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Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders characterized by androgen excess, oligo-ovulation and polycystic ovaries. Although ovaries are the main source of increased androgens in the syndrome, between 20 and 30% of patients with PCOS have adrenal androgen (AA) excess, detectable primarily by elevated dehydroepiandrosterone sulfate (DHEAS) levels. Patients with PCOS demonstrate a generalized hypersecretion of adrenocortical products, basally and in response to ACTH stimulation. The mechanisms of these abnormalities are unclear although AA excess in PCOS is likely a complex trait, modulated by both intrinsic and acquired factors. To date, no specific genetic defects have been identified. The production of AAs in response to ACTH appears to be closely related to altered factors regulating glucose-mediated glucose disposal, increased peripheral metabolism of cortisol, and to a less extent to the effects of extra-adrenal androgens, insulin resistance, hyperinsulinemia or obesity. Finally, DHEAS levels and the response of AAs to ACTH are relatively constant over time and are closely correlated between PCOS patients and their siblings suggesting that this abnormality is an inherited trait in PCOS.

Eleven healthy, eumenorrheic, nonhirsute women underwent acute adrenal stimulation testing at monthly intervals in order to measure the responses of cortisol (F), DHEA, and androstenedione, to a 60-minute acute stimulation with 1.0 mg ACTH (1-24). When the women were subdivided into either high or low responders to ACTH stimulation (relative to the group median), the relative adrenocortical response within individual subjects was found to be highly constant over time.

Objective: To test the hypothesis that adrenal steroidogenesis in nonclassic adrenal hyperplasia (NCAH) patients is, at least in part, independent of adrenocorticotropic hormone (ACTH) control., Design: Prospective controlled clinical study., Setting: Patients and healthy volunteers in an academic research environment., Patient(s): Four patients with 21-hydroxylase (21-OH) deficient NCAH and four healthy control women., Intervention(s): Patients received the long-acting gonadotropin-releasing hormone analog (GnRH-a) leuprolide acetate (3.75 mg/month IM) on weeks 0 and 4; and dexamethasone (DEX) in weekly incremented doses (0.25 mg/day, 0.50 mg/day, 1.0 mg/day, and 1.5 mg/day), beginning on weeks 4, 5, 6, and 7, respectively., Main Outcome Measure(s): The levels of 17-hydroxyprogesterone (17-HP), progesterone (P4), androstenedione (A4), dehydroepiandrosterone sulfate (DHS), and cortisol (F) were measured at the beginning of weeks 0, 4, 5, 6, 7, and at the end of the study (beginning of week 8)., Result(s): Patients and controls had a similar median age and body mass index (BMI). There were no significant decreases in the median levels of the studied hormones following 4 weeks of treatment with GnRH-a only, in either NCAH patients or controls. Analysis of individual hormonal values demonstrated that by the end of the study (after DEX of 1.5 mg/day during a week) only 2 of 4, 0 of 4, 3 of 4 and 3 of 4 NCAH patients had 17-HP, P4, A4, and DHS levels within the range of control values, respectively., Conclusion(s): Ovarian and incremental adrenal suppression did not fully suppress progestogen and androgen production in all of the study patients with 21-OH-deficient NCAH, suggesting that their production was partially independent of ACTH stimulation. Potentially in these patients subtle degrees of adrenocortical hyperplasia and/or abnormal enzymatic kinetics are responsible for the nonsupressibility.

