**3.2 Methods**

*Embryology - Theory and Practice*

**3.1 Cryoprotectants**

sive equipment like in the slow freezing technique.

the basis of many future investigations concerning CPAs.

Cryoprotectants (CPAs) act to reduce cellular damage by increasing the volume of the unfrozen residual phase. When the first cryopreservation experiments took place, two opposing methods had been developed simultaneously—a method of slow freezing of the cells and vitrification. Since these methods were very different, scientists started to compare them, in order to elucidate their benefits and drawbacks. Vitrification offers the possibility of eliminating the formation of ice crystals [16], and over the years, it has gradually replaced slow freezing as the preferred method of cryopreservation in the field of reproductive medicine. The main reason behind this fact is that vitrification achieves better survival rates, and moreover, it is less time consuming to perform and does not require highly specialized and expen-

Cryoprotectants (CPAs) are substances that protect cells/tissues from the damage that may occur during the freezing process. In order to achieve successful cryopreservation of any biological material, the freezing protocols must be optimized, starting with the correct choice of CPA and ending with the thawing process and post-thawing handling of the material. The choice of the most appropriate CPA for a certain freezing procedure is difficult to make, because it must take into consideration the CPA's toxicity, permeability, and also its physicochemical properties. CPAs are widely used to improve the cryosurvival rates, although their mechanism of action is not fully understood. One of their properties is to lower the freezing point of a certain solution, while also protecting the cell membrane during the freezing process. CPAs may also act to stabilize intracellular protein structure. As mentioned earlier, freezing an embryo is a very delicate procedure and embryos may be damaged by chilling, fracturing, ice formation (intra and extracellular), the chemical toxicity of CPAs, osmotic swelling, and osmotic shrinkage [17]. Chilling injuries can lead to changes in lipid-rich membranes and can also result in cytoskeletal disorganization. The mechanical effect of a solidified solution may cause fracture damage, especially to embryos. One of the first documented studies that introduced the concept of cryoprotectants was that of Polge et al. [7, 18], which assessed the use of glycerol in sperm freezing, and it also provided

Regarding their structure, CPAs are small molecular weight solutes possessing high aqueous solubility and polar groups that interact weakly with water [19]. CPAs are generally divided into two groups based on their ability to penetrate through the cell membrane—permeating (PM) and nonpermeating (NPM). It is important to point out that PM and NPM cryoprotectants are often used together in order to achieve a successful cryopreservation of cells and tissues. In fact, the core of a cryopreservation solution is made of a mixture of those CPAs, and it also includes various components, like some salts, pH buffers, and others. In the PM group are included some of the most studied CPAs like glycerol (G), ethylene glycol (EG), dimethyl sulfoxide (DMSO), formamide, propylene glycol (PG), and others. PM cryoprotectants are the most important component in the vitrification solution. G and EG are the most commonly used PM CPAs. NPM cryoprotectants include saccharides, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), and others. They are large molecules, usually polymers. Sucrose is the most commonly used NPM cryoprotectant. The effect of these NPM agents is the dehydration of the cells by osmosis. They also act to stabilize the cell's membrane [20] and aid the entry of PM cryoprotectants [21]. Moreover, NPM CPAs are added during the thawing process as

Nowadays, there are many freezing media produced by biotechnology companies, which are made by mixing various substances to achieve maximum

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they act to reduce the osmotic shock.

Practically, two methods for embryo cryopreservation have been used—slow freezing and vitrification. Here, we will briefly review both of them and we will discuss their positives and negatives.
