**2.5 Cryopreservation of spermatozoa for assisted reproductive techniques (ART)**

Sperm cryopreservation is widely used in combination with ART techniques such as intrauterine insemination (IUI), IVF and ICSI. Despite many years of research and the discovery of new CPAs, significant numbers of spermatozoa still do not survive cryopreservation (Morris, 1999). Both freezing and thawing can inflict irreversible injury on a proportion of human spermatozoa, marked by a significant increase in some apoptosis markers (Giraud et al., 2000). Lipid peroxidation can lead to a decrease in sperm velocity, motility, viability, and mitochondrial activity (Mossad et al. 1994; O'Connell et al. 2002). The recovery rates of intact spermatozoa are highly dependant on the pre-freezing sample quality (de Paula et al., 2006). Poor quality semen may be more prone to DNA damage and cell death after cryopreservation than normal semen samples and thus have lower fertilizing capacity (Borges et al., 2007). It has been shown that reactive oxygen species (ROS) production impacts membrane fluidity and the recovery of motile, viable spermatozoa after cryopreservation. As well, semen samples containing leukocytes may have higher DNA fragmentation. In addition, the cryopreservation process can diminish the antioxidant activity of the semen fluid making spermatozoa more susceptible to ROS-induced damage (Lasso et al., 1994). The occurrence of sperm DNA damage may also be associated with the thawing process. A rapid increase in post thaw sperm DNA fragmentation over time has been observed, with the highest rate of fragmentation occurring during the first four hours after thawing (Gosalvez et al., 2009).

Normozoospermic semen samples appear to be more resistant to damage induced by freezing and thawing compared with oligozoospermic or asthenozoospermic samples. It has been reported that motile spermatozoa can be recovered after five refreezing and thawing rounds in normozoospermic samples, but only after two rounds in cases of oligozoospermia (Verza et al., 2009). Spermatozoa of infertile men were also found to be less resistant to damage during cryopreservation compared with spermatozoa from fertile men (Donnelly et

vulnerable to osmotic changes induced by these agents (Gao et al., 1993). Despite the use of CPAs, plasma membranes can still be damaged or ruptured due to the initial extensive dehydration followed by cell swelling and osmotic stress. Gradual introduction of CPAs to the cell suspension or stepwise increase in their concentration, with a limited waiting period prior to freezing, is utilized to minimize the potential negative effects of these agents

While there are many risk factors associated with freezing of cells, thawing can also dramatically affect survival rates of spermatozoa. When frozen samples are returned to ambient temperature, a reversal of the freezing process takes place. Cells that were frozen by the slow method, are more vulnerable to rapid thawing, due to the fast influx of water into cells causing uncontrollable swelling and osmotic shock (Curry & Watson, 1994). If cells were frozen rapidly, intracellular ice crystals could re-crystallize and form larger crystals during a slow thaw. To minimize toxic effects, CPAs have to be promptly removed from the cell suspension by washing samples in isotonic solution. Therefore, the thawing process and CPAs removal technique utilized must take into account the original method that was used

**2.5 Cryopreservation of spermatozoa for assisted reproductive techniques (ART)** 

Sperm cryopreservation is widely used in combination with ART techniques such as intrauterine insemination (IUI), IVF and ICSI. Despite many years of research and the discovery of new CPAs, significant numbers of spermatozoa still do not survive cryopreservation (Morris, 1999). Both freezing and thawing can inflict irreversible injury on a proportion of human spermatozoa, marked by a significant increase in some apoptosis markers (Giraud et al., 2000). Lipid peroxidation can lead to a decrease in sperm velocity, motility, viability, and mitochondrial activity (Mossad et al. 1994; O'Connell et al. 2002). The recovery rates of intact spermatozoa are highly dependant on the pre-freezing sample quality (de Paula et al., 2006). Poor quality semen may be more prone to DNA damage and cell death after cryopreservation than normal semen samples and thus have lower fertilizing capacity (Borges et al., 2007). It has been shown that reactive oxygen species (ROS) production impacts membrane fluidity and the recovery of motile, viable spermatozoa after cryopreservation. As well, semen samples containing leukocytes may have higher DNA fragmentation. In addition, the cryopreservation process can diminish the antioxidant activity of the semen fluid making spermatozoa more susceptible to ROS-induced damage (Lasso et al., 1994). The occurrence of sperm DNA damage may also be associated with the thawing process. A rapid increase in post thaw sperm DNA fragmentation over time has been observed, with the highest rate of fragmentation occurring during the first four hours

Normozoospermic semen samples appear to be more resistant to damage induced by freezing and thawing compared with oligozoospermic or asthenozoospermic samples. It has been reported that motile spermatozoa can be recovered after five refreezing and thawing rounds in normozoospermic samples, but only after two rounds in cases of oligozoospermia (Verza et al., 2009). Spermatozoa of infertile men were also found to be less resistant to damage during cryopreservation compared with spermatozoa from fertile men (Donnelly et

(McGann & Farrant, 1976).

for freezing.

**2.4 Biological aspects of thawing** 

after thawing (Gosalvez et al., 2009).

al., 2001). Optimization of both CPAs concentrations and cryopreservation protocols will maximize survival of spermatozoa and thus improve ART outcome.
