A METHOD OF HUMAN SEMEN CENTRIFUGATION TO MINIMIZE THE IATROGENIC SPERM INJURIES CAUSED BY REACTIVE OXYGEN SPECIES

Current techniques of sperm preparation for in vitro fertilization or intrauterine insemination require centrifugation of human semen to sep arate spermatozoa from the seminal plasma. Centrifugation increases re active oxygen species (ROS) formation in semen. Moreover, high levels of ROS are associated with sperm membrane injury through spontaneous l ipid peroxidation, which may alter sperm function. We investigated the relationship between centrifugation variables (time and g-force) and ROS production to establish an optimal centrifugation protocol for spe rm preparation techniques, Semen from 38 men (24 patients and 14 norma l volunteers) was evaluated for the formation of ROS before centrifuga tion and after centrifugation at 200 g for 2 or 10 min and after 500 g for 2 or 10 min. The absence of white blood cells in semen which can also produce ROS was determined with the myeloperoxidase technique (En dtz test), All specimens were negative (< 1 x 10(6)/ml) by the Endtz t est, The formation of ROS was measured by chemiluminescence, ROS forma tion was regarded as high (positive) when the chemiluminescence respon se was at least 10 x 10(4) counted photons/min (cpm), The sperm concen tration in each sample was adjusted to 15-20 x 10(6) cells/ml before a nalysis. Eight specimens (7 patients and I donor) exhibited high level s of ROS before centrifugation, All 8 showed further, significant incr eases in ROS formation regardless of g-force or time. The increase in ROS was significantly less when semen was centrifuged for 2 as compare d to 10 min (p < 0.001). Six specimens previously ROS-negative became ROS-positive after centrifugation for 10 min at 200 and 500 g. We conc lude that the time of centrifugation is more important than g-force fo r inducing ROS formation in semen. Based on these results, we recommen d a shorter centrifugation period in the preparation of sperm for assi sted reproductive techniques.