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

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

Cryopreservation of Human Spermatozoa

by Vitrification *vs.* Slow Freezing: Canadian Experience 83

surface to enable a uniform cooling rate of the sample, 3)have proper heat exchange properties, 4)be easy to label and seal securely and 5)be available in small sterile units. When storage packaging is chosen, the possible risk of microbial or viral contamination must also be considered. The type of packaging also depends on the freezing protocol and sometimes on the quality of the sample. For conventional slow freezing, the two most common types of containers currently used are plastic screw-top vials or straws. Straws can be made of polyethylene terephthalate glycol (PETG) or ionomeric resin (CBS High Security Straws by CryoBioSystem, Paris, France). As described above, a low number or single spermatozoa have been experimentally frozen in empty animal or human zona pellucida, spheres of Volvox Globator algae, alginate agarose bead microspheres and ICSI pipettes (AbdelHafez et al., 2009; Herrler et al., 2006; Isaev et al., 2007; Just et al., 2004; Walmsley et al., 1998). For vitrification purposes different types of storing strategies have been suggested. These include: cryoloops, electron microscope copper grids, nylon meshes, open-

Vitrification is an alternative method of freezing based on the rapid coolling of water to a glassy state through extreme elevation of viscosity without intracellular ice crystallization (Fahy, 1986; Katkov et al., 2006). The relationship between the size of different cells, particularly, different spermatozoa species, and the ability of cells to be vitrified are

The earliest experiments on vitrification from the 1930s was not successful because critical rates of cooling were unachievable at that time. With the use of LN2 and the discovery of cryoprotectants, however, it became possible to vitrify many types of cells. The five basic ways to achieve vitrification have been described in details by Katkov et al.: equilibrium freezing-out of the bulk of water with the use of CPAs and storage at ultra low temperature; lyophilization using slow freezing to moderately low (-40 °C) followed by secondary drying at +30°C (mostly used in food and pharmaceutical industries); ice-free vitrification at high rates and high concentration of CPAs; ice-free vitrification at very fast rates without permeable agents ("CPAs-free vitrification"); high temperature' vitrification by air/vacuum

However, until only recently, vitrification of spermatozoa was unsuccessful, possibly due to high concentrations of permeable CPAs (30-50% compared to 5-7% with slow freezing) and low tolerance of spermatozoa to permeable agents. Even brief exposure to a high concentration of CPAs can lead to toxic and osmotic shock and would be lethal for spermatozoa. One possible strategy to lower the concentration of CPAs could be to increase the speed of cooling and warming temperatures as higher rates of cooling and warming, require lower concentrations of CPAs; these conditions can help eliminate intracellular ice crystallization, and facilitate the formation of a glassy state (Katkov et al., 2006). Another option is to add non-permeable CPAs--such as carbohydrates--to permeable CPAs to minimize osmotic shock by decreasing osmotic pressure and stabilizing the nuclear membrane. Since the intracellular matrix of human spermatozoa contains large amounts of proteins and sugars, they can be successfully frozen in the absence of permeable CPAs using

pulled straws and standard open straws (V. Isachenko et al., 2005).

discussed in details in the paper by Katkov (Katkov et al., 2006).

**3.1 Background on vitrification of spermatozoa** 

drying at temperature above 0°C (Katkov et al., 2006).

protein- and sugar-rich non-permeable agents (Koshimoto et al., 2000).

**3. Cryopreservation of human spermatozoa by vitrification** 

used (Cohen et al., 1997). While this method requires special skills, equipment, and is very labour-intensive; live births were reported using both human and hamster zona (Walmsley et al., 1998). Spermatozoa were also injected and frozen within spheres of Volvox Globator algae and recovered after thawing using an ICSI needle (Just et al., 2004). While all of these methods appear to be attractive for single spermatozoa cryopreservation, they have a number of limitations. Issues around the use of donor human zona pellucida as well as exposure of human gametes to animal or algae genetic materials present potential risks that restrict the use of such methods for human ART procedures. While in theory zona could be obtained from the female partner of men with severe male infertility, this would be unrealistic in the clinical setting, as it would require IVF egg retrieval and destruction of ooplasma to obtain empty zona pellucidae. An alternative proposal would be to use a non-biological carrier such as nontoxic polysaccharide alginate agarose to cryopreserve small numbers of sperm (Herrler et al., 2006, Isaev et al., 2007). In these studies spermatozoa were mixed with CPAs and added to the alginate before the gelatin stage and then frozen in small bead microspheres. After cryopreservation, they were dissolved in a sodium citrate solution. The residual alginic acid on the sperm membrane can reduce sperm motility with slow freezing (Herrler et al., 2006). Agarose microspheres were also frozen in 0.25 cc straws by vitrification with better recovery rates (Isaev et al., 2007). Another reported method was to divide the sample into several small aliquots of 15–20-µl and to freeze in 0.2-mm cryopreservation embryo straws cut into smaller sections, sealed on one end (Desai et al., 2004).Conventional and open-pulled straws containing 1 or 5 µl of sperm suspension frozen by vitrification has also been reported (V. Isachenko et al., 2005). However, individual spermatozoa could not be easily sequestered because of possible adherence to the walls of the straws. ICSI pipettes were suggested as a container to freeze individual spermatozoa by either the slow method or vitrification (AbdelHafez et al., 2009; Sohn et al., 2003). Cryopreservation of sperm in microdroplets containing 1 or 40 μl of spermatozoa in cryoprotectant placed on a cold surface or directly plunged into liquid nitrogen was also reported (Gil-Salom et al., 2000; Isachenko et al., 2005). Microdroplets covered by mineral oil in a plastic tissue culture dish placed in liquid nitrogen were also used to cryopreserve individual spermatozoa (Quintans et al., 2000; Sereni et al., 2008). A nylon cryoloop first introduced for embryo freezing was successfully used to cryopreserve small volumes of sperm suspension by both slow freezing and vitrification (Nawroth et al., 2002; Schuster et al., 2003; V. Isachenko et al., 2004; Desai et al., 2004). However direct placement of sperm into LN2 without a container using an 'open system' such as cryoloop or unsealed culture dish increases the risk of cross-contamination and such techniques are discouraged by regulatory agencies such as the FDA and the European Tissue Directive on Sperm.

