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Physiological function of reactive oxygen species (ROS) has been known since a long, but recently toxic effects of ROS on spermatozoa have gained much importance in male infertility. Mitochondrial DNA (mtDNA) is believed to be both source and target of ROS. mtDNA unlike nuclear DNA is not compactly packed and hence more susceptible to oxidative stress (OS) than nuclear DNA. In the present study, the role of OS in mitochondrial genome changes was studied in men with idiopathic infertility. The study included 33 infertile oligo-asthenozoospermic (OA) men and 30 fertile controls. Semen analyses were performed and OS was measured by estimating the level of malondialdehye (MDA) in the seminal plasma and ROS in the sperm. Sperm mtDNA was sequenced by standard PCR-DNA sequencing protocol for ATPase and nicotinamide adenine dinucleotide dehydrogenase (ND) groups of genes. Sperm count and progressive motility were found to be significantly lower in infertile group than the fertile controls. Semen MDA and ROS levels of infertile group were significantly higher (p<0.0001), when compared to the control group. However, catalase and glutathione peroxidase (GPx) levels were significantly lower in infertile group, compared to controls, but no significant difference in superoxide dismutase (SOD) activity was observed between control and cases. This might be due to higher expression of SOD alone in order to overcome OS in the semen. mtDNA analysis showed significant and high frequency of nucleotide changes in the ATPase (6 and 8), ND (2, 3, 4 and 5) genes of infertile cases compared to the controls. Hence excess …