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

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 (H₂O₂) 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 H₂O₂ 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 H₂O₂ levels, these changes were not significant.

Conclusion(s): In vitro sperm incubation with H₂O₂ 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.