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Adrenocortical hyperresponsiveness to corticotropin in polycystic ovary syndrome patients with adrenal androgen excess
FERTILITY AND STERILITY威 VOL. 81, NO. 1, JANUARY 2004 Copyright ©2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A.
Adrenocortical hyperresponsiveness to corticotropin in polycystic ovary syndrome patients with adrenal androgen excess Carlos Moran, M.D.,a,b Rosario Reyna, B.S.,a Larry S. Boots, M.D.,a and Ricardo Azziz, M.D.a,c The University of Alabama at Birmingham, Birmingham, Alabama
Received October 7, 2002; revised and accepted July 11, 2003. Presented in part at the 83rd Annual Meeting of the Endocrine Society, Denver, Colorado, June 20 –23, 2001. Financial support provided by the National Institutes of Health RO1-HD29364, K24-HD01346 and M01RR00032, Bethesda, Maryland. Reprint requests: Ricardo Azziz, M.D., Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, 8635 West Third Street, Suite 160, Los Angeles, California 90048 (FAX: 310-423-3470; Email: [email protected]). a Department of Obstetrics and Gynecology, The University of Alabama at Birmingham. b Health Research Council, Mexican Institute of Social Security, Mexico City, Mexico. c Department of Medicine, The University of Alabama at Birmingham. 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2003. 07.008
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Objective: To test the hypothesis that adrenal androgen (AA) excess in the polycystic ovary syndrome (PCOS) is due to a generalized exaggeration in AA output in response to adrenocorticotropic hormone (ACTH), and that this abnormality is due to an identifiable alteration in the biosynthesis of AAs. Design: Cross-sectional prospective controlled study. Setting: Academic tertiary care medical center. Patient(s): Patients with PCOS (n ⫽ 9) and without (n ⫽ 9) AA excess and controls (n ⫽ 12) without hyperandrogenism, matched for age and body mass. Intervention(s): Acute 60-minute ACTH test was performed on patients. Main Outcome Measure(s): Basal levels of dehydroepiandrosterone sulfate (DHEAS), total testosterone (T), free T, and basal (Steroid0) and the 60-minute ACTH-stimulated levels (Steroid60) of pregnenolone (PREG), progesterone (P4), 17-hydroxypregnenolone (17-HPREG), 17-hydroxyprogesterone (17-HP), dehydroepiandrosterone (DHEA), and androstenedione (A4) were measured. Adrenocortical activities of 17-hydroxylase (17-OH), 17,20-lyase, and 3-hydroxysteroid dehydrogenase were estimated from product to precursor ratio, using Steroid60 values. Result(s): Compared with PCOS patients without AA excess, PCOS patients with AA excess demonstrated significantly greater levels of DHEA0 and A460. PCOS patients with AA excess had significantly higher activity of ⌬517-OH, compared with PCOS patients without AA excess. Conclusion(s): Adrenal androgen excess in PCOS is associated with a greater ⌬517-OH activity in response to ACTH. (Fertil Steril威 2004;81:126-31. ©2004 by American Society for Reproductive Medicine.) Key Words: Androgen, hyperandrogenism, polycystic ovary syndrome, 17-hydroxylase
Approximately 4% of unselected women of reproductive age suffer from the polycystic ovary syndrome (PCOS) (1). Adrenal androgen (AA) excess, as evidenced by high levels of dehydroepiandrosterone sulfate (DHEAS), has been reported in 20% to 60% of PCOS patients (2–5). Nonetheless, because DHEAS levels decrease with age, we recently reported that only about 25% of PCOS patients actually can be characterized as having AA excess (6). The etiology of the excessive AA production in PCOS patients is unknown. Adrenal androgen production, basally and in response to adrenocorticotropic hormone (ACTH), varies widely among normal individuals (7). As such, elucidating the mechanism(s) underlying the AA excess in PCOS may aid in understand-
ing the etiology of the steroidogenic abnormalities present in PCOS. It is possible that the AA excess observed is simply the result of changes in the metabolism of precursors. For example, AA excess in PCOS may reflect the increased sulfation of dehydroepiandrosterone (DHEA), which will result in greater circulating levels of DHEAS. In fact, there is evidence to suggest that extraadrenal hyperandrogenemia alone may favor the increased production of DHEAS from DHEA. In this regard, we have observed that the administration of high doses of testosterone (T) to oophorectomized women results in an increased DHEAS/DHEA ratio, suggesting an increase in the sulfation of DHEA (8).
Alternatively, AA excess in PCOS may result from abnormalities in adrenocortical steroidogenesis, resulting from the exaggerated production of these hormones in response to ACTH stimulation. For example, we have previously reported that PCOS patients with AA excess demonstrate a supranormal ACTH-stimulated DHEA level (9). Furthermore, AA excess in PCOS is associated with a hyperresponse to ACTH stimulation, but not to an altered pituitary responsiveness to ovine corticotropin-releasing hormone (CRH) or an increased sensitivity of the adrenal cortex to ACTH (10). Finally, we have also observed that hyperandrogenic women demonstrate a positive correlation between basal DHEAS and androstenedione (A4) (11). To more precisely define the aberration in adrenocortical behavior, in various overlapping studies we have previously demonstrated that isolated defects in 3-hydroxysteroid dehydrogenase (3-HSD) (12), 17,20-lyase (13), 11-hydroxylase (11), and 21-hydroxylase (21-OH) (14) activity do not generally account for AA excess in PCOS. Taken together, these data suggest that patients with PCOS may have a generalized exaggeration in AA output in response to ACTH, which is not due to specific defects in most enzymatic pathways studied. We should note, however, that these previous studies examined one individual pathway at a time, with the studies performed at different times over a decade and in different populations. Furthermore, patients with AA excess were not always identified a priori. We now hypothesize that AA excess in PCOS is due to a generalized exaggeration in AA output in response to ACTH, and that this abnormality is due to an identifiable alteration in the biosynthesis of AAs. To test this hypothesis, while addressing our concerns with the study design of our previous studies, we prospectively studied patients with PCOS and AA excess, and compared them with PCOS patients without AA excess as well as healthy women without hyperandrogenism.
