Association of TUSC1 and DPF3 gene polymorphisms with male infertility.

PURPOSE: Recently, genome-wide association studies of a Hutterite population in the USA revealed that five single nucleotide polymorphisms (SNPs) with a significant association with sperm quality and/or function in ethnically diverse men from Chicago were significantly correlated with family size. Of these, three SNPs (rs7867029, rs7174015, and rs12870438) were found to be significantly associated with the risk of azoospermia and/or oligozoospermia in a Japanese population. In this study, we investigated whether the rs10966811 (located in an intergenic region between the TUSC1 and IZUMO3 genes) and rs10129954 (located in the DPF3 gene) SNPs, previously related to family size, are associated with male infertility. In addition, we performed association analysis between rs12348 in TUSC1 and rs2772579 in IZUMO3 and male infertility. METHODS: We genotyped 145 patients with infertility (including 83 patients with azoospermia and 62 with oligozoospermia) and 713 fertile controls by PCR-RFLP technique for polymorphism. Because rs10966811 has no restriction sites, the SNP rs12376894 with strong linkage disequilibrium was selected as an alternative to rs10966811. RESULTS: There was a statistically significant association between rs12376894 proxy SNP of rs10966811 and oligozoospermia. Also, a statistically significant association between rs10129954 and azoospermia, and oligozoospermia was observed. When we assessed the relationship between rs12348 in TUSC1 and rs2772579 in IZUMO3 and male infertility traits, we found that rs12348 in TUSC1 was significantly associated with azoospermia and oligozoospermia, but rs2772579 in IZUMO3 was not associated with male infertility. CONCLUSION: We found that the polymorphisms in TUSC1 and DPF3 displayed strong associations with male infertility.

Erratum: An independent validation study of three single nucleotide polymorphisms at the sex hormone-binding globulin locus for testosterone levels identified by genome-wide association studies.

[This corrects the article DOI: 10.1093/hropen/hox002.][This corrects the article DOI: 10.1093/hropen/hox002.].

An independent validation study of three single nucleotide polymorphisms at the sex hormone-binding globulin locus for testosterone levels identified by genome-wide association studies.

STUDY QUESTION: Are the single nucleotide polymorphisms (SNPs) rs2075230, rs6259 and rs727428 at the sex hormone-binding globulin (SHBG) locus, which were identified by genome-wide association studies (GWASs) for testosterone levels, associated with testosterone levels in Japanese men? SUMMARY ANSWER: The SNP rs2075230, but not rs6259 and rs727428, is significantly associated with testosterone levels in Japanese men. WHAT IS ALREADY KNOWN: Previous GWASs have revealed that rs2075230 is associated with serum testosterone levels in 3495 Chinese men and rs6259 and rs727428 are associated with serum testosterone levels in 3225 men of European ancestry. STUDY DESIGN SIZE AND DURATION: This is an independent validation study of 1687 Japanese men (901 in Cohort 1 and 786 in Cohort 2). PARTICIPANTS/MATERIALS SETTING AND METHOD: Cohort 1 (20.7 ± 1.7 years old, mean ± SD) and Cohort 2 (31.2 ± 4.8 years) included samples obtained from university students and partners of pregnant women, respectively. The three SNPs were genotyped using either TaqMan probes or restriction fragment length polymorphism PCR. Blood samples were drawn from the cubital vein of the study participants in the morning, and total testosterone and SHBG levels were measured using a time-resolved immunofluorometric assay. Association between each SNP and testosterone levels was evaluated by meta-analysis of the two Japanese male cohorts. MAIN RESULTS AND THE ROLE OF CHANCE: The age of the two cohorts was significantly different (P < 0.0001). We found that rs2075230 was significantly associated with serum testosterone levels (ß STD = 0.15, P = 7.2 × 10-6); however, rs6259 and rs727428 were not (ß STD = 0.17, P = 0.071; ß STD = 0.082, P = 0.017, respectively), after adjusting for multiple testing in a combined analysis of two Japanese male cohorts. Moreover, rs2075230, rs6259 and rs727428 were significantly associated with high SHBG levels (ß STD = 0.22, P = 3.4 × 10-12; ß STD = 0.23, P = 6.5 × 10-6 and ß STD = 0.21, P = 3.4 × 10-10, respectively). LARGE SCALE DATA: Not applicable. LIMITATIONS REASONS FOR CAUTION: This study had differences in the age and background parameters of participants compared to those observed in previous GWASs. In addition, the average age of participants in the two cohorts in our study also differed from one another. Therefore, the average testosterone levels, which decrease with age, between studies or the two cohorts were different. WIDER IMPLICATIONS OF THE FINDINGS: The three SNPs have a considerable effect on SHBG levels and hence may indirectly affect testosterone levels. STUDY FUNDING/COMPETING INTERESTS: This study was supported partly by the Ministry of Health and Welfare of Japan (1013201) (to T.I.), Grant-in-Aids for Scientific Research (C) (26462461) (to Y.S.) and (23510242) (to A.Ta.) from the Japan Society for the Promotion of Science, the European Union (BMH4-CT96-0314) (to T.I.) and the Takeda Science Foundation (to A.Ta.). There are no conflicts of interest to declare.

