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

Abstract

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.