MATERIALS AND METHODS Subjects We prospectively recruited 9 women with PCOS and AA excess (i.e., a DHEAS level ⬎6.8 mol/L), 9 PCOS women without AA excess (i.e., DHEAS levels ⱕ6.8 mol/L, or within normal limits), and 12 healthy control women without hyperandrogenism. None of the study subjects was taking hormonal medications, including oral contraceptives. Groups were matched for age and body mass index (BMI). Patients were recruited from the reproductive endocrinology clinic at the University of Alabama at Birmingham (UAB), and controls were recruited by word of mouth or in response to advertising. The Institutional Review Board (IRB) for Human Use at UAB approved the study, and all subjects provided written informed consent. Control women were physically normal without evidence of hirsutism, reporting regular menstrual cycles every 26 –32 FERTILITY & STERILITY威
days. In affected patients, PCOS was diagnosed by the presence of oligo-ovulation, in combination with hyperandrogenism (clinical and/or hormonal), after the exclusion of other related disorders, such as nonclassic adrenal hyperplasia (NCAH), Cushing’s syndrome, hyperprolactinemia and hypothyroidism (15). Ovulatory dysfunction was defined as menstrual cycles ⬎45 days in length, or by a luteal phase P4 level ⬍12.7 nmol/L (4 ng/mL) in conjunction with a monophasic basal body temperature chart (BBT) if menstrual cycles were less than 45 days in length. Hirsutism was defined as a modified Ferriman–Gallwey (F-G) score ⱖ6 (16). For the purpose of this study, subjects were deemed hyperandrogenemic if the level of total T was ⬎2.6 nmol/L or the level of free T was ⬎24 pmol/L. All these values were the mean ⫹3 standard deviations (SD) of the hormonal levels of control women in the present study. The 21-OH deficient NCAH was excluded by a basal follicular phase 17-hydroxyprogesterone (17-HP) level ⬍6.0 nmol/L (2 ng/ mL) or a 17-HP level following acute ACTH stimulation of ⬍30.3 nmol/L (10 ng/mL), as previously described (17); hyperprolactinemia and hypothyroidism were excluded by normal levels of PRL and TSH, respectively; and Cushing’s syndrome and androgenic tumors were excluded by appropriate testing, if suspected clinically.
Study Protocol All studies were performed between 8:00 and 10:30 A.M. in fasting conditions during the follicular phase (days 3– 8) of the menstrual cycle, with the exception of P4, which was measured in follicular and luteal phases when needed for detection of ovulation. In the event the patient was amenorrheic, tests were performed after withdrawal bleeding was induced by progesterone in oil (100 mg i.m. All PCOS patients and control women underwent an acute 60-minute stimulation test with 0.25 mg of ACTH-(1–24) i.v. (Cortrosyn, Organon, West Orange, NJ), as described previously (18). Basal levels of DHEAS, total T, free T, and sex hormone-binding globulin (SHBG) were determined. Basal and 60-minute ACTH stimulated levels of pregnenolone (PREG), P4, 17-hydroxypregnenolone (17-HPREG), 17-HP, DHEA, and A4, were measured in serum samples. During the ACTH stimulation test, the basal hormonal levels were termed as Steroid0 and the 60-minute ACTHstimulated levels were termed as Steroid60. The activities of the ⌬5 and ⌬4 pathways of 17-hydroxylase (17-OH); the ⌬5 and ⌬4 pathways of 17,20-lyase; and the 3-HSD pathways for the C21-mineralocorticoids (C21m-3-HSD), the C21- glucocorticoids (C21g-3-HSD), and the C19-sex steroids (C193-HSD) were estimated by the Product60/Precursor60 ratio. In these analyses, as the Product/Precursor ratio increases the estimated enzymatic activity increases, although this method is limited because it does not consider a decreased metabolism of product or an increased metabolism of precursor. 127
TABLE 1 Basal hormones in PCOS patients with and without AA excess, and in control women.
Steroid
PCOS patients with AA excess (n ⫽ 9)
PCOS patients without AA excess (n ⫽ 9)
Total T (nmol/L) Free T (pmol/L) DHEAS (mol/L) PREG0 (nmol/L) P40 (nmol/L) 17-HPREG0 (nmol/L) 17-HP0 (nmol/L) DHEA0 (nmol/L) A40 (nmol/L)
2.84 (1.42–5.03)a 42.64 (19.42–97.08)a 7.69 (6.87–9.70)a,b 10.78 (0.63–31.70)a 2.51 (1.24–4.01) 3.30 (0.30–29.44) 4.84 (2.69–12.16) 23.60 (12.15–97.16)a 8.73 (4.89–14.66)a
1.80 (1.35–4.23)b 19.07 (12.83–52.35)b 2.90 (0.66–3.84)a 15.21 (6.02–38.04)b 2.70 (1.65–3.34)a 1.20 (0.30–6.61) 3.42 (2.51–7.05) 11.80 (6.59–23.60)a,b 4.89 (3.14–8.38)
Control women (n ⫽ 12) 1.33 (0.60–2.08)a,b 9.36 (3.47–18.37)a,b 2.15 (1.13–4.88)b 37.25 (27.26–65.30)a,b 1.58 (0.60–3.21)a 1.65 (0.30–11.11) 3.03 (1.33–7.69) 24.64 (5.21–57.26)b 4.01 (2.09–9.77)a
Note: Data is expressed in medians, with ranges in parentheses. PCOS ⫽ polycystic ovary syndrome; AA ⫽ adrenal androgen; T ⫽ testosterone; DHEAS ⫽ dehydroepiandrosterone sulfate; PREG ⫽ pregnenolone; P4 ⫽ progesterone; 17-HPREG ⫽ 17-hydroxypregeneolone; 17-HP ⫽ 17␣-hydroxyprogesterone; DHEA ⫽ dehydroepiandrosterone; A4 ⫽ androstenedione. a,b Significant difference (P⬍.05) between pairs. Moran. Hyperresponsiveness to ACTH in PCOS. Fertil Steril 2004.
Hormonal Assays Dehydroepiandrosterone sulfate, A4, P4, and 17-HP were measured by direct radioimmunoassays (RIAs) using commercially available kits (DHEAS, A4, and P4 by Diagnostic System Laboratories, Webster, TX; and 17-HP by Diagnostic Products Corporation, Los Angeles, CA). The levels of 17-HPREG, PREG, and DHEA were assayed by in-house methods after chromatography, as previously described (10, 13, 18). Total T was measured by an in-house method after serum extraction, as previously reported (19). The activity of SHBG was determined by equilibrium dialysis, and free T was calculated, as previously described (20). All samples were batched in the same assay to eliminate interassay variations. The intraassay coefficient of variation for all steroids did not exceed 9%.
Statistical Analysis Data were expressed as median and range. Statistical software packages (Kwikstat 4; Texas Soft, Cedar Hill, TX) were used to perform clinical and hormonal comparisons among groups by the Kruskal–Wallis nonparametric analysis of variance (ANOVA). A value of P⬍.05 was considered statistically significant.
