Relationship between resazurin reduction test, reactive oxygen species generation, and gamma-glutamyltransferase

The production of reactive oxygen species (ROS) from spermatozoa has been measured by chemiluminescence in the two fractions of a Percoll gradient column (47 and 90%). Chemiluminescent signals were recorded in each fraction after addition of luminol and horse-radish peroxidase (basal state), and after stimulation with formyl-methionyl-leucyl-phenyl-alanine (FMLP) and phorbol ester (PMA). We found an inverse correlation between the reducing capacity of the semen as estimated by the resazurin test, and the production of ROS by the spermatozoa, especially after stimulation with PMA (r = 0.51, P < 0.001). gamma-glutamyltransferase activity of seminal plasma was inversely correlated with ROS (r = -0.47, P < 0.01) and the resazurin test result (r = -0.43, P < 0.001) suggesting a possible role of prostatic secretions in the protection of spermatozoa against oxygen radicals. The resazurin test has a positive predictive value of 92.5% at a criterion value of colour scale 4 to discriminate between normal and excessive ROS production by spermatozoa, with sensitivity 79% and specificity 74%. In addition, at criterion value of colour scale 3, the resazurin test could distinguish between samples with normal or low activity of gamma-glutamyltransferase, with sensitivity 57% and specificity 93%. It is concluded that the result of the resazurin test can be influenced by the oxidative stress of spermatozoa and by prostatic function.