Cryopreservation is defined as the frozen storage of sperms, eggs, embryos, or ovarian and testicular tissues (ART glossary – ESHRE).

Cryopreservation, in general, is a process where cells, whole tissues, or any other substances susceptible to damage caused by chemical reactivity or time are preserved by cooling to subzero temperatures.

Following ice formation in an aqueous solution like growth media, the ionic composition increases dramatically which is lethal to cells. Cryoprotectants are low-molecular-weight and highly permeable chemicals used to protect spermatozoa from freeze damage during ice crystallization by increasing the unfrozen fraction at a given temperature and hence reduce the ionic composition. Cryoprotectants act by decreasing the freezing point of a substance, reducing the amount of salts and solutes present in the liquid phase of the sample, and also by decreasing ice formation within the spermatozoa (Royere et al. 1996).

They can be classified as follows based on their ability to diffuse across cell membranes:

Lovelock suggested that the mechanism of action of cryoprotectants was due to their colligative properties (the collective properties that a solution has when these compounds are present). In particular, it was the reduction in salt concentration at a given temperature that allowed cells to suffer less injury at that temperature. An effective penetrating cryoprotectant should provide colligative properties in which the salt is buffered down to low temperatures. It should also be freely permeable across the cell membrane so that it can buffer the intracellular salt as well, thereby reducing the damage caused to the plasma membrane (Holt 2000; McGann 1979, 1988).

Non-penetrating cryoprotectants act by dehydrating the cell at high subfreezing temperatures, thereby allowing them to be rapidly cooled before the solution effects injury of slow cooling can lead to extensive damage. These compounds are generally polymers and form extensive hydrogen bonds with water, reducing the water activity to a greater extent.

It is necessary that the medium interacts with the cells. The effectiveness of cryoprotecting substances is a function of the time of interaction between the cryoprotectants and the cells. Glycerol is a permeating cryoprotectant most widely used for human sperm cryopreservation. It acts by depressing the freezing point and the consequent lowering of electrolyte concentrations in the unfrozen fraction at any given temperature that would help to counter the harmful “solution effects” imposed during the freezing process.

Cryopreservation has proven to be a very useful technique in the management of infertility, and the success of the technique has an impact on the reproductive outcome of the couple, though the process of cryopreservation itself has a deleterious effect on the structure and function of sperm. Cryopreservation is said to reduce sperm motility and fertility rates by the deleterious effects on sperm membranes, acrosomal structure, and acrosin activity. Many studies have proven the deleterious effect of freezing on motility.

It is said that spermatozoa are less sensitive to cryodamage when compared to other cell types. This can be attributed to the high fluidity of the membrane and low water content. Despite this, there tend to be significant deleterious changes that have been observed in sperm characteristics. It has been reported that several damaging processes can occur during the freezing and thawing of human spermatozoa due to thermal shock with the formation of intracellular and extracellular ice crystals, cellular dehydration, and osmotic shock. The intracellular ice crystals thus formed may breach the membranes and affect the functioning of the organelles. This can lead to impaired cell survival. Cryoinjury is not only limited to the freezing process but it may also occur during the thawing process. Moreover, the survival of sperm after cryopreservation exhibits large male-to-male variability.

Sperm motility is one of the important parameters that determine the success rate of cryopreservation. The motility of sperms is found to decrease after cryopreservation. The decrease in motility of the spermatozoa has been attributed to the damage caused to the mitochondrial membrane. The greatest amount of energy necessary for sperm motility is provided by ATP molecules. The ATP generated by oxidative phosphorylation in the inner mitochondrial membrane is transferred to the microtubules, to drive motility. Therefore an impairment of mitochondrial activity may explain the reduction in motility. However, with progression of time, significant recovery of sperm motility was reported during the post-thaw period. Though there is no direct link between motility and fertilizing capacity of the sperm, it is one of the important factors that affects sperm quality. The quality of motility had greater impact on the fertility outcome rather than the percentage of motility (Oberoi et al. 2014).

The morphological effects of cryopreservation that have been noticed are flattening, cupping, and wrinkling of the head and tail regions. The reasons attributed to the shrinkage are an interaction between the cooling and warming rates. When exposed to hyperosmotic solutions such as glycerol, sperms first shrink because of dehydration and then increase in volume as the glycerol permeates and water concomitantly reenters the cell. The sperm cells maintain membrane integrity in the shrunken state, and serious cell membrane damage can be detected only after returning to isotonic solutions. Also, the post-hypertonic injury of sperm was shown to be a function of time, that is, the shorter the time of exposure to hypertonic solution, the higher the percentage of cell survival (Gao et al. 1993).

There have been studies suggesting that there is significant damage to DNA leading to fragmentation following cryopreservation. (Donnelly et al. 2001) reported that only spermatozoa from subfertile males and not from fertile males demonstrated a significant increase in DNA fragmentation following cryopreservation (Baumber et al. 2003). Also, studies have suggested that morphologically abnormal sperm are more prone to cryodamage than normal sperm (Kalthur et al. 2008).

The main reasons that lead to DNA fragmentation include increase in the oxidative stress (Said et al. 2010) during cryopreservation. The alteration in the mitochondrial membrane fluidity leads to rise in the mitochondrial membrane potential and subsequent release of reactive oxygen species. This release of reactive oxygen species causes DNA damage that presents with high frequencies of single- and double-strand DNA breaks. The source of ROS include human spermatozoa and seminal leukocytes. Thus cryopreserved samples containing leukocytes are more prone to DNA damage. Besides, cryopreservation process by itself diminishes the antioxidant activity of the spermatozoa making them more susceptible for DNA damage.

At slow cooling rates, cryoinjury occurs due to solution effects (i.e., the solute/electrolyte concentration, the severe cell dehydration, and the reduction of unfrozen fraction in the extracellular space); and at high cooling rates, cryoinjury occurs due to the lethal intracellular ice formation. The optimal cooling rate for cell survival should be low enough to avoid intracellular ice formation but high enough to minimize the solution effects. Hence an optimal freezing rate has to be established in order to prevent cell damage (Mazur et al. 1972).

In the initial days of ART, treating azoospermic men involved reconstructive surgery, in the case of an obstruction or donor insemination. But with the advancement of treatment options such as intracytoplasmic sperm injection (ICSI) technique, men with azoospermia can become biological fathers using sperm obtained from their epididymis or testis (Shah 2011).

Azoospermia is the complete absence of spermatozoa in the ejaculate. The two forms of azoospermia are obstructive and nonobstructive azoospermia (Schill). Obstructive azoospermia has been attributed to a mechanical blockage that can occur anywhere along the reproductive tract, including the vas deferens, epididymis, and ejaculatory duct. On the other hand, the conditions that may cause NOA include genetic and congenital abnormalities, postinfectious issues, exposure to gonadotoxins, medications, varicocele, trauma, endocrine disorders, and idiopathic causes (Esteves and Agarwai 2013).