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From: Contemporary Endocrinology: Androgen Excess Disorders in Women:
Polycystic Ovary Syndrome and Other Disorders, Second Edition Edited by: R. Azziz et al. © Humana Press Inc., Totowa, NJ
18
Ovarian Steroidogenic Abnormalities in the Polycystic Ovary Syndrome
Denis A. Magoffin
SUMMARY
Excess androgen biosynthesis is a diagnostic feature of polycystic ovary syndrome. The excess circulating androstenedione and testosterone is produced primarily by the ovary. The ovarian theca cells, the site of de novo androgen biosynthesis, are increased in number in polycystic ovaries, and they have increased steroidogenic capacity. The increase in steroidogenic capacity is caused by overexpression of steroidogenic enzymes because of increased transcription and mRNA stability. Primary factors in the hyperstimulation of thecal androgen production appear to be increased luteinizing hormone (LH) concentrations in some women and elevated insu- lin concentrations secondary to insulin resistance. Additional contributions may be made by other intraovarian factors that can augment the stimulatory effects of LH on thecal androgen biosynthesis.
The granulosa cells in arrested follicles in polycystic ovaries fail to increase the expression of aromatase, causing markedly decreased estrogen secretion. They also express higher concentrations of 5D-reductase enzymes, leading to the production of 5D-androstane-3,17-dione, a competitive inhibitor of aromatase activity. The granu- losa cells prematurely express the cholesterol side-chain cleavage enzyme and LH receptors, hence they are overresponsive to LH and produce increased amounts of progesterone compared to granulosa cells from fol- licles at a similar developmental stage in regularly cycling control women. Thus, polycystic ovaries produce increased concentrations of androgens and progesterone, decreased concentrations of estrogens, and abnor- mally high concentrations of 5D-reduced androgens, compared to normal ovaries.
Key Words: Ovary; theca cell; granulosa cell; steroidogenesis; androgens; estrogens; progesterone; poly- cystic ovary syndrome.
1. INTRODUCTION
The polycystic ovary syndrome (PCOS) is a heterogeneous disorder in which abnormalities of steroidogenesis are inherent to the etiology. Excess circulating androgen concentrations are a cardi- nal feature of PCOS that is central to the diagnosis (1). Evidence supporting a central role for hyperandrogenism as a cause of PCOS includes the development of polycystic ovaries in female-to- male transsexuals treated with high concentrations of testosterone and in girls with excess adrenal androgen secretion because of congenital adrenal hyperplasia, and the observation that PCOS can be reversed in some women by reducing circulating androgen concentrations using insulin-sensitizing therapy. Therefore, understanding ovarian steroidogenic abnormalities in PCOS is central to under- standing the etiology of the disorder.
2. BACKGROUND
Ovarian steroidogenesis in PCOS affects androgen and estrogen—and possibly progesterone—
production.
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2.1. Abnormal Androgen Production
2.1.1. Contribution of the Ovary to Abnormal Androgen Production in PCOS
The serum concentrations of androstenedione and testosterone are elevated in women with PCOS.
In up to 50% of hyperandrogenic women, dehydroepiandrosterone sulfate is also increased (2).
Although it is clear that the ovary and the adrenal gland are the principal sources of circulating androgens in women, it is important to understand the relative contributions of each gland. Direct measurement of androgens in selectively catheterized ovarian and adrenal veins and the use of ova- rian and adrenal stimulation and suppression have not unambiguously defined the relative contribu- tions of each gland to elevated circulating androgens. However, selective suppression of ovarian androgen production with long-acting gonadotropin-releasing hormone agonists that do not affect adrenal androgen production indicate that the ovary is the principal source of the excessive andros- tenedione and testosterone in hyperandrogenic women (3). In hyperandrogenic women in whom con- genital adrenal hyperplasia involving 3E-hydroxysteroid dehydrogenase (3E-HSD), 21-hydroxylase, or 11-hydroxylase deficiency has been ruled out, up to 50% have both ovarian and adrenal hyperandrogenism, and the remainder have exclusively ovarian hyperandrogenism. These data lead to the conclusions that the ovary is the principal source of excessive androstenedione and testoster- one in hyperandrogenic women and that the ovary plays a major, although not exclusive, role in female hyperandrogenism.
