Effect of freezing method, thawing temperature and post-thaw dilution/washing on motility (CASA) and morphology characteristics of high-quality human sperm.

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.