A replication study of a candidate locus for follicle-stimulating hormone levels and association analysis for semen quality traits in Japanese men.

In men, follicle-stimulating hormone (FSH) acts on the seminiferous tubules and enhances spermatogenesis. Recently, a candidate locus (rs2414095) for FSH levels was identified by a genome-wide association study (GWAS) in Chinese men. The rs2414095 single-nucleotide polymorphism (SNP) is found on the third intron of the cytochrome P450, family 19, subfamily A, peptide 1 (CYP19A1) gene encoding an aromatase. In the present study, we performed a replication study in 1687 Japanese men (901 from cohort 1 and 786 from cohort 2) to assess whether this SNP is associated with circulating FSH levels. Furthermore, we investigated whether the rs2414095 SNP is correlated with semen quality traits in 2015 Japanese men (1224 from cohort 1 and 791 from cohort 2). The rs2414095 SNP was significantly associated with circulating FSH levels (ßSTD=0.15, P=9.7 × 10-5), sperm concentration (ßSTD=0.073, P=0.032) and total sperm number (TSN) (ßSTD=0.074, P=0.027) in a combined analysis of the two Japanese male cohorts. We successfully replicated, in Japanese men, the results of the previous GWAS for the rs2414095 SNP in Chinese men, and found that the rs2414095 SNP was related with sperm production.

An association study of four candidate loci for human male fertility traits with male infertility.

