Cryoprotectant-free vitrification of human spermatozoa in large (up to 0.5 mL) volume: a novel technology

The aim of this study was to develop and to test the aseptic technology of cryoprotectant-free vitrification of human spermatozoa in large volume (for intrauterine insemination). Spermatozoa, vitrified by this technology, are free of permeant cryoprotectants and are ready for further use immediately after warming without any additional treatment (centrifugation or separation in the gradient for removal of cryoprotectant).

Each of 52 swim up-prepared ejaculates were divided into three aliquots and distributed into three treatment groups: Group 1: non-treated control; Group 2: spermatozoa cryopreserved by slow conventional freezing with glycerol-containing medium, and Group 3: spermatozoa vitrified in 0.5 mL insemination "French" straws in culture medium with 0.25 M sucrose. Sperm motility 1, 24 and 48 hours after warming, plasma membrane integrity and capacitation-like changes (spontaneous "cryo-capacitation" and acrosome reaction) were assessed after freezing-thawing.

In contrast to conventional freezing, spermatozoa vitrified with aseptic cryoprotectant-free technology displayed superior functional characteristics. The motility rate, integrity rates of cytoplasmic, and acrosomal membranes were significantly higher after vitrification than after conventional freezing (76% vs 52%, 54% vs 28% and 44% vs 30%, respectively) (p < 0.05). However, there were no differences between vitrification and conventional freezing in the presence of glycerol in terms of percentage of spermatozoa expressing CTC-capacitation pattern (11% vs 10%, respectively) (p > 0.1).

A basic protection from cryo-injury can be achieved for human spermatozoa using the novel technology of aseptic cryoprotectant-free vitrification in large volumes.