INVESTIGATION OF SOME BIOCHEMICAL AND FUNCTIONAL-EFFECTS OF CRYOPRESERVATION OF HUMAN SPERMATOZOA USING AN AUTOMATED FREEZING-QUICK-THAWINGMETHOD

The objective of the present studies was to assess the functional inte grity of the sperm plasma membrane and metabolic and motility characte ristics of the recovered motile fraction of human spermatozoa subjecte d to an automated freezing/quick-thawing method. Sperm membrane featur es examined included progesterone-induced changes in intracellular lev els of calcium ([Ca2+](i)), as measured by the fluorescent fura-2 indi cator, and the tight binding of spermatozoa to homologous zonae pelluc idae as assesed by the hemizona assay (HZA). Basal [Ca2+](i), intracel lular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) lev els, determined using chemiluminescence with luciferin-luciferase, and motility parameters determined using a computer-aided semen analyser (CASA) were studied concomitantly as an expression of metabolic/functi onal status. Ejaculates from fertile men (donors) were evaluated after swim-up separation of the motile fraction in both fresh and cryoprese rved-thawed samples, and fractions of each ejaculate (fresh and frozen -thawed) were subjected to parallel measurements of the same parameter s at the same time frame. Basal and progesteone-induced increase in [C a2+](i), and ATP levels (up to 24 h) were similar in fresh and frozen- thawed samples. HZA results showed a modest (26%) although significant (p = 0.008) decrease in binding in frozen-thawed samples. The ratios of ATP/ADP in fresh and frozen-thawed samples were also found to be si milar. Although post-thaw sperm motility was significantly lower than that of the fresh samples, comparison of the results indicated that th e method was capable of preserving > 65% of motile spermatozoa in almo st all of the samples cryopreserved. Additionally, the swim-up rescued a motile fraction in the frozen-thawed samples that was not significa ntly impaired with regard to motility, mean linear velocity or lineari ty as compared to the fresh fractions in the first 4 h. Our results sh ow that this automated freezing-quick-thawing method results in a smal l reduction in sperm-zona binding capacity, and that the time-dependen t decline in motility parameters observed for both fresh and cryoprese rved-thawed samples cannot be related to ATP deficiency under the cond itions of our experiments. These in-vitro results are coincident with the maintenance of fertilizing capacity for donor spermatozoa in the i n-vitro fertilization (IVF) setting.