RESULTS Clinical and Hormonal Characteristics of the Study Groups As expected and by design, PCOS patients with and without AA excess, as well as controls, had similar median [range] ages (24 [21– 40] years, 29 [20 –33] years, and 28 [19 –32] years, respectively) and BMI (29.1 [18.0 –39.9] kg/m2, 32.0 [20.7–37.4] kg/m2, and 25.9 [19.6 –36.8] kg/m2, respectively). Polycystic ovary syndrome patients with and 128
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without AA excess had similar median [range] hirsutism scores (modified F-G: 8 [3–16] and 8 [7–14], respectively). Six of the nine PCOS patients with AA excess and all nine PCOS patients without AA excess were considered hirsute (F-G ⱖ6). Overall, PCOS women, whether with or without AA excess, had higher total and free T levels and lower PREG0 compared with controls (Table 1). As expected, the median DHEAS levels were higher among PCOS patients with AA excess, compared with PCOS patients without AA excess or control women. Polycystic ovary syndrome patients with AA excess had significantly greater levels of DHEA0 than PCOS patients without AA excess, but not controls. In addition, PCOS patients without AA excess had significantly lower levels of DHEA0 than controls. Polycystic ovary syndrome patients with AA excess also had significantly higher levels of A40 than controls, although the difference with PCOS patients without AA excess did not reach significance. Levels of PREG0 were lower in both PCOS women with and without AA excess, compared with controls. Levels of P40 were higher in both groups of PCOS patients compared with controls, although the difference only reached significance for PCOS patients without AA excess.
Comparing the ACTH-Stimulated Hormonal Levels Consistent with the differences in the basal hormonal profile, PCOS patients with AA excess had significantly greater levels of A460 than PCOS patients without AA excess (Table 2). Compared with controls, PCOS patients with AA excess also had significantly higher levels of A460, but significantly lower levels of PREG60. Polycystic ovary syndrome patients without AA excess had significantly lower PREG60 levels than control women. There were no other Vol. 81, No. 1, January 2004
TABLE 2 ACTH-stimulated steroid (Steroid60) levels in PCOS patients with and without AA excess, and in control women.
Steroid PREG60 (nmol/L) P460 (nmol/L) 17-HPREG60 (nmol/L) 17-HP60 (nmol/L) DHEA60 (nmol/L) A460 (nmol/L)
PCOS patients with AA excess (n ⫽ 9)
PCOS patients without AA excess (n ⫽ 9)
Control women (n ⫽ 12)
18.39 (0.63–31.70)a 5.28 (3.47–8.33) 21.93 (13.22–60.08) 13.68 (8.17–16.67) 43.72 (34.70–121.45) 11.17 (8.73–17.45)a,b
26.31 (9.19–31.70)b 5.28 (3.24–6.49) 13.82 (7.81–25.23) 11.38 (6.75–13.86) 32.97 (14.23–57.26) 7.68 (4.89–10.47)a
51.99 (30.12–84.32)a,b 4.73 (3.05–7.60) 15.17 (9.01–22.53) 12.07 (6.11–20.09) 70.44 (18.04–117.98) 6.28 (2.44–9.42)b
Note: Data is expressed in medians, with ranges in parentheses. PCOS ⫽ polycystic ovary syndrome; AA ⫽ adrenal androgen; PREG ⫽ pregnenolone; P4 ⫽ progesterone; 17-HPREG ⫽ 17-hydroxypregnenolone; 17-HP ⫽ 17␣-hydroxyprogesterone; DHEA ⫽ dehydroepiandrosterone; A4 ⫽ androstenedione. a,b Significant difference (P⬍.05) between pairs. Moran. Hyperresponsiveness to ACTH in PCOS. Fertil Steril 2004.
significant differences in the ACTH-stimulated hormonal levels among the groups.
Comparing the Estimated Adrenocortical Enzymatic Studying the enzymatic activity estimated after ACTH stimulation or peak enzymatic activity, we observed that PCOS patients with AA excess had significantly higher ⌬517-OH activity than PCOS patients without AA excess (Table 3). In addition, we found that PCOS patients with AA excess had significantly higher activities of ⌬517-OH, ⌬417,20-lyase, C21m-3-HSD, and C19-3-HSD, as well as significantly lower ⌬517,20-lyase, than controls (Table 3). Likewise, PCOS patients without AA excess had an ACTHstimulated enzymatic activity for ⌬417,20-lyase, C21m-3HSD, and C19-3-HSD that was significantly higher, and a significantly lower ⌬517,20-lyase activity in comparison with control women, although the ⌬517-OH activity was not
different from controls. Overall, the only differences between PCOS patients with and without AA excess were significantly higher stimulated ⌬517-OH activity in the former.
DISCUSSION We had hypothesized that AA excess in PCOS is due to a generalized exaggeration in the AA response to ACTH stimulation. Contrary to our hypothesis, our data suggests that the exaggerated production of AAs appears to be primarily associated with an increase in ⌬517-OH activity. All other differences, including a higher ⌬417,20-lyase, C21- and C19-3-HSD, and a lower ⌬517,20-lyase compared with controls appeared to affect both PCOS subgroups equally. Thus, it would appear that the down-regulation of ⌬517,20lyase and up-regulation of 3-HSD activation is a feature of PCOS in general. We should note that ⌬5, not ⌬417,20-lyase,
TABLE 3 Estimated adrenocortical enzymatic activities based on peak steroid levels following ACTH-(1–24) stimulation in PCOS patients with and without AA excess, and in control women. Enzymatic activity ⌬517-OH ⌬517,20-lyase ⌬417-OH ⌬417,20-lyase C21m-3-HSD C21g-3-HSD C19g-3-HSD
Steroid measures (product/precursor)
PCOS patients with AA excess (n ⫽ 9)
PCOS patients without AA excess (n ⫽ 9)
Control women (n ⫽ 12)
17-HPREG60/PREG60 DHEA60/17-HPREG60 17-HP60/P460 A460/17-HP60 P460/PREG60 17-HP60/17-HPREG60 A460/DHEA60
1.90 (0.44–21.80)a,b 2.02 (0.95–3.16)a 2.51 (1.99–3.77) 0.91 (0.71–1.07)a 0.27 (0.18–7.27)a 0.51 (0.28–1.02) 0.22 (0.12–0.40)a
0.55 (0.27–2.55)a 2.42 (1.44–4.22)b 2.14 (1.66–2.73) 0.74 (0.61–0.91)b 0.21 (0.10–0.62)b 0.71 (0.44–1.46) 0.25 (0.15–0.39)b
0.25 (0.20–0.50)b 4.52 (1.82–7.24)a,b 2.50 (2.00–3.38) 0.53 (0.40–0.65)a,b 0.09 (0.05–0.15)a,b 0.76 (0.60–1.11) 0.10 (0.06–0.19)a,b
Note: Data is expressed in medians with ranges in parentheses. ACTH ⫽ adrenocorticotropic hormone; PCOS ⫽ polycystic ovary syndrome; AA ⫽ adrenal androgen; 17-OH ⫽ 17-hydroxylase; 17-HPREG ⫽ 17-hydroxypregnenolone; PREG ⫽ pregnenolone; DHEA ⫽ dehydroepiandrosterone; P4 ⫽ progesterone; A4 ⫽ androstenedione; 3-HSD ⫽ 3-hydroxysteroid dehydrogenase; C21m represents the mineralocorticoid (17-deoxy) pathway; C21g represents the glucorticoid (17-hydroxy) pathway; C19 represents the carbon-19 (sex-steroid) pathway. Moran. Hyperresponsiveness to ACTH in PCOS. Fertil Steril 2004.