Overall reported recovery rates of a known number of frozen spermatozoa varied from 59 to 100% with reported survival rates of 8–85% and motility of 0 to 100%. The wide ranges of results depended on patient population, initial quality and number of frozen spermatozoa, as well as the type of cryopreservation device, type of cryoprotectant, and freezing and thawing protocols.

#### **2.8 Sperm packaging and relation to the method of cryopreservation**

Storage of frozen samples has to be in suitable freezing containers and at an optimal temperature to ensure long term survival. The packaging containers must meet several criteria. They must: 1)hold freezing temperatures without cracking or leaking, 2)have a large

used (Cohen et al., 1997). While this method requires special skills, equipment, and is very labour-intensive; live births were reported using both human and hamster zona (Walmsley et al., 1998). Spermatozoa were also injected and frozen within spheres of Volvox Globator algae and recovered after thawing using an ICSI needle (Just et al., 2004). While all of these methods appear to be attractive for single spermatozoa cryopreservation, they have a number of limitations. Issues around the use of donor human zona pellucida as well as exposure of human gametes to animal or algae genetic materials present potential risks that restrict the use of such methods for human ART procedures. While in theory zona could be obtained from the female partner of men with severe male infertility, this would be unrealistic in the clinical setting, as it would require IVF egg retrieval and destruction of ooplasma to obtain empty zona pellucidae. An alternative proposal would be to use a non-biological carrier such as nontoxic polysaccharide alginate agarose to cryopreserve small numbers of sperm (Herrler et al., 2006, Isaev et al., 2007). In these studies spermatozoa were mixed with CPAs and added to the alginate before the gelatin stage and then frozen in small bead microspheres. After cryopreservation, they were dissolved in a sodium citrate solution. The residual alginic acid on the sperm membrane can reduce sperm motility with slow freezing (Herrler et al., 2006). Agarose microspheres were also frozen in 0.25 cc straws by vitrification with better recovery rates (Isaev et al., 2007). Another reported method was to divide the sample into several small aliquots of 15–20-µl and to freeze in 0.2-mm cryopreservation embryo straws cut into smaller sections, sealed on one end (Desai et al., 2004).Conventional and open-pulled straws containing 1 or 5 µl of sperm suspension frozen by vitrification has also been reported (V. Isachenko et al., 2005). However, individual spermatozoa could not be easily sequestered because of possible adherence to the walls of the straws. ICSI pipettes were suggested as a container to freeze individual spermatozoa by either the slow method or vitrification (AbdelHafez et al., 2009; Sohn et al., 2003). Cryopreservation of sperm in microdroplets containing 1 or 40 μl of spermatozoa in cryoprotectant placed on a cold surface or directly plunged into liquid nitrogen was also reported (Gil-Salom et al., 2000; Isachenko et al., 2005). Microdroplets covered by mineral oil in a plastic tissue culture dish placed in liquid nitrogen were also used to cryopreserve individual spermatozoa (Quintans et al., 2000; Sereni et al., 2008). A nylon cryoloop first introduced for embryo freezing was successfully used to cryopreserve small volumes of sperm suspension by both slow freezing and vitrification (Nawroth et al., 2002; Schuster et al., 2003; V. Isachenko et al., 2004; Desai et al., 2004). However direct placement of sperm into LN2 without a container using an 'open system' such as cryoloop or unsealed culture dish increases the risk of cross-contamination and such techniques are discouraged by regulatory agencies such as the FDA and the European Tissue

Overall reported recovery rates of a known number of frozen spermatozoa varied from 59 to 100% with reported survival rates of 8–85% and motility of 0 to 100%. The wide ranges of results depended on patient population, initial quality and number of frozen spermatozoa, as well as the type of cryopreservation device, type of cryoprotectant, and freezing and

Storage of frozen samples has to be in suitable freezing containers and at an optimal temperature to ensure long term survival. The packaging containers must meet several criteria. They must: 1)hold freezing temperatures without cracking or leaking, 2)have a large

**2.8 Sperm packaging and relation to the method of cryopreservation** 

Directive on Sperm.

thawing protocols.

surface to enable a uniform cooling rate of the sample, 3)have proper heat exchange properties, 4)be easy to label and seal securely and 5)be available in small sterile units. When storage packaging is chosen, the possible risk of microbial or viral contamination must also be considered. The type of packaging also depends on the freezing protocol and sometimes on the quality of the sample. For conventional slow freezing, the two most common types of containers currently used are plastic screw-top vials or straws. Straws can be made of polyethylene terephthalate glycol (PETG) or ionomeric resin (CBS High Security Straws by CryoBioSystem, Paris, France). As described above, a low number or single spermatozoa have been experimentally frozen in empty animal or human zona pellucida, spheres of Volvox Globator algae, alginate agarose bead microspheres and ICSI pipettes (AbdelHafez et al., 2009; Herrler et al., 2006; Isaev et al., 2007; Just et al., 2004; Walmsley et al., 1998). For vitrification purposes different types of storing strategies have been suggested. These include: cryoloops, electron microscope copper grids, nylon meshes, openpulled straws and standard open straws (V. Isachenko et al., 2005).
