Quantitative prospective experimental study on time-dependent ultrastructural changes in human spermatozoa after cryopreservation

Sperm cryopreservation is a common procedure in assisted reproduction (ART) laboratories. Although generally regarded as an efficient and safe technique, a decrease in the fertilization potential has been described. Hence, a detailed ultrastructural analysis of human spermatozoa might provide important insights into cellular alterations occurring during cryopreservation. To address this question, the ultrastructure of human spermatozoa was analysed, before and after cryopreservation, for the time periods of one month (1-Mo) and seven months (7-Mo), using paired, repeated measures. Semen samples were cryopreserved with a rapid freezing protocol, using a glycerol- and raffinose-containing cryopreservation medium, allocated into cryo-straws and stored in liquid nitrogen. Ultrastructural results indicate a significant increase in anomalous sperm, with changes in the head membrane and acrosomal vesicle, after both cryopreservation periods (fresh:26.8% vs. 1-Mo:52.2%, 7-Mo:57.4%, P<0.001, partial η²=0.849), suggesting a concurrent cryo-damage to these structures. Concerning the sperm head membrane, a significant increase in total detachment was observed, after 1 and 7 months of cryopreservation (fresh:9.3%, 1-Mo:26.3%, 7-Mo:34.5%, P<0.001, partial η²=0.727); for the acrosomal vesicle, a significant increase was observed in apical detachment after 1 month (fresh:11.4%, 1-Mo:22.9%, 7-Mo:17.1%, P=0.006, partial η²=0.433) and in total detachment after 7 months (fresh:24.9%, 1-Mo:31.8%, 7-Mo:43.6%, P<0.001, partial η²=0.636). In conclusion, our results indicate that both the sperm head membrane and the acrosomal vesicle are affected by cryopreservation, with both potentially decreasing the functional competence of the spermatozoa. These data adds information about cryopreservation, highlighting the importance of detailed inspection for sperm selection during ART. (244/250)