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Departments of Endocrinology (D.G., A.P.H., J.H., C.H., M.A.) and Clinical Chemistry (K.B.), Odense University Hospital, 5000 Odense C, Denmark
Address all correspondence and requests for reprints to: Dorte Glintborg, Odense University Hospital, Department of Endocrinology, Kløvervænget 10, 3, 5000 Odense C, Denmark. E-mail: dorte.glintborg{at}ouh.fyns-amt.dk.
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Abstract |
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Methods: A total of 337 hirsute patients were evaluated by Ferriman-Gallwey score, serum testosterone, ACTH-stimulated 17OHP, and cortisol during the follicular phase. A cutoff value of 16 nmol/liter for maximum ACTH-stimulated 17OHP (M17OHP) responses was defined as the upper limit of the 95% confidence interval (CI) for the 97.5 percentile in 42 female controls. All patients were offered total screening of the CYP21 gene, and 252 healthy, premenopausal women with regular menses underwent genetic screening.
Results: Patients were divided into idiopathic hirsutism (IH) (n = 180) and polycystic ovary syndrome (PCOS) (n = 157) groups. M17OHP levels were significantly higher in IH [geometric mean value (nmol/liter ± 2 SD) 12.2 (4.6–32.3)] and PCOS [11.9 (5.3–27.2)] compared with controls [8.5 (5.1–14.2)] (P < 0.001). A similar percentage of IH and PCOS patients had elevated M17OHP (20.5 vs. 20.8%, not significant), and these also had significantly higher 30-min cortisol levels compared with controls (P < 0.05). The prevalence of CYP21 mutations in patients was 8.6% compared with 6.3% in controls (P = 0.38). Ten of 19 carriers had M17OHP levels below the cutoff limit.
Conclusion: The significantly higher ACTH-stimulated levels of cortisol and 17OHP in hirsute patients indicated adrenal hyperresponsiveness in IH and PCOS. CYP21-carrier status could not explain the observed high prevalence of abnormal ACTH-stimulated 17OHP levels in the hirsute population.
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Introduction |
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Previous studies have shown more than 20% of hirsute patients to be heterozygote CYP21 carriers (6, 7, 8). CYP21 carriers most often have elevated ACTH-stimulated 17-hydroxyprogesterone (17OHP) values between 4.3 and 9.9 µg/liter (13–30 nmol/liter) (6). A substantial overlap between healthy individuals and heterozygote CYP21 carriers is, however, known to exist, and a large propor-tion of heterozygote CYP21 carriers have normal ACTH-stimulated 17OHP levels (9). Elevated ACTH-stimulated 17OHP responses have been found in previous studies comparing patients with IH (10, 11) and PCOS (12, 13, 14) with controls. It was not established whether the increased ACTH-stimulated 17OHP levels were a result of CYP21 mutations or a hyperactive pituitary adrenal axis in IH or PCOS patients.
Most previous studies evaluating CYP21 carrier status in hirsute patients have used a screening of the most common mutations seen in homozygote individuals with adrenogenital syndrome. We, however, speculate that elevated ACTH-stimulated maximum 17OHP (M17OHP) values may be caused by mutations not necessarily seen in patients with adrenogenital syndrome and that elevated 17OHP levels during ACTH testing alternatively may be caused by a more nonspecific adrenal hyperactivity or by dysregulation of the adrenal enzyme P450c17
(13, 14, 15, 16, 17, 18, 19). Previous studies showing more than 20% of hirsute patients to be heterozygote CYP21 carriers have examined only small populations of patients with hyperandrogenemia or PCOS. To our knowledge, the evaluation of CYP21 carrier status has not been performed in IH patients. In our study, we performed full screening of the CYP21 gene to describe possible new mutations in hirsute patients. To characterize the adrenal secretory potential in our population, we furthermore established the reference intervals for basal (B17OHP) and maximum stimulated (M17OHP) 17OHP levels in 42 controls (20) and applied these results to subgroups of hirsute patients. We measured ACTH-stimulated cortisol levels to establish adrenal insufficiency in patients with CYP21 mutations and evaluate adrenal hyperactivity.
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Subjects and Methods |
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We included all patients with hirsutism as referral diagnosis in the Department of Endocrinology at Odense University Hospital during 1997–2002. None of the patients had a previously diagnosed CYP21 mutation. Three hundred and forty Caucasian, premenopausal patients participated in the study. Forty patients did not attend the ACTH test, and basal determinations were carried out. Cushing’s syndrome was excluded using 24 h urinary cortisol measurements or a short dexamethasone suppression test. All patients were screened to exclude thyroid dysfunction or hyperprolactinemia.
