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Correlation of adrenocorticotropin steroid levels between women with polycystic ovary syndrome and their sisters
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Correlation of adrenocorticotropin steroid levels between women with polycystic ovary syndrome and their sisters Mark O. Goodarzi, MD, PhD; Xiuqing Guo, PhD; Bulent O. Yildiz, MD; Frank Z. Stanczyk, PhD; Ricardo Azziz, MD, MBA, MPH OBJECTIVE: The purpose of this study was to assess the sibling correlation of adrenocorticotropic hormone–stimulated steroid hormone levels between probands with polycystic ovary syndrome and their sisters.
nificantly correlated between siblings (r ⫽ 0.47, P ⫽ .01 and r ⫽ 0.57, P ⫽ .01, respectively); a similar trend was observed for the 60minute A4 values (r ⫽ 0.29, P ⫽ .06).
STUDY DESIGN: Twenty-seven women with polycystic ovary syndrome and
CONCLUSION: Women with polycystic ovary syndrome and their sisters have significantly correlated levels of adrenocorticotropic hormone–stimulated steroids, supporting a genetic basis of the adrenal androgen excess observed in polycystic ovary syndrome.
28 of their sisters underwent an adrenocorticotropic hormone stimulation test with measurement of the steroids dehydroepiandrosterone, androstenedione, and cortisol 60 minutes later. The 60-minute values were used to calculate sister-sister correlations by regression analyses. RESULTS: The adrenocorticotropic hormone–stimulated (60-minute)
log-transformed dehydroepiandrosterone and cortisol values were sig-
Key words: adrenal, androgen excess, genetics, polycystic ovary syndrome, sibling correlation
Cite this article as: Goodarzi MO, Guo X, Yildiz BO, et al. Correlation of adrenocorticotropin-stimulated steroid levels between women with polycystic ovary syndrome and their sisters. Am J Obstet Gynecol 2007;196:398.e1-398.e6.
P
olycystic ovary syndrome (PCOS) is a highly prevalent hyperandrogenic disorder. PCOS affects approximately 7% of reproductive-aged women1 and is associated with menstrual dysfunction, polycystic ovaries, and hirsutism, and an increased risk of
infertility, diabetes mellitus (DM), and possibly cardiovascular disease (CVD). Although multiple sets of diagnostic criteria have been proposed for PCOS, a common feature of all of them is hyperandrogenism.2,3 Androgen biosyn-
From the Division of Endocrinology, Diabetes and Metabolism, Department of Medicine (Dr Goodarzi), the Department of Obstetrics and Gynecology (Drs Goodarzi and Azziz), and the Medical Genetics Institute (Drs Goodarzi and Guo), Cedars-Sinai Medical Center, Los Angeles, CA; the Departments of Medicine (Drs Goodarzi and Azziz) and Obstetrics and Gynecology (Dr Azziz), the David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA; the Division of Endocrinology and Metabolism, Department of Internal Medicine, Hacettepe University School of Medicine, Ankara, Turkey (Dr Yildiz); and the Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Southern California Keck School of Medicine, Los Angeles, CA (Dr Stanczyk). Presented at the 25th Annual Meeting of the American Gynecological and Obstetrical Society, Sept. 14-16, 2006, Williamsburg, VA. Received Oct. 21, 2006; accepted Dec. 11, 2006. Reprints: Ricardo Azziz, MD, MPH, MBA, Department of Obstetrics/Gynecology and Center for Androgen Related Disorders, Cedars-Sinai Medical Center, 8635 W Third St, Suite 160W, Los Angeles, CA 90048; [email protected]. This study was supported in part by the Helping Hand of Los Angeles, and by grants R01HD29364 and K24-HD01346 from the National Institutes of Health (R.A.) and the Cedars-Sinai General Clinical Research Center grant RR000425 (X.G.). Financial disclosure: Dr Azziz has received consulting fees from Procter and Gamble, Merck and Co, and Organon. 0002-9378/$32.00 © 2007 Mosby, Inc. All rights reserved. doi: 10.1016/j.ajog.2006.12.009
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thesis in both the ovary and the adrenal is upregulated and exaggerated in PCOS. Ovarian androgen production in response to human chorionic gonadotropin (hCG) or long-acting gonadotropinreleasing hormone analogue (GnRH-a) stimulation is exaggerated in PCOS.4,5 Similarly, adrenocortical steroidogenesis and adrenal androgen (AA) secretion are exaggerated after adrenocorticotropic hormone (ACTH) stimulation.6,7 Abnormalities of steroidogenesis may be more clearly investigated in the adrenal than the ovary. Ovarian function in vivo is confounded by various factors, including obesity, intermittent ovulation, and hyperinsulinism, whereas adrenocortical steroidogenesis appears to be primarily affected by the diurnal rhythm and only modestly affected by extraadrenal or acquired factors.7-11 Adrenocortical dysfunction and AA excess may be the result of inherited (intrinsic or extrinsic to the adrenal glands) or acquired factors. Adrenocortical function in PCOS and healthy patients likely is a complex trait, modulated by both inherited and acquired factors. Preliminary evidence suggests that adrenocortical steroidogenesis and AA excess in
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www.AJOG.org PCOS are primarily determined by inherited intrinsic factors. Studies in brothers and sisters of patients with PCOS indicate that AA levels, reflected by the levels of the AA metabolite circulating dehydroepiandrosterone sulfate (DHEAS), primarily reflect the impact of genetic factors.12,13 However, circulating DHEAS levels may not fully reflect the AA biosynthesis as measured by the adrenocortical response to acute ACTH stimulation. For example, exogenous testosterone administration increases the circulating DHEAS/DHEA ratio of oophorectomized women without altering measurable adrenocortical steroidogenesis in response to ACTH.8 Consequently, in this study, we sought to determine whether ACTH-stimulated AA steroid levels are inherited traits in families of women with PCOS. We hypothesize that adrenocortical biosynthesis in PCOS is highly heritable and, consequently, that the ACTH-stimulated steroid levels between women with PCOS and their sisters will be highly correlated.
M ATERIALS AND M ETHODS Subjects We recruited 27 PCOS probands and 28 of their sisters as part of study of the heritability of PCOS and its features. The study was approved by the Institutional Review Boards of University of Alabama at Birmingham, Birmingham, AL (UAB), Cedars-Sinai Medical Center, Los Angeles, CA (CSMC), and Haceteppe University, Ankara, Turkey, and all subjects gave written consent.
PCOS proband Patients were recruited from the reproductive or medical endocrinology clinics at UAB, CSMC, and Hacettepe University. None had used hormonal preparations, including oral contraceptives, for at least 3 months preceding the study. Patients were excluded if they were pregnant, prepubertal, or menopausal. After undergoing a history and physical examination, blood sampling was performed for the measurement of total testosterone (T), calculated free T, sex hormonebinding globulin (SHBG), DHEAS, and
17-hydroxyprogesterone (17-HP) (see below). The presence of PCOS was defined by criteria arising from a National Institutes of Child Health and Human Development–sponsored conference in 1990, including: (1) clinical evidence of hyperandrogenism and/or hyperandrogenemia, (2) oligoovulation, and (3) the exclusion of related disorders.2 These criteria are also consistent with those of a consensus conference sponsored by the European Society for Human Reproduction and Embryology and the American Society for Reproductive Medicine in Rotterdam in 2003.3 Although these newer criteria have indicated that the presence of polycystic ovaries detected on ultrasonography could also be used as criteria for PCOS, the proceedings were also clear that research criteria might be more or less inclusive. Consequently, as we have the greatest familiarity with this diagnostic scheme and because obligatory transvaginal ultrasonography may be perceived as excessively invasive and may hamper our ability to recruit patients and their sisters, we used the 1990 National Institutes of Health criteria. Oligoovulation was defined as menstrual cycles more than 45 days in length or less than 8 cycles per year, or by a luteal phase progesterone (P4) level less than 4 ng/mL in conjunction with a monophasic basal body temperature chart, if menstrual cycles were less than 45 days in length. Hirsutism was defined as a modified Ferriman-Gallwey (mF-G) score 6 or greater.1,14 For the purpose of this study, hyperandrogenemia was defined as total T greater than 83.6 ng/dL (2.96 nmol/L), free T greater than 0.81 ng/dL (0.026 nmol/L), or DHEAS greater than 2540 ng/mL (6.64 mol/L), as previously reported.1 Patients with 21-hydroxylase deficient nonclassic adrenal hyperplasia (NCAH) were excluded by a basal follicular phase 17-HP level less than 2 ng/mL (6.0 nmol/L), or a 17-HP level after acute ACTH stimulation of less than 10 ng/mL (30.3 nmol/L), as previously described.15 Hyperprolactinemia and hypothyroidism were excluded by normal levels of prolactin and thyroid-stimulating hormone, respectively; Cushing’s syndrome and andro-
genic tumors were excluded by appropriate testing, if suspected clinically.