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is the natural pathway toward androgen production in humans (21). We note that basal DHEA levels are similar between controls and PCOS patients with AA excess, while PCOS patients without AA excess have lower levels than either group. Although this does not invalidate the interpretation of the data comparing PCOS patients with and without AA excess, it does suggest that PCOS patients, in general, may have increased sulfotransferase activity such that there are proportionally higher DHEAS levels for an equivalent amount of DHEA in hyperandrogenized women. We previously suggested that an increase in sulfotransferase activity could potentially be secondary to circulating testosterone (T) (8). The suggestion that exaggerated ⌬517-OH activity is responsible for AA excess is consistent with our data and that of other investigators. For example, we previously reported in an unrelated population of 92 hyperandrogenic patients that 57% of all patients had an exaggerated basal activity of ⌬517-OH, although few, if any, had increased ⌬417-OH, ⌬517,20-lyase, or ␦417,20-lyase activities (13). Nonetheless, in that study we did not consider separately the subgroup of patients with AA excess. In agreement, Rosenfield et al. have observed that a significant proportion of their hyperandrogenic women appeared to have excessive 17-OH activity and high, yet partially down-regulated, ⌬417,20-lyase activity in response to either ovarian (with a GnRH agonist [GnRH-a]) or adrenal (with ACTH-[1–24]) stimulation (22, 23). Again, these latter investigators did not attempt to a priori recruit patients with and without AA excess. Although the finding of an exaggerated ⌬517-OH activity in PCOS patients with AA excess appears to be relatively consistent, the underlying mechanism(s) is(are) not clear. P450c17␣ is a single microsomal enzyme that catalyzes both the ⌬5 and ⌬417-OH activity for C21 steroids. This enzyme also catalyzes the ⌬5 and ⌬417,20-lyase activities that cleave the C17-C20 bond to produce C19 sex steroids, although we should note that there is strong evidence that ⌬417,20-lyase activity is relatively minimal in normal human steroidogenesis (23). Some investigators have suggested the observed differences in steroid levels are the result of a “dysregulation” of P450c17␣ (21, 22). In fact, clinical and in vitro observations indicate that 17-OH and 17,20-lyase activities of P450c17␣ are independently regulated (24). Although the relative activity of 17,20-lyase appears to be regulated by the amount of available electron donors, such as P450 oxidoreductase or cytochrome b5, 17-OH activity appears to be less readily modifiable (23, 24). Unfortunately, molecular and/or genetic studies to date do not support the concept that P450c17␣ is abnormal in PCOS. The gene CYP17 encodes for P450c17␣ in both the adrenal cortex and the ovary. Although several CYP17 mutations have been reported, all have been associated with P450c17␣ enzymatic deficiencies, and none have been noted to result in 130
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either 17-OH or 17,20-lyase hyperactivity (25). Although it is possible that genetically determined posttranscriptional modifications of P450c17␣ in vivo might result in the relative enhancement of ⌬517-OH activity in some patients, this remains to be demonstrated. It is also unlikely that the intracellular environment alters P450c17␣ function. Using skin fibroblasts from PCOS patients transfected with human P450c17␣, Martens et al. were unable to detect an abnormality in the ratio of the 17-OH to 17,20-lyase activities of the enzyme in the fibroblasts of patients with PCOS, compared with controls (26). It is tempting to suggest that the observed differences in circulating steroids arises from an abnormal function, inherited or otherwise, of the enzymes within the adrenal cortex. For example, it has been well documented that mutations of CYP21, leading to the production of an abnormal P450c21, result in very high levels of the immediate 17-hydroxy precursor, 17-HP (27). Alternatively, it is much more difficult to attribute subtle differences in circulating steroids to an alteration in enzymatic function, as the observed steroid levels in blood are many levels removed from the actual intracellular enzyme in the adrenal cortex. For example, in the present study significant differences are observed between groups in the estimated C21m-3-HSD and C19-3HSD activities. Using microsomes from yeast-expressing human type II 3-HSD, however, Lee et al. were unable to detect any kinetic differences in the activity of this enzyme for C21-17-deoxy, C21-17-hydroxy, and C19 precursors (28). Simard et al. reported similar findings in transfected COS-1 cells (29). Thus, it is not generally possible to infer from in vivo physiologic studies that the activity of a particular intracellular enzyme (e.g., P450c17␣) is altered in a subtle manner. In conclusion, although PCOS per se is associated with multiple changes in steroidogenesis that favor an exaggeration in adrenocortical secretion, AA excess in PCOS appears to be uniquely associated with an increased ⌬517-OH activity. Mechanistically, it is unlikely that the exaggerated ⌬517-OH activity observed is due to an intrinsic defect in P450c17␣; however, it is possible that alterations in the intracellular trafficking of steroids, in the degradative pathways of the steroids, or in intrinsic adrenal blood flow affecting precursor availability may be responsible.
Acknowledgments: The authors thank Walter L. Miller, M.D., for his review and constructive criticism of the manuscript.