2.1.2. Excessive Androgen Production by Theca Cells From Polycystic Ovaries
In the ovarian follicle steroidogenesis is accomplished through the cooperation of the theca and granulosa cells (Fig. 1). The theca cells are the exclusive source of androstenedione in women. The 17E-hydroxysteroid dehydrogenase enzyme is predominantly expressed in the granulosa cells; hence much of the testosterone is produced in the granulosa cells. Regardless of the site of metabolism of androstenedione to testosterone, the de novo production of androgens in the ovaries is exclusive to the theca cells.
Clinically, women with PCOS respond to human chorionic gonadotropin (hCG) challenge with increased testosterone production compared with control women. The increased responsiveness of the polycystic ovary to gonadotropin stimulation implies an increased steroidogenic capacity of the theca interna. In vitro studies confirm that theca cells from polycystic ovaries produce more andro- gen both in the unstimulated and in the gonadotropin-stimulated state (4). Thus, it is clear that the polycystic ovary has enhanced capacity to secrete androgens. Two principal factors influence the total amount of androgen secreted by the ovary: the total number of theca cells in the ovary and the steroidogenic capacity of each of the theca cells.
2.1.3. Excessive Theca-Cell Proliferation in Polycystic Ovaries
In the polycystic, unlike the normal, ovary there is an accumulation of small antral follicles 3–7 mm in diameter forming the classic “string-of-pearls” morphology. Thus, the ovaries in women with PCOS contain more antral follicles than normal. Many of the follicles in polycystic ovaries demon- strate a hypertrophied theca interna containing many more layers of differentiated steroidogenic cells than the three to five layers in the normal theca interna (Fig. 2). Because of these factors, the polycys- tic ovary contains more steroidogenic cells in the theca interna than normal, which is consistent with the excessive androgen secretion in PCOS. In contrast, the granulosa cell layers are underdeveloped, and the polycystic ovary contains fewer potential estrogen-producing cells than would be expected for the stage of follicle development.
Little is known about the factors regulating human theca-cell proliferation. Most of the data origi-
nate from studies in animals. Chronic exposure of theca cells to luteinizing hormone (LH) (or hCG)
leads to a marked increase in the number of cells in the theca interna. There is also evidence that
intraovarian growth factors can support theca-cell proliferation. Among these are activin, epidermal
growth factor or transforming growth factor (TGF)-D, insulin and insulin-like growth factor (IGF)-1,
Fig. 1. Steroidogenic capabilities of theca and granulosa cells. Under the influence of luteinizing hormone (LH) theca cells can produce androstenedione de novo. The theca cells express little 17E-hydroxysteroid dehy- drogenase and produce very little testosterone. The androstenedione is metabolized in the granulosa cells to estradiol in normal ovaries, but in polycystic ovaries the androstenedione is metabolized to testosterone, 5D- dihydrotestosterone, and 5D-androstane-3,17-dione. Gs, Stimulatory guanosine triphosphate-binding protein;
AC, adenyl cyclase; cAMP, cyclic adenosine monophosphate; CYP11A, cholesterol side-chain cleavage cyto- chrome P450; CYP17, 17D-hydroxylase/C17-20 lyase cytochrome P450; 3E-HSD, 3E-hydroxysteroid dehydro- genase; DHEA, dehydroepiandrosterone; FSH, follicle-stimulating hormone; CYP19, aromatase cytochrome P450; 17E-HSD, 17E-hydroxysteroid dehydrogenase; 5D-Red, 5D-reductase; 5D-A4, 5D-androstane-3,17-dione;
5D-DHT, 5D-dihydrotestosterone.
stem cell factor or kit ligand, and tumor necrosis factor (TNF)-D (5). There is evidence that insulin and IGF-1 can stimulate human theca-cell proliferation in vitro, but the extent to which these findings apply to normal follicle development and/or PCOS remains uncertain.
