Center for Research on Reproduction and Womens Health
(J.F.S.) University of Pennsylvania Medical Center
Philadelphia, Pennsylvania 19104
Division of Womens Health
(A.D.) Brigham and Womens Hospital and Harvard Medical School
Boston, Massachusetts 02115
INTRODUCTION
Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of women of reproductive age, affecting from 510% of women in this age group (1, 2). The symptoms of PCOS include hirsutism and acne, evidence of excessive androgen production, menstrual disturbances and, consequently, anovulation and infertility. Symptoms often appear soon after menarche, and biochemical abnormalities may be present at the time of adrenarche. A common feature of PCOS is follicular maturation arrest resulting in the accumulation of small subcortical follicle cysts and increased ovarian stromal volume, yielding a characteristic ultrasound image and a basis for the most commonly used appellation for the syndrome. Insulin resistance and pancreatic ß-cell dysfunction, both independent of obesity and unrelated to the actions of androgens on insulin dynamics, are strongly associated with PCOS (3, 4). Physicians have only recently come to appreciate the potential long-term consequences of PCOS, which include type 2 diabetes mellitus, hypertension, and cardiovascular disease (5). Moreover, PCOS women who become pregnant have an increased risk of preeclampsia (6). In addition, endometrial hyperplasia and endometrial cancer also occur frequently in women with PCOS who do not receive treatment. Therefore, PCOS is a major womens health issue with ramifications well beyond the reproductive endocrine abnormalities that usually bring women with PCOS to medical attention. Indeed, significant health risks may remain after the reproductive dysfunction has been treated by hormonal therapy or disappear as a result of reproductive aging (i.e. the menopause).
Since PCOS was identified as a gynecological syndrome more than 60 yr ago by Stein and Leventhal (7), the diagnosis and even the name of the syndrome have been topics of continuous debate. Despite the recommendations of conferences, clinicians still do not use uniform criteria to make the diagnosis of PCOS (8). The resulting heterogeneity of phenotypes included under this umbrella confounds the literature on pathophysiology and outcomes of therapeutic interventions. For the purpose of this discussion, we define PCOS as a syndrome of hyperandrogenism and anovulation/oligoovulation in the absence of other hypothalamic, pituitary, ovarian, or adrenal disease. Unfortunately, the quest to bring some order to the field has not been helped by the absence of relevant animal models. Although cystic ovaries and anovulation can be produced in laboratory and domestic animal species with various pharmacological manipulations or transgenic approaches, none of these animal models faithfully replicate the human disorder.
Theories of the pathophysiology of PCOS have implicated primary defects in hypothalamic-pituitary function, ovarian activity, and insulin action; but alas, none of these can comfortably accommodate the multiple abnormalities associated with PCOS. This state of relative confusion is not an indictment of investigators working on PCOS, but rather a reflection of the complexity of the disorder. The existing theories, however, do direct attention to specific hormones and/or processes including LH, thecal and adrenal androgen biosynthesis, and insulin/insulin-like growth factor action.
Observations from therapeutic interventions provide validation for the notions that PCOS is associated with abnormal gonadotropin secretion, defects in ovarian steroidogenesis, and insulin resistance. The symptoms of hyperandrogenism can, in most cases, be treated by suppression of LH secretion, suggesting that LH has a permissive role in augmenting ovarian androgen production in PCOS. Removal of wedges of ovarian tissue or destruction of cystic follicles causes at least a transitory reduction in androgen levels in many women with PCOS, which may restore ovulatory function. Stimulation of follicular maturation either through the use of clomiphene citrate, which augments endogenous FSH secretion, or the administration of exogenous FSH can drive follicles past the PCOS arrest, resulting in full maturation and ovulation. Reducing insulin levels in PCOS women causes a decline in circulating androgen concentrations (9). Moreover, insulin sensitizing agents, such as metformin hydrochloride (Bristol-Meyers Squibb, Princeton, NJ) and troglitazone (Park-Davis Morris Plains, NJ), reduce circulating androgen levels and restore normal menstrual cycles in amenorrheic women (10, 11, 12). They also increase the response to ovulation-inducing agents (13). The beneficial effects of these drugs are most certainly a result of reduced insulin levels and secondarily, in the case of troglitazone, to direct inhibitory effects on steroidogenic enzymes (14).
