OBJECTIVE

In patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), low levels of adrenal steroids have been repeatedly demonstrated, but the site of alteration has not been exactly described because measurements of serum pregnenolone and 17-hydroxypregnenolone (<em>17OHP</em>reg) together with other adrenal steroids have never been performed.

METHODS

We measured serum levels of adrenal hormones such as pregnenolone, <em>17OHP</em>reg, dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), progesterone (P), 17-hydroxyprogesterone (<em>17OHP</em>), androstenedione (ASD), and cortisol in 24 healthy controls, 24 patients with RA, and 24 patients with SLE.

RESULTS

Serum levels of pregnenolone were similar in RA and SLE patients as compared to healthy controls irrespective of prior prednisolone therapy. In all RA and SLE patients (including those with prior prednisolone treatment), serum levels of all measured hormones except pregnenolone were significantly lower as compared to controls. In RA patients without prior prednisolone treatment, serum levels of <em>17OHP</em>reg, DHEA, cortisol, and ASD were similar to controls, and serum levels of P, <em>17OHP</em>, and DHEAS were significantly lower as compared to controls. In SLE patients without prior prednisolone treatment, serum levels of <em>17OHP</em>reg and cortisol were similar, and serum levels of P, <em>17OHP</em>, ASD, DHEA, and DHEAS were significantly lower as compared to controls.

CONCLUSIONS

The primary hormone of the adrenal steroid cascade, pregnenolone, is almost normal in RA and SLE irrespective of corticosteroid treatment. In patients with RA, we believe that there is a near normal P450scc reaction and a normal double step P450c17 reaction. In SLE patients, the P450scc reaction also seems normal but the second step of the P450c17 reaction seems to be inhibited. In both diseases, cortisol levels remain relatively stable at the expense of other adrenal hormones. This study revealed distinct changes of steroid pathways that are related to the disease entities.

OBJECTIVE

The appearance of 21-hydroxylase autoantibodies (21OHAbs) identifies subjects with preclinical adrenal insufficiency. In 21OHAb-positive subjects, the adrenocortical function is best evaluated by peak cortisol (F) levels after the low-dose (1 micro g) ACTH stimulation test (LDT). No information is currently available on the correlation between F and other adrenocortical hormone responses to the LDT in subjects with an ongoing autoimmune adrenal process. In this study, we tested the hypothesis that the dehydroepiandrosterone (DHEA), 17alpha-hydroxyprogesterone (<em>17OHP</em>) and aldosterone (A) responses to the LDT are consensual to that of F during the preclinical phase of autoimmune adrenal insufficiency.

METHODS

We studied 12 subjects positive for 21OHAb, in the absence of clinical signs of adrenal insufficiency. On the basis of peak F levels after the LDT, and according to the lower level of normal observed in 15 healthy volunteers (510.4 nmol/l), patients were subdivided into two groups: group A, n = 6 subjects with normal F response; and group B, n = 6 subjects with impaired F response. Results were expressed as absolute delta increase (Delta) between peak and basal levels.

RESULTS

DeltaF was significantly higher in group A (314.5 +/- 115.8 nmol/l) than in group B (151.7 +/- 88.2 nmol/l) (P = 0.041). DeltaDHEA and Delta<em>17OHP</em> were also significantly higher in group A (17.0 +/- 13.5 nmol/l and 6.1 +/- 4.4 nmol/l, respectively) than in group B (0.69 +/- 2.25 nmol/l and 1.9 +/- 1.7 nmol/l, respectively) (P = 0.002 and P = 0.041). The difference in DeltaA between the two groups did not reach statistical significance (group A 321.8 +/- 272.0 pmol/l vs. group B 157.0 +/- 154.0 pmol/l). DeltaDHEA, Delta<em>17OHP</em> and DeltaA tended to correlate positively with DeltaF (P = 0.039, P = 0.039 and P = 0.044, respectively), but the correlations did not reach significance after correction of the P-value.

CONCLUSIONS

Our study demonstrates a high concordance between F and DHEA, <em>17OHP</em> and A responses to the LDT in subjects with preclinical adrenal autoimmunity, thus strengthening the concept that the LDT is an accurate test to identify early adrenal dysfunction.

A patient with a testicular interstitial cell tumor and gynecomastia is reported. The testicular tumor was capable of aromatizing 8.3% testosterone. Spermatic venous samples taken from the tumor-bearing side had marked elevations of 17 beta-estradiol (E2), progesterone, and 17-hydroxyprogesterone (170 HP). The progesterone to <em>17OHP</em> and <em>17OHP</em> to androstene-dione ratios suggested an enzymatic block of 17 alpha-hydroxylase and 17-20 lyase possibly secondary to locally produced E2. The E2 produced by the tumor appeared to suppress gonadotropin secretion. The plasma testosterone and gonadotropin levels rose within 7 days after the removal of tumor, and the gynecomastia began to decrease.

Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency (21OHD) has a worldwide incidence of 1 in 15-20,000. Affected individuals have adrenal insufficiency and androgen excess; the androgen excess begins during fetal life, typically resulting in 46,XX disordered sexual development. In 21OHD, 17-hydroxyprogesterone (<em>17OHP</em>), the steroid proximal to 21-hydroxylase, accumulates. Most industrialized countries have newborn screening programs that measure <em>17OHP</em>; such screening has permitted rapid detection of newborns with 21OHD, saving lives previously lost to mineralocorticoid deficiency and salt wasting. However, newborn screening is plagued by false positives. <em>17OHP</em> is above most "cutoff values" in the first 24 h of life, is high in otherwise normal premature infants, and in many term infants with physiologic stress from unrelated diseases. In addition, newborn <em>17OHP</em> may be elevated in other forms of CAH, including 11-hydroxylase deficiency, 3β-hydroxysteroid dehydrogenase deficiency, and P450 oxidoreductase deficiency. In 21OHD, some of the accumulated intra-adrenal <em>17OHP</em> is converted to 21-deoxycortisol (21-deoxy) by 11β-hydroxylase (CYP11B1); 21-deoxy is not elevated in premature infants or in other forms of CAH, and hence is a more specific marker for 21OHD. However, 21-deoxy assays have not been generally available until recently, hence experience is limited. We urge clinical investigators, commercial reference laboratories, and newborn screening programs to investigate replacing <em>17OHP</em> with 21-deoxy as the analyte of choice for studies of 21OHD.

OBJECTIVE

Adverse outcomes in adult congenital adrenal hyperplasia (CAH) patients are frequent. The determinants of them have not yet been established.

OBJECTIVE

To establish the prevalence of adverse outcomes and to find determining factors for each of them.

METHODS

Cross-sectional monocentric study of 104 patients with childhood onset of CAH (71 women, 33 men). Analysis established first the determinants of clinical, hormonal, genetic variables and second a composite criterion for some of the outcomes and determinants.

RESULTS

BMI was above 25 kg/m(2) in 44% of the cohort, adrenal hyperplasia and/or nodules were present in 45% of the patients, and irregular menstrual cycles and hyperandrogenism were found in 50 and 35% of the women respectively. In univariate analysis, the determinants of these outcomes were all linked to disease control, especially 17-hydroxyprogesterone (<em>17OHP</em>) and androstenedione concentrations. Low weight was a determinant of abnormal bone mineral density (BMD) (60% of the cohort). Multivariate analysis confirmed these data. A classic form (CF) of CAH was a determinant of testicular adrenal rest tumors (TARTs) (36% of the men). Total cumulative glucocorticoid dose was a determinant of BMI and TART, whereas fludrocortisone dose was a determinant of TART (P=0.03). In men, the composite criterion was associated with androstenedione concentration and CF. In women, the composite criterion was associated with total testosterone concentration.

CONCLUSIONS

The present study confirms the high prevalence of adverse outcomes in CAH patients. These are, most often, related to disease control. The impaired health status of adults with CAH could therefore be improved through the modification of treatment.

OBJECTIVE

This study investigated the prevalence and consequences of heterozygous CYP21A2 mutations in premature pubarche (PP) girls.

RESULTS

We investigated 36 French Mediterranean girls with isolated PP. We performed synacthen testing with <em>17OHP</em> and 21-deoxycortisol evaluation, along with molecular analysis of the CYP21A2 gene in girls with abnormal elevation of one of these two adrenal steroids. Three girls (8.3%) had nonclassical adrenal hyperplasia, secondary to compound heterozygosity that associated at least one severe mutation for the three girls. A heterozygous mutation of the CYP21A2 gene was confirmed by molecular biology in eight girls (22%); a deletion of the CYP21A2 gene was found in one of them. Biological hyperandrogenism was found in the prepubertal CYP21A2 mutation carriers, whereas the four heterozygous girls who were followed long enough to have reached pubertal age presented biological and clinical hyperandrogenism.

CONCLUSIONS

We underline the high prevalence of heterozygous CYP21A2 mutations in girls with PP and demonstrate the usefulness of systematic screening by synacthen testing, both to improve their future clinical management and to prevent the transmission of classical adrenal hyperplasia to future offspring. Because of the severe metabolic and cardiovascular consequences of hyperandrogenism, long-term follow-up of these heterozygous patients is mandatory.

This investigation was conducted to evaluate the effect of thyroid dysfunction on the pituitary-gonadal axis. Ten men with Graves' disease and 5 hypothyroid patients were studied; 10 normal males were studied as a control group. In untreated conditions hyperthyroidism was associated with a normal serum-free testosterone concentration, an increased serum of <em>17OHP</em> levels, a reduced testosterone response to hCG stimulation, and a hyperresponse of LH to GnRH. These abnormalities reverted after normalization of high FT4 serum levels. Untreated hypothyroid men showed a normal hormone sex response to hCG, but the LH responst to GnRH was reduced, with a tendency to improve after T4 supplementation. There was a strong and significant negative correlation between FT4 and testosterone response, expressed as an area under the curve, and a positive correlation with LH response to GnRH. Despite normal basal free testosterone concentrations, 70% of hyperthyroid and 60% of hypothyroid patients had complaints of decreased libido. The results suggest that thyroid hormones play an important dual pituitary-gonadal effect that is reflected by an impairment of testicular testosterone synthesis associated to hyperresponse to LH in hyperthyroidism and a defective LH response to GnRH in hypothyroidism.

