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Sperm is not a simple carrier of paternal genetic information but its role extends clearly beyond fertilization. Integrity of sperm genome is an essential pre-requisite for birth of healthy offspring and evaluation of sperm should entail DNA integrity analysis. DNA integrity analysis is a better diagnostic and prognostic marker of sperm reproductive potential. Conventional semen analysis emphasizes on sperm concentration, viability, motility and morphology and has been proven to be a poor indicator of reproductive potential and pregnancy outcome. To overcome the drawbacks associated with conventional semen analysis more useful fertility tests and molecular biomarkers have been explored. Among the different tests which have evolved for assessing the sperm reproductive potential, tests for sperm DNA quality are most promising. Sperm DNA damage has been closely associated with numerous indicators of reproductive health including fertilization, embryo quality, implantation, spontaneous abortion, congenital malformations and childhood diseases. It therefore has great potential as a prognostic test for both in vitro and in vivo conception. This review presents an updated account of tests that have better diagnostic and prognostic implications in the evaluation of sperm DNA damage. The basic principles, outline of methodology, advantage, disadvantage, clinical significance of each technique and implications of these tests have been discussed. The logistics of each test with respect to available resources and equipment in an andrology laboratory, the feasibility of performing these tests in routine diagnostic workup of infertile men and the opportunities and challenges provided by DNA testing in male fertility determination are also presented.

Purpose Yq microdeletions are the leading genetic cause of male infertility and its detection is clinically relevant for appropriate genetic counseling. We aimed to determine the prevalence and type of Yq microdeletions, the associated seminal phenotypes and the STS markers that are relevant for its testing in Indian population. Methods Yq microdeletion analysis was carried out in 1,636 infertile cases in our centers. Additional data was collected from published studies in Indian population leading to a total of 3,647 cases. Results In our cohort, 3.4 % (56/1,636) of infertile men had Yq microdeletions. Combining the data from other published studies identified 215/3,647 (5.8 %) infertile individuals to harbor Yq microdeletions; with 6.4 % in azoopsermia, 5.8 % in oligozoospermia and 3.2 % in oligoasthenozoospermia and teratozoospermia cases. No significant differences in the deletion frequencies were observed between idiopathic vs non idiopathic cases (5.8 vs 8.6 % respectively). Deletions of AZFc were at highest frequency (46.6 %) with double deletions most commonly observed in azoospermic subjects. With respect to the STS markers, screening with the six European Academy of Andrology (EAA) markers would miss 3

Objective To evaluate the relationship between epigenetic patterns in sperm and fecundity. Design Prospective study of couples trying to conceive, utilizing semen samples collected through the HOPE study, at the University of Utah. Setting Academic Andrology and IVF Laboratory Patients DNA methylation alterations associated with fecundity were analyzed in 124 semen samples. 27 semen samples from couples who conceived within 2 months of attempting a pregnancy and a total of 29 semen samples from couples who were unable to achieve a pregnancy within 12 months were analyzed to identify regions of interest. Interventions None. Main Outcome Measures Genome-wide assessment of differential sperm DNA methylation and standard semen analysis. Results No differences in sperm count, sperm morphology, or semen volume were observed between the patients achieving a pregnancy within 2 months of study time and those not obtaining a pregnancy within 12 months. However, using data from the Human Methylation 450k array analysis we did identify 2 genomic regions with significantly decreased (FDR <0.01) methylation and 3 genomic regions with significantly increased methylation in the “failure-to-conceive” group. Interestingly, the only two sites where decreased methylation was associated with reduced fecundity are at closely related genes known to be expressed in sperm, HSPA1L and HSPA1B. Conclusions Our data suggest that there are genomic loci where DNA methylation alterations are associated with decreased fecundity. We have thus identified candidate loci for future study to verify these results and investigate the causative or contributory relationship between altered sperm methylation and decreased fecundity.

