Fertility Preservation in Monovulatory Species

The ovary functions by periodically releasing an egg, grown within a follicle, which may then be fertilised. When the number of follicles is exhausted, the ovary ceases to function. Some medical treatments, such as chemo- and radiotherapies, destroy large numbers of follicles and prematurely cause the loss of ovarian function and fertility. One possible way of preventing this damage is to remove small pieces of ovarian tissue rich in follicles before treatment begins and preserve them by freezing to very low temperatures. The tissue can then be grafted back at a later date to resume function. Whilst this has been successful in both animals and humans, the length of time the tissue functions for is again determined by the number of follicles preserved in the graft which, in these small pieces of tissue, is limited. One way to improve this is to freeze whole ovaries, however considerable improvements are still required to minimise the damage currently seen following freeze-thawing and to achieve long-term ovarian function. Initial thoughts were that the freezing process and the lack of oxygen and nutrients supplied to the ovary during preparation for freezing and transplantation were causing this damage. However we have shown that this is not the case. As this technique is still relatively new, there remain several avenues of research to be explored and optimised to provide the most efficient method of freeze-thawing and grafting whole ovaries. This research initially aims to identify those areas which may be responsible for follicle damage and failure of frozen ovaries to being working again. This will then lead to a series of experiments looking at ways to correct the damage which may arise at each stage of the freeze, thaw and grafting processes to maximise ovarian and vascular tissue preservation. A variety of laboratory techniques will be used to study tissue structure, cell regeneration and gene expression in order to assess ovarian and vascular tissue health and damage. Finally, a study using sheep will implement all the improvements in technique developed over the course of the study and assess the relative value of the changes made in terms of the length of ovarian function, restoration of fertility and its possible clinical application in human fertility preservation. The overall aim is to improve the success of whole ovary freezing and bring it closer to becoming a viable option for the preservation of ovarian function in women.

Ovarian tissue cryopreservation and autografting have been proposed as a means to preserve and restore the ovarian function in women who have a high risk of premature ovarian failure as a result of genetic defects, surgical interventions or cancer treatments. Experimental evidence which supports this strategy has utilised cryopreservation of fragments of the ovarian cortex as a means to freeze-store a fraction of the ovarian reserve. An alternative approach which maximises reproductive longevity is to cryopreserve and transplant the whole ovary and supporting vasculature (WOCP). This approach has been evaluated in sheep, where the size and physiology of the ovary are similar to the human and this research has shown long-term patency of the ovarian vasculature after freezing and thawing and acute follicle survival, but high rates of follicle attrition following ovarian transplantation. The underlying cause of this follicles loss is unknown. This project will therefore use the sheep as a model to test the efficiency, efficacy and application of WOCP and autotransplantation as a means to preserve and restore fertility in girls and women. Our targeted approach will combine novel in vitro assays with animal experimentation to identify the primary causes of follicle loss associated with WOCP and autotransplantation and to devise strategies to overcome these limitations. The proposed research will evaluate which genes underpin follicle survival following freezing and transplantation of ovarian tissues. The expression patterns of key genes involved in angiogenic, apoptotic, DNA damage and repair and cell stress signalling pathways will be quantified in isolated ovarian cortex and whole ovaries following WOCP and autotransplantation. The efficacy of different ovarian perfusion regimens and the penetration of different cryoprotective agents (CPAs) will be measured using NMR spectroscopy. Parallel studies will utilise vascular microsphere extravasation in combination with histological (light and electron microscopy), immunohistochemical and immunoassay methods to assess ovarian and vascular tissue health and function following ovarian perfusion. Additional indices of tissue health will include evaluation of the uptake of vital dyes as well as assays of cell membrane damage following exposure to CPAs. The impact of survival promoters, such as the anti-apoptotic factor sphingosine-1-phosphate, will be investigated in vitro using cortical strip and preantral follicle cultures. Finally, the efficacy of the improved whole ovarian freezing, thawing, autografting and autotransplantation protocols developed during the course of the proposed studies will be investigated in long-term in vivo follow-up studies to evaluate the restoration of natural fertility.