Systematic investigation of the effect of chemotherapeutic agents on the ovary using ovarian follicle culture systems.

Many women undergoing chemotherapy treatment for cancer subsequently suffer from premature ovarian failure, and hence infertility. With recent advances in treatment resulting in greatly improved cancer survival rates, it is becoming increasingly important to counter adverse effects of treatment such as ovarian failure and infertility. Breast cancer patients are the most common group affected: 25% of these patients are pre-menopausal at the time of diagnosis and treatment will affect ovarian function in around half of these women. Loss of fertility can be a vital issue, but even where that is not the case, early onset of the menopause, with all of its accompanying adverse effects, is an important consideration for longer-term health care. While effects on the ovary are known to result from damage to ovarian follicles, the structures containing the eggs, it is not known what stages of follicle, or what cell types within the follicle, the chemotherapy drugs initially damage. This application proposes to use tissue culture techniques with isolated ovarian follicles, methods in which we have a great deal of expertise, to investigate this problem. We will investigate the precise stages of development of ovarian follicles at which they are most sensitive to different chemotherapy drugs, which cells within the follicle are the main site of damage from chemotherapy, and how this damage may indirectly affect other follicles growing within the ovary. We believe that this will be a powerful way forward that will, in the longer term, allow clinicians to test and compare the levels of damage to the ovary of a range of chemotherapy drugs, and provide the information necessary to facilitate ways of preventing ovarian failure.

The human ovary contains a fixed supply of germ cells, oocytes, maintained within ovarian follicles. Chemotherapeutic agents are known to damage ovarian follicles, and due to the finite nature of the follicle pool, this damage is irreversible (since germ cells cannot later be regenerated, as is possible for many other damaged tissues). As a result of this damage, premature ovarian failure (POF) and hence infertility occurs in a significant number of young female cancer patients. With recent advances in cancer treatment leading to increased survival rates, the long-term adverse affects of chemotherapy on female fertility now demand attention. We propose to investigate the effect of chemotherapeutic drugs using established follicle culture techniques of which the applicants have considerable expertise in both mouse and larger mammalian species (including humans), to determine the precise sequence of events that results from exposure to the drugs, establishing which effects are direct and which are indirect, secondary consequences. This is vital, since treatment to prevent POF would need to protect against the initial, direct effect if it is to be successful. These in vitro models allow ovarian follicle material of known developmental stages to be exposed to chemotherapeutic drugs and then monitored closely. Such analysis is not possible in vivo, because of the heterogeneity of the ovarian follicle population. This heterogeneity precludes the possibility of determining the developmental stage at exposure of any follicle subsequently found to be affected. The first part of the proposal will concentrate on using well established in vitro animal models to investigate effects of five individual chemotherapeutic agents and two of the most common drug combinations used in young patients. Experiments will be undertaken to investigate germ cell survival and follicle growth, examining site and time course of apoptosis and cell proliferation. In the second half, we will examine key drugs and doses using: (a) in vitro fertilisation carried out on cultured mouse material to determine fertilisation rate and embryo viability of oocytes previously exposed to low doses of chemotherapeutic agents; and (b) culture of human ovarian material, to check that similar effects are seen in human as in mouse. By carrying out the first, detailed screen using mouse material, we can ensure optimal use of the more limited supply of human material. In a final series of experiments, we will test key findings in vivo to assess how our findings translate into a murine preclinical model.