Objective: To determine whether the frequency of the N363S variant of the glucocorticoid receptor (GRL) was increased in women with PCOS and adrenal androgen (AA) excess., Design: Prospective case-control study., Setting: University reproductive endocrinology laboratory and outpatient clinic., Patient(s): Consecutive patients of non-Hispanic white race diagnosed with PCOS (n = 114) and healthy controls (n = 92)., Intervention(s): Blood and DNA sampling before hormonal therapy., Main Outcome Measure(s): PCOS patient and healthy control genotypes, with the N363S allele representing a variant of GRL., Result(s): Fifty-four PCOS patients with (DHEAS > or = 3000 ng/mL) and 55 without (DHEAS < or = 2,500 ng/mL) AA excess, respectively, were studied. Six of 109 (5.5%) patients studied were found to be heterozygous carriers of the A-->G base pair substitution at cDNA position 1220, resulting in the missense mutation N363S. Of these six, four had excessive AA secretion (i.e., excess DHEAS levels). There was no significant difference in the allele frequency of the GRL variant between PCOS patients with and without AA excess and controls (3.7% [95% confidence interval: 1.0%-5.7%], 1.8% [0.2%-6. 0%], and 3.3% [2.3%-6.0%]). None of the subjects were found to be homozygous for the N363S allele., Conclusion(s): The N363S variant of GRL was an uncommon occurrence in our population of healthy women and PCOS patients and did not appear to play a major role in the genetic predisposition to PCOS or to AA excess in PCOS.

We sought to determine the effects of aging on several aspects of adrenal steroidogenesis in the hopes of characterizing the possible causes of adrenal androgen deficiency in elderly women. To this end, we quantified basal morning concentrations of cortisol (F), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DS), and androstenedione (A4) and then evaluated the effects of overnight dexamethasone (DEX) suppression followed by adrenal responses to graded hourly infusions of ACTH, ranging from 20-1280 ng/1.5 m2 x h. Finally, we performed a standard 0.25-mg ACTH bolus stimulation test, with sampling at 1 h thereafter. Basal serum levels of DHEA, DS, and A4 were significantly reduced (approximately 50% each) in a group of 35 healthy postmenopausal women, 55-68 yr old, compared to those in 30 healthy, regularly menstruating women, 20-25 yr old. Post-DEX levels of these C19 steroids also were significantly lower in the older women than in the younger women; the percent decrease after DEX for A4 was greater in the older women, whereas those in DHEA and DS were not age related. Basal and post-DEX levels of F were similar in both groups. Secretory responses of DS to ACTH were not informative due to its large plasma pool and slow clearance rate. The maximally stimulated levels of DHEA after ACTH bolus were significantly lower in the older women than in younger women; those of A4 were similar in both age groups, and the maximally achieved levels of F were higher in the older women than in the younger women. The sensitivity of adrenal DHEA, A4, and F to ACTH (defined as the minimal dose of ACTH required to significantly increase the steroid levels above basal post-DEX values) was similar in older and younger women. The responsiveness of the steroids of interest to ACTH (defined as the slope of the dose-response curve over the linear portion of the dose-response curve) also was similar among younger and older women. These data demonstrate that the deficiency in adrenal androgen production in women is restricted to the delta5-pathway steroid products (DHEA and DS), whereas there is no reduction in the capacity of the adrenal to produce A4 or cortisol. As DHEA and DS are likely to be produced mainly in the zona reticularis of the adrenal cortex, we propose that these data point to an alteration in that cortical zone as the cause of adrenal androgen deficiency in aging. The reductions in A4 in aging are probably due to reduced ovarian secretion after menopause.