STUDY QUESTION: Are the four candidate loci (rs7867029, rs7174015, rs12870438 and rs724078) for human male fertility traits, identified in a genome-wide association study (GWAS) of a Hutterite population in the USA, associated with male infertility in a Japanese population? SUMMARY ANSWER: rs7867029, rs7174015 and rs12870438 are significantly associated with the risk of male infertility in a Japanese population. WHAT IS KNOWN ALREADY: Recently, a GWAS of a Hutterite population in the USA revealed that 41 single-nucleotide polymorphisms (SNPs) were significantly correlated with family size or birth rate. Of these, four SNPs (rs7867029, rs7174015, rs12870438 and rs724078) were found to be associated with semen parameters in ethnically diverse men from Chicago. STUDY DESIGN, SIZE, DURATION: This is a case-control association study in a total of 917 Japanese subjects, including 791 fertile men, 76 patients with azoospermia and 50 patients with oligozoospermia. PARTICIPANTS/MATERIALS, SETTING, METHODS: Azoospermia was diagnosed on the basis of semen analysis (the absence of sperm in ejaculate), serum hormone levels and physical examinations. Oligozoospermia was defined as a sperm concentration of <20 × 10(6)/ml. We excluded patients with any known cause of infertility (i.e. obstructive azoospermia, varicocele, cryptorchidism, hypogonadotropic hypogonadism, karyotype abnormalities or complete deletion of AZF a, b or c). The SNPs rs7867029, rs7174015, rs12870438 and rs724078 were genotyped using DNA from peripheral blood samples and either restriction fragment length polymorphism PCR or TaqMan probes. Genetic associations between the four SNPs and male infertility were assessed using a logistic regression analysis under three different comparative models (additive, recessive or dominant). MAIN RESULTS AND THE ROLE OF CHANCE: The genotypes of all four SNPs were in Hardy-Weinberg equilibrium in the fertile controls. The SNPs rs7867029 and rs7174015 are associated with oligozoospermia [rs7867029: odds ratio (OR) = 1.70, 95% confidence interval (CI) = 1.07-2.68, P = 0.024 (log-additive); rs7174015: OR = 6.52, 95% CI = 1.57-27.10, P = 0.0099 (dominant)] and rs12870438 is associated with azoospermia (OR = 10.90, 95% CI = 2.67-44.60, P = 0.00087 (recessive)] and oligozoospermia [OR = 8.54, 95% CI = 1.52-47.90, P = 0.015 (recessive)]. The association between rs7174015 and oligozoospermia under a dominant model and between rs12870438 and azoospermia under additive and recessive models remained after correction for multiple testing. There were no associations between rs724078 and azoospermia or oligozoospermia. LIMITATIONS, REASONS FOR CAUTION: Even though the sample size of case subjects was not very large, we found that three SNPs were associated with the risk of male infertility in a Japanese population. WIDER IMPLICATIONS OF THE FINDINGS: The three infertility-associated SNPs may be contributing to a quantitative reduction in spermatogenesis. STUDY FUNDING/COMPETING INTERESTS: This study was supported in part by the Ministry of Health and Welfare of Japan (1013201) (to T.I.), Grant-in-Aids for Scientific Research (C) (23510242) (to A.Ta.) from the Japan Society for the Promotion of Science, the European Union (BMH4-CT96-0314) (to T. I.) and the Takeda Science Foundation (to A.Ta.). None of the authors has any competing interests to declare.

Lack of replication of four candidate SNPs implicated in human male fertility traits: a large-scale population-based study.

STUDY QUESTION: Are the four candidate loci (rs7867029, rs12870438, rs7174015 and rs724078) for human male fertility traits, identified in a genome-wide association study (GWAS) of a Hutterite population in the USA, associated with semen quality traits in a Japanese population? SUMMARY ANSWER: The four single nucleotide polymorphisms (SNPs) rs7867029, rs12870438, rs7174015 and rs724078 have no association with semen parameters in a meta-analysis of two Japanese male cohorts. WHAT IS KNOWN ALREADY: Four (rs7867029, rs12870438, rs7174015 and rs724078) of the SNPs associated with family size or birth rate in the GWAS of a Hutterite population in the USA were associated with semen parameters in ethnically diverse men from Chicago, USA. STUDY DESIGN, SIZE, DURATION: This is a replication study in a total of 2015 Japanese subjects, including 791 fertile men and 1224 young men from the general population. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed a replication study in two cohorts to assess whether the SNPs rs7867029, rs12870438, rs7174015 and rs724078 are associated with sperm concentration, semen volume, total sperm numbers, total motile sperm numbers or sperm motility. The rs12870438 SNP was detected by restriction fragment length polymorphism PCR while rs7174015, rs724078 and rs7867029 SNPs were genotyped using TaqMan probes. MAIN RESULTS AND THE ROLE OF CHANCE: This study indicated that none of the four SNPs rs7867029, rs12870438, rs7174015 and rs724078 displayed a significant association with semen parameters in the meta-analysis of two Japanese male cohorts. LIMITATIONS, REASONS FOR CAUTION: Only four SNPs identified in the Hutterite GWAS were examined for associations with semen quality traits in a Japanese population. In addition, the linkage disequilibrium structures around the testing markers were different between ethnic groups. WIDER IMPLICATIONS OF THE FINDINGS: Locus mapping studies using a set of tagging SNPs across the loci will be necessary in populations with larger sample sizes in order to understand the contribution of specific genes to semen quality. STUDY FUNDING/COMPETING INTEREST S: This study was supported in part by the Ministry of Health and Welfare of Japan (1013201) (to T.I.), Grant-in-Aids for Scientific Research (C) (23510242) (to A.Ta.) from the Japan Society for the Promotion of Science, the European Union (BMH4-CT96-0314) (to T.I.), and the Takeda Science Foundation (to A.Ta.). None of the authors has any competing interests to declare.

