Effect of sperm washing on levels of reactive oxygen species in semen

The possibility was evaluated that the production of reactive oxygen species (ROS) by human sperm is stimulated by the repeated cycles of centrifugation and resuspension involved in conventional sperm preparation. ROS generation by human sperm was monitored before and after the washing of sperm from 55 men (43 men with suspected subfertility and 12 normal volunteers). The ROS activity of all 55 specimens before washing was inversely correlated with original sperm motility (r = .278, p < .05). The mean level of ROS activity was significantly higher after washing than before processing (p < .05) for the 26 specimens with normal sperm motility, the 20 specimens with normal sperm morphology, and the 12 specimens with both normal motility and normal morphology. In contrast, the mean ROS level was not significantly changed after washing in the 27 specimens with poor sperm motility, the 16 specimens with abnormal sperm morphology, or the 13 specimens with both abnormal motility and abnormal morphology. It would appear that repeated centrifugation, resuspension, and vortexing cause excessive generation of ROS in the motile sperm population of the washed specimen. Washing procedures involving excessive manipulation of sperm may, in fact, cause the most harm to motile sperm, i.e., those that the method is trying to select. Procedures that minimize multiple centrifugation, resuspension, and vortexing steps should therefore be used for the preparation of semen specimens for assisted-reproduction techniques.