OBJECTIVE

A previously published study has identified that anovulatory women with PCOS have an increased response of 17 alpha-hydroxyprogesterone (<em>17OHP</em>) and androstenedione to a GnRH analogue suggesting dysregulation of cytochrome P450c17 alpha. The object of this study was to compare the responses of the pituitary-ovarian axis to a single dose of a long-acting GnRH agonist (GnRHa) in both ovulatory and anovulatory women with PCOS with those in normal subjects.

METHODS

Comparative study of responses of LH, FSH and ovarian steroids to buserelin and the adrenal steroid response to synthetic ACTH in two groups of women with hyperandrogenaemia and polycystic ovaries: those with anovulatory menses or amenorrhoea and those with equally elevated serum testosterone concentrations but regular menses. Results in both groups of women with PCO were compared with those in normal subjects.

METHODS

Twenty-four women with hyperandrogenism and PCO (14 had oligo or amenorrhoea, 10 regular cycles) and 12 weight matched controls with normal ovaries, regular cycles and neither clinical nor biochemical evidence of hyperandrogenism. Subjects were given synthetic ACTH (Synacthen) 250 micrograms i.v. on day 1 of the study and blood collected at 30 and 60 minutes thereafter. On the evening of day 1, dexamethasone treatment was commenced to suppress adrenal androgens. GnRHa 100 micrograms s.c. was given on day 2 and blood samples collected at 30-minute intervals for 4 hours and once more at 24 hours after the injection.

RESULTS

The acute responses of both immunoactive and bioactive LH to GnRHa were significantly greater in the ovulatory PCO group (ovPCO) than controls but the response was greater in anovulatory women with polycystic ovaries (anovPCO) than in either ovPCO or controls, throughout the 24-hour study period. Despite the discrepancy in LH concentrations, basal serum concentrations of androstenedione were equally elevated in anovulatory and ovulatory women with PCO, compared with controls. There was a small but significant increase in androstenedione following GnRHa in both PCO groups at 24 hours but not in controls. A similar pattern was observed in the response of <em>17OHP</em> to GnRHa although the response was significantly higher than controls in anovPCO women only. By contrast, the responses of both androstenedione and <em>17OHP</em> to 250 micrograms synthetic ACTH were similar in PCO women to those in controls.

CONCLUSIONS

These data provide evidence for ovarian hypersecretion of androgens in ovulatory, as well as anovulatory women with PCO, supporting the concept of abnormal regulation of 17-hydroxylase and (17,20-lyase activity in the ovary. The finding of an equal degree of hyperandrogenaemia in ovPCO and anovPCO groups, even though LH levels were much higher in the latter, suggests that hypersecretion of LH is not the primary cause of ovarian hyperandrogenism. Hyperandrogenism in PCOs may therefore represent an intrinsic abnormality of ovarian theca-interstitial cell function.

To study ovarian and adrenal steroid profiles of women with idiopathic hirsutism, we compared sex steroid and basal and corticotropin (ACTH)-stimulated adrenal steroid levels before and after ovarian suppression induced by a long-acting gonadotropin-releasing hormone agonist analog (GnRH-a) in 24 hirsute women without hyperandrogenemia. Twelve healthy women served as controls for basal and ACTH-stimulated adrenal steroid levels. Serum levels of testosterone (T), sex hormone-binding globulin (SHBG), estradiol (E2), basal and ACTH-stimulated 17-hydroxyprogesterone (<em>17OHP</em>), dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), delta 4-androstenedione (delta 4-A), 11-deoxycortisol (S) and cortisol (F), and basal and luteinizing hormone-releasing hormone (LHRH)-stimulated gonadotropin levels were measured before and 21 days after 3.75 mg intramuscular triptorelin in hirsute women. Basal T levels and basal and ACTH-stimulated delta 4-A, DHEA, and DHEAS levels were not different in hirsute women with respect to controls. Basal and ACTH-stimulated <em>17OHP</em> was elevated, and decreased to normal after ovarian suppression with triptorelin. Although basal and ACTH-stimulated delta 4-A levels were normal, the delta delta 4-A/delta F and delta delta 4-A/delta <em>17OHP</em> ratios were elevated and remained elevated after ovarian suppression, suggesting enhanced adrenal delta 4-17,20-lyase activity. T, F, S, and DHEAS levels were not affected by ovarian suppression. Basal and ACTH-stimulated <em>17OHP</em> and delta 4-A, and stimulated DHEA concentrations were reduced with ovarian suppression, but their net increment and ratio to the increase of F in response to ACTH remained unchanged, reflecting the ovarian contribution to the secretion of these steroids. We conclude that idiopathic hirsute women with normoandrogenemia show an increase in ovarian secretion of <em>17OHP</em> and a minimally increased adrenal delta 4-17, 20-lyase activity, suggesting that mild forms of ovarian and adrenal functional hyperandrogenism may be present in these patients with otherwise unexplained hirsutism.