Y chromosome gr/gr subdeletion is associated with lower semen quality in young men from the general Japanese population but not in fertile Japanese Men.

Several case-control studies have investigated whether Y chromosome haplogroups or deletions are associated with spermatogenic failure. However, the relationships between Y chromosome haplogroups or deletions and semen quality in general population have not been elucidated. In this study, we assessed relationships between Y chromosome haplogroups or deletions and semen parameters in 791 fertile Japanese men and 1221 young men from the general Japanese population. We found that the haplogroup D2 (M55 lineage) was significantly associated with lower semen parameters, especially total motile sperm count (P = 0.00051, beta = -0.097), in men from the general population but not in fertile men. In addition, we found that the gr/gr subdeletion was associated with semen quality and in particular, strongly associated with decreased sperm motility (P = 0.00041, beta = -3.14) and total motile sperm count (P = 0.00031, beta = -0.099) in men from the general population but not in fertile men. The combined analysis of fertile Japanese men and men from the general Japanese population showed that the haplogroup D2 (M55 lineage) and the gr/gr subdeletion were strongly associated with reduced sperm motility (P = 0.00056, beta = -2.71, and P = 7.7 × 10(-5), beta = -3.05, respectively) and that haplogroup O2b1 was strongly associated with elevated sperm motility (P = 0.00089, beta = 2.94). These observations add further support for the view that the gr/gr subdeletion diminishes sperm motility that consequently may result in male infertility.

Semen quality of fertile Japanese men: a cross-sectional population-based study of 792 men.

OBJECTIVES: To establish a base line for future studies on temporal trends, to describe potential geographical differences in semen quality and reference values for studies of men from the general population. DESIGN: Cross-sectional study of fertile men from four areas in Japan. Inclusion criteria were: age 20-45 years at the time of invitation, and both the man and his mother had to be born in Japan. Additionally, the current pregnancy of the female partner had to be achieved by normal sexual relations without any fertility treatment. SETTING: Four Japanese study centres at urban areas located in Sapporo, Osaka, Kanazawa and Fukuoka. PARTICIPANTS: 792 men, median age 31.4 years, included from 1999 to 2002. OUTCOME MEASURES: Semen volume, sperm concentration, total sperm count, sperm motility and sperm morphology. RESULTS: Semen volumes, percentages of motile spermatozoa and morphologically normal spermatozoa differed slightly between the four groups, whereas no differences in sperm concentrations or total sperm counts were found. In total, 1.2% of men had a sperm concentration below 5 million/ml, 2.1% below 10 million/ml, 3.5% below 15 million/ml and 16.3% below 40 million/ml. For morphology, 14.7% had less than 5% normal spermatozoa. Reproductive hormone levels varied significantly, however, only little from a biological point of view. CONCLUSIONS: This is the first cross-sectional study on semen quality covering fertile men from the major regions of Japan. It showed that semen quality of fertile Japanese men is comparable to that of the best in European regions. The results may serve as reference values for studies of men from the general population.

Climatic influence on the reproductive characteristics of Japanese males.

We previously performed a survey of the sperm characteristics of the partners of pregnant women in four cities in Japan. In the present study, we analyzed the sperm characteristics of these subjects and the correlations between these sperm characteristics and climatic changes or Y chromosome haplogroups. Our results showed that more haplogroup D2a1 males than O2b1 males were born in the first half of the year (January to June), whereas more O2b1 males were born in the last half of the year (July to December) (P<0.05). This was agreed and correlated with the seasonal variations in their mean sperm concentrations. The haplogroup C, D* and D2a1 males displayed lower sperm concentrations from March to May, followed by an increase in their sperm concentrations starting in June or July, while the O2b1 males displayed higher sperm concentrations in the first half of the year followed by a sudden decrease from July to August (P<0.05). We hypothesize that the Japanese climate has different effects on the sperm characteristics and reproductive seasonality of males from different lineages; and therefore, has influenced the modern population of Japan.

