Optimizing sperm cryopreservation and recovery (OSCAR)

Abstract

The objective of this study is to compare three common sperm freezing methods and two common thawing methods to one another to determine if there is a superior method to enhance post-thaw sperm survival. Prospective cohort study. Twelve discarded fresh semen samples were obtained with patient consent. Each fresh sample was washed with mHTF (LifeGlobal, Guilford, CT) and centrifuged for 15 minutes at 400G. The pellet was resuspended in a 3:1 ratio of mHTF to Artic™ Sperm Cryopreservation Medium (Irvine Scientific, Santa Ana, CA). Motility was evaluated manually and each participant’s sample was divided into six aliquots containing 0.5mL of specimen. Two aliquots of each specimen underwent one of 3 different freezing methods: (a) plunge into liquid nitrogen (LN2), (b) suspension in LN2 vapor for 15 minutes followed by plunge into LN2 or (c) suspension in LN2 vapor for 1 hour followed by plunge into liquid nitrogen. Each of the freezing methods was subject to two different thaw methods: (a) 37°C dry bath for 20 minutes followed by 40 minutes at room temperature or (b) room temperature (RT) for 1 hour. Following recovery, a second evaluation of motility was performed for each aliquot as a measure of sperm viability (percent motility versus initial). Analysis by two way repeated measures ANOVA was utilized with a p-value of 0.05 to determine significance. The mean motility for plunge sperm thawed at RT and 37°C were 22.9 ± 0.04% and 20.3 ± 0.04%, respectively. The mean motility for 15 minutes vapor then plunge thawed at RT and 37°C were 31.7 ± 0.06% and 31.5 ± 0.07%, respectively. For 1 hour in vapor then plunge, the mean motility at RT and 37°C were 43.8 ± 0.07% and 48.1 ± 0.06%, respectively. Survival rates between all freezing methods varied significantly, with greater recovery noted in the 1 hour vapor phase when compared both plunge (p<0.001) and 15 minutes in vapor (p=0.005). The shorter vapor phase of 15 minutes also demonstrated statistically greater viability compared to plunge (p = 0.027). Thawing methods did not significantly affect sperm recovery. The recovery of motile sperm varied directly with the length of cooling prior to plunge in LN2 with an hour suspension in vapor resulting in greatest motility compared to the initial sample. Recovery was not dependent on the thaw method used, with either of the two methods producing similar results for a given cryopreservation method. Interestingly, it was also noted that samples plunged directly from room temperature into LN2 retained appreciable, if diminished viability