Human ovarian follicle recruitment : an in vitro approach

Abstract

Following the first reports of successful cryopreservation of human ovarian tissue, the freezing and banking of such tissue has become common practice worldwide for the preservation of fertility. Patients include those at risk of premature ovarian failure, women and girls with Turner's syndrome, and cancer patients for whom radiation or cytotoxic treatments are likely to destroy their reproductive potential. Optimal success of utilising cryopreserved human ovarian tissue for fertility preservation requires the development of defined methods with in vitro culture being one alternative. Animal models have met with some success yet methods for the human are still being developed. A tissue culture system to study follicle and oocyte development has been gradually established, investigating growth factors and hormones implicated in folliculogenesis, as well as other basic handling techniques.

In the in vitro system, small pieces of ovarian cortical tissue were cultured on inserts coated with extracellular matrix in defined media for 7 and 14 days at 37°C with 5% C02. Different culture medium supplements were investigated including growth differentiation factor-9 (GDF-9), 8-bromo cyclic GMP (cGMP), and activin A alone or in combination with follicle stimulating hormone (FSH). Furthermore, tissue dimensions and extracellular matrix composition and coating density were examined. Following culture, tissue was fixed and stained for morphological analysis of follicle development, viability, density and diameter of both the follicle and oocyte. Hormone production (oestradiol, progesterone and inhibin B) was also measured in media sampled throughout the duration of culture.

Culture of human ovarian cortical tissue allows the early stage follicles to grow within the supporting stroma thus maintaining their structural integrity. In the studies performed in this thesis, a significant activation of primordial follicles was observed with culture accompanied by a reduction in the proportion of viable follicles. Follicle viability, recruitment and early growth were enhanced by the addition of GDF-9 and cGMP, indicating the beneficial effects of these factors in culture. Activin A and FSH appeared to play differentiation roles affecting only hormone production and may therefore be important in subsequent stages of follicle development. Cultures of 7 days duration may benefit from tissue being cultured as cubes on diluted Matrigel matrix, both of which support follicle survival. Extracellular matrix composition alone had little effect on the follicles, although its components and interaction with hormones and must be considered when planning a culture system or experiment. As a result of these findings, a multi-step sequential culture media process is being designed. Different hormones and factors will be added at various time points, attempting to mimic the follicle requirements in vivo.

The research in this thesis has aided the development of an optimal method of ovarian tissue culture in vitro and may provide a solution to the successful recovery and growth of follicles from cryopreserved human ovarian tissue. Further studies investigating additional factors and sequential culture media may lead us to convert this technique from the research laboratory to clinical practice.