Handling of Human Ejaculate Affects Osmolality and Yield by Density Gradient Centrifugation and Possibly Also Sperm DNA Integrity

During routine handling in the assisted reproductive technology (ART) laboratory, the osmolality of the ejaculate increases, and live spermatozoa adapt to this increase. Preparation media are generally isotonic, having an osmolality of ~290 mOsm/kg. Therefore, when the ejaculate is placed on a density gradient, the spermatozoa are exposed to a hypotonic challenge. The aim of this study was to investigate whether osmotic challenges affect the yield of density gradient centrifugation (DGC), the DNA fragmentation index (%DFI), and the proportion of high DNA stainable sperm (%HDS). Furthermore, it was investigated if these effects could be counteracted by dilution of the ejaculate. Semen samples from healthy donors were collected and used after written consent. Four protocols of different experimental conditions were followed. These included storage at 37°C, storage at room temperature (RT), dilution after liquefaction, and ejaculation directly into a buffer. Pairwise comparisons of the studied aliquot and its own control were performed. Storage at 37°C led to an increase in osmolality to 352 mOsm/kg and halved the yield to 4.5%. Storage at RT led to an increase in osmolality to 384 mOsm/kg and halved the yield to 5.8%. The %DFI and %HDS did not change during these storage conditions. In undiluted samples, osmolality increased to 373 mOsm/kg and in diluted to 323 mOsm/kg. The %DFI and %HDS did not change. After ejaculation into a buffer, the osmolality increased only to 315 mOsm/kg after storage and the yield halved after storage to 12.9%. The %DFI was significantly lower after ejaculation into a buffer compared to dilution after liquefaction, 6% versus 11%, respectively. No difference was observed in %HDS. The challenges to sperm function induced by osmolality changes, and possibly also sperm DNA integrity, can be counteracted by collecting ejaculates directly into an isotonic buffer.