Primary azoospermia factor C duplication associated with spermatogenic impariment: a case-control study based on Y-chromosome haplogrouping in a Han Chinese population.

BACKGROUND: Azoospermia factor C (AZFc) in the male-specific region of Y-chromosome (MSY) presents wide structure variation mainly due to frequent non-allele homologous recombination, leading to significant copy number variation of the AZFc-linked coding sequences involving in spermatogenesis. A large number of studies had been conducted to investigate the association between AZFc deletions and male infertility in certain Y chromosome genetic backgrounds, however, the influence of primary AZFc duplication on spermatogenesis remained controversial and the cause of the discrepant outcomes is unknown. METHODS: In the present study, a total of 1,102 unrelated Han Chinese males without any detectable AZF deletions were recruited from 2014 to 2019, including 411 controls with normozoospermia and 691 patients with idiopathic spermatogenic failure. Using multiple paralog ratio tests (PRTs), the structure duplications were classified by the copy number of the AZFc-linked amplicons and genes. The Y-chromosome haplogroup (Y-hg) was categorized by genetyping of MSY-linked polymorphism loci. The association of primary AZFc duplication with spermatogenic phenotype was investigated in males with the same Y-hg. RESULTS: Within Y-hg O3* group, the frequency of the gr/gr duplication in patients is significantly higher than that of controls (P = 1.29×10-3 , odds ratio (OR) 7.64, 95% confidence interval (CI) 1.79-32.57). Moreover, Y-hg O3* males with the gr/gr duplication presented a significantly lower sperm production compared with non-AZFc duplicated ones (sperm concentration: P = 1.46×10-3 ; total sperm count: P = 1.82 ×10-3 ). The b2/b3 duplication were identified clustered in Y-hg Cα2*, and the significant difference in the distribution was not observed between patients with spermatogenic failure and controls. CONCLUSION: The results suggest that, in the Han Chinese population, the gr/gr duplication is a predisposing genetic factor for spermatogenic impairment in males harboring Y-hg O3* . Meanwhile, the b2/b3 duplication may be fixed on a yet-unidentified subbranch of Y-hg Cα2* without significantly deleterious effect on spermatogenesis. Our findings provide evidence that the difference in the Y-hg composition may cause the discrepancy on the association of AZFc duplication with spermatogenic failure among the studied populations.

The association of stromal antigen 3 (STAG3) sequence variations with spermatogenic impairment in the male Korean population.

The stromal antigen 3 (STAG3) gene, encoding a meiosis-specific cohesin component, is a strong candidate for causing male infertility, but little is known about this gene so far. We identified STAG3 in patients with nonobstructive azoospermia (NOA) and normozoospermia in the Korean population. The coding regions and their intron boundaries of STAG3 were identified in 120 Korean men with spermatogenic impairments and 245 normal controls by using direct sequencing and haplotype analysis. A total of 30 sequence variations were identified in this study. Of the total, seven were exonic variants, 18 were intronic variants, one was in the 5'-UTR, and four were in the 3'-UTR. Pathogenic variations that directly caused NOA were not identified. However, two variants, c.3669+35C>G (rs1727130) and +198A>T (rs1052482), showed significant differences in the frequency between the patient and control groups (P = 0.021, odds ratio [OR]: 1.79, 95% confidence interval [CI]: 1.098-2.918) and were tightly linked in the linkage disequilibrium (LD) block. When pmir-rs1052482A was cotransfected with miR-3162-5p, there was a substantial decrease in luciferase activity, compared with pmir-rs1052482T. This result suggests that rs1052482 was located within a binding site of miR-3162-5p in the STAG3 3'-UTR, and the minor allele, the rs1052482T polymorphism, might offset inhibition by miR-3162-5p. We are the first to identify a total of 30 single-nucleotide variations (SNVs) of STAG3 gene in the Korean population. We found that two SNVs (rs1727130 and rs1052482) located in the 3'-UTR region may be associated with the NOA phenotype. Our findings contribute to understanding male infertility with spermatogenic impairment.

Interaction between Y chromosome haplogroup O3* and 4-n-octylphenol exposure reduces the susceptibility to spermatogenic impairment in Han Chinese.

