DNA methylation level of spermatozoa from subfertile and proven fertile and its relation to standard sperm parameters.

The epigenetic mechanism plays an important role in spermatogenesis such as DNA methylation where this episode is represented by either switching genes on or off. Twenty-eight samples (14 case and 14 controls) were subjected to Infinium 450K BeadChip arrays to identify genomic regions that differ in sperm DNA methylation patterns in the subfertile compared to the proven fertile group. Then two CpGs were validated by deep bisulphite sequencing on 82 sperm samples. The results screening study revealed eight CpGs were significantly different in their sperm DNA methylation levels between cases and control group. The results of the validation study for the two CpGs (cg19779893 and cg19406113) showed that a significant variation in the methylation level at 2 CpGs of 3 CpGs related to cg19779893 site amplicon in cases compared to the controls. Moreover, six CpGs related to the cg19406113 site amplicon showed significant differences in sperm DNA methylation between the cases and the control group. Furthermore, there was a significant decrease in the sperm parameters in the cases compared to the control group. This study found two CpGs altered in their sperm DNA methylation levels. In addition, a strong association was found between changes in the sperm DNA methylation levels in these CpGs sites and sperm parameters.

Impact of cigarette-smoking on sperm DNA methylation and its effect on sperm parameters.

DNA methylation is an epigenetic modification of the genome. The purpose of this study was to determine the influence of cigarette-smoking on sperm DNA methylation from a genomewide survey of sperm samples and to ascertain its effect on sperm parameters. Twenty-eight sperm DNA samples (from 14 fertile smokers as a case study and 14 proven fertile nonsmokers as controls) were subjected to Infinium 450K BeadChip arrays to identify the changes in the DNA methylation level between the two groups. Then, deep bisulphite sequencing was used to validate five CpGs on 78 samples. The results from the Infinium 450K found that only 11 CpGs showed a significant difference in DNA methylation between the case and the control groups. Five CpGs of the eleven (cg00648582, cg0932376, cg19169023, cg23841288 and cg27391564) underwent deep bisulphite sequencing where cg00648582, related to the PGAM5 gene, and the cg23841288 CpGs, related to the PTPRN2 gene amplicons, showed a significant increase in their DNA methylation level in more than one CpG in the case group. In contrast, a significant decrease was found at cg19169023 and at its various neighbouring CpGs in the TYRO3 gene-related amplicons. Furthermore, this study demonstrated a significant correlation between the variation in sperm DNA methylation level and standard sperm parameters in the case group.

Cigarette smoking induces only marginal changes in sperm DNA methylation levels of patients undergoing intracytoplasmic sperm injection treatment.

DNA methylation plays important roles in genome stability and regulation of gene expression. This study was designed to determine the influence of cigarette smoking on sperm DNA methylation. From a genome-wide survey on sperm samples, differentially methylated target CpGs should be selected and subjected to local deep bisulphite sequencing. Obtained methylation data are compared to sperm parameters and (ICSI) outcome. Similar to pilot study, samples were subjected to Infinium 450K BeadChip arrays to identify alterations in sperm DNA methylation between smokers and nonsmokers males. Routine testing on a significantly altered CpG site was performed on more samples using local deep bisulphite sequencing. Of approximately 485,000 CpG sites analysed, only seven CpGs were found to show a significant DNA methylation difference of >20% with the top six CpGs overlapping common SNP sites. The remaining CpG site (cg19455396) is located in intron 12 of the TAP2 gene. The results of deep bisulphite sequencing showed only a tendency towards hypomethylation in the smoking group. This study could not detect biologically relevant CpG positions that are altered in sperm DNA methylation on the influence of cigarette smoking beyond individual-specific effects that may be caused by other environmental factors.

Spermatozoa from males with reduced fecundity exhibit differential DNA methylation patterns.

Infertility affects 10-15% of couples, and approximately 50% of cases are linked to male factor infertility. The purpose of this study was to evaluate the DNA methylation patterns in spermatozoa from males who are suffering from a reduction in fecundity. Thirty samples were subjected to 450K arrays as a screening study to evaluate the variation in sperm DNA methylation levels between cases and controls groups, and then four CpG sites (cg05799088, cg07227024, cg16338278, and cg08408433) underwent to deep bisulfite sequencing to validate the observed methylation differences in 111 samples (56 proven fertile males as 'controls' and 55 males suffering from a reduction in fecundity as 'cases'). A significant difference in the mean methylation level was found between cases and controls in the CpGs of PRICKLE2 gene-related amplicon (CpG1, p ≤ 0.002, and CpG2, p ≤ 0.004) and CpG of ALS2CR12 gene-related amplicon (CpG1, p ≤ 0.015, and CpG2, p ≤ 0.009). Besides, a significant difference was found at seven from thirteen CpGs tested in the ALDH3B2 gene amplicon CpG2, CpG6, CpG9, CpG10, CpG11, CpG12, and CpG13 (p ≤ 0.005, p ≤ 0.004, p ≤ 0.012, p ≤ 0.028, p ≤ 0.012, p ≤ 0.009, and p ≤ 0.001, respectively). In addition, the results showed that nine CpGs out of the twenty-six within the PTGIR gene-related amplicon (CpG4, CpG6, CpG8, CpG9, CpG11, CpG15, CpG19, CpG23, and CpG26) had a significant difference in their mean methylation level (p ≤ 0.006, p ≤ 0.009, p ≤ 0.003, p ≤ 0.003, p ≤ 0.007, p ≤ 0.002, p ≤ 0.018, p ≤ 0.018, and p ≤ 0.040, respectively) in the case vs. CONTROL GROUP: In conclusion, an alteration in the methylation levels of sperm DNA from males with reduced fecundity was observed. In addition, an association between changes in the methylation level for these CpGs and different semen parameters has been found.