41 Effect of vitrification on DNA integrity of human spermatozoa

Abstract

The aim of our study was to investigate the effect of vitrification with sucrose on swim-up-prepared human spermatozoa in comparison with standard, conventional manual freezing with permeable cryoprotectants. After informed consent, 35 ejaculates were obtained from 35 patients with normozoospermia who were patients of a fertility clinic. All specimens used for this study had fulfilled the following quality criteria for spermatozoa concentration and motility on IVOS (Hamilton Thorne). Semen analysis was performed according to published guidelines of the World Health Organization (WHO Laboratory Manual for the Examination and Processing of Human Semen, 2010). After swim-up, each sample was centrifuged, resuspended with the basic medium (human tubal fluid + 1% human serum albumin) to achieve a concentration of 5 × 10⁶ spermatozoa/mL, and finally aliquoted into two equal subsamples. Each of these aliquots was assigned to one of two groups: group 1 included conventionally cryopreserved spermatozoa and group 2 included spermatozoa that were vitrified. For conventional cryopreservation, freezing media (15% (vol/vol) glycerol, 20% (vol/vol) egg yolk) and citrate was added to the washed spermatozoa in a 1:2 ratio. The sperm suspension was aspirated into 0.5-mL straws (CryoBioSystem). Subsequent to the room-temperature incubation for 10 min, straws were placed horizontally in the vapour phase for 15 min and then submerged into liquid nitrogen. For thawing, cryopreserved straws were immersed in water (23°C) for 5 min. For preparation of vitrification solution, the basic medium (human tubal fluid + 1% human serum albumin) was diluted 1:1 with 0.5 M sucrose. Immediately after processing, the sperm suspension was diluted in a 1:1 ratio with the vitrification solution to reach a final sucrose concentration of 0.25 M. The vitrification and sperm solution (300 μL) were aspirated into the straws 0.5 mL. Straws were then left at room temperature (20-21°C) for 10 min and subsequently submerged horizontally into the liquid nitrogen (Isachenko et al. 2012 J. Androl. 33, 462-468; https://doi.org/10.2164/jandrol.111.013789) and stored similarly to the conventionally cryopreserved straws. To thaw, vitrified straws were immersed in a water bath (42°C) for 20 s. The DNA fragmentation was analysed using the APO DIRECT kit (BD Pharmingen^TM). The cells were stained according to the manufacturer's protocol, followed by flow cytometry analysis CyFlow (Sysmex-Partec). An analysis of variance with a significance of 0.05 for nonparametric statistical analysis to establish differences between groups was used. In our study, no statistically significant differences were observed in the total motility, progressive motility, or velocity parameters of spermatozoa (P > 0.05) post-thawing. Also, higher percentages of DNA fragmentation (35.1 ± 8.1% vs. 20.1 ± 6.8%; P < 0.05) were found in spermatozoa cryopreserved by means of vitrification with sucrose compared with conventional cryopreservation. Therefore, these methods are comparable and either can be implemented for the storage of spermatozoa to be used for future assisted-reproduction-technology procedures. Vitrification of human spermatozoa provides a simpler, faster, more cost-effective alternative to conventional cryopreservation methods.