Abstract
Cryopreservation of ovarian tissue is a promising technique to rescue animal species and breeds from extinction and to restore fertility of women after cancer treatment. Establishment of cryopreservation protocols starts by choosing the most efficient cryoprotective agents, the dimension of ovarian fragments, the volume of the cryoprotectant solution, the addition
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of extra-cellular cryoprotectant, and efficient methods to evaluate follicular quality. Based on these parameters, caprine ovarian tissue was successfully cryopreserved when cortical fragmented in pieces (~ 1mm3) exposed to a low volume (frozen in 1 ml or vitrified in 0.5 ml) of a combination of ethylene glycol and sucrose, according to analysis based on viability markers to assure follicular viability. However, cellular viability after cryopreservation does not guarantee follicular development. It was observed that cryopreserved early-staged follicles show a significant decline in quality after having them in vitro cultured for 24 h. One of the damage effects caused by cryoprotectants is called osmotic stress, because of the high osmolality of the cryoprotective agents. It has been demonstrated that high concentrations of cryoprotectants, e.g. 4.0 M EG (5242 mOsm), are not responsible for osmotic damage. On the other hand, when early-staged follicles were exposed to an ionic solution of 9 % NaCl (2800 mOsm), most of the cells (92 % ) had lost their viability, showing that ions are the cause the cellular damage and not the cryoprotectants (non-ionic solutes). The aim of cryopreservation of early-staged follicles is storage for an undetermined time without viability impairment, followed by further complete development after thawing/warming. Despite the success obtained with the complete in vitro development of cryopreserved early-staged murine follicles, including development of frozen-thawed follicles followed by successful fertilization, embryo development and healthy offspring, similar success is difficult to achieve with large mammals in vitro. There is no protocol for complete in vitro development of early-staged follicles from large mammals. Thus, the importance of transplantation to obtain complete follicular development after cryopreservation. Success of the cryopresevation procedure was demonstrated by the complete follicular development (from primordial to pre-ovulatory stages) after freezing-thawing of ovarian tissue fragments and their re-implantation in ovariectomised donor goats. Thereby, ovarian cortical fragments re-modulated into a new ovarian-like organ, from which blood supply and endocrine function (oestradiol and progesterone production) were restored. The next step before clinical application, consist in evaluate some remaining points related to cryopreservation and transplantation. For example, absence of DNA damage and high survival rate of early-staged follicles after cryopreservation, does not mean that cooling cannot affect DNA by activating genes related to diseases and malformations. Thus, more experiments should be performed in adequate animal models, such as the goat, until data can be satisfactorily repeated and normal offspring can be guaranteed. Finally, although cryopreservation of ovarian tissue should not be promoted to postpone childbearing in healthy women, such a technique may be used in the future for safeguarding maternity for women requiring chemo- or radiotherapy. In addition, it may in the near future be an important technique for the preservation of endangered breeds and species.
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