Effects of hydrogen peroxide on DNA and plasma membrane integrity of humanspermatozoa

Objective: To evaluate the effects of oxidative stress on DNA and plasma me mbrane integrity of human spermatozoa. Design: Prospective cohort study. Setting: University-based, tertiary-care infertility center. Patient(s): Men (n = 10) undergoing infertility investigation. Intervention(s): Purified populations of sperm with high motility were sepa rated using Percoll density gradients. Then, spermatozoa were incubated wit h 0, 10, 100, and 200 muM hydrogen peroxide (H2O2) under capacitating condi tions. Main Outcome Measure(s): Motion parameters were assessed by computer analys is. Genomic integrity was examined by the terminal deoxynucleotidyl transfe rase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay. Plasma membrane i ntegrity was evaluated by the annexin V-binding assay, a measure of phospha tidylserine translocation. Result(s): Under basal conditions, there was a significant and negative rel ationship between sperm motility and the percentages of sperm with DNA frag mentation and membrane translocation of phosphatidylserine. After a 2-h inc ubation, there was a significant, dose-dependent effect of H2O2 on motion p arameters (decrease) and DNA fragmentation (increase). The percentage of an nexin V- live (normal) cells declined significantly as the level of oxidati ve stress increased. Although the percentages of annexin V+ live cells (spe rm depicting translocation of phosphatidylserine) and necrotic cells increa sed at the highest H2O2 levels, these changes were not significant. Conclusion(s): In vitro sperm incubation with H2O2 induces DNA fragmentatio n in a dose-dependent fashion. The sublethal effects of oxidative stress on motion parameters were not significantly associated with membrane transloc ation of phosphatidylserine. (Fertil Steril(R) 2000;74:1200-7. (C) 2000 by American Society for Reproductive Medicine).