Exaggerated adrenal response (ExAR), i.e. hypersecretion of both 17-hydroxypregnenolone (170HPreg) and 17-hydroxyprogesterone(<em>17OHP</em>) in response to adrenocorticotropic hormone (ACTH) stimulation, is frequently found in women with polycystic ovary (PCO) syndrome who had precocious adrenarche. In an earlier study we found an abnormal adrenal response in girls with idiopathic true central precocious puberty (CPP) at early stages of puberty. On follow-up it was noted that a significant number of girls with CPP develop PCO-like syndrome at a relatively young age. The aim of the present study was to determine if there is an association between ExAR and early PCO in girls with a history of CPP. Included were 49 girls with a history of CPP, 34 of whom were treated with gonadotropin-releasing hormone (GnRH) analog. All 49 were evaluated at full maturity, at ages 12.5-14 years, 0.5-4 years after menarche or resumption of menses. Of the 49 girls, 20 had at least 3/4 clinical signs of PCO (irregular menses, hirsutism, acne and obesity) and were defined as PCO-like+, whereas 29 did not fulfil the criteria and were considered PCO-like-. Girls with a definite enzyme deficiency were excluded from the study. All participants underwent a combined iv ACTH-GnRH test at early follicular phase. The PCO-like+ girls all revealed ExAR, i.e. an elevated stimulated <em>17OHP</em>reg of 63.4 +/- 9.6 nmol/l (normal 28.6 +/- 9.2 nmol/l) and a normal stimulated <em>17OHP</em>reg/<em>17OHP</em> ratio of 7.1 +/- 1.8 (normal 6.2 +/- 2.7), whereas all the PCO-like- had a normal adrenal response (30.0 +/- 8.7 and 5.3 +/- 2.0 nmol/l, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

The purpose of this study was to evaluate the usefulness of 17 hydroxyprogesterone (<em>17OHP</em>) determination in dried filter paper blood samples from patients with congenital adrenal hyperplasia caused by 21-hydroxylase deficiency. It was hypothesized that these home samples would enhance patient treatment.

METHODS

Results of <em>17OHP</em> determination in simultaneously collected venous and dried filter paper blood samples were compared to establish assay reliability. Thereafter, parents mailed dried filter paper blood samples collected before each hydrocortisone dose.

RESULTS

The <em>17OHP</em> levels in wet and dried blood samples correlated well (r = 0.98). Results did not change when stored for 2 weeks under various conditions. Blood sampling at different times of the day provided insights into the patterns of <em>17OHP</em> secretion and identified times of inadequate adrenal suppression. Dose adjustments were then made considering the time of day when adrenal suppression was inadequate.

CONCLUSIONS

Home monitoring of <em>17OHP</em> is a reliable and practical approach for assessing adrenal steroid activity in patients with congenital adrenal hyperplasia. Considering the time of day of <em>17OHP</em> elevations also facilitates hydrocortisone dosing adjustment.

Amniotic fluid (AF) levels of 17-hydroxyprogesterone (<em>17OHP</em>) and testosterone (T) were determined at 16-17 weeks in 17 pregnancies at risk for CAH and results compared to 75 normal controls. The fetus was predicted to be unaffected in 12 cases on the findings of normal AF levels of both <em>17OHP</em> and T and the latter allowed a correct prediction of fetal sex in all instances. HLA typing confirmed normality in 12 cases revealing 5 carriers, 5 homozygous normal and 2 indeterminate. Steroid levels of the 2 groups were similar. Three fetuses were predicted to be CAH affected on unambiguously high levels of <em>17OHP</em> and T (in female only). HLA typing was in agreement, and the diagnosis was confirmed in 2 abortuses and a female newborn by physical and hormonal studies. In the last 2 cases AF levels of OHP and T were normal but HLA (A/B/C) genotypes were identical to the CAH affected siblings. Normal physical and hormonal findings in the 2 aborted fetuses would exclude the possibility of an in utero virilizing form of CAH. The discrepancy could be explained on the basis that the fetuses had an allelic form of 21-hydroxylase deficiency or on the basis of recombination (not fully tested). It is concluded that a fully informative prenatal diagnosis of CAH should not rely entirely on HLA typing but on hormonal studies.

17alpha-Hydroxyprogesterone is a metabolic precursor of cortisol; elevated levels of 17alpha-hydroxyprogesterone are indicative of congenital adrenal hyperplasia. Traditional determination by immunoassay is plagued by poor antibody specificity, resulting in significant interferences. This study explores an LC-MS/MS method for the quantitation of <em>17OHP</em> in serum. Deuterated 17alpha-hydroxyprogesterone was added as internal standard, followed by solid-phase extraction, HPLC separation with a C16-amide reverse-phase column with run time of 7 min, and quantification by MS/MS (positive electrospray ionisation) in the selected reaction monitoring mode (SRM). Transitions monitored were 331>109 for the analyte and 339>113 for the deuterated internal standard. Intra-assay precision (%R.S.D.) was 7.4% at 7 nmol/L, inter-assay precision (%R.S.D.) at 2, 7 and 27 nmol/L was 15.4, 10.0 and 7.9% and accuracy at 0.9 nmol/L was 100%. The method was linear from 0.156 to 80 nmol/L. Lower limit of quantitation was 0.2 nmol/L, providing meaningful data for patients within normal range as well as those with elevated levels.