Sisters Twenty-eight sisters were contacted and recruited after consent from the proband. Twenty-six probands had 1 sister, whereas 1 proband had 2 sisters. All completed a history and physical examination, and provided a basal blood sample, after appropriate informed written consent.
Clinical protocol Early morning blood samples were obtained from each subject between 8:00 AM and 10:30 AM after an overnight fast, in the follicular phase (days 3-8) of menstrual cycle, or after a progestogen-induced bleed. Probands with PCOS were off medication for at least 3 months before the study, whereas this was not required for sisters. The test samples from the probands and sisters were assayed in the same run, to minimize the effect of interassay variability. All subjects underwent an acute adrenal stimulation test using 0.25 mg ACTH-1-24, as previously described.16 Because of the effect of the circadian rhythm on adrenocortical function all stimulations were performed between 7:00 and 9:00 AM. After a rest period after the placement of an intravenous angiocath and heparin lock, 3 blood samples were drawn at ⫺30 minutes, ⫺15 minutes, and 0 minutes, and mixed to form the baseline sample. Afterward, 0.25 mg (1 vial) of ACTH-1-24 (Cortrosyn, Organon Co, New Orange, NJ) was then injected intravenously over 60 seconds, and a final blood sample obtained 60 minutes later. In the baseline samples we measured total and free T, SHBG, and DHEAS; in both the baseline and 60minute samples we measured DHEA, androstenedione (A4), and cortisol (F). The parameters calculated from the acute ACTH stimulation results were as previously described,16 including the basal (0 minutes), peak levels (60 minutes), and net increment (Steroid0, Steroid60, ⌬Steroid, respectively) for A4, DHEA, and F. We have previously demonstrated that these parameters are
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TABLE 1
Clinical characteristics and results of baseline and ACTH-stimulated steroids in probands and sisters Trait
Proband
Sister
P value*
Age (y)
28.3 ⫾ 10.7
27.5 ⫾ 8.3
.79
BMI (kg/m )
34.5 ⫾ 7.5
29.7 ⫾ 8.5
.037
Waist-hip ratio
0.84 ⫾ 0.11
0.83 ⫾ 0.07
.74
Total testosterone (ng/dL)
70.4 ⫾ 37.2
47.8 ⫾ 28.3
.013
Free testosterone (pg/mL)
18.6 ⫾ 10.4
10.7 ⫾ 7.3
.0017
DHEAS (g/dL)
234.7 ⫾ 140.6
155.6 ⫾ 101.5
.019
SHBG (nmol/L)
24.7 ⫾ 15.0
38.0 ⫾ 17.6
.004
A40 (ng/mL)
1.87 ⫾ 0.87
1.25 ⫾ 0.59
.003
DHEA0 (ng/mL)
7.51 ⫾ 6.68
4.54 ⫾ 3.17
.038
9.3 ⫾ 6.2
8.7 ⫾ 4.7
.70
.............................................................................................................................................................................................................................................. 2 .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. ..............................................................................................................................................................................................................................................
F (g/dL)
0 ..............................................................................................................................................................................................................................................
Steroid0 refers to basal unstimulated basal levels. * P value from unpaired 2-sided t-test.
highly stable in the short- and longterm.16,17 Alternatively, although multiple other calculations can be performed (eg, steroid ratios to estimate enzyme activities) we have observed that these parameters are more variable over time within patients16 and consequently were not be used in estimating sister correlation.
Assays Serum was frozen at ⫺70°C until assayed. We performed assays at regular intervals during this study, batching samples from the same family when analyzed. The specifics for the assays for A4, DHEA, DHEAS, total and free T, SHBG, glucose, and insulin have been previously described.9,11,17 The intraassay variances are consistently less than 10% and the interassay less than 15%.
Statistical analysis Phenotypic parameters are given as mean ⫾ SD. Mean trait values were compared between the probands and sisters
by using 2-sided unpaired t-tests. Because many of the parameters being tested demonstrated a skewed distribution, all adrenal response parameters were log-transformed for analysis. Familial correlation r values between probands and their sister pairs were estimated by regressing the Steroid60 and ⌬Steroid values in sisters against that in the corresponding probands. As a control analysis, the sister data were shuffled and then remerged to the proband data, resulting in 27 probands unrelated sister pairs; the adrenal steroid response regressions were then carried out among these unrelated subject pairs.
R ESULTS The mean age and waist-hip ratio was similar in proband and sister groups, whereas probands had a higher mean body mass index (BMI) than sisters (Table 1). Twenty-five probands were white, whereas 2 proband-sister pairs were black. The probands had higher baseline levels of total T, free T, DHEA, DHEAS,
A4, and lower SHBG than their sisters. There were no differences in baseline F levels. The ACTH-stimulated (60-minute) log-transformed DHEA and F values were significantly correlated between siblings (r ⫽ 0.47, P ⫽ .01 and r ⫽ 0.57, P ⫽ .01, respectively); a similar trend was observed for the 60-minute A4 values (r ⫽ 0.29, P ⫽ .06) (Table 2). Similarly, the log-transformed net increment in A4 (⌬A4) and F (⌬F) were significantly correlated between siblings (Table 2). A similar trend was observed for ⌬DHEA. Three sisters were receiving hormonal therapy; elimination of their data did not materially affect the correlations reported previously. Repeating the correlations after randomly scrambling the sibling-pairs yielded no significant correlations in adrenal response parameters (data not shown).
C OMMENT Although the majority of patients with PCOS demonstrate an ovarian source for their high androgen secretion, many also display AA excess. The measurement of circulating DHEAS has been often used as a marker of AA secretion (and excess), because this metabolite is as follows: (a) 97-99% of adrenocortical origin, (b) the most abundant steroid, (c) relatively stable throughout the day and the menstrual cycle, and (d) easily measured. With the use of racial and age normative values, we have determined the prevalence of absolute (ie, ⬎95th percentile of normal) DHEAS excess to be approximately 25%.18 Although DHEAS is a useful marker of AA secretion in epidemiologic studies, it reflects both AA production and DHEA sulfotransferase activity. As our studies suggest that extraadrenal factors may modulate DHEA sulfotransferase activity to a
TABLE 2
Correlation coefficients between PCOS probands and their sisters for the ACTH-stimulated steroid parameters log A460 (ng/mL)
log ⌬A4
log DHEA60 (ng/mL)
log ⌬DHEA
log F60 (g/dL)
log ⌬F
r value
0.29
0.48
0.47
0.14
0.57
0.33
P value
.06
.03
.01
.46
.01
.05
................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................
Steroid60 refers to peak ACTH-stimulated basal levels and ⌬Steroid to net increment from baseline (0 minutes) to Steroid60.