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tine FP, Merriam GR, eds. Polycystic ovary syndrome. Boston: Blackwell Scientific Publications, 1992:377–84. Hatch R, Rosenfield RL, Kim MH, Tredway D. Hirsutism: implications, etiology, and management. Am J Obstet Gynecol 1981;140:815– 30. Azziz R, Hincapie LA, Knochenhauer ES, Dewailly D, Fox L, Boots LR. Screening for 21-hydroxylase deficient nonclassic adrenal hyperplasia among hyperandrogenic women: a prospective study. Fertil Steril 1999;72:915–25. Azziz R, Bradley E Jr, Huth J, Boots LR, Parker CR, Zacur HA. Acute adrenocorticotropin-(1-24) (ACTH) adrenal stimulation in eumenorrheic women: reproducibility and effect of ACTH dose, subject weight, and sampling time. J Clin Endocrinol Metab 1990;70:1273–9. Boots LR, Potter S, Potter HD, Azziz R. Measurement of total serum testosterone level using commercially available kits: high degree of between-kit variability. Fertil Steril 1998;69:286 –92. Pearlman WH, Crepy O. Steroid-protein interaction with particular reference to testosterone binding by human serum. J Biol Chem 1967; 242:182–9. Auchus RJ, Lee TC, Miller WL. Cytochrome b5 augments the 17,20lyase activity of human P450c17 without direct electron transfer. J Biol Chem 1998;273:3158 –65. Barnes RB, Rosenfield RL, Burstein S, Ehrmann DA. Pituitary-ovarian responses to nafarelin testing in the polycystic ovary syndrome. N Engl J Med 1989;320:559 –65. Rosenfield RL, Barnes RB, Cara JF, Lucky AW. Dysregulation of cytochrome P450c17␣ as the cause of polycystic ovarian syndrome. Fertil Steril 1990;53:785–91. Miller WL, Auchus RJ, Geller DH. The regulation of 17,20 lyase activity. Steroids 1997;62:133–42. Yanase T. 17␣-Hydroxylase/17,20-lyase defects. J Steroid Biochem Mol Biol 1995;53:153–7. Martens JWM, Geller DH, Arlt W, Auchus RJ, Ossovskaya VS, Rodriguez H, et al. Enzymatic activities of P450c17 stably expressed in fibroblasts from patients with the polycystic ovary syndrome. J Clin Endocrinol Metab 2000;85:4338 –46. Speiser PW, Knochenhauer ES, Dewailly D, Fruzzetti F, Marcondes JA, Azziz R. A multicenter study of women with nonclassical congenital adrenal hyperplasia: relationship between genotype and phenotype. Mol Genet Metab 2000;71:527–34. Lee TC, Miller WL, Auchus RJ. Medroxyprogesterone acetate and dexamethasone are competitive inhibitors of different human steroidogenic enzymes. J Clin Endocrinol Metab 1999;84:2104 –10. Simard J, Rheaume E, Mebarki F, Sanchez R, New MI, Morel Y, et al. Molecular basis of human 3-hydroxysteroid dehydrogenase deficiency. J Steroid Biochem Mol Biol 1995;53:127–38.
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Adrenocortical hyperresponsiveness to corticotropin in polycystic ovary syndrome patients with adrenal androgen excess Carlos Moran, M.D.,a,b Rosario Reyna, B.S.,a Larry S. Boots, M.D.,a and Ricardo Azziz, M.D.a,c The University of Alabama at Birmingham, Birmingham, Alabama
Received October 7, 2002; revised and accepted July 11, 2003. Presented in part at the 83rd Annual Meeting of the Endocrine Society, Denver, Colorado, June 20 –23, 2001. Financial support provided by the National Institutes of Health RO1-HD29364, K24-HD01346 and M01RR00032, Bethesda, Maryland. Reprint requests: Ricardo Azziz, M.D., Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, 8635 West Third Street, Suite 160, Los Angeles, California 90048 (FAX: 310-423-3470; Email: [email protected]). a Department of Obstetrics and Gynecology, The University of Alabama at Birmingham. b Health Research Council, Mexican Institute of Social Security, Mexico City, Mexico. c Department of Medicine, The University of Alabama at Birmingham. 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2003. 07.008
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Objective: To test the hypothesis that adrenal androgen (AA) excess in the polycystic ovary syndrome (PCOS) is due to a generalized exaggeration in AA output in response to adrenocorticotropic hormone (ACTH), and that this abnormality is due to an identifiable alteration in the biosynthesis of AAs. Design: Cross-sectional prospective controlled study. Setting: Academic tertiary care medical center. Patient(s): Patients with PCOS (n ⫽ 9) and without (n ⫽ 9) AA excess and controls (n ⫽ 12) without hyperandrogenism, matched for age and body mass. Intervention(s): Acute 60-minute ACTH test was performed on patients. Main Outcome Measure(s): Basal levels of dehydroepiandrosterone sulfate (DHEAS), total testosterone (T), free T, and basal (Steroid0) and the 60-minute ACTH-stimulated levels (Steroid60) of pregnenolone (PREG), progesterone (P4), 17-hydroxypregnenolone (17-HPREG), 17-hydroxyprogesterone (17-HP), dehydroepiandrosterone (DHEA), and androstenedione (A4) were measured. Adrenocortical activities of 17-hydroxylase (17-OH), 17,20-lyase, and 3-hydroxysteroid dehydrogenase were estimated from product to precursor ratio, using Steroid60 values. Result(s): Compared with PCOS patients without AA excess, PCOS patients with AA excess demonstrated significantly greater levels of DHEA0 and A460. PCOS patients with AA excess had significantly higher activity of ⌬517-OH, compared with PCOS patients without AA excess. Conclusion(s): Adrenal androgen excess in PCOS is associated with a greater ⌬517-OH activity in response to ACTH. (Fertil Steril威 2004;81:126-31. ©2004 by American Society for Reproductive Medicine.) Key Words: Androgen, hyperandrogenism, polycystic ovary syndrome, 17-hydroxylase
Approximately 4% of unselected women of reproductive age suffer from the polycystic ovary syndrome (PCOS) (1). Adrenal androgen (AA) excess, as evidenced by high levels of dehydroepiandrosterone sulfate (DHEAS), has been reported in 20% to 60% of PCOS patients (2–5). Nonetheless, because DHEAS levels decrease with age, we recently reported that only about 25% of PCOS patients actually can be characterized as having AA excess (6). The etiology of the excessive AA production in PCOS patients is unknown. Adrenal androgen production, basally and in response to adrenocorticotropic hormone (ACTH), varies widely among normal individuals (7). As such, elucidating the mechanism(s) underlying the AA excess in PCOS may aid in understand-
ing the etiology of the steroidogenic abnormalities present in PCOS. It is possible that the AA excess observed is simply the result of changes in the metabolism of precursors. For example, AA excess in PCOS may reflect the increased sulfation of dehydroepiandrosterone (DHEA), which will result in greater circulating levels of DHEAS. In fact, there is evidence to suggest that extraadrenal hyperandrogenemia alone may favor the increased production of DHEAS from DHEA. In this regard, we have observed that the administration of high doses of testosterone (T) to oophorectomized women results in an increased DHEAS/DHEA ratio, suggesting an increase in the sulfation of DHEA (8).
Alternatively, AA excess in PCOS may result from abnormalities in adrenocortical steroidogenesis, resulting from the exaggerated production of these hormones in response to ACTH stimulation. For example, we have previously reported that PCOS patients with AA excess demonstrate a supranormal ACTH-stimulated DHEA level (9). Furthermore, AA excess in PCOS is associated with a hyperresponse to ACTH stimulation, but not to an altered pituitary responsiveness to ovine corticotropin-releasing hormone (CRH) or an increased sensitivity of the adrenal cortex to ACTH (10). Finally, we have also observed that hyperandrogenic women demonstrate a positive correlation between basal DHEAS and androstenedione (A4) (11). To more precisely define the aberration in adrenocortical behavior, in various overlapping studies we have previously demonstrated that isolated defects in 3-hydroxysteroid dehydrogenase (3-HSD) (12), 17,20-lyase (13), 11-hydroxylase (11), and 21-hydroxylase (21-OH) (14) activity do not generally account for AA excess in PCOS. Taken together, these data suggest that patients with PCOS may have a generalized exaggeration in AA output in response to ACTH, which is not due to specific defects in most enzymatic pathways studied. We should note, however, that these previous studies examined one individual pathway at a time, with the studies performed at different times over a decade and in different populations. Furthermore, patients with AA excess were not always identified a priori. We now hypothesize that AA excess in PCOS is due to a generalized exaggeration in AA output in response to ACTH, and that this abnormality is due to an identifiable alteration in the biosynthesis of AAs. To test this hypothesis, while addressing our concerns with the study design of our previous studies, we prospectively studied patients with PCOS and AA excess, and compared them with PCOS patients without AA excess as well as healthy women without hyperandrogenism.
