These methods are based on the phenomenon that dead and moribund spermatozoa are extremely sticky and will attach to glass surfaces even in the presence of relatively high concentrations of protein (16).
In this method, motile spermatozoa are separated from immotile spermatozoa by means of densely packed glass wool fibers. The principle of this technique involves both the self-propelled movement of the spermatozoa and the filtration effect of the glass wool. The method initially employed vertical Pasteur pipettes filled with glass wool fibers on to which the ejaculate was placed and allowed to filter by gravity (27). The method has evolved such that in a current variation (28), the filter is created by placing 30 mg of pre-cleaned glass wool microfibers in the barrel of a 3 mL disposable syringe and gently packing it down using the syringe plunger (minus its rubber tip). The syringe is suspended vertically in a 15 mL centrifuge tube and rinsed several times with culture medium to remove any loose wool fibers prior to filtration. Meanwhile, the ejaculate is washed with an equal volume of culture medium, pipetted into 15 mL centrifuge tubes (no more than
3 mL/tube), and centrifuged at 300 g for 3 minutes. Each resulting pellet is resuspended in 1 mL of culture medium, and centrifuged again at 300 g for 3 minutes. The pellet in one tube is resuspended with 300 mL of culture medium, and this single supernatant is sequentially added to resuspend the sperm pellet in any remaining tubes (the total volume should not exceed 400 mL). The washed sperm suspension is gently pipetted over the pre-wet glass wool column and then allowed to filter by gravity into a clean 15 mL centrifugal tube. When the dripping stops, 100 mL of culture medium is added to the filter and allowed to drip through. The filter is removed and the filtrate can be assessed for sperm concentration and motility.
The success of this method is related to the kind of glass wool used— the chemical nature of the glass, the surface structure and charge of the glass wool, and the thickness of the glass wool fibers. Glass wool from Manville Fiber Glass Corporation (Denver, CO) or SpermFertil® columns from Mello (Holzhausen, Germany) has been tested extensively in clinical practice (12). Glass wool filtration and two-layer, discontinuous density gradient centrifugation resulted in an average recovery of 50-70% of the progressively motile and about 50% of the HOS-positive spermatozoa (29). Additionally, glass wool filtration tended to be more successful than density gradient centrifugation when the ejaculates were asthenozoospermic or had an abnormal HOS test. After processing, the activity of the zona lysing enzyme acrosin increased approximately two- to threefold, but no significant improvement in the percentage of normal sperm forms occurred. Glass wool filtration was also more effective in removing non-motile and HOS-negative spermatozoa than density gradient centrifugation when the percentage of these types of spermatozoa in the ejaculate is high.
This method can use the whole volume of the ejaculate and thus yield a significantly higher total number of motile spermatozoa, which means it can be used for patients with oligozoospermia and/or asthenozoospermic. It is also possible to prepare motile spermatozoa from patients with retrograde ejaculation (12). Another advantage of glass wool filtration is the elimination of up to 90% of the leukocytes present in the ejaculate (30). As leukocytes are a major producer of ROS, elimination of a majority of leukocytes should significantly reduce ROS. Finally, glass wool filtration was also found to yield a significantly higher percentage of chromatin-condensed spermatozoa than swim-up or density gradient centrifugation (30). Disadvantages of the glass wool filtration method include the added expense of the glass wool and a filtrate that is not as clean as it is with other sperm preparation methods because remnants of debris may still be present.
Sperm separation using Sephadex beads is another filtration method (31), and a kit based on this principle (SpermPrep) is commercially available (Fertility Technologies, Inc.). Basically, liquefied semen is diluted with culture medium and centrifuged at 400 g for 6 minutes. The supernatant is discarded and the sperm pellet resuspended in culture medium to a concentration of 100 x 106sperm/mL. One milliliter of the washed semen is placed in the filter column containing hydrated filtration beads and mixed gently. The bottom cap is removed from the filter column and fluid is allowed to filter for 15 minutes. The filtrate is centrifuged at 400 g for 6 minutes and resuspended in 1 mL of culture medium before being assessed for concentration, motility, and morphology.
In a comparative study, the yield of spermatozoa post-processing was highest with SpermPrep than with swim-up or migration sedimentation in both fertile and subfertile men (19), and for that reason the authors recommended that specimens with a lower than normal sperm count but normal motility and morphology should be processed with SpermPrep. Disadvantages of Sephadex bead filtration include the added expense of the kit and a filtrate that is not as clean as it is with other sperm preparation methods because remnants of debris may still be present. In addition, the prefiltration centrifugation step might generate ROS.
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