Mitochondrial Effects, DNA Damage, and Antioxidant Enzyme Activity in Cryopreserved Human Sperm Samples: A Pilot Study

This study aimed to evaluate the impact of a vapor freezing protocol on antioxidant enzyme activity (superoxide dismutase (SOD) and glutathione reductase (GR)), sperm with active mitochondria, DNA damage, and spermatic parameters. Twenty-six semen samples from men undergoing infertility investigation were cryopreserved in liquid nitrogen (LN) vapors and plunged into LN, with (method A) and without (method B) a commercial sperm freezing medium (SFM) and inherent removal with a sperm preparation medium (SPM). Most parameters were assessed before and after freezing, except for SOD and GR activity, which were only assessed after freezing. Although method A promoted better results than method B, the percentage of spermatozoa with active mitochondria, motility, vitality, and normal morphology decreased significantly. DNA damage (determined by comet assay) increased similarly with both methods, but the percentage of spermatozoa with fragmented DNA (by TUNEL assay) remained similar to fresh values when method A was applied. GR activity was higher and SOD activity lower with method A. The addition of SFM coupled with the sperm wash with SPM seems essential to preserve the quality of most of the analyzed spermatic parameters and active mitochondria. The detrimental effects promoted by freezing were shown to depend on the quality of the fresh semen, according to correlation coefficients. Interestingly, thawed samples of both methods shared similar DNA damage. These results highlight the need to find more effective protocols, especially for the freezing of low-quality semen samples.