I-6: Follicle Development in Culture of Frozen / Thawed Human Ovarian Tissue

The primary therapeutic goal for the oncology patient is survival. Recent advances in diagnoses and treatment of neoplasia have resulted in an ever-increasing number of patients being cured and resuming a normal life. However, recognized side effects of treatments used to eradicate malignancies are temporary or permanent sterility. The cryopreservation of ovarian tissue harvested before cancer therapy may preserve the reproductive function and/or restore fertility in women and young girls. Good preservation of reproductive function, pregnancies, and births of healthy babies have been reported after ortho/heterotopic transplantation of cryopreserved ovarian tissue in patients with neoplastic disease. In addition, long term culture and in vitro development of antral follicles to produce metaphase II oocytes for in vitro fertilization (IVF) may represent another alternative to overcome iatrogenic sterility. Several developmental milestones already accomplished include follicle activation, preantral follicle growth, follicle differentiation, and oocyte maturation using cryopreserved human cortex. The length of the follicular growth phase from the primordial to Graafian stage and the changes in the trophic requirements of the cells, cellular interactions, morphogenesis, and the sheer increase in bulk as the antrum forms are major challenges for contemporary cell culture technology. At any age the human ovarian follicle reserve is composed of primordial follicles, the abundance and lack of differentiation of primordial follicles makes this population an ideal choice for in vitro growth to obtain fertilizable oocytes for potential use in asissted reproductive technologies (ART) and fertility preservation programs. The ability to develop these immature follicles fully in vitro, also, would facilitate greater understanding of the mechanisms regulating oocyte development. It is assumed that complete follicle development from primordial to the pre-ovulatory stage in humans takes up to eight months and it has been calculated that the time needed for a follicle to grow from the primary to the pre-ovulatory stage to be 84 days. However, there is no solid evidence to show that this is a continuous period of growth; indeed, it is likely that follicles grow in vivo in a ‘start-stop’ manner in response to local influences. It appears that oocyte development can be supported within a rapidly developing follicle. Indeed, this ‘‘accelerated’’ growth has been demonstrated in other (nonhuman) culture systems and fully grown oocytes have been obtained. Complete oocyte development in vitro from the primordial stage has been achieved in mice, but the larger size and longer growth period of human follicles has made the interspecies translation of these techniques difficult. The challenge now is to define the in-vitro conditions that facilitate a rate of growth that supports normal human oocyte development. Culture conditions: The development of refined complex media is ongoing, and new evidence continues to contribute to literature. When supplemented with human serum acting as a protein support, a-MEM appears to be the most suitable medium for in vitro follicular development, when compared with EBSS or Waymouth’s medium. Among the factors that regulate the initiation and progression of primordial follicle development, KL and bFGF seem to be essential for the progression from primordial stage to primary stage; VEGF and GDF-9 are fundamental for the achievement of the secondary stage follicle; insulin, IGF-I, and IGF-II act as trophic factors for follicles and simultaneously stimulate follicular growth; N-acetyl cysteine (NAC), a free-radical scavenger, working in combination with FSH, plays an important role in follicle growth; and gonadotropins, FSH, and luteinizing hormone (LH) are essential for progression from the preantral stage to the antral follicle stage. The approaches involve incubating small pieces of cortex, isolation and culture of growing follicles, and isolation and incubation of follicles isolated from cultured cortical strips. It is recognized that, whereas cortical strip culture supports human follicle activation and growth to the secondary stage, follicle integrity and oocyte survival are only maintained for a relatively short period of time. Therefore to develop further, follicles must be released from the stromal environment, but many observations appear to confirm that local ovarian factors indeed inhibit development of follicles. The question remains as to whether rapid in-vitro growth results in the production of competent oocytes. It has been noted that the presence of stromal cells is vital during initial growth. For culture of isolated follicles, preantral follicle isolation from cortical tissue is usually achieved by either mechanical dissection, enzymatic isolation, or a combination of both. Enzymatic isolation commonly uses collagenase and DNase to liberate follicles from stromal tissue, yielding many more follicles than mechanical dissection, and it has also been associated with enhanced steroidogenic potential in culture. Collagenase has been associated with follicle damage and poor survival in large mammals. Mechanical isolation, using fine needles, has the advantage of preserving follicular integrity by maintaining the basal lamina and thecal layers of the follicle but the procedure is protracted and laborious. The development progression of human follicles following isolation from the cortex is notable however isolation of follicles is much more difficult in humans than in other mammals because human cortical tissue is more compact and fibrous when compared with commonly used animal ovaries. Cortical strip culture removes follicles from the in vivo endocrine and paracrine processes regulating growth rate; however, follicles will still be subject to the effect of follicle interactions and the influence of stromal cell factors. It is clear that tissue shape and stromal density are important factors which regulate follicle growth initiation in-vitro, as solid cubes of cortical tissue show a lower rate of growth initiation. In contrast, when stromal cells are removed and the tissue is cultured as flattened ‘sheets’, the initiation rate is greater and follicles grow faster. The physical environment of the follicles within the cortical tissue affects their response to stimulatory and inhibitory factors and therefore influences their ability to grow. Once follicle growth is initiated within the strip, they can develop to multilaminar stages. At this point, the cortical strip environment becomes inhibitory to further growth. Therefore, strip culture does not support optimal development of all stages and a multi-step culture system is required to support complete development. Pre-antral follicles can be mechanically isolated from the cortex culture system after six days and placed within an individual culture system for further development to antral stages. In addition, with tissue culture systems, the use of high oxygen concentration appears to be important for facilitating oxygen diffusion to the follicles within tissue segments, as it is believed that oxygen deficiency may cause centralized necrosis. Central necrosis is thought to be the main cause of damage to tissue and cells and may reduce the ability of oocytes to continue to develop. Measures to support the three-dimensional follicular architecture and thereby maintain intra-follicular cell association in-vitro have been employed in the culture of mammalian follicles for almost 20 years and it has been demonstrated, largely using rodent systems, that the use of suspension cultures, mineral oil sheaths, hydrophobic membranes and follicle encapsulation result in the promotion of follicle growth and the attainment of developmental milestones. The ability of the system to promote human follicle growth in vitro from the earliest stages at rates that are accelerated in comparison with the in vivo environment is indeed promising but we need to know if oocytes produced by these systems are deleterious to oocyte epigenetic health and normality. Significant progress has been made but further optimization is required to routinely complete the in vitro development of the stages detailed here. Translation of any in vitro human follicle growth system into a clinical setting will require rigorous testing to determine the normality and health of in vitro grown oocytes before the application of IVF procedures.