To determine whether serum 3 alpha-androstanediol glucuronide (3AG) reflects the overall effect of integrated adrenal androgen secretion in the virilizing form of congenital adrenal hyperplasia (CVAH), circadian levels (0800, 1200, 1600, and 2000 h) of serum 3AG and 17-hydroxyprogesterone (<em>17OHP</em>) or 11-deoxycortisol (S), androstenedione (A), testosterone (T), and 24-h urinary 17-ketosteroids (17KS) were examined in seven patients (pts) with classical 21-hydroxylase deficiency (21OHD) and one pt with classical 11 beta-hydroxylase deficiency (11 beta OHD). Hormonal studies were conducted during the second day of dexamethasone (Dex) administration (2 mg/day). In five poorly controlled CVAH pts, including the 11 beta OHD pt, highly elevated baseline morning (AM) serum <em>17OHP</em> or S as well as A levels, and elevated AM T levels in three pts decreased markedly in the evening (PM), while elevated serum 3AG showed no significant circadian changes; 17KS levels were markedly elevated for age. During Dex, moderately or slightly elevated AM <em>17OHP</em>, A, or T in two to four pts with 21OHD decreased to the normal range in the PM. In the pt with 11 beta OHD, S, A, and T levels were suppressed. 3AG levels were modestly elevated or normal, without circadian changes, in these pts; 17KS levels were elevated or normal. In two other 21OHD pts, modestly elevated AM baseline <em>17OHP</em> and A levels decreased in the PM; elevated AM T decreased in one pt in the PM; modestly elevated 3AG levels showed no circadian changes; 17KS levels were modestly elevated. During Dex, normal or slightly elevated serum steroids and 17KS levels were associated with normal or high normal 3AG levels without circadian changes. In one postpubertal female with 21OHD, modestly elevated AM baseline <em>17OHP</em> levels decreased at 2000 h; normal A and T levels throughout the day and low normal 17KS were associated with slightly low 3AG levels, without circadian variation. During Dex treatment, normal <em>17OHP</em>, A, T, and low 17KS levels were associated with low 3AG levels without circadian variation. In all pts as a group, an excellent correlation (r = 0.9) was found between either 0800 h or mean, or 2000 h serum 3AG levels and 17KS. In addition, AM and PM serum 3AG levels in five normal women were similar. We conclude that the high correlation between serum 3AG and urinary 17KS and the absence of a significant circadian variation in 3AG indicate that serum 3AG, regardless of sample time, is a useful metabolic index of integrated adrenal androgen secretion in CVAH.

It has been hypothesized that progesterone (P) exerts a direct inhibitory effect on ovarian follicular development, an effect which could be mediated by P receptors located in granulosa cells. We tested this hypothesis by examining the effect of several progestins on FSH-stimulated estrogen (E), P, and 20 alpha-dihydroprogesterone (DHP) production by cultured rat granulosa cells, and correlated the results with the ability of the progestins to bind to the granulosa cell P receptor. Granulosa cells from immature hypophysectomized DES-treated rat produced 9 ng/ml E, 21 ng/ml P and 29 ng/ml DHP during a 2-day incubation in McCoy's 5a medium containing 10(-7) M androstenedione and 10 ng/ml of FSH. The FSH-induced increase in E production was inhibited by 50 and 95% following concomitant treatment with 3 x 10(-6) and 10(-5) M resp. of R5020, a potent synthetic progestin. Added R5020 at these concentrations also significantly inhibited P and DHP production. R5020 had no effect on granulosa cell viability or plating efficiency, and the inhibitory action of R50920 on E production was reversible. In studies of the specificity of the progestin inhibitory action, the relative abilities of various progestins to inhibit E production were R5020>> P>> DHP>> 17 alpha-hydroxyprogesterone (<em>17OHP</em>). The relative abilities of these progestins to bind to the ovary P receptor were also: R5020>> P>> DHP>> <em>17OHP</em>. These results indicate that exogenous progestins directly inhibit the FSH-stimulation of granulosa cell steroidogenesis in vitro and suggest that the progestin effect may be mediated by the P receptor. Such results offer a possible mechanism whereby progesterone could exert a direct but reversible inhibitory action on ovarian follicular development.

OBJECTIVE

An interaction between adiponectin, steroid synthesis or action and measures of insulin resistance (IR) have been reported in the pathogenesis of polycystic ovary syndrome (PCOS). The present study was done to determine plasma adiponectin concentration (PAC) in women with and without PCOS and to assess its correlation to the hormonal and metabolic parameters including measures of IR. The effect of Metformin for 6 months in PCOS was also evaluated.

METHODS

In total, 72 selected women were classified as follows: 17 obese (body mass index (BMI))>25 kg/m(2) with PCOS; 19 normal weight (BMI) 18-22.9 kg/m(2) with PCOS; 17 obese (BMI)>25 kg/m(2) without PCOS and 19 normal weight (BMI) 18-22.9 kg/m(2) without PCOS.

METHODS

Blood samples were collected from all women with PCOS between 0800 and 1100 h, after an overnight fast.

METHODS

Serum level of LH, FSH, TSH, total T4, testerosterone, 17-α-hydroxyprogesterone (<em>17OHP</em>), DHEAS, insulin, adiponectin and glucose. Measures of IR included fasting serum insulin (FSI), glucose-to-insulin ratio, and homeostasis model assessment (HOMA).

