Effect of repeated vitrification-thawing on sperm chromatin and morphology

Abstract

Cryoinjury in sperm after repeated cycles of thaw-refreezing is of interest in patients with few sperm, cancer patients and for couples needing same donor sperm. Presently, there is little information on sperm chromatin integrity after repeated vitrification. The null hypothesis was that this process would not disrupt sperm chromatin and alter morphology. The objectives were: (1) to analyze sperm chromatin and morphology after repeated vitrification and thawing and (2) to compare vitrification with a standard cryopreservation method for preservation of chromatin integrity. In vitro study in an IVF center. Sperm cells (normozoospermic, WHO criteria) were washed by 2-gradient centrifugation and resuspended in either: (1) modified human tubal fluid (HTF) medium and Irvine Scientific Freezing Medium 90128 (standard freezing) or, (2) HTF medium with 0.25M sucrose with 1% albumin (Isachenko vitrification method). Aliquots were analyzed (pre-freeze parameters). For comparison, sperm from an oligozoospermic case was processed in a similar manner. Sperm (standard freezing) were placed in Nunc cryovials (0.25 mL each) and frozen in liquid nitrogen (LN2). In the vitrification group, sperm were aspirated into 0.25mL cryostraws, sealed in 0.5mL cryostraws and frozen in LN2. After 1-week, sperm were thawed and one vial/straw was analyzed. The remaining vials/straws were re-frozen. Three cycles of repeated freeze-thaw were carried out. Differences were tested using ANOVA and Student's t-test. In the vitrification group, sperm chromatin integrity decreased by 9.6, 29.1 and 47.1% with each subsequent freeze-thaw cycle. Decreased chromatin integrity in the standard freeze group were 0.4, 12.9, 27.0% respectively. In the oligozoospermic specimen, chromatin integrity (vitrification) decreased by 45.3, 48.4 and 68.5% compared with standard freeze reduction of 25.8, 37.1 and 57.2%. Normal morphology in the vitrification group were 17.7+/-0.4, 7.0+/-1.4, 5.3+/-0.4, 5.6+/-0.4% (mean+/-SEM) while the standard freeze group were 18.3+/-0.7, 12.0+/-0.7, 9.3+/-0.4, 7.7+/-1.1% for pre-freeze and 3 cycles of freeze-thaw respectively. Similar decreases were seen in the oligozoospermic case. Sperm viability and motility also decreased in a similar manner. The results showed that vitrification was less optimal than standard freeze method when repeated freeze-thaw cycles were involved. Furthermore, normal morphology was greatly decreased with repeat vitrification suggesting extensive structural disruption. Interestingly, after the third freeze-thaw cycle, 46% (vitrification) or 62% (standard freeze) of sperm had normal chromatin suggesting adequacy for conserving sperm for patients.