2.1.4. Theca-Cell Overexpression of Steroidogenic Enzyme mRNAs in Polycystic Ovaries
In addition to the increase in the number of theca cells in polycystic ovaries, overexpression of
steroidogenic enzymes also contributes to ovarian hyperandrogenism. The mRNAs for LH receptor,
steroidogenesis acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme P450scc
(CYP11A), and the 17D-hydroxlase/C
17–20lyase enzyme P450scc (CYP17) are overexpressed com-
pared to theca cells from regularly cycling women (6). The relative overexpression of theca-cell
206 Magoffin
mRNAs persists, and there continues to be increased 17D-hydroxylase activity when theca cells are cultured in vitro (7), indicating that the signals regulating theca-cell steroidogenesis are increased in PCOS. The increased rate of CYP17 gene transcription in theca cells from polycystic ovaries demon- strates that both thecal differentiation and steroidogenesis are hyperstimulated in PCOS. The height- ened state of thecal differentiation appears to involve not only increased transcription of steroidogenic enzyme genes, but also increased mRNA stability, indicating multifactorial abnormalities in theca- cell regulation.
2.1.5. Abnormal Androgen Metabolism in Polycystic Ovaries
The normal ovary metabolizes the vast majority of the androstenedione produced by the theca cells to estrone and estradiol in the granulosa cells. The arrested small antral follicles in polycystic ovaries do not express the high concentrations of aromatase that are present in larger dominant fol- licles (8), hence there is an accumulation of androstenedione. Small antral follicles from normal and polycystic ovaries express both isoforms of 5D-reductase (9). At approximately the time when aromatase begins to be expressed in the granulosa cells, 5D-reductase expression declines to unde- tectable levels. In PCOS, follicle development is arrested at this stage. Hence, there is increased expression of 5D-reductase activity and the concentration of 5D-androstane-3, 17-dione is elevated in both follicular fluid and serum in PCOS (10). Although the concentration of 5D-androstane-3,17- dione in normal developing follicles has little effect, the concentrations in the follicular fluid of polycystic ovaries is high enough to suppress aromatase activity by a competitive mechanism (10), potentially contributing to the failure to develop dominant follicles.
2.1.6. Abnormalities in the Regulation of Androgen Production
Within the ovary there are many regulatory molecules that have the potential to contribute to hyperstimulation of theca-cell differentiation and androgen production in PCOS.
2.1.6.1. G
ONADOTROPINSThe literature is replete with in vivo and in vitro studies demonstrating that LH is the principal
regulator of theca-cell differentiation and function. The actions of LH are mediated through activa-
tion of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway in-
volving activation of either type I or type II PKA. LH stimulates the expression of CYP11A, 3E-HSD,
and CYP17 mRNAs and translation of the mRNAs into functional proteins. Although LH can stimu-
Fig. 2. Ovarian follicle cells in normal and polycystic ovaries. (A) Section of follicle wall from a normal ovary containing approximately three layers of healthy granulosa cells (GC) and three to four layers of theca cells (TC). (B) Section of a follicle wall from a polycystic ovary. The GC are fewer in number and loosely organized. The theca interna contains many more layers of highly differentiated theca cells.late inositol phosphate formation in granulosa and luteal cells, there is no evidence that inositol phosphate signaling is involved in mediating the actions of LH in theca cells. Direct activation of PKC with ligands such as angiotensin II sensitizes rat theca cells to LH, and depletion of PKC with long-term treatment with phorbol ester inhibits LH stimulation of thecal steroidogenesis, suggesting that protein kinase C (PKC) may play a role in modulating the sensitivity of theca cells to LH. The role of PKC in ovarian hyperandrogenism has yet to be explored. LH stimulates marked increases in progesterone and androgen production in differentiated theca cells from all mammalian species, includ- ing rats, mice, sheep, swine, cows, horses, nonhuman primates, and women. Thus, LH is a key regulator that must be considered when attempting to understand the genesis of ovarian hyperandrogenism.
2.1.6.2. I
NTRAOVARIANG
ROWTHF
ACTORSLH is clearly not the only regulator that can modulate the steroidogenic capacity of theca cells.
There are a host of stimulatory and inhibitory factors within the ovary with the potential to play a role in PCOS. Of these, only a few have been investigated with respect to their role in PCOS.
