In Vitro Models of Ovarian Toxicity

6.1

Introduction

Investigating the precise effect of chemotherapy on female fertility is a key research aspect underpinning fertility-preservation work. Protecting the ovarian follicle pool will require detailed understanding of precisely how the different chemotherapy drugs damage the ovary: whether chemotherapy drugs directly kill oocytes or whether damage is first to surrounding somatic cells; if the drugs are equally toxic to all follicles or if specific stages of follicle development are particularly vulnerable to damage. The more information that is available, the more informed will be our development of clinical treatments to protect the ovarian reserve. So far, in vitro techniques have been used relatively little for research into effects of chemotherapy treatment and potential ovarian protection against damage, but work to date indicates that these are, in general, powerful tools for reproductive toxicology studies, and so also likely to be very useful here.

6.2

Why Use Culture Systems?

Tissue culture systems have been extensively used to study key mechanisms and processes in the ovary, including follicle recruitment, oocyte–somatic cell interactions and follicle–follicle interactions. There are a variety of culture systems available, short- and long-term, most covering specific stages of follicle development. The addition of chemotherapeutic drugs into such systems allows determination of the effect of these agents on the health of various components of the ovary. Depending on the aims of the study, the molecular mechanisms of cell and follicle death can be established, along with examination of the effect on individual ovarian cell types, specific follicle stages and elucidation of primary versus secondary effects. A major consideration when studying the ovary is that the follicle population within it is heterogeneous, with follicles in varying stages of development: in postpubertal females, this includes follicles from the primordial right through to the preovulatory stage. This complicates in vivo research into the effect of chemotherapy drugs, since it is difficult to ascertain the initial site of damage following chemotherapy exposure. In vitro models can span various, yet precise, stages of follicle development, allowing for analysis of specific cell and follicle types.

While in vitro models can maintain follicle–follicle interactions and the influence of stromal cells essential for physiological ovarian function, culture does, of course, involve isolating the ovary from its neuroendocrine inputs, which also impacts on follicle recruitment and growth. In vitro models are therefore useful for determining the direct effect of chemotherapeutic agents on the ovary, but do not allow examination of indirect ovarian effects, such as occurs following hypothalamic/pituitary damage during cranial irradiation.

When using in vitro techniques to examine the effects of chemotherapeutic agents, there are several limitations that must be considered. Premenopausal women receive complicated treatment regimens often involving combinations of several different drugs, generally given repeatedly over many weeks, which can be difficult to replicate. It is also hard to extrapolate the amount of drug that reaches the ovary solely from known serum concentrations, making it challenging to determine clinically relevant dosages to use in vitro . Chemotherapeutic agents can be metabolized in vivo , often in sites away from the ovary, such as the liver or the kidney, with the metabolites potentially having differing levels of ovotoxicity. An example of this is cyclophosphamide, which is metabolized by hepatic enzymes into a range of active metabolites including phosphoramide mustard and acrolein. Utilizing the exact combination of metabolites that the ovary could be exposed to is therefore difficult to simulate in vitro , as is the metabolism of a drug if the metabolites themselves are not readily commercially available. There may also be limited information on the full range of active metabolites. Metabolites may have varying half-lives, even existing only transiently, complicating an experiment that is trying to replicate clinical scenarios. The volatility of some chemotherapy agents/metabolites is another consideration, as these can produce “next well” effects, requiring different treatment groups to be physically separated during culture.

Overall, whilst in vitro techniques are valuable, they cannot simply replace in vivo experiments. The clinical scenario will involve indirect effects and potential input from other organs. Thus, in vivo systems are required to determine secondary effects from other organs, including changes to the hypothalamic–pituitary–gonadal axis. Nonetheless, use of in vitro culture systems reduces the number of animal experimentations required and allows for screening of a large range of drugs and drug doses. In particular, high drug dosages can be investigated more easily in vitro , since the experiments are not complicated by deteriorations in animal health or subsequent mortality. Overall, therefore, in vivo and in vitro methods both have their advantages and disadvantages, and both should be used for thorough investigation of reproductive toxicology.