Ultra-rapid freezing of very low numbers of sperm using cryoloops

Abstract

Background: With the availability of ICSI, men with severe oligozoospermia (<5×106/ml) are able to reproduce. Current methods for cryopreservation of severe oligozoospermic samples are labour intensive and costly. The objective of this study was to evaluate whether freezing small numbers of motile sperm (∼100) was feasible using cryoloops. Methods: Initial tests assessed the effect of various dilutions of cryoprotectants on pre-freezing sperm motility. Several solutions were further evaluated for their ability to cryoprotect sperm during ultra-rapid freezing. Sperm were placed on cryoloops and held in liquid nitrogen vapour for 5 min prior to freezing (ultra-rapid freezing) or directly submerged into liquid nitrogen. Using the optimal cryoprotectant and technique from these experiments, ultra-rapid and standard slow-rate freezing protocols were compared. Results: Optimal sperm survival was seen when sperm in cryoloops were placed in liquid nitrogen vapour in test yolk buffer with 12% v/v glycerol versus other cryoprotectants. Using this cryoprotectant, post-thaw sperm motility is comparable between ultra-rapid and slow-rate freezing methods. Conclusion: Ultra-rapid freezing of very low numbers of sperm is feasible using cryoloops suspended in liquid nitrogen vapour for 5 min.