MORPHOLOGICAL AND FUNCTIONAL ANALYSIS OF CRYOPRESERVED HUMAN SPERM: COMPARISON OF DIFFERENT FREEZING PROTOCOLS

Background: Human semen and epididymal spermatozoa cryopreservation are crucial for men's fertility preservation, particularly for those patients facing neoplastic, autoimmune, urological, and neurological conditions where medical or surgical treatments may pose a risk to fertility or where obstructive or secretory azoospermia is documented. However, there are currently no standardized methods to assure optimal cryosurvival rates. Objective: To determine the best freezing protocol out of five selected methods based on routine sperm analysis and additional assays including cytofluorimetric analysis, comet assay, and transmission electron microscopy. Materials and methods: The study is a cross-sectional analysis of 26 fresh semen samples frozen using five different freezing protocols (or methods, M), varying in cooling phase time and temperatures, and utilizing TEST-Yolk Buffer (TYB) as a cryoprotectant. Data on sperm motility, viability, membrane integrity, DNA fragmentation, and ultrastructural shape post-thawing were collected. Results: Our findings showed that the method 1 (M1) and method 3 (M3) (involving a three-phase cooling process with a phase at +4 degree C, followed by 10 min of exposure to the gas phase of liquid nitrogen before immersion in liquid nitrogen) yielded the best protocols, resulting in minimal deterioration of semen quality. Conclusion: These results highlight the importance of a pre-freezing phase at +4 degree C when using TYB cryoprotectant on untreated semen, regardless of the duration, despite the less-than-optimal survival rate achieved. It is crucial to use a range of assays to study the effects of cryopreservation procedures, not only assessing sperm motility and viability, but also evaluating membrane integrity, DNA fragmentation, and ultrastructural shape.