Role of growth factors of the TGFbeta superfamily in aberrant follicle development in polycystic ovary syndrome

Polycystic Ovary Syndrome (PCOS) affects more than 5% of women of reproductive age and has a major impact both on fertility and on long-term health. It results in irregular menstruation, is the main reason for ovulation-related infertility and, because of its associated metabolic abnormalities, carries a high risk of developing diabetes. However we still know very little about its cause or causes, which makes diagnosis imprecise and means that we are treating the resulting symptoms rather than the underlying causes. The main problem that we are attempting to address in this application is to understand the reasons why women with PCOS ovulate infrequently or not at all.

A woman is born with a supply of eggs that must last throughout her reproductive lifespan. These eggs are surrounded by a single layer of supporting (granulosa) cells and stored in tiny follicles in the ovary. This resting pool of follicles is termed the primordial pool. The processes that prompt primordial follicles to start growing remain unclear but there is evidence that locally produced growth factors play an important part, particularly those of the so-called transforming growth factor beta (TGFbeta) family. Much of this evidence comes from studies of mouse follicle development in our own laboratory. TGFbeta growth factors are proteins that are produced by and act in follicles of all sizes, including the very smallest, primordial, follicles.
In our earlier studies of ovarian samples from women with PCOS, we have shown that the problems that lead to infrequent ovulation may lie in the very earliest stages of the life of the ovarian follicle and we have already shown that at least one of the TGFbeta family members (anti-Müllerian Hormone or AMH) is produced abnormally in very small follicles of women with PCOS. For the current proposal we will have unique access to ovarian tissue from women with and without PCOS, much of which will come from a research-orientated programme for conservation of ovary tissue in young women about to undergo treatment for cancer. Every sample that we obtain will be "spare" tissue samples that would normally be discarded. We will also be able to collect small biopsies of women with and without PCOS who are undergoing routine laparoscopy (to visualise the ovaries and Fallopian tubes) or who are having ovarian "drilling" as a procedure to stimulate ovulation.

We will determine where and when (according to developmental stage) TGFbeta growth factors (and their receptor targets) are produced, how they signal within ovarian cells, how they may interact other growth factor networks, and whether these processes differ between normal and polycystic ovaries. We will also examine the effects of added TGFbeta growth factors in small samples of cultured ovarian tissue. We expect these studies will provide important clues not only to understanding fertility problems in women with PCOS but also how they may relate to the development of metabolic disorders, including diabetes, to which these women are predisposed.

Polycystic ovary syndrome (PCOS) is the commonest endocrine disorder in women and is the major cause of anovulatory infertility. PCOS is characterized by hypersecretion of ovarian androgens but women with PCOS also carry an increased risk of metabolic dysfunction and have 2-4 fold increased risk of developing type 2 diabetes mellitus (T2DM). Anovulation in PCOS is characterized by arrest of antral follicles, and appears to a function of the abnormal endocrine environment, but there is increasing evidence for aberrations in preantral follicle development which may predispose to later, antral follicle dysfunction. Our recent studies using human ovarian tissue point to a key role for growth factors of the TGFbeta (TGFb) superfamily in development of preantral follicles. Our overall objective is to test the hypothesis that aberrant expression of TGFb ligands and their receptors plays a key role in abnormal preantral follicle development and function in PCOS. We suggest that the findings in ovarian tissue will provide insight into the putative role of aberrant TGFb signalling in metabolic dysfunction (and in predisposition to T2DM), a proposal that can be explored in parallel studies of adipose tissue. The specific aims are to study (1) the stage related pattern of gene expression of TGFb ligands, their receptors and endogenous antagonists in preantral follicles from ovaries of women with PCOS compared with that in normal ovaries (using next generation sequencing and qPCR); (2) where and when proteins of TGFb ligands, receptors, antagonists, and Smads are expressed in polycystic compared with normal ovaries; (3) the effects of TGFb ligands and antagonists on early follicle development in cultured slices of human normal and PCOS ovarian cortex; (4) the effect of androgen treatment on expression of TGFb ligands, receptors, antagonists and Smads in mouse ovaries and (5) Do other key growth factors (IGFs, EGFs) in the ovary affect TGFb signalling pathways?

Despite the profound effects of polycystic ovary syndrome (PCOS) on ovarian and metabolic function, little is known about the underlying pathogenic mechanisms. Increased understanding of the processes by which local growth factors influence activation of follicle growth provides a basis for better-targeted therapies for both reproductive and metabolic effects of PCOS.

ACADEMIC IMPACT
Our principal aim is to understand the regulation of activation of follicle growth in PCOS. Our recent studies of expression of TGFbeta family members and their receptors in follicles from normal human ovaries indicate that this growth factor network also has a key role in early follicle development in women. With an established track record in top-ranking research in ovarian physiology, we are now in a position to make significant advances in understanding aberrant ovarian function in PCOS.
The potential impact on women's health includes (1) Improving understanding and treatment of infertility and menstrual disturbances in PCOS: The mechanism of ovulatory dysfunction in PCOS remains unclear and current methods of induction of ovulation are empirical and symptomatic and have remained essentially unchanged for decades. There is a clear need for more specific modes of management. It is therefore important to understand the abnormalities of follicle development that underlie anovulation in PCOS. (2) Improving understanding and treatment of metabolic disorders in PCOS: Women with PCOS are at high risk of developing diabetes. Metabolic dysfunction in PCOS is characterised by insulin resistance and abnormal adipocyte function. TGFbeta growth factors have been implicated in development of insulin resistance, adipose tissue dysfunction and diabetes; thus abnormalities of TGFbeta signalling may play a part both in reproductive and metabolic dysfunction. Studies of expression and action of TGFbeta signalling in ovary will inform our understanding of their role in abnormal metabolic function.

ECONOMIC AND SOCIETAL IMPACT
Charities: The outcome of the work proposed is relevant to human fertility in general and to PCOS in particular. SF is a member of the Medical Advisory Board of Infertility Network UK and is medical advisor to Verity, the PCOS patient support group. He contributes to the quarterly magazine of both charities and speaks at their regular meetings.
Education: An important aim is to motivate young people by providing information about, and promoting interest in biomedical research. Links with local schools have already been established; KH has spoken at Latymer Upper School in Hammersmith and is involved in a number of its science activities.
General Public: We wish to promote public engagement in science. SF has provided information to media about reproductive science and its impact on society, both directly to journalists and via the Imperial College Press Office or the Society for Endocrinology.

Planned activities that will contribute to realising these aims
Charities: The Genesis Research Trust (of which SF and KH are trustees) relies on a committed team of fund-raisers. KH and SF will continue to inform participants about Trust-funded research. SF will make regular contributions to the publications and meetings of Verity and The Infertility Network.
Education & General Public: KH and SF will identify and establish links with Biology Departments in local state schools in West London, with activities ranging from giving motivational talks on careers in science to seminars on specific research topics.