The sperm preparation method used for the first IVF cases involved dilution of the semen with culture medium (usually at 2-10 times the volume) and separation of the spermatozoa by centrifugation. After removal of the supernatant, the pellet is resuspended in another aliquot of culture medium. Repeat centrifugation, usually two or three times in total, is often used to ensure removal of contaminating seminal plasma. The centrifugation is usually performed at 200-300 g and it should certainly be performed at centrifugal forces less than 800 g (8). Advantages of this method are that it is the simplest and the least expensive to perform. One disadvantage of this technique is nonviable, and immotile spermatozoa as well as any leukocytes, squamous epithelial cells, or non-cellular debris that contaminated the original semen sample will still be present in the washed sample. Another disadvantage is the concern about potential damage caused by centrifugation.
Aitken and Clarkson (9) reported that techniques involving the repeated centrifugation of unselected populations of human spermatozoa generate cell suspensions with significantly reduced motility. Moreover, these detrimental effects of centrifugation were associated with a sudden burst of ROS produced by a discrete subpopulation of cells characterized by significantly diminished motility and fertilizing capacity. The ROSs were found to impair the functional competence of normal spermatozoa in the same suspension, reflected in impaired capacity for sperm-oocyte fusion. It has also been shown that ROS can cause DNA damage in human spermatozoa when exposed for time periods consistent with clinical sperm preparation techniques for ICSI or IVF (10). Thus, sperm preparation techniques that involve a washing step in which semen is diluted with culture medium and centrifuged have mostly been abandoned for alternative techniques such as direct swim-up from semen or density gradient centrifugation.
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