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www.AJOG.org greater degree than AA production in response to ACTH,7-11 we chose to focus the current investigation on the sibling correlation of ACTH-stimulated steroid levels. ACTH-stimulated steroids are most reflective of adrenal secretory function and also appear to be elevated in PCOS.6,7 We found substantial sibling-sibling correlation for ACTH-stimulated steroids, including DHEA, A4, and F. We noted that the sister correlation was highest for F, possibly a reflection of the need for precisely regulating F levels to maintain optimal health and immune function. The correlations for these 3 steroids appears higher than the correlation between sisters for DHEAS alone (0.28),12 consistent with our suggestion that DHEAS varies more in response to acquired or extraadrenal factors compared with ACTH-stimulated steroid levels.7-11 These data strongly suggest that genetic factors play an important role in determining the response of adrenal steroids to ACTH stimulation, although the population size must be expanded to confirm this. Besides contributing to the hyperandrogenism in PCOS, AA excess is relevant in that it may be a risk factor for PCOS. Various data support the concept that AA excess, particularly when evident during pubertal development, increases the risk for developing PCOS. For example, patients with premature adrenarche are at higher risk for the development of PCOS than their nonaffected peers.19 Furthermore, other investigators have suggested that stress resulting in exaggerated AA secretion increases the risk of developing PCOS.20 Most evidence currently indicates that the effect of extraadrenal factors on adrenocortical dysfunction and AA excess in PCOS is modest at best. By using GnRH-a suppression9 and oophorectomy,11 exogenous estrogen and testosterone replacement,8 and correlation studies with measures of insulin secretion or action,10 we have observed that extraadrenal factors play only a small role, if any, in determining the response of the adrenal to ACTH in normal and PCOS patients, and primarily modestly alter sulfotransferase activity and thus
the circulating levels of the AA metabolite DHEAS levels. Overall, the modest role of extraadrenal modulators on the adrenal in PCOS supports the hypothesis of an underlying genetic cause in the abnormal adrenocortical steroidogenesis and metabolism, and AA excess, of PCOS. Various data support the concept of an inherited basis for AA excess in PCOS. First, it is clear that the levels of the AA metabolite DHEAS are under significant genetic control as evidenced in twin20 and family21,22 studies. The brothers (n ⫽ 119) of 87 unrelated PCOS women had significantly elevated DHEAS levels compared with control men, and a positive linear relationship between DHEAS in probands and their brothers was observed.13 In PCOS proband–sister pairs we have also observed a significant correlation in DHEAS levels and the AA response to ACTH stimulation between the proband and her untreated sister.12 Despite the number of studies examining heritability of DHEAS, we are not aware of any study that has examined heritability or sister correlation of ACTH-stimulated steroid levels. In terms of what actual genes may be driving AA excess, a small number of studies have examined candidate genes for association with AA, mostly DHEAS, levels, with largely negative results.23,24 We recently discovered association of single nucleotide polymorphisms in the gene for DHEA sulfotransferase (SULT2A1) with circulating DHEAS in a population of approximately 300 PCOS and approximately 200 controls.25 Alternatively, other investigators have observed an association between the gene for 11-hydroxysteroid dehydrogenase, type 1 (HSD11B1), which primarily catalyzes the conversion of cortisone to F, and DHEAS levels in PCOS.26 Overall, these data provide further support, and an underlying explanation, for the role of inherited factors and genetic variants in determining adrenocortical function and DHEAS levels in PCOS. In this study, we present sister-sister correlation data as evidence of inheritance. Another commonly used parameter to quantify the inherited nature of a trait is heritability (h2). In accordance
with established quantitative genetic theory, the total phenotypic variance in a trait (P2) can be partitioned into the variance because of the additive effects of genes (G2) and the variance caused by environmental effects (E2). Heritability (h2) can be estimated by the proportion of the phenotypic variance caused by the additive effects of genes, ie, G2/P2 and tested by using maximum likelihood methods. However, in the current study we were unable to directly calculate heritability in this manner given the limited sample size and the lack of control data from unrelated subjects; however, our data allow an estimation of the upper bound of heritability, which is double the sibling correlation value. Thus, the upper bounds of heritability of DHEA60, A460, and F60 are 0.94, 0.58, and 1.0, respectively, indicating a majority of variance in these traits is due to genetic factors. We should note that correlation or heritability estimates cannot distinguish between inherited factors (genetic effects) vs effects arising from shared environmental factors. Shared environmental factors among siblings can inflate these estimates. Our data support the hypothesis that adrenocortical steroidogenesis is under significant genetic control and likely reflects variations in the genes controlling their biosynthesis and/or metabolism. Which actual genes may be driving the AA excess remain to be determined, and large-scale genotype-phenotype association studies, evaluating ACTH-stimulated steroid values as the relevant phenotype, are needed. f REFERENCES 1. Knochenhauer ES, Key TJ, Kahsar-Miller M, Waggoner W, Boots LR, Azziz R. Prevalence of the polycystic ovary syndrome in unselected black and white women of the southeastern United States: a prospective study. J Clin Endocrinol Metab 1998;83:3078-82. 2. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril 2004;81: 19-25. 3. Zawadzki JK, Dunaif A. Diagnostic criteria for polycystic ovary syndrome: towards a rational approach. In: Dunaif A, Givens JR, Haseltine F, Merriam GR, eds. Polycystic ovary syndrome. Cambridge, England: Blackwell Scientific Publications; 1992.
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AGOS Papers 4. Piltonen T, Koivunen R, Perheentupa A, Morin-Papunen L, Ruokonen A, Tapanainen JS. Ovarian age-related responsiveness to human chorionic gonadotropin in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2004;89:3769-75. 5. Rosenfield RL, Barnes RB, Ehrmann DA. Studies of the nature of 17-hydroxyprogesterone hyperresonsiveness to gonadotropin-releasing hormone agonist challenge in functional ovarian hyperandrogenism. J Clin Endocrinol Metab 1994;79:1686-92. 6. Moran C, Reyna R, Boots LS, Azziz R. Adrenocortical hyperresponsiveness to corticotropin in polycystic ovary syndrome patients with adrenal androgen excess. Fertil Steril 2004;81:126-31. 7. Azziz R, Rittmaster RS, Fox LM, Bradley EL Jr, Potter HD, Boots LR. Role of the ovary in the adrenal androgen excess of hyperandrogenic women. Fertil Steril 1998;69:851-9. 8. Chang WY, Stanczyk F, Bartolucci A, Azziz R. Effect of bilateral oophorectomy on basal and adrenocorticotropin (ACTH)-stimulated adrenal androgen (AA) secretion in polycystic ovary syndrome (PCOS). Paper presented at: 86th Annual Meeting of the Endocrine Society; June 16-19, 2004; New Orleans, LA. 9. Azziz R, Gay FL, Potter SR, Bradley E Jr, Boots LR. The effects of prolonged hypertestosteronemia on adrenocortical biosynthesis in oophorectomized women. J Clin Endocrinol Metab 1991;72:1025-30. 10. Slayden SM, Crabbe L, Bae S, Potter HD, Azziz R, Parker CR Jr. The effect of 17 betaestradiol on adrenocortical sensitivity, responsiveness, and steroidogenesis in postmenopausal women. J Clin Endocrinol Metab 1998;83:519-24. 11. Farah-Eways LA, Reyna R, Knochenhauer ES, Bartolucci A, Azziz R. Glucose action and adrenocortical biosynthesis in the polycystic ovary syndrome. Fertil Steril 2004;81:120-5. 12. Azziz R, Trader BT, Pall M, Yildiz BO, Stanczyk F. Heritability of adrenal androgen (AA) secretion in sisters of women with polycystic ovary syndrome (PCOS). Paper presented at: 88th Annual Meeting of the Endocrine Society; June 24-27, 2006; Boston, MA. 13. Legro RS, Kunselman AR, Demers L, Wang SC, Bentley-Lewis R, Dunaif A. Elevated dehydroepiandrosterone sulfate levels as the reproductive phenotype in the brothers of women with polycystic ovary syndrome. J Clin Endocrinol Metab 2002;87:2134-8. 14. Hatch R, Rosenfield RL, Kim MH, Tredway D. Hirsutism: implications, etiology, and management. Am J Obstet Gynecol 1981;140: 815-30. 15. Azziz R, Hincapie LA, Knochenhauer ES, Dewailly D, Fox L, Boots LR. Screening for 21hydroxylase-deficient nonclassic adrenal hyperplasia among hyperandrogenic women: a prospective study. Fertil Steril 1999;72:915-25. 16. Azziz R, Bradley E Jr, Huth J, Boots LR, Parker CR Jr, Zacur HA. Acute adrenocorticotropin-(1-24) (ACTH) adrenal stimulation in eu-
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www.AJOG.org menorrheic women: reproducibility and effect of ACTH dose, subject weight, and sampling time. J Clin Endocrinol Metab 1990;70:1273-9. 17. Azziz R, Yildiz BO, Woods KS, Stanczyk FZ, Bartolucci AA. Long-term variability of adrenal androgen (AA) levels and their response to corticotropin in the polycystic ovary syndrome (PCOS). Paper presented at: 86th Annual Meeting of the Endocrine Society; June 16-19, 2004; New Orleans, LA. 18. Kumar A, Woods KS, Bartolucci AA, Azziz R. Prevalence of adrenal androgen excess in patients with the polycystic ovary syndrome (PCOS). Clin Endocrinol (Oxf) 2005;62:644-9. 19. Ibanez L, Potau N, Virdis R, et al. Postpubertal outcome in girls diagnosed of premature pubarche during childhood: increased frequency of functional ovarian hyperandrogenism. J Clin Endocrinol Metab 1993;76: 1599-603. 20. Akamine Y, Kato K, Ibayashi H. Studies on changes in the concentration of serum adrenal androgens in pubertal twins. Acta Endocrinol (Copenh) 1980;93:356-64. 21. Rotter JI, Wong FL, Lifrak ET, Parker LN. A genetic component to the variation of dehydroepiandrosterone sulfate. Metabolism 1985; 34:731-6. 22. Rice T, Sprecher DL, Borecki IB, Mitchell LE, Laskarzewski PM, Rao DC. The Cincinnati Myocardial Infarction and Hormone Family Study: family resemblance for dehydroepiandrosterone sulfate in control and myocardial infarction families. Metabolism 1993;42: 1284-90. 23. Kahsar-Miller M, Boots LR, Bartolucci A, Azziz R. Role of a CYP17 polymorphism in the regulation of circulating dehydroepiandrosterone sulfate levels in women with polycystic ovary syndrome. Fertil Steril 2004;82:973-5. 24. Witchel SF, Kahsar-Miller M, Aston CE, White C, Azziz R. Prevalence of CYP21 mutations and IRS1 variant among women with polycystic ovary syndrome and adrenal androgen excess. Fertil Steril 2005;83:371-5. 25. Goodarzi MO, Antoine HJ, Azziz R. Genes for enzymes that metabolize DHEA influence levels of DHEA-sulfate in polycystic ovary syndrome (PCOS). Paper presented at: 4th Annual Meeting of the Androgen Excess Society; June 23, 2006; Boston, MA. 26. Gambineri A, Vicennati V, Genghini S, et al. Genetic variation in 11beta-hydroxysteroid dehydrogenase type 1 predicts adrenal hyperandrogenism among lean women with polycystic ovary syndrome. J Clin Endocrinol Metab 2006;91:2295-302.