IT ISNT SIMPLY LH
LH is characteristically elevated in women with PCOS, the result of increased pulse frequency and amplitude of LH secretion (15). Because LH stimulates thecal steroidogenesis, a primary abnormality in gonadotropin secretion has been an appealing explanation for the symptoms of hyperandrogenism associated with PCOS. Indeed, suppression of LH secretion by GnRH superagonists results in a reduction of ovarian androgen production in PCOS. However, chronic elevations of LH alone do not appear to be capable of sustaining increased ovarian androgen synthesis. Women with mutant FSH ß-subunit genes or inactivating mutations of the FSH receptor, whose ovaries contain only primordial and primary follicles, are not hyperandrogenic despite persistently elevated LH levels (16). Activating mutations of the LH receptor that cause Leydig cell hyperplasia and precocious puberty in males fail to produce symptoms of hyperandrogenemia and amenorrhea in women (17). These clinical observations suggest that 1) LH is permissive for the excessive ovarian androgen secretion in PCOS; 2) some follicular development is required for the theca to gain steroidogenic competence; and 3) in normal follicles there is a check on LH-driven thecal cell steroidogenesis that prevents excessive androgen formation. Thus, there is likely to be some factor in addition to LH that leads to increased ovarian androgen formation in PCOS. This additional factor may also affect adrenal androgen production, which is elevated in a number of women with PCOS. Finally, because ovarian wedge resection and follicle drilling, as well as treatments that reduce insulin levels, diminish androgen production and can restore ovulatory cycles, it is likely that the alterations in LH secretory dynamics characteristic of PCOS are set in motion by the disturbance in ovarian function rather than the converse situation.
PRIMARY DEFECTS IN STEROIDOGENESIS?
It has been postulated that PCOS is caused by an abnormality in
ovarian and adrenal androgen biosynthesis localized to the
17-hydroxylase/17,20-desmolase reaction, catalyzed by a single
enzyme, CYP17 (18, 19). Increased in vivo flux through CYP17
has been suggested from results of 17
-hydroxyprogesterone
measurements in PCOS women challenged with a GnRH agonist or human
(h)CG after GnRH agonist-induced pituitary suppression. Because the
relative ratio of 17
-hydroxylase to lyase activity of CYP17
represents a key control locus for androgen biosynthesis, a shift in
this ratio could account for aberrations in ovarian and adrenal
androgen production. Therefore, it is interesting that cleavage of
phosphate from serine residues on CYP17 by alkaline phosphatase
treatment reduces lyase activity without affecting 17
-hydroxylase
activity (20). This observation raises the possibility that
posttranslational modification of CYP17 influences androgen synthesis.
However, the serine residues in CYP17 that are phosphorylated and the
kinase that mediates this phosphorylation remain to be identified, so
that it has not been possible to reverse the in vitro
effects of alkaline phosphatase treatment on lyase activity or
manipulate lyase activity in intact cells through a phosphorylation
mechanism.
The concept of a primary defect in androgen biosynthesis in PCOS is
supported by in vitro studies on enzymatically dispersed
human theca cells (21). Increased basal and LH-stimulated
androstenedione production was found with theca cells from PCOS women
compared with thecal cells from normal ovaries. Progesterone production
by PCOS thecal cells was also increased. While these findings seem to
support the idea of an intrinsic abnormality in thecal androgen
synthesis, the possibility that the in vitro behavior of the
dispersed cells reflected their prior in vivo milieu could
not be discounted. Indeed, the in vivo studies suggesting
augmented CYP17 activity mentioned above predict that freshly isolated
thecal cells would be capable of increased androgen production. More
recently, McAllister and colleagues (22) studied long-term cultures of
replicating thecal cells originally isolated from size-matched
follicles collected from PCOS and normal women. In this study, the PCOS
theca cells secreted significantly greater amounts of progesterone, 17
-hydroxyprogesterone, and testosterone in response to forskolin
stimulation than theca cells from normal ovaries, despite the fact that
both the PCOS and control cells had been maintained in LH-free culture
media through multiple passages. McAllister and co-workers also found
increased activities of 3ß-hydroxysteroid dehydrogenase and
17ß-hydroxysteroid dehydrogenase in addition to 17
-hydroxylase and
17,20-desmolase activities in PCOS theca cells and increased
forskolin-stimulated CYP11A and CYP17 mRNA expression. The clear
message from these in vitro studies is that PCOS theca cells
display enhanced steroidogenic potential that encompasses more than
CYP17. This in vitro biochemical phenotype may be the result
of a stable metabolic imprint obtained in vivo or an
intrinsic genetic variation.
Abnormalities in granulosa cell function are also present in PCOS (23). Follicular atresia was originally thought to be a significant feature of PCOS, possibly due to the increased intraovarian concentrations of androgens. It is now evident that the small follicles that accumulate in the ovaries contain healthy granulosa cells, albeit reduced in number and in capacity to convert the abundant thecal androgens into estradiol. Nevertheless, the granulosa cells in PCOS follicles respond in vivo to increased endogenous FSH levels or exogenous FSH. In addition, when studied in vitro, they are highly responsive to gonadotropins and produce large amounts of steroid hormone. Indeed, PCOS granulosa cells appear to gain reactivity to LH at an earlier stage of follicular development than granulosa cells from normal follicles (24).