CONCLUSIONS

Waist-hip ratio (WHR), insulin, and HOMA index were significantly higher in the lower adiponectin group than in the higher adiponectin group. By using stepwise multiple regression analysis, in model 1 (including BMI, FSI, fasting plasma glucose (FPG) with other variables such serum as testerosterone and DHEAS), the weight and contributions from other variables, namely FSI and FPG were significant independent determinants of fasting PAC (adjusted r(2)=0.66); and in model 2 (including BMI, HOMA, FPG only as an index of IR with other variables such as serum testerosterone and DHEAS), BMI, and HOMA were significant independent determinants of fasting PAC (adjusted r(2)=0.59). FPG, HOMA index and FSI were significantly lower after Metformin treatment in both obese and non-obese PCOS while adiponectin levels increased significantly.

It has been shown that growth hormone (GH) and insulin-like growth factor-1 (IGF1) enhance steroidogenesis responsiveness to ACTH in cultured adrenal cells. To investigate the GH effect on adrenal steroidogenesis in non-GH-deficient subjects, we studied 9 girls with Turner syndrome (chronological age 5.5-7.2 years; bone age 5-7 years). In all subjects an ACTH test (Synacthen depot, 0.25 mg i.v. with blood samples at 0 and 60 min) was performed basally at 8-9 a.m. and 6 months after GH therapy (1 IU/kg/week). 17-Hydroxypregnenolone (17PGN), 17-hydroxyprogesterone (<em>17OHP</em>), dehydroepiandrosterone (DHA), its sulfate (DHA-S), androstenedione and cortisol were evaluated by radioimmunoassay. Two groups of normal girls were selected as controls: group A age-matched the patients at the start of the study, and group B age-matched the patients at the end of the study. The responsiveness of each hormone to ACTH was expressed as the difference between stimulated and basal values. A p value of < 0.01 was considered to indicate significance. There were no significant differences between pre- and posttreatment basal values of 17PGN, <em>17OHP</em>, DHA, androstenedione and cortisol in the Turner syndrome patients, whereas a significant increase was observed for basal DHA-S (1.57+/-0.31; 1.89+/-0.43 micromol/l, p < 0.01). Comparison of increments before and after GH treatment showed a significant increase in responsiveness to ACTH after GH therapy DHA (p < 0.01). The increase in 17PGN was evident (p < 0.02), but the established significant p value was not reached. No differences for <em>17OHP</em>, androstenedione and cortisol were found. The stimulated 17PGN/<em>17OHP</em> ratio was significantly higher (p < 0.01) after GH, whereas the <em>17OHP</em>/androstenedione ratio was considerably lower, but the p value was < 0.02. No differences between pretreatment values with the control group androstenedione was found, whereas basal and stimulated posttreatment values of DHA and stimulated values of 17PGN were higher in patients after GH therapy than in control group B. No differences between the 2 control groups were found. In conclusion our study showed that adrenal steroid responsiveness to ACTH increases in Turner syndrome after long-term treatment with high GH doses. An increase in the number of ACTH adrenal receptors and/or a modulation of enzyme activities may be suggested. The positive or negative pharmacological implications of these data remain to be determined especially when taking into consideration the wide use of GH therapy in non-GH-deficient subjects.

OBJECTIVE

Previous reports of endocrinological profiles in children presenting with premature adrenarche have not shown consistent abnormalities. We therefore aimed to review the clinical and biochemical aspects of a large number of patients presenting with premature adrenarche without virilization and determine the relation between clinical and biochemical characteristics and the frequency of adrenal steroid disorders.

METHODS

Eighty-eight patients presenting with adrenarche without virilization during 1985-1992 were retrospectively reviewed. There were 72 girls and 16 boys. All were normotensive and had either prepubertal breasts or testes < 4 ml. In patients with high adrenal androgen levels, adrenal tumours had been excluded by either adrenal ultrasound or CT scan.

METHODS

We recorded clinical manifestations, auxological data, bone age, biochemical results including basal 17OH-progesterone (b<em>17OHP</em>), dehydroepiandrosterone sulphate (DHEAS), androstenedione (delta 4A), testosterone, cortisol and stimulated <em>17OHP</em> and cortisol. ACTH stimulation tests (using soluble Synacthen 250 micrograms intramuscularly and collecting blood at 0, 30 and 60 minutes) were performed when clinically indicated. 17OH-Pregnenolone (<em>17OHP</em>reg) was also measured during ACTH stimulation tests in 13 individuals to look for abnormalities of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD).

RESULTS

The age of onset ranged from 3 to 9.5 years (mean 6.8 +/- 1.3). There were no significant differences by sex for height SDS, weight SDS or % ideal body weight, but bone age advancement was greater in males (P < 0.02). The most common presenting clinical manifestation was premature appearance of pubic hair in 93.8%, the other 6.2% presenting with body odour, acne and/or hirsutism. Twelve patients had b<em>17OHP</em>>> 6 nmol/l of whom 5 were diagnosed as having congenital adrenal hyperplasia (CAH) resulting from 21-hydroxylase deficiency after ACTH stimulation tests. A further 33 patients who had b<em>17OHP</em> < 6 nmol/l had normal <em>17OHP</em> and cortisol responses to ACTH stimulation. Patients, after excluding those with CAH, were divided on the basis of their DHEAS levels into prepubertal (< 1.5 mumol/l), pubertal (1.5-6 mumol/l) and above pubertal range >> 6 mumol/l). The 8 patients with DHEAS values above the pubertal range were described as having 'exaggerated adrenarche'. There were no significant clinical differences between these 3 groups, but significant differences were found for bone age advancement and the steroids, b<em>17OHP</em>, delta 4A and testosterone. There was a strong correlation between DHEAS and delta 4A (r = 0.623, P < 0.001). The 'exaggerated adrenarche' group had higher 17 OHPreg/<em>17OHP</em> ratios at 60 minutes after stimulation but these were not diagnostic for 3 beta-HSD deficiency.