D ISCUSSION Rogerio Lobo, MD. 1. The adrenocorticoptropin (ACTH)stimulated responses of cortisol (F) and androstenedione (A4) were correlated in women with
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2.
3.
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5.
6. 7.
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the polycystic ovary syndrome (PCOS) and their sisters. Please explain why this supports the claim of a genetic basis for adrenal androgen (AA) excess in PCOS. Other biochemical indices are correlated on a biochemical basis. Why were there no normal controls to determine how many women with PCOS and how many sisters had exaggerated responses to ACTH? Why were other steroids (eg, 17hydroxyprogesterone and 17-hydroxypregnenolone) not measured to gain insight into enzymatic dysregulation? Why were the sisters allowed to use oral contraceptive pills (OCPs)? This would affect basal levels of androgens. Why were other methods of determining ACTH responses not considered in light of lowered basal levels in users of OCPs? What was the cut-off value for an elevated DHEAS? Were the data normally distributed? Were parametric statistics used? How many sisters may have had PCOS (this could not be determined given the protocol used)? Can the authors speculate on whether these data may relate to other phenotypes of PCOS?
Dr Goodarzi (closing). There is variability (a range of values) of the stimulated hormone levels in the population. The premise is that traits that are genetically determined will correlate between related individuals, but not between unrelated individuals. Correlation of a trait between siblings is related to the heritability of a trait (which is the proportion of the total variance of the trait that is explained by genetic factors). Thus, if one sibling has a high dehydroepiandrosterone (DHEA) response to ACTH, we would expect other siblings to also have a high response if they tend to share the same genes that determine DHEA response. To rule out the possibility that the correlations we have observed are not re-
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www.AJOG.org lated to heritability, we scrambled the proband and sister data and then calculated the trait correlations between unrelated individuals. For all traits, there were no correlations between unrelated subjects. Notwithstanding, we agree that correlation (or even heritability) analyses only support the suggestion that the adrenal response to ACTH stimulation is inherited. More extensive family studies, and linkage studies when specific genetic markers of adrenal steroidogenesis are identified, are needed to confirm this hypothesis. We did not include healthy controls because we did not need to establish an “upper normal limit (UNL),” as we have previously established these ranges using large populations of healthy controls.1-6 In addition, we consider the ACTH response to represent a continuum, and we prefer not to artificially subdivide patients into those with and without an exaggeration in response above the UNL. We did not measure other steroidogenic or AA precursors because the primary endpoints were AA production, namely, DHEA) and A4; measuring other precursors may help understand the steroidogenic mechanisms underlying the excess. However, we have done this repeatedly previously,3-5 and there are limitations in resources to consider. We could not control whether sisters were or were not on OCPs, although we note that disorder itself, namely, polycystic ovary syndrome (PCOS), predisposes to the use of OCPs. We should note that OCP or other hormonal therapy would, if anything, decrease the degree of association, which we did not observe in our prior study analyzing the heritability of DHEAS.7 In agreement, in the current study only 3 sisters were receiving hormonal therapy at the time of their study, and their removal from the analysis did not change the results. We have found that using the peak (maximum) value poststimulation gives the most precise and reproducible value8; calculations of net increment, which could be done, in our experience increases significantly the
intrasubject variation, to as high as 60%. Nonetheless, we calculated the net increment (change from 0-60 minutes), which supported the findings using peak values. The UNL value for DHEAS used was 2750 ng/mL. However, as we have indicated, we now prefer to consider DHEAS as a continuous value, and not arbitrarily separate into normal and supranormal based on a percentile, because this belays: (a) the observation that PCOS women, in general, demonstrate varying degrees of hyperresponsivity, and (b) the fact that DHEAS is not only the product of AA secretion in response to ACTH, but also the result of DHEA sulfotransferase (DHEA-ST) activity, and, consequently, is only loosely related to the AA response to ACTH stimulation. We tested the data for skewness or kurtosis and found that about 60% of parameters were normally distributed. To account for the remainder, we log-transformed the data before analysis. We agree that with the protocol used we could not determine how many sisters had PCOS. We should note that we previously reported that 40-50% of sisters are affected with PCOS.9 However, it is possible that the prevalence of PCOS in the current cohort of sisters may be less, as they had significantly lower levels of androgens and body mass index. The fact that a significant association in adrenal response was found regardless further emphasizes the heritability of the ACTH-stimulated adrenal steroid production. We have previously observed that approximately 10% of PCOS patients solely have an elevated DHEAS.10 We have also observed that women with elevations in DHEAS tend to be younger, as would be expected, and thinner.11 Furthermore, some of these women may not have overt ovulatory dysfunction.11 Alternatively, the degree of adrenocortical dysfunction in response to ACTH, in contrast to basal levels of DHEAS, may possibly mirror the degree of ovarian dysfunction, and thus may not necessarily indicate a less affected individual. This hypothesis remains to be determined, although Ehrmann et al12 did not find a high degree of correlation between the results of stimulation with ACTH and the long-acting gonadotropin-releasing
hormone agonist nafarelin when studying 40 consecutive women with hyperandrogenism who had oligomenorrhea, hirsutism, or acne. REFERENCES 1. Azziz R, Boots LR, Parker CR Jr, Bradley E Jr, Zacur HA. 11-hydroxylase deficiency in hyperandrogenism. Fertil Steril 1991;55:733-41. 2. Azziz R, Zacur HA, Parker CR Jr, Bradley E Jr, Boots LR. Effect of obesity on the response to acute adrenocorticotropin stimulation in eumenorrheic women. Fertil Steril 1991;56: 427-33. 3. Azziz R, Bradley EL Jr, Potter HD, Boots LR. 3-Hydroxysteroid dehydrogenase deficiency in hyperandrogenism. Am J Obstet Gynecol 1993;168:889-95. 4. Azziz R, Bradley EL Jr, Potter HD, Boots L. Adrenocortical hyperactivity and androgen excess in hyperandrogenism: lack of role for 17hydroxylase and 17,20-lyase dysregulation. J Clin Endocrinol Metab 1995;80:400-5. 5. Moran C, Potter HD, Reyna R, Boots LR, Azziz R. Prevalence of 3-hydroxysteroid dehydrogenase deficient non-classic adrenal hyperplasia in hyperandrogenic women with adrenal androgen excess. Am J Obstet Gynecol 1999;181:596-600. 6. Azziz R, Fox LM, Zacur HA, Parker CR Jr, Boots LR. Adrenocortical secretion of dehydroepiandrosterone in healthy women: highly variable response to ACTH. J Clin Endocrinol Metab 2001;86:2513-7. 7. Yildiz BO, Goodarzi MO, Guo X, Rotter JI, Azziz R. Heritability of dehydroepiandrosterone sulfate among polycystic ovary syndrome women and their sisters. Fertil Steril 2006;86:1688-93. 8. Azziz R, Bradley E Jr, Huth J, Parker CR Jr, Boots LR, 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. 9. Kahsar-Miller M, Nixon C, Boots LR, Go RCP, Azziz R. Prevalence of the polycystic ovary syndrome (PCOS) among first degree relatives of patients with PCOS. Fertil Steril 2001;75:53-8. 10. Chang W, Knochenhauer ES, Bartolucci AA, Azziz R. Phenotypic spectrum of the polycystic ovary syndrome (PCOS): clinical and biochemical characterization of the major clinical subgroups. Fertil Steril 2005;83: 1717-23. 11. Azziz R, Sanchez LA, Knochenhauer ES, et al. Androgen excess in women: experience with over 1000 consecutive patients. J Clin Endocrinol Metab 2004;89:453-62. 12. Ehrmann DA, Rosenfield RL, Barnes RB, Brigell DF, Sheikh Z. Detection of functional ovarian hyperandrogenism in women with androgen excess. N Engl J Med 1992;327:157-62.
