**2.3 Cryoprotective agents (CPAs)**

Most cells would not survive cryopreservation without CPAs, which can minimize cryoinjury of cells. CPAs are low molecular weight chemicals that serve to protect spermatozoa from freezing damage or ice crystallization by decreasing the freezing point of materials. There are two categories of CPAs, and they differ in their ability to penetrate the plasma membrane. Firstly, permeating CPAs such as dimethylacetaldehyde; dimethyl sulfoxide, glycerol, glycol, ethylene and methanol, stabilize cell plasma membrane proteins and reduce concentrations of electrolytes (Arakawa et al., 1990). In contrast, nonpermeating CPAs such as albumins, dextrans, egg yolk citrate, hydroxyethyl, polyethylene glycols, polyvinyl pyrollidone and sucrose, minimize intracellular crystallization by increasing viscosity of the sample. CPAs themselves can be toxic if used at high concentrations and spermatozoa are

Cryopreservation of Human Spermatozoa

by Vitrification *vs.* Slow Freezing: Canadian Experience 81

al., 2001). Optimization of both CPAs concentrations and cryopreservation protocols will

Couples with male factor infertility represent 30 to 40% of the infertile population. Azoospermia accounts for 10% of cases of confirmed male infertility, and often requires surgical retrieval of spermatozoa. Since the introduction of ICSI, many cases of severe male infertility can now be successfully treated. Cryopreservation of surgically retrieved spermatozoa is a valuable component in the effective management of male infertility, reducing the necessity of repeat surgeries. Diagnostic sperm retrieval prior to IVF has several benefits including the possibility of freezing spermatozoa for future use, or if none are retrieved, initiation of the IVF stimulation cycle can be postponed or avoided. Testicular spermatozoa have been utilized to achieve pregnancy in couples with severe male factor infertility, with reported pregnancy rates similar to ejaculated spermatozoa, according to a meta-analysis study (Nicopoullos et al., 2004). In the case of obstructive azoospermia, recovery of spermatozoa by aspirations varies from 45 to 97% (Craft et al., 1995; Lania et al., 2006). In cases of non obstructive azoospermia recovery depends on the degree of testicular pathology and varies from 0 to 64% (Schlegel et al., 1997; Hauser et al., 2006). A second or third surgery can increase the chance of complications including hematomas, inflammation, testicular devascularization, fibrosis and permanent testicular damage (Schlegel and Su, 1997). To avoid this, if pregnancy is not achieved during the first ICSI attempt, a repeat of the surgical procedure would not be required if a portion of the surgical specimen has been banked. Cryopreservation of surgically retrieved spermatozoa can also aid the coordination of oocyte retrieval and avoids the pressure of having the urologist available on the day of the ICSI procedure. Usually the number of spermatozoa obtained during a surgical procedure is limited, and in the case of testicular sperm they may not be fully matured. In the future, if no mature spermatozoa are recovered, spermatogonial stem cells or early germs cells could potentially be matured in vitro and used for fertility treatments (Hwang & Lamb, 2010).

There are significant technical challenges for successful cryopreservation of testicular tissue due to its complex structure and intracellular interactions. Different cells of testicular tissue will have dissimilar responses to cryopreservation and require different concentration of CPAs. Freezing larger pieces of tissue is not advisable as it would increase resistance of heat transfer and penetration of CPAs leading to variation in cooling rates within different parts of the tissue. In addition, seminiferous tubules capture liquid and increase chances of ice formation (Woods et al., 2004). To avoid these difficulties, cryopreservation of smaller tissue

The idea of cryopreservation of low numbers or individual spermatozoa was introduced more than a decade ago (Cohen et al., 1997). While this approach remains very attractive, there are multiple biological and technical issues to overcome. Early attempts to freeze individual spermatozoa were performed by placing them in empty animal or human zona pellucida prefilled with CPAs (Walmsley et al., 1998). Data from these studies suggested lower recovery and fertilization rates with human zona in comparison to hamster, possibly due to the presence of the ZP3 binding protein and induced acrosome reactions when human zona were

fragments or mincing tissue prior to freezing has been advocated (Hovatta, 2003).

**2.7 Cryopreservation of low number or single spermatozoa** 

maximize survival of spermatozoa and thus improve ART outcome.

**2.6 Cryopreservation of epididymal and testicular spermatozoa** 

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 (McGann & Farrant, 1976).
