Ovarian tissue cryopreservation followed by in vitro follicle maturation has been highlighted as the main option for preserving fertility in prepubertal girls and cancer patients requiring immediate treatment. The inability of the conventional cellular culture system (two dimensional- 2D) to sustain the in vivo-like follicle shape in large mammals has limited the success of the follicle in vitro culture to generate mature oocytes. Although many studies have shown that a calcium alginate-based matrix supports the three-dimensional (3D) architecture of mice follicles permitting the in vitro development of pre-antral follicles to the antral stage and leading to mature oocyte and live births, the success of such technique in large species still unknown. Similarities on ovarian physiology and reproductive cycle dynamic between humans and cattle as well as the easy collection of ovarian samples in the slaughterhouses make the latter an excellent experimental model for reproductive studies. Although the ovarian tissue cryopreservation technique has been already patronized in the bovine, the retrieval and culture of immature follicles remains to be determined. In the present study we aim to validate the use of the 3D system to culture bovine pre-antral follicles in vitro derived from cryopreserved ovarian tissue. Using this model we also intent to evaluate the physiological mechanisms related with follicular growth and oocyte maturation by analyzing follicular development during IVM evaluating follicular and oocyte diameter, stage of maturation, cellular viability, steroidogenic capacity an reproductive potential based on in vitro fertilization techniques. Thus we intend to generate data for the further development of an adequate milieu for the human pre-antral follicle in vitro development.
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