👤 Singh Rajender

To study peripheral blood DNA differential methylation in oligozoospermic infertile men in comparison with normozoospermic fertile controls. Case-control study. Reproductive biology laboratory. Azoospermic and oligozoospermic infertile patients (n = 6) and normozoospermic fertile controls (n = 6) in the discovery phase, and oligo/asthenozoospermic infertile men (n = 11) and normozoospermic fertile controls (n = 10) in the validation phase. Blood samples drawn from all participants, DNA isolation and methylation analysis. DNA methylation values analyzed using genomewide methylation 450K BeadChip array, followed by deep sequencing of selected regions for methylation analysis in the neighborhood regions of differentially methylated CpGs. We found 329 differentially methylated CpG spots, out of which 245 referred to the genes, representing 170 genes. Deep-sequencing analysis confirmed the methylation pattern suggested by 450K array. A thorough literature search suggested that 38 genes play roles in spermatogenesis (PDHA2, PARP12, FHIT, RPTOR, GSTM1, GSTM5, MAGI2, BCAN, DDB2, KDM4C, AGPAT3, CAMTA1, CCR6, CUX1, DNAH17, ELMO1, FNDC3B, GNRHR, HDAC4, IRS2, LIF, SMAD3, SOD3, TALDO1, TRIM27, GAA, PAX8, RNF39, HLA-C, HLA-DRB6), are testis enriched (NFATC1, NMNAT3, PIAS2, SRPK2, WDR36, WWP2), or show methylation differences between infertile cases and controls (PTPRN2, RPH3AL). We found a statistically significant correlation between peripheral blood DNA methylation and male infertility, raising the hope that epigenome-based blood markers can be used for screening male infertility risk. The study also identified new candidates for spermatogenesis and fertility. Show less

Do methylation changes in sperm DNA correlate with infertility? Loss of spermatogenesis and fertility was correlated with 1680 differentially-methylated CpGs (DMCs) across 1052 genes. Methylation changes in a number of genes have been correlated with reduced sperm count and motility. This case-control study used spermatozoal DNA from 38 oligo-/oligoastheno-zoospermic infertile patients and 26 normozoospermic fertile men. Genome-wide methylation analysis was undertaken using 450 K BeadChip on spermatozoal DNA from six infertile and six fertile men to identify DMCs. This was followed by deep sequencing of spermatozoal DNA from 32 infertile patients and 20 fertile controls. A total of 1680 DMCs were identified, out of which 1436 were hypermethylated and 244 were hypomethylated. Classification of DMCs according to the genes identified BCAN, CTNNA3, DLGAP2, GATA3, MAGI2 and TP73 among imprinted genes, SPATA5, SPATA7, SPATA16 and SPATA22 among spermatogenesis-associated genes, KDM4C and JMJD1C, EZH2 and HDAC4 among genes which regulate methylation and gene expression, HLA-C, HLA-DRB6 and HLA-DQA1 among complementation and immune response genes, and CRISPLD1, LPHN3 and CPEB2 among other genes. Genes showing significant differential methylation in deep sequencing, i.e. HOXB1, GATA3, EBF3, BCAN and TCERG1L, are strong candidates for further investigations. The role of chance was ruled out by deep sequencing of select genes. N/A. Genome-wide analyses are fairly accurate, but may not be exactly validated in replication studies across all DMCs. We used the 't' test in the genome-wide methylation analysis, whereas other tests could provide a more robust and powerful analysis. DMCs can serve as markers for inclusion in infertility screening panels, particularly those in the genes showing differential methylation consistent with previous studies. The genes validated by deep sequencing are strong candidates for investigations of their roles in spermatogenesis. The study was funded by the Council of Scientific and Industrial Research (CSIR), Govt. of India with grant number BSC0101 awarded to Rajender Singh. None of the authors has any competing interest to declare. Show less