Effects of hydrogen peroxide on DNA and plasma membrane integrity of human spermatozoa

Abstract

Objective: To evaluate the effects of oxidative stress on DNA and plasma membrane 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 separated using Percoll density gradients. Then, spermatozoa were incubated with 0, 10, 100, and 200 μM hydrogen peroxide (H 2O 2) under capacitating conditions. Main Outcome Measure(s): Motion parameters were assessed by computer analysis. Genomic integrity was examined by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end–labeling (TUNEL) assay. Plasma membrane integrity was evaluated by the annexin V-binding assay, a measure of phosphatidylserine translocation. Result(s): Under basal conditions, there was a significant and negative relationship between sperm motility and the percentages of sperm with DNA fragmentation and membrane translocation of phosphatidylserine. After a 2-h incubation, there was a significant, dose-dependent effect of H 2O 2 on motion parameters (decrease) and DNA fragmentation (increase). The percentage of annexin V − live (normal) cells declined significantly as the level of oxidative stress increased. Although the percentages of annexin V + live cells (sperm depicting translocation of phosphatidylserine) and necrotic cells increased at the highest H 2O 2 levels, these changes were not significant. Conclusion(s): In vitro sperm incubation with H 2O 2 induces DNA fragmentation in a dose-dependent fashion. The sublethal effects of oxidative stress on motion parameters were not significantly associated with membrane translocation of phosphatidylserine.