Comparison between computerized slow-stage and static liquid nitrogen vapour freezing methods with respect to the deleterious effect on chromatin andmorphology of spermatozoa from fertile and subfertile men

The purpose of this study was to determine the negative effects (cryodamage ) human spermatozoa after freeze-thawing and to determine whether freeze-th awing of spermatozoa with a programmed slow freezer is better than freezing with liquid nitrogen vapour (rapid freezing) with regard to alterations in sperm chromatin and morphology in semen from fertile (donor) and subfertil e, IVF/ICSI, patients. Ninety-five semen samples were obtained either from patients attending our IVF unit for treatment (n = 34) or from donors (n = 25) with proven fertility and normal sperm quality according to WHO guideli nes, Each semen sample was divided into two parts after liquefaction and ad dition of the cryoprotectant. The first part was frozen using a programmed biological freezer and the second part was frozen by means of liquid nitrog en vapour. Smears were made before the freezing and after the thawing proce dure to assess morphology (strict criteria) and chromatin condensation (Acr idine Orange test). The mean percentage of chromatin condensed spermatozoa in the samples front donors (control group) was 92.4 +/- 8.4% before freezi ng and this decreased significantly (p < 0.0001) to 88.7 +/- 11.2% after fr eeze-thawing with the computerized slow-stage freezer and to 87.2 +/- 12.3% after using static liquid nitrogen vapour (p < 0.001). The corresponding v alues for semen obtained from patients was 78.9 +/- 10.3% before freezing w hich decreased to 70.7 +/- 10.8 and 68.5 +/- 14.8%, respectively (p < 0.001 ). On the other hand, the mean percentage of normal sperm morphology in the control group decreased from 26.3 +/- 7.5% before freezing to 22.1 +/- 6.4 % (p < 0.0001) after thawing with the computerized slow-stage freezer and t o 22.2 +/- 6.6% (p < 0.0001) after the use of static liquid nitrogen vapour , In the patient group, the mean percentage of normal morphology decreased from 11.7 +/- 6.1% after freezing with the biological freezer to 9.3 +/- 5. 6% and to 8.0 +/- 4.9% after freezing with static liquid nitrogen vapour. This study demonstrates that chromatin packaging and morphology of human sp ermatozoa decrease significantly after the freeze-thawing procedure, not on ly after the use of static liquid nitrogen vapour but also after the use of a computerized slow-stage freezer. However, the chromatin of semen samples with normal semen parameters (donor sperm) withstand the freeze-thaw injur y better than those with low quality semen samples. Therefore, the computer ized slow stage freezer could be recommended for freezing of human spermato zoa, especially for subnormal semen samples, for example, ICSI and ICSI/TES E candidates and from patients with testicular tumours or Hodgkin's disease , in order to avoid further damage to the sperm chromatin structure.