**Review on Ovarian Cryopreservation in Large Animals and Non-Human Primates**

Milan Milenkovic, Cesar Díaz-Garcia and Mats Brännström *Department of Obstetrics and Gynecology, Sahlgrenska Academy University of Gothenburg Sweden* 

#### **1. Introduction**

186 Current Frontiers in Cryopreservation

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> The ultimate aim of ovarian cryopreservation research is naturally to increase the effectiveness of this fertility preservation procedure in female cancer victims and much of this research on whole ovary cryopreservation, ovarian cortex cryopreservation and transplantation has been performed in animal models. However, ovarian cryopreservation could also be used in the future in programs with the purpose to rescue endangered species (Santos et al., 2010) and certain specific strains of animals (Dorsch et al., 2004). Due to the ethical barriers in the research on human tissue and the shortage of human premenopausal ovarian tissue for research purposes, there is a need to find animal models that are reasonably analogous to the human. As a general rule, animal models have to be comparable in biochemical, physiological and anatomical characteristics to the human so that the results can be applicable to human conditions (VandeBerg, 2004). Regarding research of ovarian cryopreservation for human fertility preservation, a similar tissue architecture and size of the ovary (Table 1) as well as being a mono-ovulatory species with the primordial follicles located superficially in the cortex would be of advantage (Gerritse et al., 2008). The previous research on ovarian cryopreservation using bovine, porcine, sheep and non-human primate models will be presented in this chapter.


Table 1. Ovarian characteristics of different large animal models

Review on Ovarian Cryopreservation in Large Animals and Non-Human Primates 189

While the study by Celestino and colleagues (Celestino et al., 2008) showed increased toxicity with rising concentration of DMSO, the other study (Lucci et al., 2004) showed slightly higher proportion of normal follicles in the 3 M DMSO group when assessed by conventional histology. However, ultrastructural analysis by transmission electron microscopy (TEM) revealed some irregularities in the cytoplasm of granulosa cells when 3

There is one study on bovine ovarian cortical strips comparing slow freezing with vitrification and this study demonstrated higher efficiency of slow-freezing (Gandolfi et al., 2006). Furthermore, another study also demonstrated advantages of the slow-freezing method as compared to vitrification, when bovine ovarian cortical pieces were used, but on the other hand an advantage of the vitrification method was seen when whole ovaries with vasculature were used as the model system (Zhang et al., 2011). In the latter study, the effectiveness of the different cryo techniques was evaluated by Trypan blue test, histology as well as E2 and progesterone levels obtained from supernatant after in vitro culture of the tissue. The same research group (Zhang et al., 2011) performed controlled-rate slow freezing with DMSO of whole bovine ovary and compared different cooling rates and ice seeding temperatures. The cooling rate of 0.2ºC/min and ice seeding temperature of -5ºC showed superiority in comparison to different combinations of the cooling rates of 2ºC/min and

Additionally, one study on bovine ovarian tissue was designed to evaluate the effect of the thickness of the ovarian cortex strip on follicular morphology after incubation for 20 min and slow freezing with 1.5 M propylene glycol (PROH) (Ferreira et al., 2010). Ovarian cortex pieces of 10 x 3 mm, with a thickness of either 2 or 4 mm, were compared and considerably higher proportion of normal follicles were found in the 2 mm group compared to the 4 mm in both fresh and cryopreserved tissue. This result may be explained by superior tissue impregnation with CPA in the 2 mm group, but the exact mechanisms remain to be clarified.

The pig is a species that has been used in biomedical research, particularly regarding development and training of surgical techniques for later use in the human. The reproductive cycle of the pig lasts for 18-24 days and generally 8-15 oocytes are released from each ovary at ovulation (Soede et al., 2011). The size of the pig ovary is about 7.3 (+/- 2.2) cm3 (Gerritse et al., 2008), which is comparable to the human ovarian size of 6.5 (+/- 2.9) cm3 (Munn et al., 1986). The equal ovarian size of the sow and human female, as well as the possibility to get fresh pig ovaries from slaughterhouses, renders the pig as a good model for ovarian cryopreservation research. Nevertheless, a fairly low number of studies in this area with the pig ovary as an experimental model have been performed, as shown below.

One recent study on pig ovarian tissue evaluated whether the size of ovarian cortical pieces is important for the cryopreservation outcome (Jeremias et al., 2003). Cortical strips were all of 1 mm thickness but either 1x1 mm or 5x1 mm in surface area. The pieces were cryopreserved by slow freezing in 1.5 M DMSO and after rapid thawing the size of the surviving primordial follicle pool, was compared to fresh tissue (1x1 mm) (Jeremias et al., 2003). The freezing method was uncontrolled-rate freezing with the cryovials containing

0.1ºC/min and the ice-seeding temperatures of -2ºC, -5ºC and -8ºC.

M DMSO was applied (Lucci et al., 2004).

**3. Pig** 
