Evaluation of chemiluminescence and flow cytometry as tools in assessing production of hydrogen peroxide and superoxide anion in human spermatozoa

Objective: To examine simultaneously the levels of hydrogen peroxide (H2O2) and superoxide (O-2(-center dot)) using chemiluminescence and flow cytometry. Design: Prospective laboratory study. Setting: Reproductive research lab in a tertiary hospital. Patient(s): Semen samples from 18 healthy male volunteers. Intervention(s): Sperm preparation and measurement of reactive oxygen species (ROS) by chemiluminescence using luminol and lucigenin before and after H2O2 exposure and by flow cytometry using dichlorofluorescin diacetate (DCFH-DA) for H2O2 and dihydroethidium (DHE) for O-2(-center dot). Main Outcome Measure(s): Sperm count, motility, viability, and ROS levels. Result(s): Immature sperm fractions showed significantly higher levels of ROS measured by either luminol or lucigenin compared with the neat and mature fraction. ROS levels were detectable by flow cytometry in chemiluminescence-negative samples. Both mature and immature sperm fractions had a significantly higher percentage of cells positive for H2O2 compared with neat semen. On the other hand, the percentage of O-2(-center dot)-positive cells in neat semen was significantly higher compared with the percentage found in mature fractions but significantly lower than that in the immature sperm fractions. Conclusion(s): We recommend ROS measurement by flow cytometry on the basis that it requires a lower sperm count, is comparable to chemiluminescence, and has higher specificity for intracellular ROS in viable spermatozoa. Samples tested negative by chemiluminescence still may have high intracellular H2O2 generation that can be detected by flow cytometry. (Fertil Steril (R) 2009;92:819-27.(c) 2009 by American Society for Reproductive Medicine.)