Density gradients may be either continuous or discontinuous although the discontinuous gradients have been used almost exclusively since the late 1980s (21). Discontinuous gradients are usually prepared with two or three layers. Colloidal silica with covalently bound silane molecules is probably the most common density gradient material currently used for clinical IVF and andrology. PureSperm® (NidaCOn International AB, Goteborg, Sweden), Isolate® (Irvine Scientific, Santa Ana, California, U.S.A.), IxaPrep (MediCult, Copenhagen, Denmark), and Enhance® (Conception Technologies, San Diego, California, U.S.A.) are examples of silane-coated silica particle solutions that can be used for discontinuous gradients. These products are made isosmotic by the inclusion of polysucrose; they have very low toxicity, are nonirritating, and are approved for human in vivo use. As with any product, it is important to follow the manufacturer's recommendation for proper use and application.
In the discontinuous density gradient method, the ejaculate is placed on top of the density gradient medium and is centrifuged at 300-400 g for 15-30 minutes. As the density gradient medium is a colloid rather than a solution, it has low viscosity and it does not retard the sedimentation of spermatozoa due to centrifugation (21). Highly motile spermatozoa move actively in the direction of the sedimentation gradient and can penetrate the boundary faster than poorly motile or immotile spermatozoa (12). Thus, the soft pellet at the bottom is enriched for highly motile spermatozoa. The pellet is washed with culture medium and centrifuged at 200 g for 4-10 minutes. The wash and centrifugation is then repeated to ensure removal of contaminating density gradient medium. The final pellet is resuspended in culture medium so that concentration and motility can be determined.
Density gradient centrifugation usually results in a clean fraction of highly motile spermatozoa. As the whole volume of the ejaculate is used in density gradient centrifugation (as it is in the swim-up techniques), it yields a significantly higher total number of motile spermatozoa and it can be used for patients with varying degrees of suboptimal semen parameters (e.g., oligozoospermia and asthenozoospermia). Other advantages of density gradient centrifugation include the elimination of leukocytes and the significant reduction of ROS (12). Additionally, Nicholson et al. (22) demonstrated that centrifugation through one brand of silane-coated silica particles (PureSperm) efficiently reduces bacterial contamination. Hamma-deh et al. (23) reported that another advantage of the density gradient method is the recovery of a higher percentage of morphologically normal spermatozoa than found in conventional swim-up or glass wool filtration. The technique has also been shown to yield sperm populations with better DNA quality and chromatin packaging (24,25). Further, preliminary reports suggest that specimens known to be contaminated with sexually transmissible viruses can effectively be "cleaned up'' using density gradient centrifugation and the isolated spermatozoa can be used for therapy with exceptionally low risk for horizontal disease transmission (26). One disadvantage of density gradient centrifugation is that the density gradient medium is a bit more expensive than either of the swim-up techniques.
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