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Correlation of adrenocorticotropin steroid levels between women with polycystic ovary syndrome and their sisters Mark O. Goodarzi, MD, PhD; Xiuqing Guo, PhD; Bulent O. Yildiz, MD; Frank Z. Stanczyk, PhD; Ricardo Azziz, MD, MBA, MPH OBJECTIVE: The purpose of this study was to assess the sibling correlation of adrenocorticotropic hormone–stimulated steroid hormone levels between probands with polycystic ovary syndrome and their sisters.
nificantly correlated between siblings (r ⫽ 0.47, P ⫽ .01 and r ⫽ 0.57, P ⫽ .01, respectively); a similar trend was observed for the 60minute A4 values (r ⫽ 0.29, P ⫽ .06).
STUDY DESIGN: Twenty-seven women with polycystic ovary syndrome and
CONCLUSION: Women with polycystic ovary syndrome and their sisters have significantly correlated levels of adrenocorticotropic hormone–stimulated steroids, supporting a genetic basis of the adrenal androgen excess observed in polycystic ovary syndrome.
28 of their sisters underwent an adrenocorticotropic hormone stimulation test with measurement of the steroids dehydroepiandrosterone, androstenedione, and cortisol 60 minutes later. The 60-minute values were used to calculate sister-sister correlations by regression analyses. RESULTS: The adrenocorticotropic hormone–stimulated (60-minute)
log-transformed dehydroepiandrosterone and cortisol values were sig-
Key words: adrenal, androgen excess, genetics, polycystic ovary syndrome, sibling correlation
Cite this article as: Goodarzi MO, Guo X, Yildiz BO, et al. Correlation of adrenocorticotropin-stimulated steroid levels between women with polycystic ovary syndrome and their sisters. Am J Obstet Gynecol 2007;196:398.e1-398.e6.
P
olycystic ovary syndrome (PCOS) is a highly prevalent hyperandrogenic disorder. PCOS affects approximately 7% of reproductive-aged women1 and is associated with menstrual dysfunction, polycystic ovaries, and hirsutism, and an increased risk of
infertility, diabetes mellitus (DM), and possibly cardiovascular disease (CVD). Although multiple sets of diagnostic criteria have been proposed for PCOS, a common feature of all of them is hyperandrogenism.2,3 Androgen biosyn-
From the Division of Endocrinology, Diabetes and Metabolism, Department of Medicine (Dr Goodarzi), the Department of Obstetrics and Gynecology (Drs Goodarzi and Azziz), and the Medical Genetics Institute (Drs Goodarzi and Guo), Cedars-Sinai Medical Center, Los Angeles, CA; the Departments of Medicine (Drs Goodarzi and Azziz) and Obstetrics and Gynecology (Dr Azziz), the David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA; the Division of Endocrinology and Metabolism, Department of Internal Medicine, Hacettepe University School of Medicine, Ankara, Turkey (Dr Yildiz); and the Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Southern California Keck School of Medicine, Los Angeles, CA (Dr Stanczyk). Presented at the 25th Annual Meeting of the American Gynecological and Obstetrical Society, Sept. 14-16, 2006, Williamsburg, VA. Received Oct. 21, 2006; accepted Dec. 11, 2006. Reprints: Ricardo Azziz, MD, MPH, MBA, Department of Obstetrics/Gynecology and Center for Androgen Related Disorders, Cedars-Sinai Medical Center, 8635 W Third St, Suite 160W, Los Angeles, CA 90048; [email protected]. This study was supported in part by the Helping Hand of Los Angeles, and by grants R01HD29364 and K24-HD01346 from the National Institutes of Health (R.A.) and the Cedars-Sinai General Clinical Research Center grant RR000425 (X.G.). Financial disclosure: Dr Azziz has received consulting fees from Procter and Gamble, Merck and Co, and Organon. 0002-9378/$32.00 © 2007 Mosby, Inc. All rights reserved. doi: 10.1016/j.ajog.2006.12.009
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thesis in both the ovary and the adrenal is upregulated and exaggerated in PCOS. Ovarian androgen production in response to human chorionic gonadotropin (hCG) or long-acting gonadotropinreleasing hormone analogue (GnRH-a) stimulation is exaggerated in PCOS.4,5 Similarly, adrenocortical steroidogenesis and adrenal androgen (AA) secretion are exaggerated after adrenocorticotropic hormone (ACTH) stimulation.6,7 Abnormalities of steroidogenesis may be more clearly investigated in the adrenal than the ovary. Ovarian function in vivo is confounded by various factors, including obesity, intermittent ovulation, and hyperinsulinism, whereas adrenocortical steroidogenesis appears to be primarily affected by the diurnal rhythm and only modestly affected by extraadrenal or acquired factors.7-11 Adrenocortical dysfunction and AA excess may be the result of inherited (intrinsic or extrinsic to the adrenal glands) or acquired factors. Adrenocortical function in PCOS and healthy patients likely is a complex trait, modulated by both inherited and acquired factors. Preliminary evidence suggests that adrenocortical steroidogenesis and AA excess in
AGOS Papers
www.AJOG.org PCOS are primarily determined by inherited intrinsic factors. Studies in brothers and sisters of patients with PCOS indicate that AA levels, reflected by the levels of the AA metabolite circulating dehydroepiandrosterone sulfate (DHEAS), primarily reflect the impact of genetic factors.12,13 However, circulating DHEAS levels may not fully reflect the AA biosynthesis as measured by the adrenocortical response to acute ACTH stimulation. For example, exogenous testosterone administration increases the circulating DHEAS/DHEA ratio of oophorectomized women without altering measurable adrenocortical steroidogenesis in response to ACTH.8 Consequently, in this study, we sought to determine whether ACTH-stimulated AA steroid levels are inherited traits in families of women with PCOS. We hypothesize that adrenocortical biosynthesis in PCOS is highly heritable and, consequently, that the ACTH-stimulated steroid levels between women with PCOS and their sisters will be highly correlated.
M ATERIALS AND M ETHODS Subjects We recruited 27 PCOS probands and 28 of their sisters as part of study of the heritability of PCOS and its features. The study was approved by the Institutional Review Boards of University of Alabama at Birmingham, Birmingham, AL (UAB), Cedars-Sinai Medical Center, Los Angeles, CA (CSMC), and Haceteppe University, Ankara, Turkey, and all subjects gave written consent.
PCOS proband Patients were recruited from the reproductive or medical endocrinology clinics at UAB, CSMC, and Hacettepe University. None had used hormonal preparations, including oral contraceptives, for at least 3 months preceding the study. Patients were excluded if they were pregnant, prepubertal, or menopausal. After undergoing a history and physical examination, blood sampling was performed for the measurement of total testosterone (T), calculated free T, sex hormonebinding globulin (SHBG), DHEAS, and
17-hydroxyprogesterone (17-HP) (see below). The presence of PCOS was defined by criteria arising from a National Institutes of Child Health and Human Development–sponsored conference in 1990, including: (1) clinical evidence of hyperandrogenism and/or hyperandrogenemia, (2) oligoovulation, and (3) the exclusion of related disorders.2 These criteria are also consistent with those of a consensus conference sponsored by the European Society for Human Reproduction and Embryology and the American Society for Reproductive Medicine in Rotterdam in 2003.3 Although these newer criteria have indicated that the presence of polycystic ovaries detected on ultrasonography could also be used as criteria for PCOS, the proceedings were also clear that research criteria might be more or less inclusive. Consequently, as we have the greatest familiarity with this diagnostic scheme and because obligatory transvaginal ultrasonography may be perceived as excessively invasive and may hamper our ability to recruit patients and their sisters, we used the 1990 National Institutes of Health criteria. Oligoovulation was defined as menstrual cycles more than 45 days in length or less than 8 cycles per year, or by a luteal phase progesterone (P4) level less than 4 ng/mL in conjunction with a monophasic basal body temperature chart, if menstrual cycles were less than 45 days in length. Hirsutism was defined as a modified Ferriman-Gallwey (mF-G) score 6 or greater.1,14 For the purpose of this study, hyperandrogenemia was defined as total T greater than 83.6 ng/dL (2.96 nmol/L), free T greater than 0.81 ng/dL (0.026 nmol/L), or DHEAS greater than 2540 ng/mL (6.64 mol/L), as previously reported.1 Patients with 21-hydroxylase deficient nonclassic adrenal hyperplasia (NCAH) were excluded by a basal follicular phase 17-HP level less than 2 ng/mL (6.0 nmol/L), or a 17-HP level after acute ACTH stimulation of less than 10 ng/mL (30.3 nmol/L), as previously described.15 Hyperprolactinemia and hypothyroidism were excluded by normal levels of prolactin and thyroid-stimulating hormone, respectively; Cushing’s syndrome and andro-
genic tumors were excluded by appropriate testing, if suspected clinically.
