title: Cryopreservation-Thawing of fractionated human spermatozoa is associated with membrane phosphatidylserine externalization and not DNA fragmentation.
abstract: 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-mediated 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.
authors.affiliation: The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk 23507, USA.
authors.forename: N K
authors.initials: NK
authors.lastname: Duru
authors.forename: M S
authors.initials: MS
authors.lastname: Morshedi
authors.forename: A
authors.initials: A
authors.lastname: Schuffner
authors.forename: S
authors.initials: S
authors.lastname: Oehninger
mesh_terms: D017304:Annexin A5 / Q000378:metabolism / Q000494:pharmacology; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D015925:Cryopreservation; D053938:DNA Fragmentation; D006801:Humans; D020287:In Situ Nick-End Labeling; D008297:Male; D010718:Phosphatidylserines / Q000032:analysis / Q000378:metabolism*; D012662:Semen Preservation; D013081:Sperm Motility
chemical_list: D017304:Annexin A5; D010718:Phosphatidylserines