MATERIALS AND METHODS Subjects We prospectively recruited 9 women with PCOS and AA excess (i.e., a DHEAS level ⬎6.8 mol/L), 9 PCOS women without AA excess (i.e., DHEAS levels ⱕ6.8 mol/L, or within normal limits), and 12 healthy control women without hyperandrogenism. None of the study subjects was taking hormonal medications, including oral contraceptives. Groups were matched for age and body mass index (BMI). Patients were recruited from the reproductive endocrinology clinic at the University of Alabama at Birmingham (UAB), and controls were recruited by word of mouth or in response to advertising. The Institutional Review Board (IRB) for Human Use at UAB approved the study, and all subjects provided written informed consent. Control women were physically normal without evidence of hirsutism, reporting regular menstrual cycles every 26 –32 FERTILITY & STERILITY威
days. In affected patients, PCOS was diagnosed by the presence of oligo-ovulation, in combination with hyperandrogenism (clinical and/or hormonal), after the exclusion of other related disorders, such as nonclassic adrenal hyperplasia (NCAH), Cushing’s syndrome, hyperprolactinemia and hypothyroidism (15). Ovulatory dysfunction was defined as menstrual cycles ⬎45 days in length, or by a luteal phase P4 level ⬍12.7 nmol/L (4 ng/mL) in conjunction with a monophasic basal body temperature chart (BBT) if menstrual cycles were less than 45 days in length. Hirsutism was defined as a modified Ferriman–Gallwey (F-G) score ⱖ6 (16). For the purpose of this study, subjects were deemed hyperandrogenemic if the level of total T was ⬎2.6 nmol/L or the level of free T was ⬎24 pmol/L. All these values were the mean ⫹3 standard deviations (SD) of the hormonal levels of control women in the present study. The 21-OH deficient NCAH was excluded by a basal follicular phase 17-hydroxyprogesterone (17-HP) level ⬍6.0 nmol/L (2 ng/ mL) or a 17-HP level following acute ACTH stimulation of ⬍30.3 nmol/L (10 ng/mL), as previously described (17); hyperprolactinemia and hypothyroidism were excluded by normal levels of PRL and TSH, respectively; and Cushing’s syndrome and androgenic tumors were excluded by appropriate testing, if suspected clinically.
Study Protocol All studies were performed between 8:00 and 10:30 A.M. in fasting conditions during the follicular phase (days 3– 8) of the menstrual cycle, with the exception of P4, which was measured in follicular and luteal phases when needed for detection of ovulation. In the event the patient was amenorrheic, tests were performed after withdrawal bleeding was induced by progesterone in oil (100 mg i.m. All PCOS patients and control women underwent an acute 60-minute stimulation test with 0.25 mg of ACTH-(1–24) i.v. (Cortrosyn, Organon, West Orange, NJ), as described previously (18). Basal levels of DHEAS, total T, free T, and sex hormone-binding globulin (SHBG) were determined. Basal and 60-minute ACTH stimulated levels of pregnenolone (PREG), P4, 17-hydroxypregnenolone (17-HPREG), 17-HP, DHEA, and A4, were measured in serum samples. During the ACTH stimulation test, the basal hormonal levels were termed as Steroid0 and the 60-minute ACTHstimulated levels were termed as Steroid60. The activities of the ⌬5 and ⌬4 pathways of 17-hydroxylase (17-OH); the ⌬5 and ⌬4 pathways of 17,20-lyase; and the 3-HSD pathways for the C21-mineralocorticoids (C21m-3-HSD), the C21- glucocorticoids (C21g-3-HSD), and the C19-sex steroids (C193-HSD) were estimated by the Product60/Precursor60 ratio. In these analyses, as the Product/Precursor ratio increases the estimated enzymatic activity increases, although this method is limited because it does not consider a decreased metabolism of product or an increased metabolism of precursor. 127
TABLE 1 Basal hormones in PCOS patients with and without AA excess, and in control women.
Steroid
PCOS patients with AA excess (n ⫽ 9)
PCOS patients without AA excess (n ⫽ 9)
Total T (nmol/L) Free T (pmol/L) DHEAS (mol/L) PREG0 (nmol/L) P40 (nmol/L) 17-HPREG0 (nmol/L) 17-HP0 (nmol/L) DHEA0 (nmol/L) A40 (nmol/L)
2.84 (1.42–5.03)a 42.64 (19.42–97.08)a 7.69 (6.87–9.70)a,b 10.78 (0.63–31.70)a 2.51 (1.24–4.01) 3.30 (0.30–29.44) 4.84 (2.69–12.16) 23.60 (12.15–97.16)a 8.73 (4.89–14.66)a
1.80 (1.35–4.23)b 19.07 (12.83–52.35)b 2.90 (0.66–3.84)a 15.21 (6.02–38.04)b 2.70 (1.65–3.34)a 1.20 (0.30–6.61) 3.42 (2.51–7.05) 11.80 (6.59–23.60)a,b 4.89 (3.14–8.38)
Control women (n ⫽ 12) 1.33 (0.60–2.08)a,b 9.36 (3.47–18.37)a,b 2.15 (1.13–4.88)b 37.25 (27.26–65.30)a,b 1.58 (0.60–3.21)a 1.65 (0.30–11.11) 3.03 (1.33–7.69) 24.64 (5.21–57.26)b 4.01 (2.09–9.77)a
Note: Data is expressed in medians, with ranges in parentheses. PCOS ⫽ polycystic ovary syndrome; AA ⫽ adrenal androgen; T ⫽ testosterone; DHEAS ⫽ dehydroepiandrosterone sulfate; PREG ⫽ pregnenolone; P4 ⫽ progesterone; 17-HPREG ⫽ 17-hydroxypregeneolone; 17-HP ⫽ 17␣-hydroxyprogesterone; DHEA ⫽ dehydroepiandrosterone; A4 ⫽ androstenedione. a,b Significant difference (P⬍.05) between pairs. Moran. Hyperresponsiveness to ACTH in PCOS. Fertil Steril 2004.
