PCOS is widely recognized as an endocrine disorder with numerous metabolic consequences. Unfortunately, the metabolic consequences of PCOS are interwoven with the metabolic consequences of obesity and causal relationships are therefore hard to define (i.e. while PCOS is classified as an endocrine disorder, obesity will affect the endocrine parameters of this disorder). Insulin resistance is one of the metabolic diseases associated with PCOS, and is perhaps the most well documented symptom [8]. The clinical manifestation of insulin resistance at a patient level reflects the complexities described above. Approximately half the women with PCOS are obese [9] and therefore prone to the presentation of insulin resistance [8]. However, insulin resistance is also observed in women with PCOS of a normal body weight [10], together indicating that the hyperinsulinemia associated with this syndrome is not specific to the stressors associated with obesity. The relationship between glucose and insulin is also somewhat patient-specific and affected by age, race, and physical condition. Therefore, insulin resistance/glucose tolerance is a metabolic disease associated with PCOS that can manifest itself in more severe outcomes such as diabetes. Cardiovascular risk is increased in patients with PCOS [11-14] and hypertension [15] is the most easily recognized manifesta tion. This metabolic disorder is also associated with obesity and again, insulin resistance is a known cardiovascular risk factor [11]. Insulin affects myocardial and skeletal energy metabolism and physical activity improves the insulin sensitivity of skeletal muscle [10,16-18]. Downstream effects of exercise-dependent changes in insulin include better blood flow, more efficient glycogen synthase activity and glucose transportation [18]. Similar to the individuality of glucose/insulin tolerances, measures of cardiovascular function such as maximal O₂ consumption (VO_2max) are also subject to age, physical condition and other diseases with VO_2max positively correlated to both testosterone and insulin sensitivity [19].

The relationship between steroids, lipids and glucose metabolism is not easily defined. Interactions between these parameters are known and aberrations of one will affect the other as a metabolic consequence will modulate the endocrine environment. As such, many of the symptoms and features of PCOS play on one another to exacerbate the presentation of the others. The interconnection of the symptoms of PCOS has thus made it a large challenge to pinpoint the causal factor(s) as well as those that are secondary to that initial aberration.

PCOS is a very complex disorder with heterogeneity of clinical as well as endocrine features. To treat symptoms caused by the hyperandrogenemia of PCOS (hirsutism, acne, seborrhea and hair loss), antiandrogens such as flutamide and spironolactone are routinely administered [20-27]. Most of all, however, treatment of PCOS patients has been centered on inducing ovulation. Due to an inability to naturally ovulate, PCOS patients receive either pharmacological or surgical treatments to induce ovulation together with other assisted reproductive technology (ART) procedures [28-34]. The most commonly used procedures are to increase sensitivity to insulin, induce the gonadotropin surge, or lower circulating testosterone levels. For each of the symptoms, the following treatments are used.

While the initiating factor of PCOS has yet to be determined, we can evaluate the collective pool of known and suspected features of PCOS to better clarify how each symptom relates and what may be driving the symptoms as a whole. In Figure 2, we summarize the known extraovarian and intraovarian factors, and propose causal relationships among them and their effects on inducing hyperandrogenemia and polycystic ovary (arrows). In this particular 'hypothetical' diagram, theca cell hyperplasia, an established cause of PCOS, is proposed as a driving factor for the development of hyperandrogenemia and polycystic ovaries. Of the extraovarian factors, metabolic disorders (abnormal regulation of insulin and/or IGFs) and obesity may stimulate thecal hyperplasia, noting that approximately half of PCOS patients are obese [9]. Hypergonadism caused by dysregulation of LH secretion may also induce thecal hyperplasia, as LH stimulates theca cell proliferation [61-63]. Concurrently, intraovarian factors such as defects in steroidogenesis, regulation of the cell cycle, or ovulatory defects may result in thecal hyperplasia and hyperandrogenemia.

In regard to the mechanism of how hyperandrogenism may lead to ovulatory defect and therefore formation of follicular cyst in the ovary, it has been proposed that elevated testosterone disrupts the regulation of GnRH secretion [64]. In support of this, either GnRH agonists or LH treatment induces ovulation in PCOS patients. A few animal studies have shown that androgen suppresses progesterone receptor (PR) expression in the hypothalamus [65-67]. As progesterone down-regulates GnRH secretion by activating PR activity in the hypothalamus [68-70], androgen-mediated suppression of PR expression may ameliorate GnRH secretion, and therefore LH secretion (Fig. 3). This altered release of gonadotropins will lead to defects in follicle development, the development may arrest outright leading to failure to ovulate (anovulation) or be reduced leading to irregular ovulation (oligo-ovulation). The arrested follicle may thus form a cystic structure, leading to the polycystic ovarian phenotype and loss of fertility in PCOS patients.