Sperm DNA integrity is a prerequisite for normal spermatozoal function. The aim of the study was to evaluate the role of sperm chromatin damage, its cut-off level and its effect on sperm parameters in men with idiopathic infertility by analyzing 100 idiopathic infertile men and 50 fertile controls. Semen samples were analyzed as per WHO 1999 guidelines and sperm chromatin structure assay (SCSA) was applied to measure DNA fragmentation index (DFI) in sperm. The mean DFI of infertile men (35.75) was significantly (P < .0001) higher as compared to controls (26.22). The threshold level of 30.28% was obtained as cut-off value to discriminate infertile men from fertile controls. Sperm count, forward motility, and normal morphology found to be negatively associated with DFI in overall study subjects. Infertile men with severe oligozoospermia had higher mean DFI (40.01 ± 11.31) than infertile men with oligozoospermia (35.11 ± 10.05) and normal sperm count (33.99 ± 9.96). Moreover 64% of infertile men have DFI > 30 against 6% of fertile controls (P < .0001). Higher sperm DNA fragmentation may be the underlying cause for poor semen quality in idiopathic infertile men and the threshold value of 30.28% is a clear discriminator to distinguish infertile men from fertile men of Indian population. Thus, DFI is a good prognostic marker as cases with higher sperm DFI may have poor success rate even after assisted conception and may experience recurrent pregnancy loss (RPL) and should be counseled accordingly.

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SUMMARYSemen analysis is commonly used as a tool to assess the fertility potential of a male, despite its relatively low predictive power. In this study, we have assessed associations between semen analysis findings (low count, low motility, low viability, poor sperm penetration assay results, poor morphology, and increased DNA damage) and DNA methylation patterns in mature spermatozoa. DNA methylation patterns in the mature spermatozoa are thought to be indicative of patterns in the adult germline stem cells and may offer insight into potential perturbations to cellular pathways involved in spermatogenesis. In this study, sperm DNA methylation at >480,000 CpGs was assessed in 94 men using the Illumina 450k HumanMethylation Array and compared to standard measures of sperm quality. We did not identify any global changes to methylation profiles that were associated with reduced semen parameters. Similarly, we found no significant difference in methylation variability that was associated with any abnormal semen analysis parameter, although sperm displaying abnormal parameters tended to have an increased coefficient of variability, suggesting that, in some samples, this may be a contributing factor. Analysis of methylation at single CpGs and genomic regions did identify associations for low viability and low motility, and to a smaller extent, low count. Interestingly, based on GO Term analysis, differentially methylated regions associated with low viability were over-represented in regions important in meiosis, spermatogenesis, and genomic imprinting. These results suggest that while there are not global alterations to the sperm methylome associated with semen abnormalites, some viability associated regional alterations do exist that may be indicative of perturbed cellular pathways during spermatogenesis.

Reactive oxygen species (ROS) levels in semen are believed to play both physiological and pathological roles in male fertility. The study was aimed to find the clinical significance of ROS levels in infertile Indian men. This pilot study included 33 idiopathic infertile men and 18 proven fertile controls. ROS levels in the washed sperm were measured using chemiluminescence assay and expressed as 10(6) cpm per 20 million spermatozoa. Sperm count, percent sperm motility, and percent normal sperm morphology were found to be significantly (P < 0.0001) reduced in infertile men compared with the controls. Median (minimum, maximum range) ROS levels of the infertile group [24.90 (6.89, 44.71)] were found to be significantly (P < 0.0001) elevated compared with the fertile controls [0.167(0.15, 2.78)]. No significant correlation was seen between ROS levels and semen parameters. Elevated ROS levels in the idiopathic Indian infertile men may be one of the underlying reasons for impaired fertility. Therefore measurement of seminal ROS levels may be used in Indian infertile men for better understanding of the aetiology and selection of antioxidant regimen in the treatment of male infertility. However, large studies may be urgently warranted to find out the role of antioxidants in ROS elevated Indian infertile men through randomised, controlled clinical study.