2.1.6.2.1. The Transforming Growth Factor Family
The TGF-E family of proteins includes TGF-E inhibin, activin, bone morphogenetic protein (BMP)-4, and growth differentiation factor (GDF)-9. The role of these molecules, and related pro- teins such as follistatin, have been studied with respect to PCOS. Both TGF-E1 and TGF-E2 are secreted in the human ovary, with TGF-E1 being present in both theca and granulosa cells and TGF- E2 being localized exclusively in theca cells. All of the inhibin subunits (D, E
A, and E
B) are expressed in human follicles, indicating that various inhibin isoforms are produced, but there is no evidence that follistatin is produced in the ovary. GDF-9 is produced exclusively by the oocyte.
TGF-E inhibits StAR protein expression in human theca tumor cells in vitro and blocks LH-depen- dent androgen production in rat theca cells through a direct noncompetitive inhibition of CYP17 activity (11). Inhibin stimulates, whereas activin inhibits thecal CYP17 mRNA expression and andro- gen production (12). GDF-9 has been reported to promote the proliferation of theca cells and inhibit androgen production (13). BMP-4 inhibits androgen production and CYP17 mRNA expression in human theca tumor cells (14). Thus, the evidence is consistent with a potential role for members of the TGF-E family in the genesis of steroidogenic abnormalities in PCOS.
There appears to be consensus that circulating inhibin B concentrations are not abnormal in women with PCOS in aggregate, but lean women with PCOS appear to have increased circulating inhibin B concentrations in contrast to obese women with PCOS (15). This observation does not appear to be relevant to PCOS because there is a negative effect of obesity on inhibin B concentrations indepen- dent of PCOS per se (16). On the other hand, the pulsatility of inhibin B secretion in regularly cycling women is not present in PCOS. It is unclear whether there are differences in circulating activin A concentrations in PCOS. Activin concentrations were found to be similar between blood and follicu- lar fluid. Neither inhibin B nor activin A were altered in follicles from PCOS, but there was a decrease in inhibin A in PCOS follicles compared to similar sized follicles in regularly cycling women (17). Follistatin in the circulation is increased in PCOS whether the women were lean or obese (18), but the alterations in circulating concentrations do not occur in the follicular fluid, raising doubts as to the role of follistatin in steroidogenic abnormalities in PCOS. Taken together, they do not support a major role for inhibin and activin in causing ovarian hyperandrogenism.
It has been reported that GDF-9 is decreased in polycystic ovaries (19), but the significance of this finding is uncertain. Considering that GDF-9 appears to be essential for the formation of the theca interna, a decrease in GDF-9 would be inconsistent with the theca hypertrophy observed in PCOS.
On the other hand, a decrease in GDF-9 would be in agreement with the increased androgen produc-
tion. Thus, the possibility exists that stage-specific alterations in GDF-9 secretion may contribute to
altered steroidogenesis. Further research needs to be performed before a role for GDF-9 can be estab-
lished in PCOS.
208 Magoffin
2.1.6.2.2. Transforming Growth Factor-D
TGF-D is an inhibitor of thecal androgen biosynthesis that has been implicated in PCOS. TGF-D has been localized to both theca and granulosa cells in developing human follicles, but there are no differences between TGF-D concentrations in follicular fluid and polycystic ovaries, and the produc- tion of TGF-D in vitro is comparable between tissues from polycystic ovaries and control ovaries (20). The concentrations of TGF-D in follicular fluid vary considerably, but they are always less than 1 ng/mL. The available evidence does not support a role for TGF-D in PCOS.
2.1.6.2.3. Tumor Necrosis Factor-D
The ovary, and the theca interna in particular, contains a population of resident macrophages that play an important role in a variety of ovarian functions. TNF-D is a cytokine with potent inhibitory effects on LH-dependent androgen production by theca cells. TNF-D is not present in theca cells, and it is uncertain whether granulosa cells synthesize TNF-D, but TNF-D is produced by oocytes and resident ovarian macrophages. Although the concentration of TNF-D in serum increases with increas- ing obesity in both control and women with PCOS (21), women with PCOS have increased serum TNF-D concentrations compared to weight-matched controls. In contrast, there are no differences in follicular fluid TNF-D concentrations between women with PCOS and regularly cycling controls (22). TNF-D secretion by human ovarian tissues in vitro was undetectable (22). Thus, the evidence does not support a role for intraovarian effects of TNF-D in the genesis of ovarian hyperandrogenism.
