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

Abstract

The purpose of this study was to determine the negative effects (cryodamage) on human spermatozoa after freeze-thawing and to determine whether freeze-thawing 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 subfertile, 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 guidelines. Each semen sample was divided into two parts after liquefaction and addition of the cryoprotectant. The first part was frozen using a programmed biological freezer and the second part was frozen by means of liquid nitrogen vapour. Smears were made before the freezing and after the thawing procedure to assess morphology (strict criteria) and chromatin condensation (Acridine Orange test). The mean percentage of chromatin condensed spermatozoa in the samples from donors (control group) was 92.4 +/- 8.4% before freezing and this decreased significantly (p < 0.0001) to 88.7 +/- 11.2% after freeze-thawing with the computerized slow-stage freezer and to 87.2 +/- 12.3% after using static liquid nitrogen vapour (p < 0.001). The corresponding values for semen obtained from patients was 78.9 +/- 10.3% before freezing which 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 to 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 spermatozoa decrease significantly after the freeze-thawing procedure, not only 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 injury better than those with low quality semen samples. Therefore, the computerized slow stage freezer could be recommended for freezing of human spermatozoa, especially for subnormal semen samples, for example, ICSI and ICSI/TESE candidates and from patients with testicular tumours or Hodgkin's disease, in order to avoid further damage to the sperm chromatin structure.

Evaluation of motility, membrane status and DNA integrity of frozen–thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation

“…Damage to sperm DNA can be caused by intrinsic factors like protamine deficiency, mutations that compromise DNA packaging, aging, reactive oxygen species and an incomplete process of apoptosis during spermatogenesis (Gatewood et al 1990, Sakkas et al 2003, Singh et al 2003. External factors that result in sperm DNA damage include heat (Paul et al 2008), chemotherapeutics (Delbes et al 2007), radiation (Sailer et al 1995, Aitken et al 2005, pollution (Rubes et al 2005) and age (Singh et al 2003) as well as in vitro semen processing factors like type of extender, prolonged incubation time (Krzyzosiak et al 2000) and cryopreservation (Hammadeh et al 2001).…”

DNA Fragmentation Dynamics in Fresh Versus Frozen Thawed Plus Gradient-Isolated Human Spermatozoa

“…This clarification is necessary given the ambiguous results found in the literature. Some have suggested that the highly stable DNA is fragmented by freeze-thawing [Royere et al 1988;Sakkas et al 1999;Hammadeh et al 2001]. However, other authors have differed [Steele et al 2000].…”

Effect of freezing rate on acrosome and chromatin integrity in ram semen

“…Acridine-Orange Staining Assay: Sperm DNA integrity was assessed using the AO fluorescence method (11). Air-dried slides were fixed overnight in freshly prepared Carney's solution (three parts of methanol and one part of glacial acetic acid) and allowed to air dry for a few minutes.…”

“…The success of cryopreservation of cells is affected by the rate of freezing (10,11) and the composition of the solution where the cells are frozen (11,16). When the cells are rapidly cooled, water is not lost fast enough to maintain the equilibrium; the cells become increasingly supercooled and freeze intracellulary, and this process might affect the integrity of chromatin and the morphology of spermatozoa (11).…”

“…When the cells are rapidly cooled, water is not lost fast enough to maintain the equilibrium; the cells become increasingly supercooled and freeze intracellulary, and this process might affect the integrity of chromatin and the morphology of spermatozoa (11).…”