THE INSULIN CONNECTION
Insulin and insulin-like growth factors are known to increase thecal androgen synthesis and to amplify the actions of LH (25). They also augment FSH actions on granulosa cells. The level at which insulin and insulin-like growth factors (IGFs) act on ovarian cells to promote steroidogenesis has not been fully elucidated, but it appears to encompass both transcriptional and posttranscriptional actions including increases in the stability of specific mRNAs (26, 27). However, specific insulin or IGF-responsive elements have yet to be identified in steroidogenic enzyme genes. Insulin is also known to rapidly increase mRNA translation, but there is no information available as to whether insulin or IGFs increase translation of mRNAs encoding steroidogenic proteins. Given the intense interest in elucidating the intracellular signaling cascades initiated by the binding of insulin and IGF to their receptors, it is remarkable that we still know very little about the signaling pathways activated in ovarian cells.
Severe insulin resistance caused by mutations that interfere with insulin receptor binding or signaling, as well as autoantibodies to the insulin receptor, is associated with marked hyperandrogenemia of ovarian origin (28). The impressive hyperinsulinemia in these states is thought to lead to activation of ovarian IGF type I receptors, and consequently increased androgen production as a result of synergistic interactions between IGF and LH action. As noted above, insulin resistance is prevalent in both obese and thin women with PCOS. However, this insulin resistance rarely causes increases in insulin concentrations to those found in individuals with insulin receptor mutations (1, 4). Thus, it is unlikely that the spill-over activation of IGF type I receptors accounts for the increased ovarian androgen synthesis in PCOS.
If insulin does have a role in promoting thecal androgen production, how can it do so in the face of insulin resistance? This apparent paradox may be explained by the following: 1) insulin and IGFs have multiple actions; 2) they signal through multiple intracellular pathways; and 3) insulin can regulate ovarian steroidogenesis through its cognate receptor (29, 30). Insulins metabolic and mitogenic actions are known to be mediated through distinct postreceptor signaling pathways (31 31A ). Thus, selective insulin resistance can be produced experimentally, leaving mitogenic actions intact (32). Therefore, a defect in insulin action in muscle leading to reduced glucose disposal might not disrupt or could even enhance the steroidogenic effects on theca cells and suppress sex hormone-binding globulin production by hepatocytes, leading to a hyperandrogenemic state. This hypothesis of tissue-specific alterations in insulin action is supported by several observations. Insulin can modulate gonadotropin-stimulated steroidogenesis by PCOS granulosa cells in vitro despite the presence of insulin resistance in vivo (29, 30). Evidence for insulin-stimulated thecal androgen biosynthesis via a signal transduction mechanism involving inositolglycan mediators, a signaling mechanism that is distinct from the known pathway regulating GLUT-4 translocation, has been presented (33). Finally, selective defects in skin fibroblasts from PCOS women that result in metabolic, but not mitogenic, alterations in insulin action have been documented (34).
The mechanism of the insulin resistance in PCOS is not yet known with certainty. Several studies have failed to identify mutations in exons encoding the insulin receptor in PCOS women (35, 36), effectively ruling out genetic variation in the insulin receptor as the explanation for insulin resistance in the majority of PCOS cases. There is, however, evidence for a stable phenotypic abnormality in insulin receptor phosphorylation in cells from PCOS women. Dunaif et al. (37) described increased insulin-dependent serine phosphorylation of the insulin receptor ß-subunit in skin fibroblast cultures from 50% of the women with PCOS studied, compared with fibroblasts from controls (37). A similar excessive serine phosphorylation of the receptor ß-subunit was found in skeletal muscle. The serine-phosphorylated insulin receptor had reduced ability to tyrosine phosphorylate an artificial substrate, suggesting that insulin receptor serine phosphorylation may impair signal transduction, accounting for a postbinding defect in insulin action. This alteration in insulin signaling could possibly be due to a variant serine kinase or activation of a kinase as a result of an autocrine factor. The latter idea is consistent with the suggestion of Ciaraldi et al. (38) that insulin resistance in PCOS is caused by abnormalities in adenosine modulation of insulin action. The model of insulin signaling abnormalities resulting from serine phosphorylation of the insulin receptor compliments the suggestion that serine phosphorylation of CYP17 increases lyase activity, potentially linking variations in insulin action and androgen biosynthesis to a common mechanism.
IS IT ALL IN THE FAMILY?
