Evaluation of cryoinjury of spermatozoa after slow (programmed biological freezer) or rapid (liquid nitrogen vapour) freeze-thawing techniques

Purpose: This study was initiated to determine the negative effect (cryodam age) on human spermatozoa after freeze-thawing and to find out whether free zing of spermatozoa with a computerized biological freezer is more advantag eous than freezing above static liquid nitrogen vapour with regard to sperm atozoa vitality, chromatin normality, morphology, and membrane integrity. M ethods: Forty-four semen samples were obtained front patients attending and rology laboratory, and each sample was divided into two aliquots. One aliqu ot was frozen using static liquid nitrogen vapour (G.II) and the second wit h a computerized biological freezer (G.III). Acridine orange was used for a ssessment of chromatin cryoinjury, whereas the morphology was evaluated acc ording to WHO criteria. Hypoosmotic swelling test was used to identify rnem brane integrity and eosin-nigrosin staining was used to determine the vital ity, of spermatozoa. Results: The rnean percentage of normally condensed ch romatin in the native semen sample (G.I) decreased significantly, (p<.001) after freeze-thawing by using either liquid nitrogen vapour (G.II), or a bi ological freezer (G.III), which was significantly higher (p <.001) after fr eezing with liquid nitrogen vapour than after freezing with the biological programmed freezer. Morphologically normal spermatozoa decreased significan tly (p <.001) in both freezing methods in comparison to the native semen sa mples. In addition, membrane integrity, of spermatozoa (HOS-test positive) was significantly, lower (p<.001) after the freeze-thawing procedure in G.I I and 6.III compared to G.I. In both these parameters the deterioration was similar among the two freezing procedures. Finally, the mean percentage of live spermatozoa decreased significantly (p<.001) in both freezing techniq ues in relation to the mean value in the neat semen samples. Conclusions: F reeze-thawing procedure has a detrimental effect on chromatin, morphology, membrane integrity, and vitality of human spermatozoa not only by, freezing above static liquid nitrogen vapour but even by using a computerized biolo gical freezer: Howe rev; the chromatin deterioration rates are significantl y, higher by freezing above static liquid nitrogen vapour in comparison to freezing with a programmed biological freezer: Therefore, we recommend the use of this technique for freeing semen especially, when ICSI technique is considered as the main therapeutic procedure.