Evaluation of chemiluminescence and flow cytometry as tools in assessing production of hydrogen peroxide and superoxide anion in human spermatozoa
Received 25 March 2008; received in revised form 12 May 2008; accepted 27 May 2008. published online 19 August 2008.
Objective
To examine simultaneously the levels of hydrogen peroxide (H2O2) and superoxide (O2−•) 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 O2−•.
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 O2−•-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.
aCenter for Reproductive Medicine, Glickman Urological and Kidney Institute, and Ob-Gyn and Woman's Health Institute, Cleveland Clinic, Cleveland, Ohio
bDepartment of Urology, Medical University of Vienna, Austria
cLiverpool Women's Hospital, Liverpool, United Kingdom
Reprint requests: Ashok Agarwal, Ph.D., H.C.L.D., Professor and Director, Center for Reproductive Medicine, Cleveland Clinic, 9500 Euclid Avenue, Desk A19.1 Cleveland, Ohio 44195 (FAX: 216-636-3118).
R.M. has nothing to disclose. R.S. has nothing to disclose. J.L. has nothing to disclose. N.A. has nothing to disclose. A.A. has nothing to disclose.
Presented at the 63rd Annual Meeting of the American Society of Reproductive Medicine, which was held in Washington, D.C., on October 13–19, 2007.
Supported by a research grant from the Research Programs Committee, Cleveland Clinic (RPC no. 07549).
ABSTRACT