Certain genetic background (mainly Y chromosome haplogroups, Y-hg) may modify the susceptibility of certain environmental exposure to some diseases. Compared with respective main effects of genetic background or environmental exposure, interactions between them reflect more realistic combined effects on the susceptibility to a disease. To identify the interactions on spermatogenic impairment, we performed Y chromosome haplotyping and measurement of 9 urinary phenols concentrations in 774 infertile males and 520 healthy controls in a Han Chinese population, and likelihood ratio tests were used to examine the interactions between Y-hgs and phenols. Originally, we observed that Y-hg C and Y-hg F* might modify the susceptibility to male infertility with urinary 4-n-octylphenol (4-n-OP) level (Pinter = 0.005 and 0.019, respectively). Subsequently, based on our results, two panels were tested to identify the possible protective sub-branches of Y-hg F* to 4-n-OP exposure, and Y-hg O3* was uncovered to interact with 4-n-OP (Pinter = 0.019). In conclusion, while 4-n-OP shows an adverse effect on spermatogenesis, Y-hg O3* makes individuals more adaptive to such an effect for maintaining basic reproductive capacity.

Diethylstilbestrol, flutamide and their combination impaired the spermatogenesis of male adult zebrafish through disrupting HPG axis, meiosis and apoptosis.

Both diethylstilbestrol (DES, an environmental estrogen) and flutamide (FLU, an anti-androgen) are found to impair spermatogenesis by disrupting hypothalamic-pituitary-gonadal (HPG) axis and altering androgen levels through different mechanisms/modes of action in fish with poorly understood underlying mechanisms. Furthermore, it is not known whether and how a combined exposure of DES and FLU has a stronger effect than the compounds alone. In this study, male zebrafish adults were exposed to DES, FLU and their combination (DES+FLU) for 30days, and their effects on histological structure and sperm count in testis, androgen level in plasma, as well as the mRNA levels of genes involved in HPG axis, meiotic regulation and apoptosis were analyzed. After exposure, DES and FLU disrupted spermatogenesis in zebrafish, and their combination resulted in even more severe impairment, indicating the inhibitory roles of these chemicals on spermatogenesis and their additive effects on zebrafish. The different regulation of vtg1 expression in the liver in response to DES and FLU further confirmed the different modes of action of these drugs. Gene expression and plasma steroid level analyses demonstrated the suppressed mRNA levels of the key genes (such as gnrh3, fshß and lhß in brain and dmrt1, sf1, cyp17a1 and cyp11b2 in testis) in HPG axis and decreased 11-ketotestosterone (11-KT) levels in plasma. The declined level of 11-KT was thus supposed to be closely related to the down-regulation of cyp26a1 (encoding the catabolic enzyme of retinoic acid) and suppression of genes involved in meiotic regulation (nanos1, dmc1 and sycp3). In fish exposed to DES and DES+FLU, enhanced apoptosis (elevated bax/bcl-2 expression ratio) was also observed. The suppression of meiotic regulation in response to all the exposures and enhanced apoptosis in response to DES were thus supposed to result in the spermatogenic impairment in zebrafish. The present study greatly extends our understanding on the mechanisms underlying of reproductive toxicity of environment estrogens and anti-androgens in fish.

Gene copy number alterations in the azoospermia-associated AZFc region and their effect on spermatogenic impairment.

The azoospermia factor c (AZFc) region in the long arm of human Y chromosome is characterized by massive palindromes. It harbors eight multi-copy gene families that are expressed exclusively or predominantly in testis. To assess systematically the role of the AZFc region and these eight gene families in spermatogenesis, we conducted a comprehensive molecular analysis (including Y chromosome haplogrouping, AZFc deletion typing and gene copy quantification) in 654 idiopathic infertile men and 781 healthy controls in a Han Chinese population. The b2/b3 partial deletion (including both deletion-only and deletion-duplication) was consistently associated with spermatogenic impairment. In the subjects without partial AZFc deletions, a notable finding was that the frequency of DAZ and/or BPY2 copy number alterations in the infertile group was significantly higher than in the controls. Combined patterns of DAZ and/or BPY2 copy number abnormality were associated with spermatogenic impairment when compared with the pattern of all AZFc genes with common level copies. In addition, in Y chromosome haplogroup O1 (Y-hg O1), the frequency of copy number alterations of all eight gene families was significantly higher in the case group than that in the control group. Our findings indicate that the DAZ, BPY2 genes may be prominent players in spermatogenesis, and genomic rearrangements may be enriched in individuals belonging to Y-hg O1. Our findings emphasize the necessity of routine molecular analysis of AZFc structural variation during the workup of azoospermia and/or oligozoospermia, which may diminish the genetic risk of assisted reproduction.

