Selection of spermatozoa before their use for assisted reproductive techniques is an important step in therapy of human infertility. The DNA fragmentation index of sperm plays a major role in pregnancy rates following IVF and intracytoplasmic sperm injection (ICSI). Sperm analyses and standard sperm selection methods in many cases do not eliminate a sufficient proportion of sperm with apoptosis and DNA fragmentation. Magnetic-activated cell sorting (MACS) is a selection method that eliminates apoptotic spermatozoa based on the presence of externalized phosphatidylserine residues. The aim of our study was to evaluate the effect of MACS on human sperm motility and DNA fragmentation index (DFI) in a patient population. The participants were 63 male patients of an IVF clinic, 34 to 45 years old, with 3 years of primary infertility due to male factor. Semen analysis was performed according to the World Health Organization guidelines (2010) and revealed oligoasthenoteratozoospermia in 63 patients. The DFI of fresh semen samples was evaluated using the sperm chromatin structure assay (SCSA) test and revealed DFI 32.4±5.9%. The SCSA test was done on a flow cytometer CyFlow Space (Sysmex-Partec; Evenson 2016 Anim. Reprod. Sci. 169, 56-75; https://doi.org/10.1016/j.anireprosci.2016.01.017). Sperm motility was studied on Hamilton Thorne IVOS. For MACS, we used the MACS® ART Annexin V system (Miltenyi Biotec). The semen sample was diluted to a concentration 10×106 spermatozoa mL−1. After double-density gradient centrifugation, the pellet was resuspended in 100µL of MACS Art Annexin V reagent and added MACS Art Binding Buffer (BB) to 500µL. The sample was gently mixed and incubated for 15min at room temperature. After rinsing the column with BB, the sperm-bead suspension was added on top with BB and, immediately after that, the annexin V-negative and annexin V-positive fractions were obtained (MiniMACS; Miltenyi Biotec). Data were evaluated by ANOVA Student’s t-test. Fresh semen samples collected from the patients had an average sperm concentration of 29.7±5.7×106 mL−1, motility of 32.7±5.9%, and DFI of 32.4±5.9%. Motility of spermatozoa after MACS for the annexin-negative fraction was 47.2±6.3% and for the annexin-positive fraction was 3.5±2.3% (P<0.003). Similarly, the annexin-negative spermatozoa had a lower DFI (10.5±3.8%) rate than did the annexin-positive fraction (67.8±5.9%; P<0.003). The MACS technique allowed a significant reduction of DNA fragmentation levels (from 32.4% for the original sample to 10.5% for the annexin-negative; P<0.01). The separation of a distinct population of nonapoptotic spermatozoa with intact membranes may optimize outcomes from IVF and ICSI procedures. Magnetic-activated cell sorting of human sperm using annexin-V microbeads results in selection of a population with enhanced motility and reduced DFI rates.

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 10min, straws were placed horizontally in the vapour phase for 15min and then submerged into liquid nitrogen. For thawing, cryopreserved straws were immersed in water (23°C) for 5min. For preparation of vitrification solution, the basic medium (human tubal fluid + 1% human serum albumin) was diluted 1:1 with 0.5M 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 0.25M. The vitrification and sperm solution (300μL) were aspirated into the straws 0.5mL. Straws were then left at room temperature (20-21°C) for 10min 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 20s. The DNA fragmentation was analysed using the APO DIRECT kit (BD PharmingenTM). 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.

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