Investigation of some biochemical and functional effects of cryopreservation of human spermatozoa using an automated freezing–quick‐thawing method

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Abstract
The objective of the present studies was to assess the functional integrity of the sperm plasma membrane and metabolic and motility characteristics of the recovered motile fraction of human spermatozoa subjected to an automated freezing/quick-thawing method. Sperm membrane features examined included progesterone-induced changes in intracellular levels of calcium ([Ca2+]i), as measured by the fluorescent fura-2 indicator, and the tight binding of spermatozoa to homologous zonae pellucidae as assessed by the hemizona assay (HZA). Basal [Ca2+]i intracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels 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/functional status. Ejaculates from fertile men (donors) were evaluated after swim-up separation of the motile fraction in both fresh and cryopreserved-thawed samples, and fractions of each ejaculate (fresh and frozen-thawed) were subjected to parallel measurements of the same parameters at the same time frame. Basal and progesterone-induced increase in [Ca2+]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 similar. Although post-thaw sperm motility was significantly lower than that of the fresh samples, comparison of the results indicated that the method was capable of preserving > 65% of motile spermatozoa in almost all of the samples cryopreserved. Additionally, the swim-up rescued a motile fraction in the frozen-thawed samples that was not significantly impaired with regard to motility, mean linear velocity or linearity as compared to the fresh fractions in the first 4 h. Our results show that this automated freezing-quick-thawing method results in a small reduction in sperm-zona binding capacity, and that the time-dependent decline in motility parameters observed for both fresh and cryopreserved-thawed samples cannot be related to ATP deficiency under the conditions of our experiments. These in-vitro results are coincident with the maintenance of fertilizing capacity for donor spermatozoa in the in-vitro fertilization (IVF) setting.