Comparison of Different Sperm Isolation Strategies for Fresh and Cryopreserved Human Spermatozoa Using a Simple Method for Simultaneously Assessing Sperm Vitality and DNA Fragmentation

BACKGROUND: This study compares three different methods of sperm isolation using a novel laboratory test that permits the parallel assessment of vitality and DNA integrity in the same sperm cell. METHODS: Three different sperm isolation procedures were compared including the electrophoretic Felix System, swim-up and density gradient centrifugation (DGC), each of which utilized a different separation principle comprising net charge, intrinsic motility and isopycnic density, respectively. These sperm isolation methods were also challenged with the more difficult task of isolating spermatozoa following cryopreservation, which compromises sperm vitality, motility and DNA integrity. To facilitate this comparison, a novel assessment technique was developed wherein a live/dead stain that formed covalent bonds with intracellular amines in non-viable spermatozoa was combined with the Sperm Chromatin Dispersal (SCD) assay for DNA integrity. RESULTS: Overall, Felix generated populations of spermatozoa that were superior to DGC (p < 0.001), and equivalent to swim-up, in terms of optimizing the proportion of 'live/DNA intact' cells and rejecting the 'dead/DNA intact' and 'dead/DNA damaged' subpopulations. DISCUSSION AND CONCLUSIONS: The development of a novel method for the parallel assessment of vitality and DNA damage facilitated comparison of different sperm isolation techniques and may find applications in other areas of diagnostic andrology.