**3. Ovarian tissue cryopreservation**

To preserve ovarian tissue for a lengthy period of time, it should be stored in liquid nitrogen at a temperature of -196°C. However, ovarian tissue cryopreservation is complex and requires preservation of multiple cell types, thus the ability to successfully cryopreserve would be a powerful clinical tool in an assisted reproductive laboratory.

As reported by Bakhach in 2009, is easy to imagine the cell damage when temperatures fall from +37°C to -196°C. There is a loss of about 95% of intracellular water, an increase of electrolyte concentrations in both intra and extracellular media, and ice formation in the intracellular spaces that deform cells and destroy intracellular structures (Bakhach, 2009).

Hovatta in 2005 published an interesting work on the different methods of ovarian tissue freezing which emphasizes the importance of trying to preserve most of the cellular tissue components, including even the small proportion of ovarian medulla which contain blood vessels and nerves so important for ovarian tissue function recovery after reimplantation (Hovatta, 2005). Recently, Donnez reported that revascularization of grafts depends also on the preservation of vessels in grafted tissue, and not only on neoangiogenesis from the host (Donnez et al., 2011-b).

It should be stressed that the protocol of ovarian tissue freezing is still not standardized. So, there are several freezing procedures that range from slow freezing/rapid thawing, vitrification or ultrarapid-freezing. Freezing protocols differ also depending on the cryoprotectants used, dehydration/rehydration time and temperature, different protein support, and on the containers used for storage such us cryovials, strips or other tubes.

Approaches to human ovarian tissue cryopreservation are currently characterised by the use of slow freezing/rapid thawing methods using dimethylsulphoxide (DMSO) or propanediol (PROH) as a cryoprotectant. The slow freezing requires the use of a machine that slowly decreases the temperature, so the freezing programme so takes a few hours. This procedure has the disadvantage that it can lead to the formation of ice crystals, affecting correct tissue preservation. This disadvantage is avoided by using the protocol of vitrification, where high concentrations of cryopotectants are used, which increased ovarian toxicity too. Ultra-rapid freezing should be a method somewhere between the previous two using the advantages given by vitrification with a lower concentration of cryoprotectant to act on increasing the cooling rate.

In 2003 Shaw published an interesting paper on the terminology associated with equilibrium cooling procedures or 'slow cooling' and non-equilibrium protocols such as 'vitrification', 'rapid cooling' and 'ultrarapid cooling' that is helpful in clarifying the terms often used inappropriately (Shaw & Jones, 2003).

The positive results achieved by oocyte vitrification, has been discussed in recent years, but at the present time this procedure applied to ovarian tissue has given conflicting results.

Isachenko has worked extensively on the ovarian tissue vitrification. Her paper of 2007 showed a better preservation of ovarian tissue by slow freezing in which the quality of follicles was higher compared to the rapid freezing (Isachenko et al., 2007; Isachenko et al., 2009).

In 2007 Li demonstrated that the vitrification method for cryopreservation of human ovarian tissue is effective and simple (Li et al., 2007). Kagawa in 2009 found no difference in oocyte viability between fresh and vitrified human ovarian cortical tissue (kagawa, 2009). In another paper ovarian stroma was shown to be significantly better preserved by vitrification compared to slow freezing (Keros et al., 2009).

However, data on cryopreservation of human ovarian tissues by vitrification are still modest and controversial. This may be due to the fact that the same cryopreservation procedure is often used, such as slow freezing or vitrification, but different freezing protocol (types of cryoprotectant, concentration used and time of diffusion, etc.), this creates significant bias that must be considered.

In addition vitrification presupposes direct contact with liquid nitrogen, which is a potential source of microbial contamination, as reported by Isachenko (Isachenko et al., 2009).

In conclusion, an ideal ovarian cryopreservation method has not yet been established.
