Aberrant methylation of mitochondrial genes as a link between oxidative phosphorylation dysregulation and the pathogenesis of hypospadias: a multiomics and clinical sample study.

Background: Hypospadias is among the most prevalent congenital malformations in male newborns. Despite its clinical significance, the molecular etiology of hypospadias has not extensively characterized. Although environmental exposures, epigenetic dysregulation, and mitochondrial dysfunction have been identified as potential contributing factors, the role of mitochondrial DNA (mtDNA) methylation in the pathogenesis of hypospadias has not been systematically examined. Therefore, this study aimed to investigate the potential involvement of mtDNA methylation in hypospadias and to explore its association with mitochondrial gene expression and oxidative phosphorylation-related pathways. Methods: To investigate mtDNA-related epigenetic alterations in hypospadias, an integrated analysis of transcriptomic and methylomic data was conducted. Alongside control specimens, preputial tissue samples were obtained from patients diagnosed with distal, midshaft, and proximal hypospadias. RNA sequencing (RNA-seq) was performed to profile gene expression. Subsequent functional enrichment analyses were conducted to identify the key disturbed biological pathways. Additionally, mtDNA methylation patterns were examined via publicly available methylation datasets, and this was followed by targeted validation with bisulfite pyrosequencing in order to facilitate the quantification of site-specific methylation levels of the selected mitochondrial genes. Results: RNA-seq identified 96 differentially expressed genes (DEGs), of which 87 were upregulated and 9 downregulated. Subsequent functional enrichment analysis indicated that oxidative phosphorylation (OXPHOS)- and reactive oxygen species (ROS)-related pathways were the most significantly affected biological processes. Further analysis of mitochondrial gene transcriptomes revealed broadly similar upregulation patterns across different hypospadias subtypes, with Conclusions: Our findings indicate that aberrant mtDNA methylation is associated with altered mitochondrial gene expression and disrupted OXPHOS in hypospadias. This type of epigenetic dysregulation may impair mitochondrial energy metabolism and redox balance, thereby contributing to abnormal urethral development. These results constitute novel evidence linking mitochondrial epigenetic modifications to the pathogenesis of hypospadias and highlights potential of molecular targets in future risk assessment, early diagnosis, and preventive interventions.