To maximize the total number of spermatozoa which can be retrieved from a testicular biopsy, four mechanical methods for preparation were compared. In all, 17 biopsies were divided into four equal fractions, which were weighed individually and prepared by rough shredding (control), fine mincing, vortexing and crushing (electrical Potter). After resuspension in an erythrocyte-lysing buffer, removal of the remaining tissue pieces, washing and centrifugation, the following parameters were evaluated: number of spermatozoa per 100 mg tissue, percentage motility, percentage vitality and percentage normal morphology (strict Kruger criteria). After further purification by discontinuous Percoll centrifugation, the same parameters were assessed again. This procedure was efficient in obtaining only spermatozoa in the final fraction. Before Percoll centrifugation, only minor differences between methods were observed. Percoll centrifugation generally resulted in a pronounced loss of sperm cells, partly because of the abnormal dimensions of the eliminated cells. After Percoll centrifugation, treatment of the testicular tissue by fine mincing was the most effective method in terms of the total number of motile spermatozoa and percentage normal morphology.

Sixteen semen samples, 12 donor and four patient samples of high initial quality, were processed to compare the effect of two freezing methods, two thawing temperatures and the effect of dilution and washing on sperm motility and morphology characteristics. Sperm samples were divided in two equal parts and frozen either by fast vapour freezing or by slow computer-controlled freezing. For each freezing method, half of the straws were thawed at room temperature (22 degrees C), the other half were thawed at 37 degrees C. From each freeze-thawing treatment, one straw was evaluated immediately post-thawing; another straw was washed to remove the cryoprotectant solution. In this way, each semen sample was subjected to eight freeze-thawing treatments. No effect of the freezing method and thawing temperature was observed on motility characteristics evaluated by computer-assisted semen analysis, nor on light-microscopical morphology parameters. Post-thaw dilution and washing, however, exerted a deleterious effect on sperm motility, by reducing percentage motility by 50% compared to unwashed thawed specimens. Linearity and percentage of morphologically normal spermatozoa were obviously impaired, while percentage of abnormal tails and beat cross frequency increased significantly. In general, freeze-thawing was most successful when rapid vapour freezing was followed by 37 degrees C thawing, and when slower computer-controlled freezing was combined with 22 degrees C thawing, causing significant interactions between the freezing method and the thawing temperature. For semen samples of high initial quality, vapour and computer-controlled freezing were equally effective in terms of recovery of morphologically normal, motile spermatozoa.

The efficiency of spermatozoa prepared by swim-up or by Percoll centrifugation was assessed in an in-vitro fertilization programme on 71 semen samples of a well-defined quality [total number of type A (WHO criteria) motile spermatozoa]: category I (n = 21) with > 100 x 10(6), II (n = 31) with 15-100 x 10(6), III (n = 11) with 5-15 x 10(6) and IV (n = 8) with < 5 x 10(6) type A motile spermatozoa. Oocytes were inseminated 4 h after oocyte retrieval, alternately with spermatozoa derived from swim-up and Percoll preparation. Both selection procedures resulted in a significantly higher (P < 0.001) percentage motility as compared to fresh semen. For low-quality samples (III and IV), however, swim-up was more effective in selecting highly motile (P = 0.004) and morphologically normal spermatozoa (P < 0.05). For high-quality samples, this difference might have been masked by introducing a swim-up step to remove Percoll particles. Regardless of the initial sperm quality, the mean fertilization rate was significantly higher (P = 0.003) when Percoll-treated spermatozoa were used for insemination (51.3 versus 37.8%). For semen of groups I and II, no difference in fertilization capacity was observed according to the sperm preparation method. Despite the lower percentage motility and normal morphology for the Percoll compared to the swim-up treatment in groups III and IV, fertilizing capacity was significantly (P < 0.001) in favour of this selection method (65.3 versus 26.5% in group III, 47.6 versus 11.6% in group IV).(ABSTRACT TRUNCATED AT 250 WORDS)

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