Low pre-radiotherapy prostate-specific antigen level is a significant predictor of treatment success for postoperative radiotherapy in patients with prostate cancer.

BACKGROUND: The optimal role of postoperative radiotherapy for patients with prostate cancer remains undefined. MATERIALS AND METHODS: The medical records of 70 patients (median age: 66 years), who had received radical radiotherapy (RT) between the years 1996 and 2004 after radical prostatectomy (RP), were analyzed. Fifteen patients had received immediate adjuvant RT, while the other 55 patients had received salvage therapy. Hormonal therapy had been performed in 28 patients before RT and continued in two of them concurrently with RT. A median dose of 60 Gy was delivered to the prostate bed. Pelvic node irradiation was performed in all patients. RESULTS: After a median follow-up period of 23 months, 21 patients had experienced biochemical failure. Actuarial 3- and 5-year biochemical relapse-free survival estimates were 67.4%. No patient had local failure, although distant metastases with biochemical failure were found in five patients. On univariate analysis, the following were significant for biochemical failures: seminar vesicle involvement, serum PSA level >1 ng/ml before RT, pathological pelvic node involvement, RT indication (adjuvant vs. salvage) and Gleason score. However, only the serum PSA level before RT was significant on multivariate analysis. CONCLUSION: Postoperative RT with a pre-RT PSA level <0.1 ng/ml seemed to be effective in patients with prostate cancer.

Efficacy of modest dose irradiation in combination with long-term endocrinal treatment for high-risk prostate cancer: a preliminary report.

BACKGROUND: Although radiotherapy in combination with endocrinal manipulation has been identified as an effective treatment for patients with high-risk prostate cancer, the optimal dose for locoregional control of prostate cancer in combination with hormonal therapy has not yet been determined. METHODS: The efficacy of modest doses of irradiation (60-62 Gy) combined with long-term endocrinal treatment for patients with high-risk prostate cancer (defined as a pretreatment prostate-specific antigen (PSA) level greater than 20 ng/ml or a Gleason's score of 8-10 or T3-T4 disease) was analyzed in 60 Japanese patients. The patients included in this study had received radical radiotherapy with long-term endocrinal manipulation in the period between 1993 and 2000. The median age of the patients was 70 years (range, 56-83). Neoadjuvant hormonal therapy with a median duration of 3.9 months was performed prior to radiotherapy, and hormonal therapy was continued until recurrence. A median dose of 61.4 Gy (range, 44-71.4) was delivered to the prostate. Pelvic node irradiation was performed in 49 patients (81.6%). RESULTS: After a median follow-up period of 28.5 months, the overall survival, cause-specific survival and biochemical relapse-free survival at 3 years were 94.4%, 96% and 89.8%, respectively. Local failure was observed in one patient, distant metastases were observed in three patients and a late toxic effect greater than Grade 2 was not observed in any patients. CONCLUSIONS: This study, though preliminary due to a short-term follow-up period, reveals the possibility that modest doses of irradiation combined with long-term endocrinal treatment could be an effective means of achieving excellent local control of high-risk prostate cancer.

c-Abl tyrosine kinase regulates the human Rad9 checkpoint protein in response to DNA damage.

The ubiquitously expressed c-Abl tyrosine kinase is activated in the apoptotic response of cells to DNA damage. The mechanisms by which c-Abl signals the induction of apoptosis are not understood. Here we show that c-Abl binds constitutively to the mammalian homolog of the Schizosaccharomyces pombe Rad9 cell cycle checkpoint protein. The SH3 domain of c-Abl interacts directly with the C-terminal region of Rad9. c-Abl phosphorylates the Rad9 Bcl-2 homology 3 domain (Tyr-28) in vitro and in cells exposed to DNA-damaging agents. The results also demonstrate that c-Abl-mediated phosphorylation of Rad9 induces binding of Rad9 to the antiapototic Bcl-x(L) protein. The regulation of Rad9 by c-Abl in the DNA damage response is further supported by the demonstration that the interaction between c-Abl and Rad9 contributes to DNA damage-induced apoptosis. These findings indicate that Rad9 is regulated by a c-Abl-dependent mechanism in the apoptotic response to genotoxic stress.