Selection of viable human spermatozoa with low levels of DNA fragmentation from an immotile population using density gradient centrifugation and magnetic‐activated cell sorting

We aimed to determine whether density gradient centrifugation and magnetic‐activated cell sorting (DGC‐MACS) could select viable spermatozoa, with lower levels of DNA fragmentation, from an immotile population. Analysis involved sixteen patients, each with a sperm count ≥10⁷/mL. All samples were immotile despite exhibiting a live population >40%. Spermatozoa were prepared using DGC‐MACS and selected spermatozoa evaluated for membrane and DNA integrity using the hypo‐osmotic swelling (HOS) test, vital staining and the TUNEL test. The mean proportion of spermatozoa with an intact membrane in control, DGC and DGC‐MACS populations, was 52.5 ± 12.21%, 69.38 ± 7.87% and 81.81 ± 5.29%. The mean proportion of live spermatozoa in control, DGC and DGC‐MACS populations, was 65.88 ± 12.77%, 77.25 ± 7.39% and 85.81 ± 5.2%. DGC‐MACS reduced the within‐sample discrepancy between HOS test and vital stain results from 13.18% to 4.12%. The mean proportion of spermatozoa exhibiting DNA damage in control, DGC and DGC‐MACS populations, was 9.56 ± 3.39%, 5.25 ± 1.61% and 2.75 ± 1.13%. Finally, analysis showed that 71.23% of the DNA‐fragmented spermatozoa in unprocessed samples were removed following DGC‐MACS and that the addition of MACS to an existing DGC protocol reduced fragmented spermatozoa by a further 26.15% compared to DGC alone. Consequently, DGC‐MACS is a clinically viable method able to select viable spermatozoa with lower levels of DNA fragmentation from an immotile population.