Aseptic Technology for Cryoprotectant-Free Vitrification of Human Spermatozoa by Direct Dropping into Clean Liquid Air: Apoptosis, Necrosis, Motility, and Viability.

Mengying Wang,Vladimir Isachenko,Igor I Katkov,Evgenia Isachenko,Gohar Rahimi,Peter Mallmann,Plamen Todorov

doi:10.1155/2020/2934315

Abstract

This study aimed to compare the quality of human spermatozoa vitrified by direct plunging into liquid nitrogen vs. liquid air. Spermatozoa were divided into three groups: fresh spermatozoa (Group F) were used as a control. Spermatozoa suspension (20 μl) was vitrified in open granules by direct dropping into liquid nitrogen (Group LN) or clean liquid air (Group LA). After warming at 37°C, the progressive motility rate of Group F was reduced from 65.9 ± 2.5% to 34.0 ± 1.9% (Group LN) and 38.1 ± 2.3% (Group LA), respectively (P1-2,3 < 0.05). The reductions in viability were 65.6 ± 2.2%, 29.0 ± 1.8%, and 36.6 ± 2.6% for Groups F, LN, and LA, respectively (P1-2,3 < 0.05). Comparing spermatozoa vitrified in liquid nitrogen vs. liquid air, no significant differences were detected in motility (34.0 ± 1.9% vs. 38.1 ± 2.3%), viability (29.0 ± 1.8% vs. 36.6 ± 2.6%), early apoptosis (13.8 ± 1.5% vs. 14.3 ± 1.8%), late apoptosis (45.5 ± 1.8% vs. 43.7 ± 2.2%), and necrosis (19.5 ± 2.0% vs. 15.0 ± 1.8%; p > 0.01 for all respective differences). There was a statistical tendency for increasing rates of “progressive motility” and “viability” and decreasing rates of “apoptosis” and “necrosis” when comparing spermatozoa vitrified in liquid air vs. liquid nitrogen. It is concluded that cryoprotectant-free vitrification by the direct dropping of human spermatozoa in a clean cooling agent (liquid air) is a good alternative to the use of nonsterile liquid nitrogen and can be used to cool cells while minimising the risk of microbial contamination.

Highlights

Cryopreservation of spermatozoa is an important technology of reproductive medicine [1, 2]
Since data on the cryopreservation of human spermatozoa in the presence of cryoprotectants were first published in the late 1950s [3], several cryopreservation methods have been introduced, including conventional freezing and vitrification techniques [4,5,6]
Negative impacts of conventional cryopreservation on spermatozoa functions might include intracellular ice crystal formation, cellular dehydration, osmotic injury, cytoplasm damage, functional destabilisation, and mutagenesis [8,9,10,11]. is method can lead to the alteration of membrane permeability [12,13,14]. To avoid these detrimental effects, cryoprotectant-free vitrification technology has been developed [13, 15,16,17,18]. It was noted the high effectiveness of technology for cryoprotectant-free vitrification by direct dropping of human spermatozoa suspension into liquid nitrogen [6, 19,20,21]

Summary

Introduction

Cryopreservation of spermatozoa is an important technology of reproductive medicine [1, 2]. Since data on the cryopreservation of human spermatozoa in the presence of cryoprotectants were first published in the late 1950s [3], several cryopreservation methods have been introduced, including conventional freezing and vitrification (cryopreservation by direct plunging into liquid nitrogen) techniques [4,5,6]. Is method can lead to the alteration of membrane permeability [12,13,14] To avoid these detrimental effects, cryoprotectant-free vitrification technology has been developed [13, 15,16,17,18]. To avoid the risk of microbial contamination, for cooling of cells, clean liquid air was used (bench-top device CLAir, FertileSafe, Nes Ziona, Israel) [27]. Is study aimed to compare the motility, viability, and rates of apoptosis and necrosis of human spermatozoa vitrified in open granules, without permeable cryoprotectants, comparing direct plunging of the spermatozoa droplets into liquid nitrogen vs. liquid air

Materials and Methods

Conclusion

Findings

Disclosure