REACTIVE OXYGEN SPECIES GENERATION IN HUMAN SPERM - LUMINOL AND LUCIGENIN CHEMILUMINESCENCE PROBES

The objective of this study was to compare measurements of reactive ox ygen species (ROS) generation from human spermatozoa in vitro using th e Luminol and lucigenin chemiluminescent probes. Luminol reacts with a variety of reactive oxygen species (H2O2, O-2(-), OH) and allows both intra- and extracellular ROS to be measured. Lucigenin, however, yiel ds a chemiluminescence that is more specific for superoxide anions rel eased extracellularly. Therefore, measurements made with both probes o n the same samples should allow the intra- and extracellular component s of ROS generation to be identified. Sperm samples from 47 men were d ivided into two equal aliquots, then processed by centrifugation and s wim-up. Following further division into aliquots and the addition of t he two chemiluminescent probes, Phorbol 12-myristate 13-acetate was ad ded to trigger ROS release. Forty-three percent of the sperm samples g enerated detectable levels of ROS. In the centrifuged preparations lum inol produced a significantly higher peak luminescence than lucigenin. However, the sperm prepared by swim-up showed no significant differen ces in peak luminescence between luminol and lucigenin. The higher lev el of ROS generation produced by centrifugation may be due to membrane disruption or possibly the use of unfractionated cell suspensions. Ex tracellular ROS generation is more clinically important because surrou nding healthy spermatozoa may be damaged. Therefore the Lucigenin prob e may be a more useful diagnostic tool than luminol for identifying sp erm at risk of peroxidative damage after swim up preparation. The pati ents identified in this way may benefit from the addition of ROS scave ngers to the culture medium in order to protect healthy sperm from col lateral damage.