Hormonal Assays Dehydroepiandrosterone sulfate, A4, P4, and 17-HP were measured by direct radioimmunoassays (RIAs) using commercially available kits (DHEAS, A4, and P4 by Diagnostic System Laboratories, Webster, TX; and 17-HP by Diagnostic Products Corporation, Los Angeles, CA). The levels of 17-HPREG, PREG, and DHEA were assayed by in-house methods after chromatography, as previously described (10, 13, 18). Total T was measured by an in-house method after serum extraction, as previously reported (19). The activity of SHBG was determined by equilibrium dialysis, and free T was calculated, as previously described (20). All samples were batched in the same assay to eliminate interassay variations. The intraassay coefficient of variation for all steroids did not exceed 9%.
Statistical Analysis Data were expressed as median and range. Statistical software packages (Kwikstat 4; Texas Soft, Cedar Hill, TX) were used to perform clinical and hormonal comparisons among groups by the Kruskal–Wallis nonparametric analysis of variance (ANOVA). A value of P⬍.05 was considered statistically significant.
RESULTS Clinical and Hormonal Characteristics of the Study Groups As expected and by design, PCOS patients with and without AA excess, as well as controls, had similar median [range] ages (24 [21– 40] years, 29 [20 –33] years, and 28 [19 –32] years, respectively) and BMI (29.1 [18.0 –39.9] kg/m2, 32.0 [20.7–37.4] kg/m2, and 25.9 [19.6 –36.8] kg/m2, respectively). Polycystic ovary syndrome patients with and 128
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without AA excess had similar median [range] hirsutism scores (modified F-G: 8 [3–16] and 8 [7–14], respectively). Six of the nine PCOS patients with AA excess and all nine PCOS patients without AA excess were considered hirsute (F-G ⱖ6). Overall, PCOS women, whether with or without AA excess, had higher total and free T levels and lower PREG0 compared with controls (Table 1). As expected, the median DHEAS levels were higher among PCOS patients with AA excess, compared with PCOS patients without AA excess or control women. Polycystic ovary syndrome patients with AA excess had significantly greater levels of DHEA0 than PCOS patients without AA excess, but not controls. In addition, PCOS patients without AA excess had significantly lower levels of DHEA0 than controls. Polycystic ovary syndrome patients with AA excess also had significantly higher levels of A40 than controls, although the difference with PCOS patients without AA excess did not reach significance. Levels of PREG0 were lower in both PCOS women with and without AA excess, compared with controls. Levels of P40 were higher in both groups of PCOS patients compared with controls, although the difference only reached significance for PCOS patients without AA excess.
Comparing the ACTH-Stimulated Hormonal Levels Consistent with the differences in the basal hormonal profile, PCOS patients with AA excess had significantly greater levels of A460 than PCOS patients without AA excess (Table 2). Compared with controls, PCOS patients with AA excess also had significantly higher levels of A460, but significantly lower levels of PREG60. Polycystic ovary syndrome patients without AA excess had significantly lower PREG60 levels than control women. There were no other Vol. 81, No. 1, January 2004
TABLE 2 ACTH-stimulated steroid (Steroid60) levels in PCOS patients with and without AA excess, and in control women.
Steroid PREG60 (nmol/L) P460 (nmol/L) 17-HPREG60 (nmol/L) 17-HP60 (nmol/L) DHEA60 (nmol/L) A460 (nmol/L)
PCOS patients with AA excess (n ⫽ 9)
PCOS patients without AA excess (n ⫽ 9)
Control women (n ⫽ 12)
18.39 (0.63–31.70)a 5.28 (3.47–8.33) 21.93 (13.22–60.08) 13.68 (8.17–16.67) 43.72 (34.70–121.45) 11.17 (8.73–17.45)a,b
26.31 (9.19–31.70)b 5.28 (3.24–6.49) 13.82 (7.81–25.23) 11.38 (6.75–13.86) 32.97 (14.23–57.26) 7.68 (4.89–10.47)a
51.99 (30.12–84.32)a,b 4.73 (3.05–7.60) 15.17 (9.01–22.53) 12.07 (6.11–20.09) 70.44 (18.04–117.98) 6.28 (2.44–9.42)b
Note: Data is expressed in medians, with ranges in parentheses. PCOS ⫽ polycystic ovary syndrome; AA ⫽ adrenal androgen; PREG ⫽ pregnenolone; P4 ⫽ progesterone; 17-HPREG ⫽ 17-hydroxypregnenolone; 17-HP ⫽ 17␣-hydroxyprogesterone; DHEA ⫽ dehydroepiandrosterone; A4 ⫽ androstenedione. a,b Significant difference (P⬍.05) between pairs. Moran. Hyperresponsiveness to ACTH in PCOS. Fertil Steril 2004.
significant differences in the ACTH-stimulated hormonal levels among the groups.
Comparing the Estimated Adrenocortical Enzymatic Studying the enzymatic activity estimated after ACTH stimulation or peak enzymatic activity, we observed that PCOS patients with AA excess had significantly higher ⌬517-OH activity than PCOS patients without AA excess (Table 3). In addition, we found that PCOS patients with AA excess had significantly higher activities of ⌬517-OH, ⌬417,20-lyase, C21m-3-HSD, and C19-3-HSD, as well as significantly lower ⌬517,20-lyase, than controls (Table 3). Likewise, PCOS patients without AA excess had an ACTHstimulated enzymatic activity for ⌬417,20-lyase, C21m-3HSD, and C19-3-HSD that was significantly higher, and a significantly lower ⌬517,20-lyase activity in comparison with control women, although the ⌬517-OH activity was not
different from controls. Overall, the only differences between PCOS patients with and without AA excess were significantly higher stimulated ⌬517-OH activity in the former.
