Cryopreservation of Human Spermatozoa Using Penetrating Cryoprotectant-Free Vitrification Medium Exhibits an Altered Proteomic Profile Compared to the Conventional Rapid Freezing Method.

The present study aimed to assess the functional and proteomic changes in human spermatozoa cryopreserved in a vitrification medium without a penetrating cryoprotectant. Leftover ejaculates (n = 71) from men visiting the andrology laboratory for routine semen analysis were used. The liquefied semen samples were cryopreserved using rapid freezing and vitrification for a minimum of seven days. Post-thaw motility, mitochondrial function, DNA damage, and acrosomal integrity of spermatozoa were similar between the cryoprotectant-free vitrification and rapid freezing methods. However, the head morphology of spermatozoa was better preserved when cryopreserved by vitrification method. Proteomic analysis led to the identification of a total of 4378 proteins in spermatozoa. Proteomic profiling identified 760 and 1661 significantly altered proteins in the spermatozoa subjected to rapid freezing and vitrification, respectively, compared with fresh spermatozoa. Gene Ontology (GO) analysis revealed considerable overlapping functional enrichment patterns between rapid freezing and vitrification, indicating that both cryopreservation methods influence several common functional pathways in spermatozoa. Both methods resulted in differential abundance of proteins involved in cytoskeletal organization, oxidative stress response, energy metabolism, and fertilization. Although functional analyses demonstrated that the functional properties of spermatozoa cryopreserved by penetrating cryoprotectant-free vitrification is similar to those subjected to rapid freezing, proteomic analysis revealed distinct molecular alterations, warranting further investigation to establish the possible differences in fertilizing potential, long-term safety aspects and reproductive outcomes.