Status of sperm mitochondrial functions and DNA methylation in infertile men with clinical varicocele before and after treatment

Abstract

Varicocele has been associated with reduced male fertility potential. Treatment modalities for varicocele improve semen parameters, yet more than 50% of cases remain infertile. Varicocele-induced heat and hypoxia stress may affect sperm mitochondrial functions, possibly leading to aberrant epigenetic modifications. This study includes 30 fertile men and 40 infertile men with clinical varicocele. The effect of varicocele treatment (antioxidant supplementation and or varicocelectomy) was evaluated after 3 months of treatment. Mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (iROS) were measured by flow cytometry using JC-1 and DCFDA, respectively. mtDNA copy number and deletions were determined by PCR. DNA methylation was analysed by pyrosequencing. Present investigations suggest that infertile men with varicocele have abnormal semen parameters; significantly low MMP, high iROS, and high mtDNA copy number. Semen parameters were improved in a subset of men of both the treatment modalities; however, it was noted that varicocelectomy helped better in improving sperm parameters compared to antioxidant treatment. Both treatment modalities helped in reducing iROS and mtDNA copy number significantly; however, they were noneffective in improving MMP. Altered DNA methylation at mitochondria D loop and mitochondrial structure and function genes UQCRC2, MIC60, TOM22, and LETM1 (promoter region) were observed in varicocele group. The DNA methylation levels were restored after varicocele treatment; however, the restoration was not consistent at all CpG sites. Both the treatment modalities helped in restoring the altered DNA methylation levels of mitochondrial genes but the restoration is nonhomogeneous across the studied CpG sites.