CONCLUSIONS

The value of assessing basal steroids in children presenting with premature adrenarche is demonstrated in this series with 5.7% being diagnosed with 21-hydroxylase deficiency and 9.1% with 'exaggerated adrenarche'. No relation was found between adrenal steroids and clinical features except for the acceleration of bone age. The relation between 'exaggerated adrenarche' and future ovarian hyperandrogenism needs further evaluation.

The effect of flutamide on basal and ACTH-stimulated plasma levels of adrenal androgens was investigated in 6 patients with untreated advanced prostate cancer, aged 52-75 yr. Flutamide was administered (250 mg three times daily) for 10 days; before and after treatment, a synthetic ACTH1-24 stimulation test (250 micrograms im, with blood sampling immediately before and 60 min after the stimulus) was performed. Basal plasma 17OH-pregnenolone (delta 5-<em>17OHP</em>), 170H-progesterone (delta 4-<em>17OHP</em>), androstenedione (A), dehydroepiandrosterone (DHEA) and its sulphate (DHEAS) were unchanged by flutamide treatment. In contrast, basal plasma testosterone (T) concentrations significantly increased (p less than 0.05). The response of cortisol delta 4-<em>17OHP</em>, delta 5-<em>17OHP</em>, A and DHEA to ACTH, as well as the ACTH-stimulated delta 5-<em>17OHP</em>/delta 4-<em>17OHP</em>, delta 5-<em>17OHP</em>/DHEA, delta 4-<em>17OHP</em>/A and DHEA/A ratios, were unchanged by flutamide treatment. These findings indicate that: a) Short-term flutamide administration enhances testicular steroidogenesis, via augmented LH pulse frequency; b) Adrenal steroidogenesis seems to be not affected by the drug, since ACTH-stimulated plasma levels of adrenal androgens and precursors/products ratios were unchanged.

BACKGROUND

The primary concern related to congenital adrenal hyperplasia (CAH) newborn screening (NBS) is the high rate of false-positive results (FPR) associated with prematurity; false-negative results (FNR) can also occur due to precocious sample collection.

OBJECTIVE

To determine the neonatal 17-hydroxyprogesterone (N<em>17OHP</em>) normal range in newborns in Sao Paulo using different references according to age and birthweight (BW) and to establish the optimal NBS cut-off levels.

METHODS

Neonatal 17-hydroxyprogesterone levels from 271 810 newborns (NBs) according to sample collection time (G1: 48-<72 h and G2: ≥72 h) and BW (≤1500 g, 1501-2000 g, 2001-2500 and >2500 g) were evaluated. N<em>17OHP</em> was measured by an fluoroimmunoassay, and serum <em>17OHP</em> was measured by liquid chromatography-mass spectrometry. Affected and asymptomatic NBs with persistently increased <em>17OHP</em> levels were submitted to CYP21A2-sequencing.

RESULTS

Neonatal 17-hydroxyprogesterone levels in G1 were lower than G2 in all BW groups (P < 0·001). The FPR rate in G1/G2 was 0·2% using the 99·8th and 0·5% using the 99·5th percentile. The 99·8th percentile N<em>17OHP</em> value was the best cut-off for distinguishing between unaffected and affected NBs. Forty-four salt wasters, and five simple virilisers were diagnosed; N<em>17OHP</em> levels ranged from 93·3 to 2209·8 nmol/l, and no affected neonates with FNR were identified. The positive predictive value in G1 and G2 using the 99·8th percentile was 5·6% and 14·1%, respectively, and 2·3% and 7%, respectively, using the 99·5th percentile. Molecular tests identified two NBs with the nonclassical form among the 29 FPR.

CONCLUSIONS

Neonatal 17-hydroxyprogesterone levels adjusted to sample collection age and birthweight reduced the FPR, and the use of N<em>17OHP</em> values based upon the 99·8th percentile improved the NBS efficacy.