2.1.6.3. A
DIPOCYTOKINES2.1.6.3.1. Leptin
Obesity is commonly associated with PCOS and ovarian hyperandrogenism. The amount of body fat in humans is strongly correlated with circulating leptin concentrations, raising the question of whether leptin might play a role in abnormal steroidogenesis. The follicular fluid concentration of leptin is equivalent to the plasma concentration, but there is less leptin binding in the follicular fluid than in plasma. The circulating concentration of leptin in women with PCOS is equivalent to the concentration in control women when adjusted for obesity (23), demonstrating that there are no abnormalities of leptin secretion associated with PCOS. There do not appear to be direct effects of leptin alone on ovarian steroidogenesis, but leptin inhibits the stimulatory effects of IGF-1 on thecal androstenedione production and granulosa estrogen production with no effects on progesterone syn- thesis (24). This combination of effects, taken together with the lack of alteration of leptin concentra- tions in PCOS, indicates that leptin does not play a significant role in the steroidogenic abnormalities in PCOS.
2.1.6.3.2. Resistin
Resistin is a recently described protein secreted by cells in the adipose tissue. In mice, treatment with recombinant resistin caused impaired insulin action and glucose intolerance, whereas adminis- tration of antiresistin antibodies improved insulin action, thus linking resistin with insulin resistance.
Because of the association between insulin resistance and PCOS, resistin is a candidate to play a role with respect to abnormal steroidogenesis in PCOS.
The human homolog of resistin exhibits 53% homology to murine resistin and resides on chromo- some 19p13.3 in a region not previously linked to obesity, insulin resistance, or diabetes. Several investigations have shown that resistin concentrations are elevated in the circulation of women with PCOS, and a recent study demonstrated a direct stimulatory action of resistin on thecal 17D-hydroxy- lase activity in vitro (25). Although a role for resistin in PCOS has yet to be established, these data indicate that elevated resistin may play a role in ovarian hyperandrogenism in PCOS.
2.1.6.4. I
NSULINInsulin resistance associated with PCOS (1) causes a mild compensatory increase in insulin con-
centrations in order to control blood glucose concentrations within normal limits. There is a large
body of evidence supporting the concept that the increase in insulin concentrations is related to the
elevated androgen production characteristic of PCOS. The elevated insulin in vivo is thought to con- tribute to the elevated basal androgen secretion by cultured human theca cells from polycystic ova- ries, and it is clear that the stimulatory effect of insulin on thecal steroidogenesis is augmented in PCOS (26). These data suggest that theca cells may be hyperresponsive to insulin in PCOS.
Insulin hyperresponsiveness of the theca cells in otherwise insulin-resistant women seems to present a paradox. The concept that increased insulin could activate the IGF-1 receptor and thus overstimulate androgen production is not supported by the evidence. Although the theca cells contain IGF-1 receptors, the affinity for insulin is significantly lower than for the insulin receptor. Thus, hyperinsulinemia greater than that observed in most women with PCOS would be required to signifi- cantly activate the type 1 receptor. The evidence shows that the effects of insulin on thecal androgen biosynthesis are mediated through the insulin receptor and that blocking the IGF-1 receptor with neutralizing antibody does not prevent the insulin stimulation of androgen production (27). Interest- ingly, theca cells from polycystic ovaries appear to have increased expression of insulin receptor substrates 1 and 2, suggesting that there may be an abnormal amplification of insulin signaling in PCOS (28).
There is growing consensus that insulin per se has an important contribution to excess androgen production in PCOS. Inhibition of insulin secretion with diazoxide causes a decline in circulating androgen concentrations, indicating that insulin has a direct effect on ovarian androgen production in vivo. Further evidence is provided by clinical studies with the insulin-sensitizing agents such as metformin and the thiazolidinediones. Metformin reduces fasting plasma glucose concentrations and increases oral glucose tolerance in women with PCOS, decreases plasma insulin and improves insu- lin sensitivity in type 2 diabetic subjects, and improves insulin-stimulated glucose transport in skel- etal muscle from subjects with type 2 diabetes. Recent studies demonstrate that metformin not only improves peripheral insulin sensitivity in women with PCOS, but also decreases androgen concentra- tions in both lean and obese women with PCOS and can restore menses in approximately one-third of previously amenorrheic women (29).
