Probing the impact of commercial cryoprotectants and freezing technique on the motility of human spermatozoa cryopreserved onto extremely water-repellent soot-coated surfaces

Abstract

The cryopreservation of human spermatozoa is an integral part of cryobiology, aiming to support the in-vitro fertilization. The latter relies on the availability of as much as possible reproductively active spermatozoa, whose number after thawing decreases due to the accompanied freezing injury and the cytotoxicity of cryoprotectants. An innovative option to circumvent these obstacles is to make the freezing interface non-wettable, by coating it with rapeseed oil soot possessing intrinsic cryoprotective properties, delaying the ice formation and possibly providing identical rates of intracellular dehydration and extracellular crystallization. It may mean that this technique can reduce or avoid the need of harmful cryoprotective agents, but to reach such a developmental stage, the synergistic effect of certified cryoprotectants and cooling velocities on the efficiency of soot-mediated sperm cryopreservation must be clarified. With the intention to address this research gap, we reveal that the slow freezing/thawing of distinct mixtures of three cryoprotectants (SpermFreeze™, CryoSperm™ and DMSO) and the human semen of nine patients equalizes the percent of survived spermatozoa, but declines their curvilinear velocity. At instant freezing (∼7–20 s) and slow thawing, via specially-designed soot fabric-coated sheet metal cryoboxes, the inclusion of 10 % DMSO is noxious, but the post-thaw motility reaches 74–100 % independently of the cryoprotective solutes. These surprising findings are ascribed to the formation of a quasivitrified semen, whose complete freezing ensures a fraction of extracellular ice matching that from the equilibrium phase diagram, eluding the osmotic shocks and paving the path for future replacement of the classic vitrification.