Sisters Twenty-eight sisters were contacted and recruited after consent from the proband. Twenty-six probands had 1 sister, whereas 1 proband had 2 sisters. All completed a history and physical examination, and provided a basal blood sample, after appropriate informed written consent.
Clinical protocol Early morning blood samples were obtained from each subject between 8:00 AM and 10:30 AM after an overnight fast, in the follicular phase (days 3-8) of menstrual cycle, or after a progestogen-induced bleed. Probands with PCOS were off medication for at least 3 months before the study, whereas this was not required for sisters. The test samples from the probands and sisters were assayed in the same run, to minimize the effect of interassay variability. All subjects underwent an acute adrenal stimulation test using 0.25 mg ACTH-1-24, as previously described.16 Because of the effect of the circadian rhythm on adrenocortical function all stimulations were performed between 7:00 and 9:00 AM. After a rest period after the placement of an intravenous angiocath and heparin lock, 3 blood samples were drawn at ⫺30 minutes, ⫺15 minutes, and 0 minutes, and mixed to form the baseline sample. Afterward, 0.25 mg (1 vial) of ACTH-1-24 (Cortrosyn, Organon Co, New Orange, NJ) was then injected intravenously over 60 seconds, and a final blood sample obtained 60 minutes later. In the baseline samples we measured total and free T, SHBG, and DHEAS; in both the baseline and 60minute samples we measured DHEA, androstenedione (A4), and cortisol (F). The parameters calculated from the acute ACTH stimulation results were as previously described,16 including the basal (0 minutes), peak levels (60 minutes), and net increment (Steroid0, Steroid60, ⌬Steroid, respectively) for A4, DHEA, and F. We have previously demonstrated that these parameters are
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TABLE 1
Clinical characteristics and results of baseline and ACTH-stimulated steroids in probands and sisters Trait
Proband
Sister
P value*
Age (y)
28.3 ⫾ 10.7
27.5 ⫾ 8.3
.79
BMI (kg/m )
34.5 ⫾ 7.5
29.7 ⫾ 8.5
.037
Waist-hip ratio
0.84 ⫾ 0.11
0.83 ⫾ 0.07
.74
Total testosterone (ng/dL)
70.4 ⫾ 37.2
47.8 ⫾ 28.3
.013
Free testosterone (pg/mL)
18.6 ⫾ 10.4
10.7 ⫾ 7.3
.0017
DHEAS (g/dL)
234.7 ⫾ 140.6
155.6 ⫾ 101.5
.019
SHBG (nmol/L)
24.7 ⫾ 15.0
38.0 ⫾ 17.6
.004
A40 (ng/mL)
1.87 ⫾ 0.87
1.25 ⫾ 0.59
.003
DHEA0 (ng/mL)
7.51 ⫾ 6.68
4.54 ⫾ 3.17
.038
9.3 ⫾ 6.2
8.7 ⫾ 4.7
.70
.............................................................................................................................................................................................................................................. 2 .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. ..............................................................................................................................................................................................................................................
F (g/dL)
0 ..............................................................................................................................................................................................................................................
Steroid0 refers to basal unstimulated basal levels. * P value from unpaired 2-sided t-test.
highly stable in the short- and longterm.16,17 Alternatively, although multiple other calculations can be performed (eg, steroid ratios to estimate enzyme activities) we have observed that these parameters are more variable over time within patients16 and consequently were not be used in estimating sister correlation.
Assays Serum was frozen at ⫺70°C until assayed. We performed assays at regular intervals during this study, batching samples from the same family when analyzed. The specifics for the assays for A4, DHEA, DHEAS, total and free T, SHBG, glucose, and insulin have been previously described.9,11,17 The intraassay variances are consistently less than 10% and the interassay less than 15%.
Statistical analysis Phenotypic parameters are given as mean ⫾ SD. Mean trait values were compared between the probands and sisters
by using 2-sided unpaired t-tests. Because many of the parameters being tested demonstrated a skewed distribution, all adrenal response parameters were log-transformed for analysis. Familial correlation r values between probands and their sister pairs were estimated by regressing the Steroid60 and ⌬Steroid values in sisters against that in the corresponding probands. As a control analysis, the sister data were shuffled and then remerged to the proband data, resulting in 27 probands unrelated sister pairs; the adrenal steroid response regressions were then carried out among these unrelated subject pairs.
R ESULTS The mean age and waist-hip ratio was similar in proband and sister groups, whereas probands had a higher mean body mass index (BMI) than sisters (Table 1). Twenty-five probands were white, whereas 2 proband-sister pairs were black. The probands had higher baseline levels of total T, free T, DHEA, DHEAS,
A4, and lower SHBG than their sisters. There were no differences in baseline F levels. The ACTH-stimulated (60-minute) log-transformed DHEA and F values were significantly correlated between siblings (r ⫽ 0.47, P ⫽ .01 and r ⫽ 0.57, P ⫽ .01, respectively); a similar trend was observed for the 60-minute A4 values (r ⫽ 0.29, P ⫽ .06) (Table 2). Similarly, the log-transformed net increment in A4 (⌬A4) and F (⌬F) were significantly correlated between siblings (Table 2). A similar trend was observed for ⌬DHEA. Three sisters were receiving hormonal therapy; elimination of their data did not materially affect the correlations reported previously. Repeating the correlations after randomly scrambling the sibling-pairs yielded no significant correlations in adrenal response parameters (data not shown).
C OMMENT Although the majority of patients with PCOS demonstrate an ovarian source for their high androgen secretion, many also display AA excess. The measurement of circulating DHEAS has been often used as a marker of AA secretion (and excess), because this metabolite is as follows: (a) 97-99% of adrenocortical origin, (b) the most abundant steroid, (c) relatively stable throughout the day and the menstrual cycle, and (d) easily measured. With the use of racial and age normative values, we have determined the prevalence of absolute (ie, ⬎95th percentile of normal) DHEAS excess to be approximately 25%.18 Although DHEAS is a useful marker of AA secretion in epidemiologic studies, it reflects both AA production and DHEA sulfotransferase activity. As our studies suggest that extraadrenal factors may modulate DHEA sulfotransferase activity to a
TABLE 2
Correlation coefficients between PCOS probands and their sisters for the ACTH-stimulated steroid parameters log A460 (ng/mL)
log ⌬A4
log DHEA60 (ng/mL)
log ⌬DHEA
log F60 (g/dL)
log ⌬F
r value
0.29
0.48
0.47
0.14
0.57
0.33
P value
.06
.03
.01
.46
.01
.05
................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................
Steroid60 refers to peak ACTH-stimulated basal levels and ⌬Steroid to net increment from baseline (0 minutes) to Steroid60.
