**1. Introduction**

76 Current Frontiers in Cryobiology

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Many advances in reproductive medicine in the past five decades have made cryopreservation of human spermatozoa an invaluable tool for the clinical management of infertility and sperm banking. The advent of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with microsurgical sperm handling techniques along with advances in female gamete acquisition have resulted in an increased demand for the cryopreservation of semen and tissue samples, often containing a very limited number of spermatozoa. Sperm cryopreservation also makes it possible for cancer patients to preserve their fertility prior to gonadotoxic chemotherapy or radiation. Applications of sperm banking are not limited to cancer patients but extend to patients undergoing certain types of pelvic or testicular surgeries; those who suffer from degenerative illnesses such as diabetes or multiple sclerosis; spinal cord disease or injury; and persons in occupations where a significant risk of gonadotoxicity prevails. Sperm cryopreservation is also available to men undergoing surgical sterilization such as vasectomy, in the event that children may be desired in the future. Another use for semen cryopreservation is to allow donor semen samples to be quarantined while appropriate screening is performed to prevent the transmission of infectious pathogens during therapeutic donor insemination (TDI).

Although major improvements have been made in sperm cryopreservation, there are many unresolved technical issues. Since freezing protocols differ between types of cells, the ideal conditions for human sperm freezing and thawing need to be perfected. To add more complexity, samples with abnormal semen parameters, such as severe oligospermia or high seminal fluid viscosity, often require unique cryopreservation conditions. For example, the particular cryoprotectants can affect cooling rates. In addition, storage temperature can significantly influence cryopreservation outcome. Liquid nitrogen (LN2) can offer long-term survival of spermatozoa due to essentially absent metabolic activity, such as chemical reactions, genetic modification or aging of cells (Mazur, 1984). A conventional slow freezing protocol has been in use for many years and very little has changed in terms of

Cryopreservation of Human Spermatozoa

proteins, pH shifts and potential cell death.

glass-like condition (Katkov et al., 2006).

vitrification technique.

**2.3 Cryoprotective agents (CPAs)** 

by Vitrification *vs.* Slow Freezing: Canadian Experience 79

of solutes within extracellular water. Due to the permeability of the plasma membrane, this chemical imbalance sets up the diffusion of solutes into the cell, forcing water out of the cell. Cells thus undergo excessive dehydration, losing up to 95% of their intracellular water content. This increases the intracellular concentration of solutes, resulting in denaturation of

Since velocity of cooling is crucial, inaccurate cooling rates can negatively affect sperm survival, motility, plasma membrane integrity and mitochondrial function (Henry et al., 1993). When cooling is slow enough, there is sufficient time for intracellular water efflux and balanced dehydration. If cooling is too slow, damage may occur due to exposure of cells to high concentrations of intracellular solutes. Extreme cellular dehydration leads to shrinkage of cells below the minimum cell volume necessary to maintain its cytoskeleton, genomerelated structures, and ultimately cellular viability (Mazur, 1984). On the other hand, if cooling rates are too fast, external ice can induce intracellular ice formation and potential rupture of the plasma membrane and damage intracellular organelles. In addition, mechanical damage of cells is possible due to of extracellular ice compression and close proximity of frozen cells can result in cellular deformation and membrane damage (Fujikawa & Miura, 1986). In contrast, with ultra rapid cooling, the amount of ice formation is insignificant and the entire cell suspension undergoes vitrification. At this stage water transitions, ice formation slows, molecular diffusion and aging stops, and liquids turn into a

Despite the relative insensitivity of human sperm to freezing, optimal cooling rates are needed to ensure appropriate sperm recovery. Currently, there are two types of slow freezing, either static vapour phase freezing to a certain temperature, or the multistep approach using nonlinear controlled-rate freezers, followed by plunging into LN2. Most laboratories and sperm banks adapt simple static vapour phase cooling in order to avoid induction of ice nucleation by seeding. For this technique samples are lowered into a vapour phase just above the LN2 level, allowing them to cool for 15-20 minutes before being plunged into LN2. Alternately, controlled rate freezers can be used to cryopreserve human semen. Most of these protocols utilize a "no seeding" option where samples are cooled from room temperature to -4°C at the rate of 2°C/min, followed by an increase of the cooling rate to 10°C/min until -100°C is reached, and finally plunging into LN2 (Morris et al., 1999). In contrast to these slow freezing techniques, single step ultra rapid cooling is used for the

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

methodology and reagents. While freezing aims to preserve cells it can also easily destroy them if certain precautionary steps are not taken into consideration. During cryopreservation cells and tissue undergo dramatic transformation in chemical and physical characteristics as the temperature drops from +37 to -196°C. The cells can lose up to 95% of their intracellular water. The concentration of solutes increases considerably, triggering the possibility of osmotic shock. Moreover, potential intracellular ice crystallization and mechanical deformation by extracellular ice may cause significant injury leading to cell death. Furthermore, if cells survive freezing, they might sustain additional damage during the thawing process due to osmotic shock, uncontrollable swelling and ice re-crystallization (Woods, et al., 2004).

Recently scientists have begun to re-investigate the utility of ultra rapid freezing in the search for alternative methods of sperm cryopreservation. Slow freezing of sperm utilizes cooling rates of 1–10°C/min, while the rapid freezing, or vitrification, technique allows for cooling rates to reach more than 40-1000°C/min in order to avoid intracellular ice formation. As new techniques are perfected, there is a potential for sperm cryopreservation to greatly improve in the future.
