The cryopreservation of spermatozoa is an important reproductive technology for the preservation of fertility in man and animals. Since the serendipitous discovery of glycerol as an effective cryoprotectant in 1947, sperm cryopreservation has undergone many changes in terms of the freezing methods employed, the rates at which samples are frozen and thawed, and the media used to preserve sperm functionality and DNA integrity. An extensive literature survey has been conducted addressing the cryoprotectants employed for both animal and human semen and the freezing protocols utilised. The results indicate that glycerol remains the dominant cryoprotective agent, usually incorporated into a balanced salt solution containing energy substrates, buffers, osmolytes and protein in the form of human serum albumin (human) or skimmed milk (animal). Realisation that some of the damage observed in cryostored cells involves the generation of reactive oxygen species during the thawing process, has prompted many studies to assess the relative merits of incorporating antioxidants into the cryopreservation media. However, in the absence of systematic comparisons, there is currently no consensus as to which antioxidant combination might be the most effective. Utilising our fundamental understanding of cryodamage to optimise cryopreservation protocols for each species will be important in the future.

Cryopreservation of human spermatozoa is a necessity for males suffering from infertility who cannot produce fresh semen for insemination. However, current ART cryopreservation protocols are associated with losses of sperm motility, vitality and DNA integrity, which are thought to be linked to the induction of oxidative damage and the toxic properties of commercial cryoprotectants (CPAs). Preventing or mitigating these losses would be hugely beneficial to sperm survival during ART. Therefore, in this in vitro investigation, lipid peroxidation, production of reactive oxygen species, movement characteristics, antioxidant capacity, vitality, and DNA integrity were examined in semen samples both pre- and post-cryopreservation with CPA supplementation. The findings revealed a 50% reduction in antioxidant capacity with CPA addition, which was accompanied by significant increases in generation of reactive oxygen species and formation of lipid aldehydes. These changes were, in turn, correlated with reductions in sperm viability, motility and DNA integrity. Antioxidant supplementation generated bell-shaped dose-response curves with both resveratrol and vitamin C, emphasising the vulnerability of these cells to both oxidative and reductive stress. At the optimal dose, vitamin C was able to significantly enhance vitality and reduce DNA damage recorded in cryopreserved human spermatozoa. An improvement in sperm motility did not reach statistical significance, possibly because additional pathophysiological mechanisms limit the potential effectiveness of antioxidants in rescuing this aspect of sperm function. The vulnerability of human spermatozoa to reductive stress and the complex nature of sperm cryoinjury will present major challenges in creating the next generation of cryoprotective media.

Purpose: To create a novel medium that retained human sperm quality following cryopreservation at a higher level than that seen with currently available commercial cryoprotectants. Methods: Cryopreservation was achieved via 1:1 dilution with cryoprotectant followed by slow-programmed freezing. A NaCl-free cryopreservation carrier medium based on the use of histidine as the major osmolyte was designed that was capable of sustaining human sperm motility over 6 days at ambient temperature. This medium was supplemented with ethylene glycol, glycerol, and DMSO to create the basis for a novel cryopreservation medium. Dose-dependent studies with various supplements were then conducted to optimize the effectiveness of this formulation including assessments of vitamin C, EDTA, crocin, zinc, ergothioneine, and myo-inositol, as well as the potential replacement of DMSO by Cyrene™. Post-thaw samples were assessed for motility, vitality, and DNA integrity and then reassessed following sperm isolation with the Felix™ System. Results: The completed cryopreservation formulation comprised 4.5% ethylene glycol, 4.5% glycerol, 1% DMSO in a carrier medium supplemented with 0.4 mM vitamin C, 1 mM EDTA, and 22 mM myo-inositol. Spermatozoa frozen in this medium and isolated using the Felix™ System had significantly greater total motility, progressive motility, vitality, and DNA integrity than spermatozoa frozen in a commercially available product that is widely used in infertility clinics. Conclusion: A novel cryopreservation medium has been developed in this study that represents a significant improvement over existing technologies.