Adrenal androgen (AA) excess, primarily in the form of dehydroepiandrosterone sulfate (DHEAS), affects over 50% of women with the polycystic ovary syndrome (PCOS). Nonetheless, it is unclear what role AA excess plays in the PCOS-associated oligo-ovulation. We have hypothesized that AAs are important in the maintenance of the ovulatory dysfunction of women with PCOS and AA excess, which can be improved by glucocorticoid suppression. To test our hypothesis we prospectively studied 36 unselected women, ages 18-40 yr, with PCOS; i.e. oligomenorrhea (cycles > 35 days in length), and clinical/ biochemical evidence of hyperandrogenism (i.e. hirsutism and/or hyperandrogenemia), after the exclusion of related disorders. After informed consent, all patients underwent an acute ACTH-(1-24) stimulation test, measuring androstenedione, dehydroepiandrosterone (DHEA) and cortisol (F), and were then treated with dexamethasone 0.5 mg/day for four cycles. Ovulatory function was assessed before and during treatment using a basal body temperature calendar and day 22-24 progesterone (P4) levels. If patients were anovulatory (P4 < 4 ng/mL), a withdrawal bleed was induced by the administration of 100 mg P4 in oil i.m. Before and during treatment the levels of total and free testosterone (T), sex hormone-binding globulin, androstenedione, DHEA, DHEAS, cortisol, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were monitored. With therapy, all patients demonstrated a significant decrease in all androgens (-40-60%), a 24% increase in sex hormone-binding globulin, and no change in LH/FSH. Mean body weight increased by over 4 kg (4.4%) during treatment. Of the 138 cycles monitored, 78% remained anovulatory. Twenty-five percent, 17%, 14%, and 20% of the first, second, third, and fourth treatment cycles, were ovulatory, respectively (P = 0.381). Of the 36 patients studied, 18 (50%) did not demonstrate a single ovulatory cycle (i.e. a day 22-24 P4 level > 4 ng/mL); and of the remaining, 10 (28%) had only one, five (14%) had two, and three (8%) had three ovulatory cycles. There were no significant differences either in physical features, basal hormones, adrenal response to ACTH stimulation, or hormonal levels at the end of treatment, between those women ovulating and those not. Finally, there were no differences in ovulatory response to dexamethasone therapy between women with (n = 14) and without (n = 22) DHEAS excess (i.e. DHEAS > 2750 ng/mL). In conclusion, the data from this prospective study do not suggest that continuous dexamethasone suppression results in consistent ovulation in any PCOS patient, regardless of basal DHEAS levels. Furthermore, this treatment is associated with significant side-effects, notably weight gain. Finally, these data suggest that, while AA may be an important risk factor for PCOS, once the syndrome is established, they play a limited role in the associated ovulatory dysfunction.

The responses of several adrenal steroids to adrenocorticotropin (ACTH) were investigated in a group of healthy young men. The protocol consisted of overnight adrenal suppression with dexamethasone followed by 1 hr infusions of ACTH in increasing (4X) doses ranging from 30-30,720 ng/1.5M2 followed by 0.25 mg ACTH/1.5M2. Whereas cortisol, androstenedione and androstenediol concentrations tended to plateau at the higher ACTH infusion rates, those of dehydroepiandrosterone (DHEA) and 17-hydroxyprogesterone (17-OH P) did not. The cortisol concentration achieved after the highest dose of ACTH was over 50 fold higher than that at baseline. DHEA levels rose to values about 13 times baseline whereas the other steroids increased to lesser extents (3-5 fold). The ACTH infusion rate required to significantly increase each steroid over the baseline level was extrapolated from dose response curves and was considered to be an index of the sensitivity of each steroid to ACTH. Cortisol was the most sensitive, rising significantly at approximately 35 ng ACTH/1.5M2, followed by androstenediol, DHEA, androstenedione and 17-OH P.

Some investigators have suggested that "dysregulation" of cytochrome P450c17 alpha may result in the exaggerated secretion of ovarian androgens in hyperandrogenism. Although the majority of hyperandrogenic (HA) patients demonstrate an ovarian source for their androgens, approximately 50% also display adrenocortical hyperactivity and adrenal androgen excess. To determine whether 17-hydroxylase (17-OH) and/or 17,20-lyase dysregulation is responsible for the adrenocortical abnormalities noted in many HA patients, we studied 92 consecutive women with hirsutism and/or HA oligomenorrhea; 26 healthy eumenorrheic nonhirsute women served as controls. The basal levels of total and free testosterone (T), sex hormone-binding globulin, and dehydroepiandrosterone sulfate were measured, and pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, and androstenedione were measured 0 and 60 min after the acute iv administration of ACTH-(1-24). Controls and HA patients did not differ in mean age or body mass, but HA women had higher basal T, free T, dehydroepiandrosterone sulfate, androstenedione, and LH/FSH and lower sex hormone-binding globulin levels. The mean estimated basal 17-OH activity was higher among HA patients than in controls. Although 52 HA patients demonstrated solely an exaggerated basal delta 5-17-OH activity estimate, few HA patients had an exaggerated estimate for either basal delta 4-17-OH or ACTH-stimulated 17-OH activity. No HA patient demonstrated an exaggerated 17,20-lyase basal activity, whereas 14 demonstrated an exaggerated delta 4-17,20-lyase ACTH-stimulated activity only. There was no association between these estimates of 17-OH and 17,20-lyase activities and the circulating adrenal androgen levels in HA women. Importantly, none of the patients demonstrated an increase in the basal activities of both 17-OH and 17,20-lyase, and only 4 patients demonstrated an exaggerated ACTH-stimulated activity of both 17-OH and 17,20-lyase. In conclusion, the steroidogenic profile observed in this population of HA women before and after ACTH-(1-24) stimulation is not consistent with dysregulation of cytochrome P450c17 alpha and probably represents a generalized alteration of adrenocortical control or biosynthesis.