Pathogenic variants screening in five non-obstructive azoospermia-associated genes.

Non-obstructive azoospermia (NOA) is one of the most severe forms of male infertility and a recent, genome-wide association study (GWAS) has identified four risk loci associated with NOA. However, a large portion of the heritability of NOA has not been well explained by GWAS. By hypothesizing that rare, low-frequency and common genetic variants might point toward a causal relation between candidate genes and NOA, we performed a two-stage study including deep exon sequencing in 96 NOA cases and 96 healthy controls and a replication study in a larger population containing 522 NOA cases and 484 healthy controls. In the solexa sequencing stage, a total of two rare mutations (chr20. 1902132 and chr20. 1902301 in SIRPA), four common mutations (rs1048055 and rs2281807 in SIRPG, rs11046992 and rs146039840 in SOX5) were identified by using next generation sequencing (NGS). In the validation stage, subjects in the NOA group had a significantly decreased frequency of the heterozygous GA genotype in SIRPA (4.23%, 22 out of 520) than that in the control group (8.60%, 41 out of 477) [odds ratios (OR) 0.47, 95% confidence intervals (CI) 0.28-0.80] (P = 6.00 × 10(-3)). The rs1048055 in SIRPG was associated with a significantly increased risk of spermatogenic impairment, compared with the CC genotype (OR 3.93, 95% CI 1.59-9.70) (P = 3.00 × 10(-3)). Our study provides evidence of independent NOA risk alleles driven by variants in the protein-coding sequence of two of the genes (SIRPA and SIRPG) discovered by GWAS. Further investigation in larger populations and functional characterizations are needed to validate our findings.

DAZ duplications confer the predisposition of Y chromosome haplogroup K* to non-obstructive azoospermia in Han Chinese populations.

STUDY QUESTION: What are the genetic causes for the predisposition of certain Y chromosome haplogroups (Y-hgs) to spermatogenic impairment? SUMMARY ANSWER: The AZFc(azoospermia factor c)/DAZ (deleted in azoospermia) duplications might underlie the susceptibility of Y-hg K* to spermatogenic impairment. WHAT IS KNOWN ALREADY: The roles of Y chromosomal genetic background in spermatogenesis are controversial and vary among human populations. Individuals in predisposed Y-hgs may carry some genetic factors, which might be a potential genetic modifier for the Y-hg-specific susceptibility to spermatogenic impairment. STUDY DESIGN, SIZE, DURATION: A total of 2444 individuals with azoospermia or oligozoospermia and 2456 healthy controls were recruited to this study from March 2004 and January 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed a two-stage association study to investigate the risk and/or protective Y-hgs for spermatogenic impairment. In addition, the genetic causes for the predisposition of certain Y-hg to spermatogenic impairment were investigated. Deletion typing and DAZ gene copy number quantification were performed for individuals in predisposed Y-hgs. MAIN RESULTS AND THE ROLE OF CHANCE: Y-hgs K* and O3e* showed significantly different distribution between cases and controls consistently in two-stage studies. Combined analyses identified significant predisposition to non-obstructive azoospermia in Y-hg K* [odds ratio (OR) 8.58; 95% confidence interval (CI) 3.31-22.28; P = 1.40 × 10⁻5], but a protecting effect in Y-hg O3e* (OR 0.64; 95% CI 0.53-0.78; P = 4.20 × 10⁻5). Based on the dynamic nature of the Y chromosome, we hypothesized that Y-hgs K* and O3e* may be accompanied by modifying genetic factors for their predisposing or protecting effects in spermatogenesis. Accordingly, we quantified the multi-copy DAZ gene, which has variable copy numbers between individuals and plays an important role in spermatogenesis. In combined analysis, we found that the over-dosage of DAZ was significantly more frequent in Y-hg K* than in O3e* (OR 4.79; 95% CI 1.67-13.70; P = 6 × 10⁻³). LIMITATIONS, REASONS FOR CAUTION: Owing to the inconsistency of genetic background, it remains to be determined whether the results derived from Han Chinese populations are applicable to other ethnic groups. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study can advance the etiology of spermatogenic impairment, and also shed new light on Y chromosome evolution in human populations. Y-hg-specific genetic factors of modifying spermatogenic phenotypes deserve further investigation in larger and diverse populations.