**2.2 Biological aspects of freezing**

Living cells have an isotonic condition with a melting point of their intracellular water of approximately -0.6°C. When cells are cooled below this standard freezing point, supercooling takes place and remains in a metastable state up to -5°C (Katkov et al., 2006; Mazur et al., 1972). Water crystallization and ice formation begin between -5 and -15°C, beginning with the formation of an ice nucleus (seed crystal) in the extracellular water. This 'nucleation' can be induced at a higher temperature by the planned external facilitation of ice formation, often referred to as "seeding". Prior to that stage, water remains unfrozen inside the cell as the membrane prevents ice crystals from intracellular penetration (Woods et al., 2004). Solutes are excluded from ice formation which results in rising concentrations

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

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

Remarkably, the first reference of empirical sperm freezing dates as far back as the late 16th century, but it was only with the discovery in 1937 by Bernstein and Petropavlovski that glycerol can aid spermatozoa in surviving long term freezing, that sperm cryopreservation became practical. Expansion of artificial insemination for the dairy industry led to further important research in the field of cryobiology (E. Isachenko, 2003, as sited in Bernstein & Petropavlovski, Polge et al., 1949). Shortly after these practices were initiated with animals, the first pregnancies were reported in humans after insemination with frozen spermatozoa. The next milestone was the discovery of the possibility to store human spermatozoa in LN2 at -196°C, resulting in superior recovery rates compared to storage at higher temperatures between -20 and -75°C. After the era of empirical freezing; cryobiology matured to its fundamental stage, focusing on the biophysical and biochemical principals of cryopreservation, further advancing the field (Mazur et al., 1972. A comprehensive review of the historical background of sperm freezing was recently published and is recommended

Living cells have an isotonic condition with a melting point of their intracellular water of approximately -0.6°C. When cells are cooled below this standard freezing point, supercooling takes place and remains in a metastable state up to -5°C (Katkov et al., 2006; Mazur et al., 1972). Water crystallization and ice formation begin between -5 and -15°C, beginning with the formation of an ice nucleus (seed crystal) in the extracellular water. This 'nucleation' can be induced at a higher temperature by the planned external facilitation of ice formation, often referred to as "seeding". Prior to that stage, water remains unfrozen inside the cell as the membrane prevents ice crystals from intracellular penetration (Woods et al., 2004). Solutes are excluded from ice formation which results in rising concentrations

(Woods, et al., 2004).

improve in the future.

**2. Cryopreservation of human spermatozoa 2.1 History of human spermatozoa cryopreservation** 

for readers looking for more details (Katkov et al., 2006).

**2.2 Biological aspects of freezing** 

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 proteins, pH shifts and potential cell death.

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

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 vitrification technique.