Objective: Deficient adrenocortical 3 beta-hydroxysteroid dehydrogenase activity has been reported in 5% to 30% of hyperandrogenic women. Our objective was to determine the incidence and degree of 3 beta-hydroxysteroid dehydrogenase deficiencies in hyperandrogenism., Study Design: A prospective study of adrenal function in patients with hyperandrogenism was performed in a tertiary care university medical center. Eighty-six consecutive patients with hirsutism or hyperandrogenic oligomenorrhea were studied; 26 healthy eumenorrheic women served as controls. All subjects underwent serum sampling at rest and a 1-hour adrenal stimulation test with 1 mg of intravenously corticotropin-(1-24). Dehydroepiandrosterone sulfate, androstenedione, sex hormone-binding globulin, total and free testosterone, and luteinizing and follicle-stimulating hormones were measured in basal serum; dehydroepiandrosterone, 17-hydroxyprogesterone, and 17-hydroxypregnenolone were measured in basal and corticotropin-stimulated serum. On the basis of experience with genetically defined 21-hydroxylase late-onset adrenal hyperplasia, patients were presumed to suffer from 3 beta-hydroxysteroid-deficient late-onset adrenal hyperplasia if they demonstrated a dehydroepiandrosterone or 17-hydroxypregnenolone response to corticotropin-(1-24) stimulation (absolute poststimulation level or net increment) greater than threefold the upper 95th percentile of controls., Results: Three women of two families (2.3%) had a 17-hydroxyprogesterone response consistent with 21-hydroxylase-deficient late-onset adrenal hyperplasia and were excluded from further study. Eighteen (21%) of the remaining patients had a 17-hydroxypregnenolone poststimulation increment above the upper 95th percentile of controls (13.9 nmol/L), and two had an elevated dehydroepiandrosterone increment (> 19.5 nmol/L). However, no patient exceeded threefold the upper control limit for either steroid response. Patients with an exaggerated dehydroepiandrosterone or 17-hydroxypregnenolone increment had higher circulating dehydroepiandrosterone sulfate levels but similar basal total and free testosterone, sex hormone-binding globulin, luteinizing and follicle-stimulating hormone concentrations, basal or stimulated androstenedione, dehydroepiandrosterone/androstenedione, and 17-hydroxypregnenolone/17-hydroxyprogesterone than their less responsive counterparts., Conclusions: Although an exaggerated response of 17-hydroxypregnenolone to adrenal stimulation is common in hyperandrogenism, a response severe enough to merit consideration as 3 beta-hydroxysteroid dehydrogenase-deficient late-onset adrenal hyperplasia was not encountered in this unselected patient population, suggestive of the rarity of this disorder.