Insulin resistance and obesity themselves are also known to negatively affect the secretion of pituitary hormones, and therefore follicular development [71-73]. It is not known whether altered steroidogenesis, pituitary hormone regulation or the defects in follicular development lead to an alteration of metabolism such as insulin resistance and obesity.

To identify the complex nature of this disease, suitable animal models are needed. Researchers during the past three decades have identified different animal models that mimic many of the features of PCOS in women (these are summarized in Table 1). These models have afforded valuable information into complex nature of PCOS. Currently, although a genetic component to PCOS has been identified, a specific "PCOS gene" has not, and therefore a specifically targeted gene deletion for PCOS as an animal model is not available. Therefore, the majority of PCOS models that are available to date rely upon external chemical treatments with steroids, steroid precursors or steroid receptor antagonist to achieve the pathology. Many of these models do not produce consistent results or cease to produce PCOS like symptoms once the treatment stops. Recently, Hill et al. [74] noted that their model for cardiovascular disease, the hypothalamic pro-opiomelanocortin (POMC) specific leptin-insulin double receptor knockout mouse exhibited some similar features to PCOS. Our recently developed theca specific Esr1 specific knockout mouse model reproduces the clinical pathologic features of PCOS in 100% of animals.

The unraveling of a biological pathway for PCOS and assigning etiologies to this syndrome requires the development of a novel animal model that consistently present PCOS phenotypes. We therefore launched a project to develop a mouse model of PCOS that is genetically modified and thus stably displays PCOS phenotypes. In doing so, we utilized the underlying concept of a vicious cycle [95-97], where the abnormalities are reinforced. As a way to initiate this cycle, we targeted the ovarian steroidogenic pathway to induce a preference to androgen synthesis and therefore create hyperandrogenemia.

The ovary is comprised of follicles at different stages of development. During follicular growth, the oocyte becomes surrounded by the granulosa cells, a basement membrane forms, and layers of theca cells develop, surrounding the follicle. The two cell layers act cooperatively in the production of androgens and estrogens. Theca cells produce androgens that traverse the basement membrane to the granulosa cells, where aromatase converts the androgens to estrogens. The estrogens in turn negatively feedback to the theca cells causing a decrease of androgen production. This negative feedback action of estrogen is mediated by estrogen receptor 1 (Esr1; ERα) in the theca cells, the prime site of Esr1 expression in the ovary. Upon activation by the binding of estrogen, Esr1 suppresses the expression of Cyp17, a critical enzyme that catalyzes a rate-limiting step in androgen production (Fig. 4), resulting in the decrease of androgen production. Therefore, deleting Esr1 gene would be a logical choice for achieving sustained high levels of androgens because loss of Esr1 will exclude negative regulation of Cyp17 gene expression by estrogen. This will ultimately result in increased production of androgens. The deletion of Esr1 gene has to be, however, limited to theca cells because Esr1 is expressed in so many different tissues/organs including all of the tissues of the hypothalamic-pituitary-ovary-uterus axis [98,99]. Otherwise, the Esr1 deletion in non-theca cells would cause other defects, making it difficult to isolate the Esr1 deletion effect in the theca cells.

This goal was achieved by selectively knocking out Esr1in the steroidogenic theca cells while keeping the gene intact elsewhere. In principle, this approach would result in the increased expression of Cyp17 gene that encodes a late-limiting enzyme (17apha-hydroxylase) in androgen synthesis pathway, because deletion of Esr1 will relieve Cyp17 transcription from the well-established suppression of this gene by Esr1. We achieved this aim by first generating a mouse line that expresses Cre recombinase under the regulation of the Cyp17 promoter (Cyp17^iCre) [100] and then breeding the mice successively with floxed Esr1 (Esr1^flox) mice [89]. The offspring with the genotype Esr1^flox/flox Cyp17^iCre, as was expected, expresses higher level of Cyp17 mRNA expression in the ovary and maintains higher serum testosterone levels (1.5-2 folds over wild type littermates) (ref). The Esr1^flox/flox Cyp17^iCre mice display other phenotypic symptoms of PCOS patients: (i) irregular estrous cycles, (ii) an age-dependent decrease in ovulatory capacity and fertility defect and (iii) arrest of follicular development at the early antral stage (Figs. 5, 6). This novel animal model generated from the proof-of-principal experiment should bring a new opportunity for the study of PCOS as well as for the study of hyperandrogenemia itself.

In contrast to the seriousness of the PCOS in women's health, the pathogenesis of this disease is poorly understood. In recent years, efforts by multiple laboratories have led to the development of novel animal models for this complicated disorder. In the next few years, use of these animal models and those to be newly developed will undoubtedly accelerate our understanding on PCOS pathogenesis, and possibly lead to the development of new therapeutic protocols for treating and preventing PCOS in women.