Abstract
OBJECTIVE: Sperm preparation involves separation of normal motile sperm from seminal plasma, debris and immotile/abnormal sperm to optimize fertilization in vitro. IVF clinics use established density gradient separation (DGS) procedures to isolate sperm; though DGS manufacturers are not in concert regarding processing temperatures. We hypothesize that an important variable affecting recovery/competence could be temperature of the density gradient. We compared DGS performance at 3 temperatures to determine the impact of thermal variation on sperm. DESIGN: Prospective procedure analysis. MATERIALS AND METHODS: Sperm from 3 donors were pooled and washed with Wash Media (WM) (SAGE). Samples were processed using 3 density gradient temperatures (4°C, 25°C or 37°C) with 3-layers: 95%/70%/45% (Suprasperm, Origio). DGS, samples were washed 2X, with final pellets suspended in 0.3mL WM for monitoring of motility and progression immediately, 3, 24 and 48 hrs post preparation. Two complete replicates were averaged and analyzed using χ2. RESULTS: Drops in motility were observed at 3 and 24 hrs across each temperature treatment, and significantly different (p<0.05) only after 48 hrs. The 4°C gradient resulted in an 82.6% drop in motility after 48 hrs compared to 65.4% and 63.0% decreases for 37°C and 25°C, respectively. CONCLUSION: DGS with 4°C gradient significantly effected sperm motility only after 48 hrs. It is possible cold gradient placed increased stress on the oxidative metabolism and energy needed for motility over an extended period. This study demonstrated the importance of validating manufacturer protocols and ensuring the quality of their products in producing acceptable results. We also demonstrated the importance of temperature QC during procedures. No differences were noted between experimental replicates testing the repeatability of sample processing. We continue longitudinal analyses for temperature related changes in motility, progression and recovery during sperm processing.
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