Development of an improved medium for the preservation of human spermatozoa

Abstract

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.