Differential DNA methylation profiling of spermatogenesis-associated genes in patients with idiopathic hypospermatogenesis: a hospital-based case-control study

Hypospermatogenesis is a histopathological subtype of non-obstructive azoospermia and is characterized by a decrease in the total number of germ cells within the seminiferous tubule as a result of spermatogenic failure. Germ cell epigenetic reprograming during development shows that the methylation status of testicular DNA is highly distinct in which hypomethylated foci predominates. A unique testicular DNA methylation pattern needs to be established for various clinical and histopathological subtypes of azoospermia, in order to delineate the epigenetic factors associated with the condition. This study aim to explore the differential methylation profile in idiopathic hypospermatogenesis patients using methylation microarray technique from testicular tissue DNA. A comprehensive clinical history, blood sample, semen analysis parameters, and reproductive endocrine evaluation reports of 51 hypospermatogenesis patients were collected. The known genetic causes were investigated for exclusion using XY fluorescent in-situ hybridization and Yq microdeletion techniques. DNA methylation microarray was performed, on 40 idiopathic hypospermatogenesis and 10 normospermatogenesis patient to identify differential DNA methylation patterns in spermatogenesis associated genes. A genome-wide differential DNA methylation profiling of 40 hypospermatogenesis patients identified 1,666 differentially methylated regions localised predominantly at promotor regions. A significant proportion (70%) of these differentially methylated genomic regions exhibited hypermethylation, suggesting widespread transcriptional repression of spermatogenesis-associated genes. The transcription factor protein-protein interaction analysis identified six clusters viz., TBP, AR, SP3, TP53, CCNT2 and SIN3A, indicating their potential role in the epigenetic mechanisms of the clinical condition of male infertility. Differential methylation burden analysis showed 4/29 high burden and 5/29 intermediated burden patients. Functional enrichment analysis of promoter-associated differentially methylated spermatogenic genes were found implicated in crucial molecular functions including RNA binding, DNA binding, transcription regulation, and spermatid differentiation. The findings suggest that male infertility may, in part, be driven by the silencing of genes that are not just transcriptionally active, but that also act as nodal regulators in transcription factor complexes essential for germ cell viability, hormone responsiveness, and sperm maturation. Our analysis also demonstrated methylation aberrations display a non-uniform distribution, highlighting patient-specific epigenetic patterns, suggesting a high degree of inter-individual variability in the epigenomic contributors to hypospermatogenesis.