There is substantial interest in the genetics of PCOS, an enthusiasm engendered by the hope that the linkage of genes to PCOS will shed much needed light on the underlying disease process and permit secure ascertainment of PCOS for therapeutic trials and outcomes research. The evidence that PCOS has a genetic component includes reports of familial clustering of PCOS and hyperandrogenemia (1, 39, 40, 41). Although none of the published family studies permit a valid segregation analysis because the male counterpart of PCOS is unknown (see below) and parental phenotypes are difficult to ascertain, the existing observations are consistent with a high prevalence of PCOS and hyperandrogenemia in female first-degree relatives, possibly consistent with an autosomal dominant pattern of inheritance. Even though these studies fail to present a completely convincing argument for a mode of inheritance, they do form a basis for pursuing linkage studies using affected sib pair analysis or related methods for establishing linkage of genes to disease susceptibility when the mode of inheritance, penetrance, and prevalence of the disorder are unknown.
Preliminary information from linkage and association studies has been reported by several groups, but it is evident that the study populations have been far too small to yield any definitive results (42, 43, 44, 45). The problems arising from insufficient study populations are exemplified by an early report suggesting an association between a polymorphism in the CYP17 promoter and PCOS, a conclusion that was subsequently retracted when a larger cohort of subjects was studied and when other investigators examined this polymorphism and found no significant association with PCOS (42). Further studies with some additional families led to the suggestion of linkage between elevated androgen levels and the CYP11A gene on 15q24, and association with a polymorphism in the CYP11A promoter (43). Linkage of polycystic ovaries with the insulin gene and association with a VNTR 5' to the insulin gene located at 11p15.5 have also been proposed (44). The latter assertion has not been confirmed by others who studied an even larger number of PCOS sib pairs, but linkage to other loci was suggested (45). These discrepancies may reflect erroneous conclusions regarding association or linkage based on inadequate sample sizes and flawed statistical analysis (1, 46). Alternatively, the discrepancies may be due to differences in the phenotypes used to assign affected status among these studies.
ONLY A WOMENS HEALTH PROBLEM?
Is PCOS a health problem restricted to women? If PCOS does indeed have a genetic component, then males must harbor the susceptibility genes as well. But what is the male phenotype of PCOS? Premature male pattern balding and increased pilosity have been proposed to be male symptoms of PCOS (40, 41). This idea is rooted in the notion that PCOS is a disorder of androgen production. However, premature male-pattern balding has not been found to cluster with PCOS in all family studies. Moreover, evidence for hyperandrogenemia in fathers or brothers of PCOS women has not been offered in the literature. Perhaps insulin resistance would be a better phenotype to pursue in males from PCOS families. It is noteworthy that insulin resistance may be associated with lower total serum testosterone levels as a result of reductions in sex hormone-binding globulin production by the liver secondary to hyperinsulinemia, which on the surface is contrary to the biochemical phenotype that might have been anticipated to be found in males in PCOS families. The issue of a male phenotype in PCOS families is clearly deserving of intensive scrutiny since it may reveal the same potential long-term health problems mentioned above. The secure identification of a male phenotype would also facilitate genetic linkage studies.
SOME PREDICTIONS
Since there is no shortage of hypotheses about the genesis of PCOS, there seems to be no harm in offering another, which is less a new formulation than a set of predictions, a task we fancy as these musings have been collected on and around Groundhog Day. Assembling the extensive literature on PCOS pathophysiology into a working model of how genetic factors could produce disease susceptibility leads us to conclude that the most likely candidate genes encode proteins involved in signal transduction, particularly proteins that modify insulin and gonadotropin action or the action of molecules downstream from the insulin and gonadotropin receptors. Moreover, we believe that the candidate genes most likely encode proteins that are widely expressed to account for metabolic alterations in multiple organs including the ovaries, pituitary, adrenals, pancreas, muscle, and fat. For example, dysregulation in the action of an autocrine/paracrine modulator of insulin and gonadotropin action could yield phenotypes in multiple organ systems in vivo and stable biochemical alterations in vitro. The susceptibility genes could already be known or be among the 70,000 some uncharacterized genes in the human genome. But how many genes are involved and what are their interactions? Maybe we should ask Punxsutawney Phil who, given the current state of knowledge, may be as qualified as any endocrinologist to make these pronouncements. Even if the groundhog does not have the answer today, we remain optimistic that it will come through perseverance in molecular genetic studies. Although the search for disease genes using genome-wide screening approaches is arduous and has yet to be consistently successful when applied to common and complex disorders, this approach holds the promise of ultimately shedding definitive light on the pathophysiology of PCOS.
FOOTNOTES
Address requests for reprints to: Jerome F. Strauss, III, M.D., Ph.D., 778 Clinical Research Building, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104. E-mail: jfs3{at}mail.med.upenn.edu
Research in the authors laboratories has been supported by the National Cooperative Program in Infertility Research (U54 HD-34449), R01 DK-40609 (A.D.), and P01 HD-06274 (J.F.S.).
Received for publication February 16, 1999. Revision received February 25, 1999. Accepted for publication March 1, 1999.
REFERENCES