Cryopreservation‐Thawing of Fractionated Human Spermatozoa Is Associated With Membrane Phosphatidylserine Externalization and Not DNA Fragmentation

The objective of these studies was to evaluate the effect of cryopreservation‐thawing of human spermatozoa on DNA fragmentation and membrane integrity. This was a prospective, controlled cohort study, performed at a university‐based infertility center. Ejaculates were examined from 5 donors and 16 men undergoing infertility evaluation. Purified sperm populations 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, and terminal deoxynucleotidyl transferase‐me‐diated dUTP nick end labeling (TUNEL) was utilized for the evaluation of DNA fragmentation. The results were as follows: the percentage of live cells with intact membranes (annexin V−, live) was significantly reduced after cryopreservation‐thawing. On the other hand, the percentages of live cells with phosphatidylserine translocation (annexin V+, live) and of necrotic (dead) cells increased significantly after thawing. TUNEL revealed percentages of cells with DNA fragmentation in the prefreeze and postthaw samples that were not significantly different. In a further attempt to examine differences in response to various cryoprotection protocols, experiments were carried out using no cryoprotection, glycerol alone, or TYB‐G. Samples frozen with TYB‐G demonstrated significantly higher percentages of live cells without phosphatidylserine translocation than the other conditions. We concluded that cryopreservation‐thawing of human sperm from patients and donors was associated with membrane change, as revealed by membrane translocation of phosphatidylserine, while having no major impact on DNA fragmentation.