**4.1 Slow freezing**

The protocol for slow freezing of spermatozoa was suggested by Behram and Salewa in 1966 [31]. It is based on slow dehydration of the cells. Slow freezing could be performed either manually or by using programmed freezer. The sperm samples are cooled in a stepwise manner by lowering the temperature and adding cryoprotectants. Initially the temperature is lowered by 0.5°C/min and it should go down from room temperature to +5°C. The second step is to freeze the samples from +5°C to −80°C by lowering the temperature by 1–10°C/min. Finally the semen samples are transferred into liquid nitrogen (LN2) at −196°C.

Automatic freezers have been reported as reliable when freezing sperm is performed. One of the advantages of these devices is the perfect control over temperature changes as the process is performed through software. The three step protocol of slow freezing takes about 40 min [32].

In slow freezing, changes in lipid phase transition and increase in lipid peroxidation, consequent to saturation with cryoprotectants, could cause intracellular and extracellular physical and/or chemical damage to sperm membranes. Susceptible to cryopreservation induced damage are the viability, motility and the morphology of the post-thawed spermatozoa. Mitochondrial function, as well as DNA integrity, could be affected [33–36].

#### **4.2 Rapid freezing: vitrification**

Vitrification, as a method for cryopreservation, has been primarily used for cryobanking of oocytes and embryos. This method is based on direct exposure of the gametes to liquid nitrogen at −196°C. In comparison to slow freezing, where formation of intracytoplasmic ice crystals could damage different cell structures, during vitrification the liquid components of the cells set into a glass like amorphous solid and ice crystal structures are avoided (**Figure 1**) [37].

Regardless the protocol used for cryopreservation of spermatozoa, cryoprotective medium must be inset in order to reduce the stress induced while freezing or thawing cells.

**Figure 1.** *Temperature changes in slow freezing and vitrification.*

*Permeable* cryoprotectants can penetrate through cell membranes. They have intra- and extracellular activity. By forming osmotic gradient, water is ejected outside the cell, preventing it to form crystals. The cryoprotecting agents forms non-frozen channels in the medium in which sperm cells can be positioned while frozen. Glycerol, ethylene glycol, dimethyl sulfoxide (DMSO) and 1, 2 propanediol (PROH) are commonly used permeable cryoprotectants.

*Non-permeable* cryoprotectants cannot penetrate through the cell membrane. They can induce dehydration of the cells by increasing the concentration of extracellular solutes. Osmotic gradient is formed and the intracellular water is derived. Various sugars (raffinose, mannose, and trehalose) and proteins (lipoprotein, egg yolk) can be inset as *Non-permeable* cryoprotectants [38, 39].

#### **5. Rare sperm freezing**

Rare sperm freezing could be defined as a separate branch in sperm cryobiology. It has formed an important direction in the development of freezing protocols, methods and devices. The need for efficient freezing protocol for single sperm cells was evident at the very beginning, when pregnancies from epididymal and testicular sperm were reported [40–42]. In cases, where percutaneous epididymal aspiration (PESA) or testicular sperm extraction (TESE) is performed, freezing sperm cells would be of great benefit to the patient, as these procedures are traumatic and stressful to the organism. Most of the conditions described at *3. Conditions requiring sperm preservation* and poor semen quality are to request freezing of sporadic sperm cells.

Rare sperm freezing has some major advantages to standard protocols. In one hand this method gives chances for reliable fertility preservation in patient with severe male factor and sperm alterations. On the other hand, the successful freezing of single sperm cells and revolutionary methods such as intracytoplasmic sperm injection (ICSI), represent the opportunity for storage of larger quantity of samples for every men, as this premise increases the chances for fertilization of more oocytes retrieved in a cycle or secures larger number of ART cycles.