One to 2% of hyperandrogenic women demonstrate a 17-hydroxyprogesterone (17-HP) level greater than 36.3 nmol/L (1200 ng/dL) after acute ACTH-(1-24) adrenal stimulation, consistent with 21-hydroxylase (21-OH) deficient late-onset adrenal hyperplasia (LOAH). The following study was undertaken to endocrinologically and genetically define hyperandrogenic patients with an exaggerated 17-HP response to ACTH stimulation, and which do not represent LOAH. Of 265 consecutive patients suffering from hirsutism and/or hyperandrogenic oligomenorrhea, 23 (8.7%) demonstrated a 17-HP level 30 min post stimulation greater than 9.6 nmol/L or 316 ng/dL (the upper 95th percentile in 41 eumenorrheic nonhirsute healthy control women). Seven patients or five separate families (1.8% of total) demonstrated poststimulation 17-HP levels consistent with LOAH. Of the remaining 16 patients, the net increment in 17-HP (delta 17-HP0-30) was within normal limits in seven (2.6%) and these women were assumed to have a normal 17-HP adrenocortical response superimposed on an elevated basal level of nonadrenal (e.g. ovarian) origin. In the remaining nine hyperandrogenic patients (3.4%) various abnormalities of adrenal response were noted in all but one patient, consistent with adrenal hyper-responsiveness. One patient demonstrated an 11-deoxycortisol poststimulation level greater than 3-fold the upper 95th percentile of normal, consistent with 11-hydroxylase LOAH and was excluded from further study. Six of these women were available for further genetic characterization, all Caucasian and unrelated. Three were heterozygotes for HLA-B14, three for B40, and one for B35 antigen, HLA-types associated with the inheritance of 21-OH deficiencies. Although, normally there are two 21-OH genes (a pseudogene and a functional gene) present in a 1:1 ratio, we have previously reported a high frequency of 21-OH gene ratio abnormalities in LOAH. All but one of our patients demonstrated an abnormal 21-OH gene ratio. In conclusion, 3.4% of our hyperandrogenic population demonstrated an exaggerated 17-HP increment after ACTH stimulation, not consistent with LOAH or increased extraadrenal 17-HP production. The increased prevalence of HLA alleles known to be linked to inherited defects of 21-OH function and the increased frequency in 21-OH gene ratio abnormalities suggest that a majority of these individuals may be carriers for these genetic disorders. However, the adrenocortical abnormalities noted were more consistent with a generalized hyperreactivity of the adrenal to ACTH stimulation, than a specific enzyme deficiency, implying that carrier status for 21-OH deficiency may be incidental to the hyperandrogenism.

To determine the presence of structural abnormalities of the adrenal in late-onset adrenal hyperplasia, four consecutive patients were studied by MRI before beginning glucocorticoid replacement therapy. Three women were diagnosed as 21-hydroxylase deficient late-onset adrenal hyperplasia by a 17-OHP level greater than 1,000 ng/dL 30 minutes after acute adrenal stimulation, and one patient was diagnosed as 11-hydroxylase deficient late-onset adrenal hyperplasia when her 11-deoxycortisol level was threefold the upper 95th percentile of normal. Two patients with 21-hydroxylase deficient late-onset adrenal hyperplasia had normal adrenal glands on MRI. Another 21-hydroxylase deficient late-onset adrenal hyperplasia patient was noted to have a 2.5 x 3.3-cm left adrenal nodule, which had been documented some 4 years earlier on CT scan and had not changed in size during that interval. This patient was 40 years of age when the diagnosis of late-onset adrenal hyperplasia was established. The patient with 11-hydroxylase deficient late-onset adrenal hyperplasia demonstrated a diffuse enlargement of the left adrenal gland consistent with hyperplasia, with no focal lesions. In conclusion, although patients with late-onset adrenal hyperplasia may often demonstrate nodular or diffuse adrenocortical hyperplasia on MRI, not all patients with endocrinologically evident disease demonstrate such abnormalities, consistent with a lesser degree of ACTH stimulation compared with women with classical congenital adrenal hyperplasia.

Ten patients affected with 21-hydroxylase (21-OH) deficient late-onset adrenal hyperplasia were studied to determine the prevalence of a mutation at codon 281 of the functional 21-OH gene (CYP21B) that results in a valine to leucine amino acid shift. This mutation has been reported in eight unrelated late-onset adrenal hyperplasia patients of Ashkenazi Jewish descent possessing the human leukocyte antigen-B14,DR1 haplotype. Normally, there are two 21-OH genes; a pseudogene (CYP21A) and a functional gene (CYP21B). The aberrant codon 281 sequence is normally present only in CYP21A. In all of our late-onset adrenal hyperplasia patients, hybridization of an oligonucleotide probe specific for this mutation was demonstrated to CYP21A but not to CYP21B. The mutation at codon 281 of CYP21B does not appear to be a ubiquitous genetic marker for 21-OH deficient late-onset adrenal hyperplasia, suggesting that this disorder may demonstrate the same molecular heterogeneity as congenital adrenal hyperplasia.