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AGOS Papers
www.AJOG.org greater degree than AA production in response to ACTH,7-11 we chose to focus the current investigation on the sibling correlation of ACTH-stimulated steroid levels. ACTH-stimulated steroids are most reflective of adrenal secretory function and also appear to be elevated in PCOS.6,7 We found substantial sibling-sibling correlation for ACTH-stimulated steroids, including DHEA, A4, and F. We noted that the sister correlation was highest for F, possibly a reflection of the need for precisely regulating F levels to maintain optimal health and immune function. The correlations for these 3 steroids appears higher than the correlation between sisters for DHEAS alone (0.28),12 consistent with our suggestion that DHEAS varies more in response to acquired or extraadrenal factors compared with ACTH-stimulated steroid levels.7-11 These data strongly suggest that genetic factors play an important role in determining the response of adrenal steroids to ACTH stimulation, although the population size must be expanded to confirm this. Besides contributing to the hyperandrogenism in PCOS, AA excess is relevant in that it may be a risk factor for PCOS. Various data support the concept that AA excess, particularly when evident during pubertal development, increases the risk for developing PCOS. For example, patients with premature adrenarche are at higher risk for the development of PCOS than their nonaffected peers.19 Furthermore, other investigators have suggested that stress resulting in exaggerated AA secretion increases the risk of developing PCOS.20 Most evidence currently indicates that the effect of extraadrenal factors on adrenocortical dysfunction and AA excess in PCOS is modest at best. By using GnRH-a suppression9 and oophorectomy,11 exogenous estrogen and testosterone replacement,8 and correlation studies with measures of insulin secretion or action,10 we have observed that extraadrenal factors play only a small role, if any, in determining the response of the adrenal to ACTH in normal and PCOS patients, and primarily modestly alter sulfotransferase activity and thus
the circulating levels of the AA metabolite DHEAS levels. Overall, the modest role of extraadrenal modulators on the adrenal in PCOS supports the hypothesis of an underlying genetic cause in the abnormal adrenocortical steroidogenesis and metabolism, and AA excess, of PCOS. Various data support the concept of an inherited basis for AA excess in PCOS. First, it is clear that the levels of the AA metabolite DHEAS are under significant genetic control as evidenced in twin20 and family21,22 studies. The brothers (n ⫽ 119) of 87 unrelated PCOS women had significantly elevated DHEAS levels compared with control men, and a positive linear relationship between DHEAS in probands and their brothers was observed.13 In PCOS proband–sister pairs we have also observed a significant correlation in DHEAS levels and the AA response to ACTH stimulation between the proband and her untreated sister.12 Despite the number of studies examining heritability of DHEAS, we are not aware of any study that has examined heritability or sister correlation of ACTH-stimulated steroid levels. In terms of what actual genes may be driving AA excess, a small number of studies have examined candidate genes for association with AA, mostly DHEAS, levels, with largely negative results.23,24 We recently discovered association of single nucleotide polymorphisms in the gene for DHEA sulfotransferase (SULT2A1) with circulating DHEAS in a population of approximately 300 PCOS and approximately 200 controls.25 Alternatively, other investigators have observed an association between the gene for 11-hydroxysteroid dehydrogenase, type 1 (HSD11B1), which primarily catalyzes the conversion of cortisone to F, and DHEAS levels in PCOS.26 Overall, these data provide further support, and an underlying explanation, for the role of inherited factors and genetic variants in determining adrenocortical function and DHEAS levels in PCOS. In this study, we present sister-sister correlation data as evidence of inheritance. Another commonly used parameter to quantify the inherited nature of a trait is heritability (h2). In accordance
with established quantitative genetic theory, the total phenotypic variance in a trait (P2) can be partitioned into the variance because of the additive effects of genes (G2) and the variance caused by environmental effects (E2). Heritability (h2) can be estimated by the proportion of the phenotypic variance caused by the additive effects of genes, ie, G2/P2 and tested by using maximum likelihood methods. However, in the current study we were unable to directly calculate heritability in this manner given the limited sample size and the lack of control data from unrelated subjects; however, our data allow an estimation of the upper bound of heritability, which is double the sibling correlation value. Thus, the upper bounds of heritability of DHEA60, A460, and F60 are 0.94, 0.58, and 1.0, respectively, indicating a majority of variance in these traits is due to genetic factors. We should note that correlation or heritability estimates cannot distinguish between inherited factors (genetic effects) vs effects arising from shared environmental factors. Shared environmental factors among siblings can inflate these estimates. Our data support the hypothesis that adrenocortical steroidogenesis is under significant genetic control and likely reflects variations in the genes controlling their biosynthesis and/or metabolism. Which actual genes may be driving the AA excess remain to be determined, and large-scale genotype-phenotype association studies, evaluating ACTH-stimulated steroid values as the relevant phenotype, are needed. f REFERENCES 1. Knochenhauer ES, Key TJ, Kahsar-Miller M, Waggoner W, Boots LR, Azziz R. Prevalence of the polycystic ovary syndrome in unselected black and white women of the southeastern United States: a prospective study. J Clin Endocrinol Metab 1998;83:3078-82. 2. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril 2004;81: 19-25. 3. Zawadzki JK, Dunaif A. Diagnostic criteria for polycystic ovary syndrome: towards a rational approach. In: Dunaif A, Givens JR, Haseltine F, Merriam GR, eds. Polycystic ovary syndrome. Cambridge, England: Blackwell Scientific Publications; 1992.
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AGOS Papers 4. Piltonen T, Koivunen R, Perheentupa A, Morin-Papunen L, Ruokonen A, Tapanainen JS. Ovarian age-related responsiveness to human chorionic gonadotropin in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2004;89:3769-75. 5. Rosenfield RL, Barnes RB, Ehrmann DA. Studies of the nature of 17-hydroxyprogesterone hyperresonsiveness to gonadotropin-releasing hormone agonist challenge in functional ovarian hyperandrogenism. J Clin Endocrinol Metab 1994;79:1686-92. 6. Moran C, Reyna R, Boots LS, Azziz R. Adrenocortical hyperresponsiveness to corticotropin in polycystic ovary syndrome patients with adrenal androgen excess. Fertil Steril 2004;81:126-31. 7. Azziz R, Rittmaster RS, Fox LM, Bradley EL Jr, Potter HD, Boots LR. Role of the ovary in the adrenal androgen excess of hyperandrogenic women. Fertil Steril 1998;69:851-9. 8. Chang WY, Stanczyk F, Bartolucci A, Azziz R. Effect of bilateral oophorectomy on basal and adrenocorticotropin (ACTH)-stimulated adrenal androgen (AA) secretion in polycystic ovary syndrome (PCOS). Paper presented at: 86th Annual Meeting of the Endocrine Society; June 16-19, 2004; New Orleans, LA. 9. Azziz R, Gay FL, Potter SR, Bradley E Jr, Boots LR. The effects of prolonged hypertestosteronemia on adrenocortical biosynthesis in oophorectomized women. J Clin Endocrinol Metab 1991;72:1025-30. 10. Slayden SM, Crabbe L, Bae S, Potter HD, Azziz R, Parker CR Jr. The effect of 17 betaestradiol on adrenocortical sensitivity, responsiveness, and steroidogenesis in postmenopausal women. J Clin Endocrinol Metab 1998;83:519-24. 11. Farah-Eways LA, Reyna R, Knochenhauer ES, Bartolucci A, Azziz R. Glucose action and adrenocortical biosynthesis in the polycystic ovary syndrome. Fertil Steril 2004;81:120-5. 12. Azziz R, Trader BT, Pall M, Yildiz BO, Stanczyk F. Heritability of adrenal androgen (AA) secretion in sisters of women with polycystic ovary syndrome (PCOS). Paper presented at: 88th Annual Meeting of the Endocrine Society; June 24-27, 2006; Boston, MA. 13. Legro RS, Kunselman AR, Demers L, Wang SC, Bentley-Lewis R, Dunaif A. Elevated dehydroepiandrosterone sulfate levels as the reproductive phenotype in the brothers of women with polycystic ovary syndrome. J Clin Endocrinol Metab 2002;87:2134-8. 14. Hatch R, Rosenfield RL, Kim MH, Tredway D. Hirsutism: implications, etiology, and management. Am J Obstet Gynecol 1981;140: 815-30. 15. Azziz R, Hincapie LA, Knochenhauer ES, Dewailly D, Fox L, Boots LR. Screening for 21hydroxylase-deficient nonclassic adrenal hyperplasia among hyperandrogenic women: a prospective study. Fertil Steril 1999;72:915-25. 16. Azziz R, Bradley E Jr, Huth J, Boots LR, Parker CR Jr, Zacur HA. Acute adrenocorticotropin-(1-24) (ACTH) adrenal stimulation in eu-
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www.AJOG.org menorrheic women: reproducibility and effect of ACTH dose, subject weight, and sampling time. J Clin Endocrinol Metab 1990;70:1273-9. 17. Azziz R, Yildiz BO, Woods KS, Stanczyk FZ, Bartolucci AA. Long-term variability of adrenal androgen (AA) levels and their response to corticotropin in the polycystic ovary syndrome (PCOS). Paper presented at: 86th Annual Meeting of the Endocrine Society; June 16-19, 2004; New Orleans, LA. 18. Kumar A, Woods KS, Bartolucci AA, Azziz R. Prevalence of adrenal androgen excess in patients with the polycystic ovary syndrome (PCOS). Clin Endocrinol (Oxf) 2005;62:644-9. 19. Ibanez L, Potau N, Virdis R, et al. Postpubertal outcome in girls diagnosed of premature pubarche during childhood: increased frequency of functional ovarian hyperandrogenism. J Clin Endocrinol Metab 1993;76: 1599-603. 20. Akamine Y, Kato K, Ibayashi H. Studies on changes in the concentration of serum adrenal androgens in pubertal twins. Acta Endocrinol (Copenh) 1980;93:356-64. 21. Rotter JI, Wong FL, Lifrak ET, Parker LN. A genetic component to the variation of dehydroepiandrosterone sulfate. Metabolism 1985; 34:731-6. 22. Rice T, Sprecher DL, Borecki IB, Mitchell LE, Laskarzewski PM, Rao DC. The Cincinnati Myocardial Infarction and Hormone Family Study: family resemblance for dehydroepiandrosterone sulfate in control and myocardial infarction families. Metabolism 1993;42: 1284-90. 23. Kahsar-Miller M, Boots LR, Bartolucci A, Azziz R. Role of a CYP17 polymorphism in the regulation of circulating dehydroepiandrosterone sulfate levels in women with polycystic ovary syndrome. Fertil Steril 2004;82:973-5. 24. Witchel SF, Kahsar-Miller M, Aston CE, White C, Azziz R. Prevalence of CYP21 mutations and IRS1 variant among women with polycystic ovary syndrome and adrenal androgen excess. Fertil Steril 2005;83:371-5. 25. Goodarzi MO, Antoine HJ, Azziz R. Genes for enzymes that metabolize DHEA influence levels of DHEA-sulfate in polycystic ovary syndrome (PCOS). Paper presented at: 4th Annual Meeting of the Androgen Excess Society; June 23, 2006; Boston, MA. 26. Gambineri A, Vicennati V, Genghini S, et al. Genetic variation in 11beta-hydroxysteroid dehydrogenase type 1 predicts adrenal hyperandrogenism among lean women with polycystic ovary syndrome. J Clin Endocrinol Metab 2006;91:2295-302.
