Cryo-banking of human spermatozoa by aseptic cryoprotectants-free vitrification in liquid air: Positive effect of elevated warming temperature.

Cryoprotectant-free vitrification is a common method for spermatozoa cryopreservation by direct plunging into liquid nitrogen. However, the commercial liquid nitrogen could be potentially contaminated by microorganisms. Warming temperature plays an essential role for quality of human spermatozoa after vitrification. This study aimed to evaluate comparatively a quality spermatozoa after vitrification in liquid nitrogen and clean liquid air as well as with two warming rates: at 42 °C and 45 °C. After performing of routine swim-up of normozoospermia samples, spermatozoa from the same ejaculate were divided into two groups: vitrified in liquid nitrogen (LN) and sterile liquid air (LA). Spermatozoa of LN group were warmed at 42 °C, and spermatozoa of LA groups were divided and warmed at 42 °C (LA42) and 45 °C (LA45). Then spermatozoa motility, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), reactive nitrogen species (RNS), and viability were assessed. It was no found significant differences in quality of spermatozoa from LN and LA groups in the motility, ROS, MMP, RNS rates after warming at 42 °C. A tendency to obtain better spermatozoa quality was found with using of warming by 42 °C in comparison with 45 °C. It was concluded that cryoprotectant-free vitrification by direct dropping of human spermatozoa into clean liquid air can be used as an alternative to cooling in liquid nitrogen. Warming of spermatozoa at 42 °C allows to preserve the spermatozoa physiological parameters.