On osmolality and sperm function during processing for assisted reproduction

Sammanfattning: Deep basic knowledge about sperm physiology is relevant and important to optimize the outcome of procedures used during Assisted Reproductive Technologies (ART) to select spermatozoa for fertilization. More specifically, this study examined osmolality changes and consequences for sperm motility and sperm selection in the laboratory. What kind of environmental changes occur and what challenges must the spermatozoa endure after leaving the body? How do these challenges affect the spermatozoa’s functions, fertilizing potential and the make-up of the genetic material they will deliver to the oocyte? In study I, the objective was to measure the changes in osmolality that occur after collecting the ejaculate in the laboratory. After ejaculation, the sample is mixed in order to make it homogenous. This will cause the different fractions that make up the semen sample to mix. A total of 348 individual ejaculates, 5 split ejaculates and 6 ejaculate pools were studied, and it appeared that there was an individual pattern of change in osmolality over time. At 3 hours after the ejaculation, the change in osmolality ranged from 2 mOsm/kg to 164 mOsm/kg. Furthermore, it was evident that the change in osmolality was temperature dependent. Samples stored at 37°C increased significantly more in osmolality than samples stored at 18- 22°C, than samples stored at 4-7°C and than samples stored at -20°C. Denaturising temperature (100°C) blocked any further increment in osmolality. One probable cause of the increase in osmolality is that the enzymes, which are abundant in the prostatic fluid, are degrading macro-molecules, such as the proteins that are abundant in the seminal vesicular fluid. When these two secretions are mixed, the enzymatic degradation can start (Mann and Lutwak–Mann, 1981). In study II, the markers for the different fractions of the ejaculate were measured in order to relate to the change in osmolality. As well as containing high levels of proteins, the seminal vesicular fluid also contains relatively high levels of fructose. Similarly, the prostatic fluid contains high levels of zinc. It was shown that 19% of the variation in semen osmolality covaried with the relative contribution of the prostatic fluid marker, zinc, and the seminal vesicular marker, fructose, while the epididymal marker neutral α-glucosidase did not covary. Furthermore, the results show that after removing sperm from the ejaculate, the osmolality still increased, thus, the sperm did not have an effect on the increase. In addition to the challenge of the osmotic increase occurring in the ejaculate, the preparation of the sperm for ART presents yet another challenge. Most commercial sperm preparation media, such as density gradients or swim-up media have an adjusted osmolality of 290- 300mosm/kg. Thus, depending on the individual increase in osmolality of the samples, the sperm will be exposed to varying sudden decreases in osmolality during preparation. In study III and IV, it was examined how a hypo-osmotic challenge could affect sperm motility and the outcome of sperm selection when using density gradient centrifugation. Sperm motility was assessed by Computer Assisted Sperm Analysis (CASA). When the spermatozoon was exposed to a sudden decrease in osmolality, it took up water and swelled, causing the tail to coil and fold. This in turn, resulted in a decreased motility (VCL) with as much as 20%. Furthermore, it appears that the greater the decrease in osmolality, the lower the yield was after selection of spermatozoa by density gradient centrifugation. In contrast, with further investigation, it was shown that the DNA-Fragmentation-Index (DFI), measured by flow cytometry of acridine-orange stained spermatozoa was not affected by longer incubation times. However, spermatozoa ejaculated directly into a buffer had lower values for DFI% compared to samples diluted with buffer shortly after ejaculation. The negative effect on the yield was eliminated when the ejaculate was diluted soon after ejaculation or collected directly in a buffered solution. Since the increase in osmolality in vitro is so variable, one standardized procedure for sperm preparation would not work for all ejaculates. However, if increasing osmolality can be minimized by early dilution of all samples, then the negative effects can in large be eliminated.