D ISCUSSION Rogerio Lobo, MD. 1. The adrenocorticoptropin (ACTH)stimulated responses of cortisol (F) and androstenedione (A4) were correlated in women with
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the polycystic ovary syndrome (PCOS) and their sisters. Please explain why this supports the claim of a genetic basis for adrenal androgen (AA) excess in PCOS. Other biochemical indices are correlated on a biochemical basis. Why were there no normal controls to determine how many women with PCOS and how many sisters had exaggerated responses to ACTH? Why were other steroids (eg, 17hydroxyprogesterone and 17-hydroxypregnenolone) not measured to gain insight into enzymatic dysregulation? Why were the sisters allowed to use oral contraceptive pills (OCPs)? This would affect basal levels of androgens. Why were other methods of determining ACTH responses not considered in light of lowered basal levels in users of OCPs? What was the cut-off value for an elevated DHEAS? Were the data normally distributed? Were parametric statistics used? How many sisters may have had PCOS (this could not be determined given the protocol used)? Can the authors speculate on whether these data may relate to other phenotypes of PCOS?
Dr Goodarzi (closing). There is variability (a range of values) of the stimulated hormone levels in the population. The premise is that traits that are genetically determined will correlate between related individuals, but not between unrelated individuals. Correlation of a trait between siblings is related to the heritability of a trait (which is the proportion of the total variance of the trait that is explained by genetic factors). Thus, if one sibling has a high dehydroepiandrosterone (DHEA) response to ACTH, we would expect other siblings to also have a high response if they tend to share the same genes that determine DHEA response. To rule out the possibility that the correlations we have observed are not re-
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www.AJOG.org lated to heritability, we scrambled the proband and sister data and then calculated the trait correlations between unrelated individuals. For all traits, there were no correlations between unrelated subjects. Notwithstanding, we agree that correlation (or even heritability) analyses only support the suggestion that the adrenal response to ACTH stimulation is inherited. More extensive family studies, and linkage studies when specific genetic markers of adrenal steroidogenesis are identified, are needed to confirm this hypothesis. We did not include healthy controls because we did not need to establish an “upper normal limit (UNL),” as we have previously established these ranges using large populations of healthy controls.1-6 In addition, we consider the ACTH response to represent a continuum, and we prefer not to artificially subdivide patients into those with and without an exaggeration in response above the UNL. We did not measure other steroidogenic or AA precursors because the primary endpoints were AA production, namely, DHEA) and A4; measuring other precursors may help understand the steroidogenic mechanisms underlying the excess. However, we have done this repeatedly previously,3-5 and there are limitations in resources to consider. We could not control whether sisters were or were not on OCPs, although we note that disorder itself, namely, polycystic ovary syndrome (PCOS), predisposes to the use of OCPs. We should note that OCP or other hormonal therapy would, if anything, decrease the degree of association, which we did not observe in our prior study analyzing the heritability of DHEAS.7 In agreement, in the current study only 3 sisters were receiving hormonal therapy at the time of their study, and their removal from the analysis did not change the results. We have found that using the peak (maximum) value poststimulation gives the most precise and reproducible value8; calculations of net increment, which could be done, in our experience increases significantly the
intrasubject variation, to as high as 60%. Nonetheless, we calculated the net increment (change from 0-60 minutes), which supported the findings using peak values. The UNL value for DHEAS used was 2750 ng/mL. However, as we have indicated, we now prefer to consider DHEAS as a continuous value, and not arbitrarily separate into normal and supranormal based on a percentile, because this belays: (a) the observation that PCOS women, in general, demonstrate varying degrees of hyperresponsivity, and (b) the fact that DHEAS is not only the product of AA secretion in response to ACTH, but also the result of DHEA sulfotransferase (DHEA-ST) activity, and, consequently, is only loosely related to the AA response to ACTH stimulation. We tested the data for skewness or kurtosis and found that about 60% of parameters were normally distributed. To account for the remainder, we log-transformed the data before analysis. We agree that with the protocol used we could not determine how many sisters had PCOS. We should note that we previously reported that 40-50% of sisters are affected with PCOS.9 However, it is possible that the prevalence of PCOS in the current cohort of sisters may be less, as they had significantly lower levels of androgens and body mass index. The fact that a significant association in adrenal response was found regardless further emphasizes the heritability of the ACTH-stimulated adrenal steroid production. We have previously observed that approximately 10% of PCOS patients solely have an elevated DHEAS.10 We have also observed that women with elevations in DHEAS tend to be younger, as would be expected, and thinner.11 Furthermore, some of these women may not have overt ovulatory dysfunction.11 Alternatively, the degree of adrenocortical dysfunction in response to ACTH, in contrast to basal levels of DHEAS, may possibly mirror the degree of ovarian dysfunction, and thus may not necessarily indicate a less affected individual. This hypothesis remains to be determined, although Ehrmann et al12 did not find a high degree of correlation between the results of stimulation with ACTH and the long-acting gonadotropin-releasing
hormone agonist nafarelin when studying 40 consecutive women with hyperandrogenism who had oligomenorrhea, hirsutism, or acne. REFERENCES 1. Azziz R, Boots LR, Parker CR Jr, Bradley E Jr, Zacur HA. 11-hydroxylase deficiency in hyperandrogenism. Fertil Steril 1991;55:733-41. 2. Azziz R, Zacur HA, Parker CR Jr, Bradley E Jr, Boots LR. Effect of obesity on the response to acute adrenocorticotropin stimulation in eumenorrheic women. Fertil Steril 1991;56: 427-33. 3. Azziz R, Bradley EL Jr, Potter HD, Boots LR. 3-Hydroxysteroid dehydrogenase deficiency in hyperandrogenism. Am J Obstet Gynecol 1993;168:889-95. 4. Azziz R, Bradley EL Jr, Potter HD, Boots L. Adrenocortical hyperactivity and androgen excess in hyperandrogenism: lack of role for 17hydroxylase and 17,20-lyase dysregulation. J Clin Endocrinol Metab 1995;80:400-5. 5. Moran C, Potter HD, Reyna R, Boots LR, Azziz R. Prevalence of 3-hydroxysteroid dehydrogenase deficient non-classic adrenal hyperplasia in hyperandrogenic women with adrenal androgen excess. Am J Obstet Gynecol 1999;181:596-600. 6. Azziz R, Fox LM, Zacur HA, Parker CR Jr, Boots LR. Adrenocortical secretion of dehydroepiandrosterone in healthy women: highly variable response to ACTH. J Clin Endocrinol Metab 2001;86:2513-7. 7. Yildiz BO, Goodarzi MO, Guo X, Rotter JI, Azziz R. Heritability of dehydroepiandrosterone sulfate among polycystic ovary syndrome women and their sisters. Fertil Steril 2006;86:1688-93. 8. Azziz R, Bradley E Jr, Huth J, Parker CR Jr, Boots LR, 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. 9. Kahsar-Miller M, Nixon C, Boots LR, Go RCP, Azziz R. Prevalence of the polycystic ovary syndrome (PCOS) among first degree relatives of patients with PCOS. Fertil Steril 2001;75:53-8. 10. Chang W, Knochenhauer ES, Bartolucci AA, Azziz R. Phenotypic spectrum of the polycystic ovary syndrome (PCOS): clinical and biochemical characterization of the major clinical subgroups. Fertil Steril 2005;83: 1717-23. 11. Azziz R, Sanchez LA, Knochenhauer ES, et al. Androgen excess in women: experience with over 1000 consecutive patients. J Clin Endocrinol Metab 2004;89:453-62. 12. Ehrmann DA, Rosenfield RL, Barnes RB, Brigell DF, Sheikh Z. Detection of functional ovarian hyperandrogenism in women with androgen excess. N Engl J Med 1992;327:157-62.
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