DISCUSSION We had hypothesized that AA excess in PCOS is due to a generalized exaggeration in the AA response to ACTH stimulation. Contrary to our hypothesis, our data suggests that the exaggerated production of AAs appears to be primarily associated with an increase in ⌬517-OH activity. All other differences, including a higher ⌬417,20-lyase, C21- and C19-3-HSD, and a lower ⌬517,20-lyase compared with controls appeared to affect both PCOS subgroups equally. Thus, it would appear that the down-regulation of ⌬517,20lyase and up-regulation of 3-HSD activation is a feature of PCOS in general. We should note that ⌬5, not ⌬417,20-lyase,
TABLE 3 Estimated adrenocortical enzymatic activities based on peak steroid levels following ACTH-(1–24) stimulation in PCOS patients with and without AA excess, and in control women. Enzymatic activity ⌬517-OH ⌬517,20-lyase ⌬417-OH ⌬417,20-lyase C21m-3-HSD C21g-3-HSD C19g-3-HSD
Steroid measures (product/precursor)
PCOS patients with AA excess (n ⫽ 9)
PCOS patients without AA excess (n ⫽ 9)
Control women (n ⫽ 12)
17-HPREG60/PREG60 DHEA60/17-HPREG60 17-HP60/P460 A460/17-HP60 P460/PREG60 17-HP60/17-HPREG60 A460/DHEA60
1.90 (0.44–21.80)a,b 2.02 (0.95–3.16)a 2.51 (1.99–3.77) 0.91 (0.71–1.07)a 0.27 (0.18–7.27)a 0.51 (0.28–1.02) 0.22 (0.12–0.40)a
0.55 (0.27–2.55)a 2.42 (1.44–4.22)b 2.14 (1.66–2.73) 0.74 (0.61–0.91)b 0.21 (0.10–0.62)b 0.71 (0.44–1.46) 0.25 (0.15–0.39)b
0.25 (0.20–0.50)b 4.52 (1.82–7.24)a,b 2.50 (2.00–3.38) 0.53 (0.40–0.65)a,b 0.09 (0.05–0.15)a,b 0.76 (0.60–1.11) 0.10 (0.06–0.19)a,b
Note: Data is expressed in medians with ranges in parentheses. ACTH ⫽ adrenocorticotropic hormone; PCOS ⫽ polycystic ovary syndrome; AA ⫽ adrenal androgen; 17-OH ⫽ 17-hydroxylase; 17-HPREG ⫽ 17-hydroxypregnenolone; PREG ⫽ pregnenolone; DHEA ⫽ dehydroepiandrosterone; P4 ⫽ progesterone; A4 ⫽ androstenedione; 3-HSD ⫽ 3-hydroxysteroid dehydrogenase; C21m represents the mineralocorticoid (17-deoxy) pathway; C21g represents the glucorticoid (17-hydroxy) pathway; C19 represents the carbon-19 (sex-steroid) pathway. Moran. Hyperresponsiveness to ACTH in PCOS. Fertil Steril 2004.
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is the natural pathway toward androgen production in humans (21). We note that basal DHEA levels are similar between controls and PCOS patients with AA excess, while PCOS patients without AA excess have lower levels than either group. Although this does not invalidate the interpretation of the data comparing PCOS patients with and without AA excess, it does suggest that PCOS patients, in general, may have increased sulfotransferase activity such that there are proportionally higher DHEAS levels for an equivalent amount of DHEA in hyperandrogenized women. We previously suggested that an increase in sulfotransferase activity could potentially be secondary to circulating testosterone (T) (8). The suggestion that exaggerated ⌬517-OH activity is responsible for AA excess is consistent with our data and that of other investigators. For example, we previously reported in an unrelated population of 92 hyperandrogenic patients that 57% of all patients had an exaggerated basal activity of ⌬517-OH, although few, if any, had increased ⌬417-OH, ⌬517,20-lyase, or ␦417,20-lyase activities (13). Nonetheless, in that study we did not consider separately the subgroup of patients with AA excess. In agreement, Rosenfield et al. have observed that a significant proportion of their hyperandrogenic women appeared to have excessive 17-OH activity and high, yet partially down-regulated, ⌬417,20-lyase activity in response to either ovarian (with a GnRH agonist [GnRH-a]) or adrenal (with ACTH-[1–24]) stimulation (22, 23). Again, these latter investigators did not attempt to a priori recruit patients with and without AA excess. Although the finding of an exaggerated ⌬517-OH activity in PCOS patients with AA excess appears to be relatively consistent, the underlying mechanism(s) is(are) not clear. P450c17␣ is a single microsomal enzyme that catalyzes both the ⌬5 and ⌬417-OH activity for C21 steroids. This enzyme also catalyzes the ⌬5 and ⌬417,20-lyase activities that cleave the C17-C20 bond to produce C19 sex steroids, although we should note that there is strong evidence that ⌬417,20-lyase activity is relatively minimal in normal human steroidogenesis (23). Some investigators have suggested the observed differences in steroid levels are the result of a “dysregulation” of P450c17␣ (21, 22). In fact, clinical and in vitro observations indicate that 17-OH and 17,20-lyase activities of P450c17␣ are independently regulated (24). Although the relative activity of 17,20-lyase appears to be regulated by the amount of available electron donors, such as P450 oxidoreductase or cytochrome b5, 17-OH activity appears to be less readily modifiable (23, 24). Unfortunately, molecular and/or genetic studies to date do not support the concept that P450c17␣ is abnormal in PCOS. The gene CYP17 encodes for P450c17␣ in both the adrenal cortex and the ovary. Although several CYP17 mutations have been reported, all have been associated with P450c17␣ enzymatic deficiencies, and none have been noted to result in 130
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either 17-OH or 17,20-lyase hyperactivity (25). Although it is possible that genetically determined posttranscriptional modifications of P450c17␣ in vivo might result in the relative enhancement of ⌬517-OH activity in some patients, this remains to be demonstrated. It is also unlikely that the intracellular environment alters P450c17␣ function. Using skin fibroblasts from PCOS patients transfected with human P450c17␣, Martens et al. were unable to detect an abnormality in the ratio of the 17-OH to 17,20-lyase activities of the enzyme in the fibroblasts of patients with PCOS, compared with controls (26). It is tempting to suggest that the observed differences in circulating steroids arises from an abnormal function, inherited or otherwise, of the enzymes within the adrenal cortex. For example, it has been well documented that mutations of CYP21, leading to the production of an abnormal P450c21, result in very high levels of the immediate 17-hydroxy precursor, 17-HP (27). Alternatively, it is much more difficult to attribute subtle differences in circulating steroids to an alteration in enzymatic function, as the observed steroid levels in blood are many levels removed from the actual intracellular enzyme in the adrenal cortex. For example, in the present study significant differences are observed between groups in the estimated C21m-3-HSD and C19-3HSD activities. Using microsomes from yeast-expressing human type II 3-HSD, however, Lee et al. were unable to detect any kinetic differences in the activity of this enzyme for C21-17-deoxy, C21-17-hydroxy, and C19 precursors (28). Simard et al. reported similar findings in transfected COS-1 cells (29). Thus, it is not generally possible to infer from in vivo physiologic studies that the activity of a particular intracellular enzyme (e.g., P450c17␣) is altered in a subtle manner. In conclusion, although PCOS per se is associated with multiple changes in steroidogenesis that favor an exaggeration in adrenocortical secretion, AA excess in PCOS appears to be uniquely associated with an increased ⌬517-OH activity. Mechanistically, it is unlikely that the exaggerated ⌬517-OH activity observed is due to an intrinsic defect in P450c17␣; however, it is possible that alterations in the intracellular trafficking of steroids, in the degradative pathways of the steroids, or in intrinsic adrenal blood flow affecting precursor availability may be responsible.
Acknowledgments: The authors thank Walter L. Miller, M.D., for his review and constructive criticism of the manuscript.
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