Cryopreservation of fractionated, highly motile human spermatozoa: effect on membrane phosphatidylserine externalization and lipid peroxidation

INTRODUCTION: This study investigated lipid peroxidation (LPO) and membrane integrity following cryopreservation-thawing. METHODS: Infertile men (stud y group) and donors (control group) were examined. Purified populations of highly motile spermatozoa were cryopreserved using TEST-yolk buffer and gly cerol (TYB-G) followed by quick thaw. LPO was measured by a spectrophotomet ric assay, with and without a ferrous ion promoter. Annexin V binding was u sed to assess membrane translocation of phosphatidylserine (PS). RESULTS: P re-freeze LPO was significantly higher in the study than in the control gro up (P = 0.03). In both groups, LPO measurements after thawing were signific antly higher than the pre-freeze samples not exposed to TYB-G (P = 0.002 an d P = 0.001 respectively). However, when the pre-freeze samples with TYB-G were compared with the post-thaw samples (all exposed to TYB-G), these diff erences were not significant. There was a significant increase in PS extern alization following cryopreservation in both groups (P = 0.02 and P = 0.003 respectively). In donors, pre-freeze LPO concentrations had a significant positive correlation with thawed spermatozoa depicting PS externalization ( r = 0.77, P = 0.04). CONCLUSION: Although patients had higher basal LPO tha n donors, LPO did not differ between fresh and cryopreserved-thawed fractio nated motile spermatozoa. Freezing-thawing was associated with translocatio n of PS to the external membrane leaflet.