**5. Non-human primates**

A number of non-human primate species have been used in research involving reproductive physiology and development of methods later used clinically in reproductive medicine. The benefit of these experimental animals is that the physiology and anatomy are similar to the human (Stevens, 1997; Weinbauer et al., 2008), although the ovarian size of the most studied cynomolgus monkey is considerably smaller (0.258+/- 0.159 cm3) (Jones et al., 2010) as compared to the human. Even if a lot of procedures are introduced in the human without appropriate tests in non-human primate species, it is advisable to include these animal models in preclinical research.

There is one study on non-human primate that has assessed the function of cryopreserved ovarian cortex tissue in vivo. The ovarian pieces were cryopreserved by controlled-rate freezing with 1.5 M DMSO and both fresh and frozen-thawed strips were grafted subcutaneously to the upper arm of cynomolgus macaque monkeys (Schnorr et al., 2002). Regained menstrual cycle and ovarian steroidogenesis were demonstrated in 80% of the animals after fresh ovarian cortex transplantation and in 50% of the animals after cryopreserved transplants, demonstrating that the cryopreservation technique may have damaging effect on the tissue. Additionally, only one mature oocyte was aspirated from subcutaneous ovarian tissue after gonadotropin stimulation performed in four animals. In the other part of this study (Schnorr et al., 2002) an attempt to enhance angiogenesis was performed by local administration of VEGF for two weeks after grafting, but no beneficial effect could be demonstrated.

There is one study on ovarian cortex from macaques when vitrification (3.4 M GLY, 4.5 M EG) and slow freezing (1.5 M EG) were compared and evaluated whether post-thawing coculture on feeder cells (mouse fetal fibroblast monolayer) with addition of follicle stimulating hormone, insulin, transferrin and selenium, would increase viability (Yeoman et al., 2005). The post-thaw viability, as assessed by live-dead fluorescent staining, was comparable (around 70%) in the two groups, which was only marginally lower than the follicular viability of the fresh tissue (76%). Interestingly, follicular viability was increased after post-thawing co-culture, indicating rescue of partly damaged follicles and possible beneficial effect of co-culture.

In another study on ovarian cortex tissue of rhesus macaques, controlled-rate slow-freezing with 1.5 M EG was done and expressions of activin subunits as well as the phosphorylated form of the signalling protein were investigated (Jin et al., 2010). Activin subunits and the phosphorylated form of the signalling protein are markers for early follicular development. Immunohistochemistry revealed that these proteins were regularly distributed in primordial and primary follicles of both cryopreserved and fresh cortex. One interesting finding was that a higher rate of post thawing apoptosis was found in the stromal cells as compared to oocytes and granulosa cells. Another recently published study was able to demonstrate follicular development after in vitro three dimensional culture of cryopreserved secondary follicles from rhesus macaque (Ting et al., 2011). The ovarian cortex from rhesus macaque was cryopreserved by slow freezing (1.5 M EG) and vitrification (3 M GLY and 4.5 M EG). After thawing, the secondary follicles were mechanically isolated, encapsulated in 0.25% alginate and cultured for five weeks in MEM at 37ºC. The development of antral follicles, although delayed and functionally compromised as compared to fresh tissue was demonstrated after both cryopreservation methods with slightly better results obtained after vitrification in comparison to slow freezing protocol. This observation again supports the need for further improvement of cryopreservation techniques.
