Received August 11, 2001
Accepted March 12, 2001

The objectives of these studies were:(1) to evaluate the effect of cryopreservation-thawing of human spermatozoa on DNA fragmentation and membrane integrity; and(2) to examine time dependent effects on membrane integrity and motion parameters of sperm incubated under capacitating conditions. This was a prospective, controlled cohort study. ln experiment one, ejaculates from 16men undergoing infertility evaluation(patients) and from 5 donors were examined. Purified sperm populations with high motility were prepared by gradient centrifugation, cryopreserved using a manual method and TEST-yolk buffer and glycerol (TYB-G), followed by quick thaw. Annexin V binding was used for assessing membrane translocation of phosphatidylserine (PS) and terminal deoxynucIeotidyl transferase-mediated dUTP nick end labeling (TUNEL) was utilized for the evaluation of DNA fragmentation. ln experiment two, 11ejaculates from patients and 5 from donors were examined. ln experiment three, we studied ejaculates from 16 patients and 5 donors; . The results were as follows:Experiment1: the percentage of live cells with intact membranes(annexin V,live) was significantly reduced after cryopreservation-thawing, and cells with PS translocation. TUNEL revealed percentages of cells with DNA fragmentation in the pre-freeze and post-thaw samples that were not significantly different. ln experiment2: the percentages of live cells with PS translocation and of necrotic cells increased significantly after thawing in both fractions; however, such induction of PS translocation was significantly higher in the fractions with high sperm motility. ln experiment 3: the percentage of live cells with intact membranes was significantly reduced mainly at 6-8hours of incubation and cells with PS translocation and necrotic increased significantly. We concluded that cryopreservation-thawing of human sperm from patients and donors was associated with membrane change as revealed by membrane translocation of PS while having no major impact on DNA fragmentation. Prolonged incubation of human sperm was associated with membrane PS translocation and a significant time-dependent motility loss.