The effect of sperm separation on sperm chromatin decondensation and motility at 0 and 24 hours of culture

Abstract

OBJECTIVE: The objective of this study was to determine which sperm separation technique yields the lowest level of sperm DNA fragmentation. DESIGN: Experimental. MATERIALS AND METHODS: Semen samples were collected, liquefied and divided into the following treatments: 1) Semen mixed v/v with 2% H2O2 (control), 2) Fresh sample, 3) Sperm washed and centrifuged in HTFH (wash) 4) Swim-up (SU) from washed pellet of sperm 5) 45/90% ISolate® density gradient separation (DG) and 6) Density gradient separation followed by swim-up (DG/SU). Following sperm separation, DNA integrity was measured and 100,000 sperm/ml from treatments 2-6 were cultured in HTF + 10% SSS at 37° C in 5% CO2 for 24 h. Following culture, sperm were analyzed using the sperm chromatin decondensation assay (SCD) and motility was recorded. Results of sperm DNA fragmentation and motility were analyzed using ANOVA and Turkey's test for means. Significance was determined at P<0.05. RESULTS: DNA fragmentation in sperm processed using SU, a DG and a DG/SU (8, 7, and 4%, respectively) were significantly lower than fresh and washed samples (19, 16%), respectively. After 24 h there were no differences in DNA fragmentation between washed and SU (26 and 20%), respectively. DNA fragmentation was significantly lower in the DG (12%) compared with the wash and SU treatment. There was significantly less DNA fragmentation in DG/SU (6%) than all other treatments analyzed. There was significantly higher sperm motility in the DG/SU treatment (84%) compared to all other treatments analyzed. There were no differences in sperm motility between the wash and SU treatments (38, 53%), respectively. Sperm separated with a DG had 67% motility and was significantly higher than washed semen (P<0.5). CONCLUSIONS: Immediately following sperm processing, SU, DG and DG/SU had significantly reduced levels of DNA fragmentation compared to other separation methods. These results demonstrate the strength of these techniques for ICSI, as the chance for selecting a fragmented sperm is greatly reduced. Results from the 24 h study demonstrate the strength of using DG/SU for IVF as sperm from this treatment has greatly reduced DNA fragmentation and higher rates of motility compared to all other sperm separation methods examined.