17alpha-hydroxypregnenolone (<em>17OHP</em>reg) has heretofore been considered to be the major cause of the false elevated 17alpha-hydroxyprogesterone (<em>17OHP</em>) value in the immunoassay-based newborn screening for congenital adrenal hyperplasia (CAH). To verify this point, we developed a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method that enables the simultaneous quantification of <em>17OHP</em>reg and <em>17OHP</em> in the dried blood filter papers and measured their blood levels in infants, especially in infants with low birth weights. Steroids were extracted from the filter papers with methanol, purified using a Strata-X cartridge, derivatized with 2-hydrazinopyridine and subjected to LC-MS/MS. Validation tests proved that this method was specific and reproducible; endogenous steroids did not interfere with the quantifications, and the intra- and inter-assay coefficients of variation were below 5.2%. The limits of quantitation were 1.0 and 0.5 ng/mL for <em>17OHP</em>reg and <em>17OHP</em>, respectively, when 3 disks (3 mm in diameter) of the filter papers (corresponding to 8 microL of whole blood) were used. The blood <em>17OHP</em>reg level was elevated in the very low birth weight (1000-1500 g) infants and extremely low birth weight (<1000 g) infants, compared to those in the normal birth weight (>2500 g) infants (P<0.05). However, the <em>17OHP</em>reg concentration was not high enough to cause the false positive results in the enzyme immunoassay-based screening, and it was considered that the false positive results come from other endogenous components rather than <em>17OHP</em>reg.

BACKGROUND

The clinical differential diagnosis of classic 21-hydroxylase deficiency (C21OHD) and cytochrome P450 oxidoreductase deficiency (PORD) is sometimes difficult, because both deficiencies can have similar phenotypes and high blood concentrations of 17α-hydroxyprogesterone (<em>17OHP</em>). The objective of this study was to identify biochemical markers for the differential diagnosis of C21OHD, PORD, and transient hyper 17α-hydroxyprogesteronemia (TH<em>17OHP</em>) in Japanese newborns. We established a 2-step biochemical differential diagnosis of C21OHD and PORD.

METHODS

We recruited 29 infants with C21OHD, 9 with PORD, and 67 with TH<em>17OHP</em>, and 1341 control infants. All were Japanese and between 0 and 180 days old; none received glucocorticoid treatment before urine sampling. We measured urinary pregnanetriolone (Ptl), the cortisol metabolites 5α- and 5β-tetrahydrocortisone (sum of these metabolites termed THEs), and metabolites of 3 steroids, namely dehydroepiandrosterone, androstenedione (AD4), and 11β-hydroxyandrostenedione (11OHAD4) by GC-MS.

RESULTS

At a cutoff of 0.020, the ratio of Ptl to THEs differentiated C21OHD and PORD from TH<em>17OHP</em> and controls with no overlap. Among metabolites of DHEA, AD4, and 11OHAD4, only 11β-hydroxyandrosterone (11HA), a metabolite of 11OHAD4, showed no overlap between C21OHD and PORD at a cutoff of 0.35 mg/g creatinine.

CONCLUSIONS

A specific cutoff for the ratio of Ptl to THEs can differentiate C21OHD and PORD from TH<em>17OHP</em> and controls. Additionally, the use of a specific cutoff of 11HA can distinguish between C21OHD and PORD.

17-hydroxyprogesterone (<em>17OHP</em>) is the most important plasma parameter for diagnosing and monitoring congenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency. A rapid, simple, and specific method based on microbore high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (micro-HPLC/ESI-MS/MS) was developed to determine the presence of <em>17OHP</em> on dried filter-paper blood samples from patients with CAH caused by 21-hydroxylase deficiency. <em>17OHP</em> from dried blood spots formed by the action of Girard reagent P (GirP) turned out to be a water-soluble hydrazone complex. Derivatization with GirP led to higher ESI sensitivity for <em>17OHP</em>. The LC/MS/MS detection of GirP-derivatized <em>17OHP</em> (GirP-<em>17OHP</em>) was rapid (<3 min). The method is repeatable and reproducible, with CVs <7% and 12%, respectively. This new method was used for direct determination of <em>17OHP</em> in dried blood specimens obtained from abnormal children and infants of various ages with a detection limit of 10 ng/mL ( approximately 12 microL blood). The method described allows for rapid and reliable measurements of <em>17OHP</em> in dried blood specimens from patients affected by CAH.

OBJECTIVE

To investigate the adrenal response in terms of allopregnanolone secretion in a group of hyperinsulinemic patients with polycystic ovary syndrome (PCOS).

METHODS

Controlled clinical study.

METHODS

Patients with PCOS in a clinical research environment.

METHODS

Twenty-two overweight patients with PCOS with hyperinsulinism were enrolled after informed consent.

METHODS

All patients underwent hormonal evaluations, oral glucose tolerance test (OGTT) and adrenocorticotropic hormone (ACTH) test before and after 4 months of metformin administration (500 mg p.o. bi-daily). Ultrasound examinations and Ferriman-Gallway score were also performed. Main outcome measures. plasma luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), estradiol, 17-hydroxy-progesterone (<em>17OHP</em>), androstenedione (A), testosterone (T), allopregnanolone, glucose, insulin, C peptide concentrations, body mass index (BMI).

RESULTS

Metformin administration reduced significantly LH, A, T, insulin and BMI, while allopregnanolone was significantly increased with no change in progesterone plasma levels. Insulin response to OGTT decreased and allopregnanolone response to ACTH stimulation before while this was restored after the treatment interval. The Ferriman-Gallway score as well as the ovarian volume was significantly decreased after 4 months of metformin therapy.

CONCLUSIONS

In overweight patients with PCOS with hyperinsulinism, allopregnanolone secretion is impaired and metformin administration restored normal allopregnanolone concentrations modulating both steroid syntheses from the ovaries and from adrenal gland.