Human sperm were highly purified through the use of a discontinuous Percoll density gradient placed in an inner column of a centrifuge tube. Six ml of 80% Percoll solution were poured into a centrifuge tube with an inner column containing successive 1.0-ml layers of 70, 60, and 40% Percoll solutions. Diluted semen was placed on top of the gradient, and the tube was centrifuged at 600 X g for 30 min using a swing-out rotor. After centrifugation, the majority of the progressive motile sperm were isolated in the sediment; they had a mean motility of 93 +/- 4.1% (n = 10). Other cellular components, including bacteria, remaining in the inner column. The level of bacterial contamination in the purified sperm fraction was below detection for most of the species quantified. The purified sperm were found to be more than 92 +/- 3.2% viable, as judged by dye exclusion, and abnormal sperm were reduced to 5.2 +/- 1.4%. Because of the use of the inner column, the contamination by seminal plasma was negligible in the purified sperm, as estimated by residual protein, fructose, and acid phosphatase activity.
To increase the fertilizability of sperm for use in artificial insemination with husband's semen, it is effective to concentrate progressively motile sperm from whole ejaculated semen. The simple procedure, a continuous-step density gradient technique, was developed to selectively concentrate progressively motile sperm. The ejaculated semen was placed on 6.0 ml of 80% Percoll solution, and the density gradient was formed by mixing the semen and Percoll with an L-shaped rod for two or three strokes. After centrifugation at 400 x g for 30 min, progressively motile sperm were concentrated in the sediment, whereas the immotile sperm and other types of cells remained in the upper part of the density gradient. In most specimens sperm motility was improved by more than 80% regardless of the original motility. Thus, the fertility index (sperm concentration/ml x percentage of motility x 10(-8)). was increased to 7.7 +/- 3.7 times (n = 15).
Human sperm for use in artificial insemination with husband's semen (AIH) were prepared by two methods: the monolayer Percoll method and the discontinuous Percoll density gradient of four steps. The former focused mainly on the oligozoospermic semen concentration, with the sperm concentration and motility being improved 4.29 i 3.7 times and 1.92 * 1.1 times, respectively (n -41). The latter was used principally for the selective isolation of forward motile sperm, with the sperm motility being increased from 42.3 * 29.5% to 76.3 i 11.0.% after centrifugation. The clinical application of these procedures resulted in 35 (the monolayer Percoll method) and 10 (the discontinuous Percoll density gradient) successful deliveries with greater enhancement of sperm qualities. The number of females was predominate over the males in the delivered cases: 31: 14 (100:45.1) females to males.
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