Assessment of adrenal reserve and the diagnosis of adrenal insufficiency by acute adrenocortical stimulation with ACTH-(1-24) has been well established. Alternatively, estimation of adrenocortical enzymatic activities by this method for the detection of inherited or acquired biosynthetic abnormalities has been less well characterized. Some of the discrepancies between studies estimating adrenocortical enzymatic activities in different pathological conditions (e.g. hyperandrogenism) may result from the different stimulation protocols used. The objective of this prospective study was to establish the inherent variability of the adrenal response to acute ACTH-(1-24) stimulation and to determine the effect of sampling time, stimulation dose, and subject weight on the same. Forty-one normal female volunteers were recruited (mean age, 29.1 yr), 30 within 90-110% ideal body weight and 11 weighing more than 120% ideal body weight. Three protocols were designed to study 1) the effects of sampling time, ACTH-(1-24) dose, and subject weight on adrenal response; 2) the effect of time of the day on the variability of basal steroid levels and the adrenal response to stimulation; and 3) the long term reproducibility of the adrenal response to ACTH-(1-24). Androstenedione, 17-hydroxyprogesterone, 11-deoxycortisol, dehydroepiandrosterone, and cortisol were measured in serum under basal and stimulated conditions. All subjects had normal basal levels of testosterone, androstenedione, dehydroepiandrosterone sulfate, and PRL. The acute iv administration of 0.10, 0.25, and 1.0 mg ACTH-(1-24) elicited similar and maximal steroid responses, with all steroid levels reaching a plateau 60-90 min poststimulation regardless of subject weight. Sampling of basal steroid levels every 5 min in the morning (AM; beginning 0700-0900 h) or evening (PM; 1500-1700 h) did not reveal any difference in steroid variability. Only the mean basal cortisol level was higher in AM than PM testing (P less than 0.03). Although the mean levels of dehydroepiandrosterone and 17-hydroxyprogesterone 60 min after stimulation were significantly higher in AM than PM studies, these differences were minimal. Ten volunteers underwent an average of four (range, 2-6) adrenal stimulation studies using 1.0 mg ACTH-(1-24) over a 1-yr period. The long term coefficient of variation (CV) for basal steroid levels ranged from 15-28%. Calculations of net adrenal response (delta steroid O-T and area delta steroid O-T) were less reproducible (CV, 0-82%) than measures of absolute response (steroid T, area steroid T, and %steroid T; CV, 7-32%). This difference in CV between the measures of net and absolute adrenal responses was significant for all steroids except androstenedione.(ABSTRACT TRUNCATED AT 400 WORDS)

Late-onset adrenal hyperplasia caused by 21-hydroxylase deficiency leads to hyperandrogenic symptoms in 1% to 6% of hyperandrogenic women. Normally there are two 21-hydroxylase genes present in a 1:1 ratio. Gene CYP21A is a nonfunctional pseudogene, whereas CYP21B is an active gene. Abnormalities of the CYP21A/CYP21B gene ratio may serve as a marker for late-onset adrenal hyperplasia. Eight hyperandrogenic patients with late-onset adrenal hyperplasia and five control subjects were studied by evaluation of autoradiograms of Taq I and Kpn I digests by means of laser densitometry. Seven of eight (87%) patients with late-onset adrenal hyperplasia had an abnormal CYP21A/CYP21B gene ratio on laser densitometry, suggestive of CYP21A gene duplication, CYP21B gene deletion, or the conversion of a CYP21B gene to a CYP21A gene. One of the five control subjects had a heterozygous deletion of the CYP21A gene. The CYP21A/CYP21B gene ratio may serve as a useful genetic marker for late-onset adrenal hyperplasia in a non-high-risk population.