Thiazolidinediones are a class of insulin-sensitizing agents that act by a different mechanism from metformin, potentially involving peroxisome proliferator-activated receptor-J, which is involved in stimulating transcription of factors related to glucose disposal in skeletal muscle. Thiazolidinediones decrease hyperglycemia in subjects with type 2 diabetes and increase glucose uptake in skeletal muscle. In PCOS there are dramatic reductions in androgen excess and insulin resistance and improvement in metabolic and endocrine dysfunctions (29).
On the other hand, reduction of androgen concentrations yields only a modest improvement in insulin resistance. Neither long-term treatment with gonadotropin-releasing hormone analogs nor use of anti-androgens was able to normalize insulin sensitivity in women with PCOS. Thus, reduc- tion of androgen secretion or blocking androgen action has little effect on insulin sensitivity, imply- ing that excess androgen secretion is secondary to increased insulin concentrations in insulin-resistant women with PCOS.
3. ABNORMAL ESTROGEN PRODUCTION
In normally cycling women the predominant site of estrogen production is the granulosa cells of the ovarian follicle. In ovulatory cycles in PCOS, the dominant follicle secretes estradiol in sufficient quantities to trigger the midcycle surge of LH, but in the anovulatory cycles typical of PCOS, follicle development is arrested at the small antral stage when selection of a dominant follicle would nor- mally occur and the aromatase enzyme would be expressed (30). Consequently, the granulosa cells fail to express aromatase, and estrogen production from the ovary is minimal.
PCOS, however, is not a hypoestrogenic condition. The high concentrations of androstenedione
secreted by the theca cells are metabolized to estrone in the peripheral tissues, in particular the adi-
pose stromal cells, leading to a chronic elevation of estrogen in the circulation. The chronic elevation
of estrogen in PCOS contributes to the frequently observed increase in LH-to-follicle- stimulating
hormone (FSH) ratio and can promote the development of reproductive tract cancer.
210 Magoffin
4. ABNORMAL PROGESTERONE PRODUCTION
Progesterone production by the ovary in PCOS has not been thoroughly studied. Progesterone can be produced by theca and granulosa cells in developing follicles and by the corpus luteum in ovula- tory cycles. It is likely that progesterone production from the theca cells is increased in PCOS because of the increase in steroidogenic capacity of the theca cells.
There is evidence that the granulosa cells in arrested follicles in polycystic ovaries prematurely express genes associated with luteinization, namely cytochrome P450scc and the LH receptor (8).
The premature luteinization causes the granulosa cells to respond to LH and produce excessive amounts of progesterone compared to cells from control ovaries at a similar developmental stage (31). It is unclear whether abnormal progesterone production plays an important role in the patho- physiology of PCOS.
5. FUTURE AVENUES OF INVESTIGATION
Our current understanding of steroidogenic abnormalities in PCOS is the result of recent studies that have characterized the phenotype of the polycystic ovary. However, little is known regarding the relative contributions of various regulatory molecules to the steroidogenic abnormalities, and the molecular mechanisms that are dysregulated in PCOS have not been worked out. Active areas of investigation include intracellular signaling mechanisms by which transcription and translation of steroidogenically relevant genes are controlled. Additional work is needed that defines the mecha- nisms and regulators, both intraovarian and extragonadal, that cause hyperandrogenism. An area that has not received much attention is the mechanism of how elevated androgen concentrations lead to follicle arrest. Finally, research into the mechanisms of granulosa cell dysfunction would be a fruitful area of investigation. It would be helpful to understand if there are unifying defects in theca and granulosa cells that lead to the abnormal phenotypes of these cells in PCOS.
KEY POINTS
• The ovarian theca cells in PCOS appear to be hyperstimulated by abnormal concentrations of LH and insulin.
• The increased number and steroidogenic capacity of the theca cells in PCOS leads to excessive production of androstenedione, which is metabolized to testosterone and 5D-androstane-3,17-dione rather than estro- gen by the granulosa cells.
• The granulosa cells in PCOS fail to express adequate amounts of aromatase, but instead prematurely express LH receptors and produce progesterone.
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