Improved techniques for separating motile spermatozoa from human semen

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Abstract
A simple and atraumatic method for concentrating washed, motile spermatozoa from normal and subnormal semen specimens is described. It incorporates a modified technique of centrifugation in which sperm are spun onto a soft, fluid cushion, thereby minimizing mechanical damage. Following initial semen dilution to 5-10 ml in an artificial medium, the mixture is transferred to a test tube and layered onto 1 ml of oily contrast medium (Lipiodol). After centrifugation at 300 g for 10 min all but 0.5 ml of the supernatant is discarded, and the unshaken test tube is incubated at 37.degree. C for 15-20 min, during which time the motile sperm migrate into the upper 0.5 ml. After this incubation, 0.3 ml of the upper layer is removed which is sufficent for most IVF [in vitro fertilization] and AIH [artificial insemination] purposes. It contains concentrated, washed, motile spermatozoa that are free of debris and most abnormal forms. No change in pH or osmolarity and no diffusion of any iodine from the oil base into the top layer were detected.