertile couples. Fertil Steril 79:287–291.
- Lakpour, N., Mahfouz, R.Z., Akhondi, M.M., Agarwal, A., Kharrazi, H., Zeraati, H., (2012) Relationship of seminal plasma antioxidants and serum male hormones with sperm chromatin status in male factor infertility. Syst Biol Reprod Med 58:236–244.
- Lu, C.C. and Hsieh, C.J. (2012) The importance of measuring macroprolactin in the differential diagnosis of hyperprolactinemic patients. Kaohsiung J Med Sci 28:94–99.
- Lu, N.X., Xia, Y.K., Gu, A.H., Liang, J., Wang, S.L. and Wang, X.R. (2007) Lack of association between polymorphisms in 53 gene and spermatogenetic failure in a Chinese population. Andrologia 39:223–228.
- Mashayekhi, F. and Hadiyan, S.P. (2012) A single-nucleotide polymorphism in TP53 may be a genetic risk factor for Iranian patients with idiopathic male infertility. Andrologia 44 Suppl 1:560–564.
- Murphy, M.E. (2006) Polymorphic variants in the 53 pathway. Cell Death Differ 13:916–920.
- Poongothai, J., Gopenath, T.S. and Manonayaki, S. (2009) Genetics of human male infertility. Singapore Med J 50:336–347.
- Rotter, V., Schwartz, D., Almon, E., Goldfinger, N., Kapon, A., Meshorer, A., (1993) Mice with reduced levels of 53 protein exhibit the testicular giant-cell degenerative syndrome. Proc Natl Acad Sci USA 90:9075–9079.
- Sakkas, D., Mariethoz, E. and St John, J.C. (1999) Abnormal sperm parameters in humans are indicative of an abortive apoptotic mechanism linked to the Fas-mediated pathway. Exp Cell Res 251:350–355.
- Thomas, M., Kalita, A., Labrecque, S., Pim, D., Banks, L. and Matlashewski, G. (1999) Two polymorphic variants of wild-type 53 differ biochemically and biologically. Mol Cell Biol 19:1092–1100.
- WHO (2010) World Health Organization Laboratory manual for the examination and processing of human semen, 5th ed., World Health Organization Press, Geneva, Switzerland.
- Yoon, Y.J., Chang, H.Y., Ahn, S.H., Kim, J.K., Park, Y.K., Kang, D.R., (2008) MDM2 and 53 polymorphisms are associated with the development of hepatocellular carcinoma in patients with chronic hepatitis B virus infection. Carcinogenesis 29:1192–1196