One patient was diagnosed with a pituitary adenoma, one had an androgen-producing ovarian tumor, and one patient was diagnosed with Cushing’s syndrome (for details see Ref. 1). These three patients were excluded.
Forty-two healthy, Caucasian, premenopausal women were included as controls to establish a reference interval for 17OHP responses during the ACTH test (20). All controls underwent physical examination before inclusion in the study. All controls had regular menses every 26th to 34th day, received no medication at the time of evaluation, and had total testosterone levels less than 1.8 nmol/liter and a maximum Ferriman-Gallwey score of one.
In patients and controls, oral contraceptive pills were paused for a minimum of 3 months
The protocol was approved by the local ethical committee of the counties of Vejle and Funen, Denmark. All subjects gave signed, voluntary, informed consent before participation in the study.
CYP21 genotyping
All patients were offered CYP21 genotyping. The above-mentioned 42 healthy females and 210 female blood donors were included as reference population. The blood donors were included consecutively after completing a questionnaire concerning menstrual cycles and hirsutism. No blood donors had complaints of hirsutism, and all denied plucking/removing terminal hair on chin/upper lip. All blood donors were premenopausal with regular menses.
PCOS was defined according to the criteria proposed by The Rotterdam PCOS Consensus Group 2003 (21). These criteria included two of the following three criteria: 1) oligo- or anovulation, 2) clinical and/or biochemical hyperandrogenemia, and 3) polycystic ovaries and exclusion of other etiologies. Because all patients were diagnosed with hirsutism, PCOS could be defined as patients with irregular menses or regular menses with polycystic ovaries. IH was defined as patients with regular menses and normal transvaginal ultrasound.
Experimental design
Controls used for the B17OHP and M17OHP reference intervals and all hirsute patients underwent a thorough clinical examination before inclusion in the project. Hirsutism was evaluated using Ferriman Gallwey score (22). ACTH tests were performed in the follicular phase (cycle d 2–8) in all controls and in patients having a cycle length shorter than 3 months; in patients having cycle length longer than 3 months the test was performed on a random day. All tests were performed between 0800 and 1000 h. After basal (t = 0) measurements of androgens, LH, FSH, estradiol, and cortisol, an iv bolus of 0.25 mg ACTH (Synachten 0.25 mg/ml, Novartis Healthcare, Copenhagen, Denmark) was administered. At 30 and 60 min, cortisol and 17OHP were measured. In each patient, the M17OHP value was established as the peak value of 17OHP. 
17OHP was calculated as M17OHP –B17OHP in each individual.
Hormonal assays
LH, FSH, estradiol, and cortisol were analyzed by time-resolved fluoroimmunoassay using commercial kits (AutoDELFIA, Wallac Oy, Turku, Finland). The FSH intraassay variation was 1.0–1.4%, and the interassay variation was 2.1–3.7%. Corresponding values for LH were 1.8–9.4% (intraassay variation) and 2.0–3.9% (interassay variation). Values were 3.1–5.2 and 1.8–8.5% for estradiol and 2.7–3.6 and 0.8–1.9% for cortisol. Reference values in follicular phase were 2.2–6.5 U/liter for FSH, 1.6–9.3 U/liter for LH, and 21.8–215.2 pg/ml (80–790 pmol/liter) for estradiol. Reference values for cortisol were 7.2–25.4 µg/dl (200–700 nmol/liter). 17OHP was analyzed by RIA using a commercial kit (Coat-A-Count, Diagnostic Products Corp., Los Angeles, CA). Intraassay variation was 3.5–7.1%, and interassay variation was 5.0–11%. During 1997–2000, total and free testosterone and sex hormone-binding globulin (SHBG) were analyzed using specific RIAs and extraction methods, as previously described (23). Reference values were 0.16–0.52 ng/ml (0.55–1.8 nmol/liter) for total testosterone, 0.0017–0.01 ng/ml (0.006–0.034 nmol/liter) for free testosterone, and 1.2–4.9 µg/dl (41–170 nmol/liter) for SHBG. All androgens in controls were analyzed according to this method. During 2000–2001, total testosterone and SHBG were analyzed by time-resolved immunoassay using AutoDELFIA commercial kit (Wallac Oy). The total testosterone intraassay variation was 2.1–4.2%, and the interassay variation was 5.2–7.3%. Corresponding values for SHBG were 3.3–5.2% (intraassay variation) and 4.0–5.7% (interassay variation). Reference values were less than 1.0 ng/ml (<3.5 nmol/liter) for total testosterone and 1.2–4.3 µg/dl (140–150 nmol/liter) for SHBG. Free testosterone was calculated as total testosterone/SHBG with reference interval 0.003–0.04.
CYP21 genotyping procedures
Genomic DNA was extracted from EDTA-treated whole blood using a QIAamp DNA Maxi Kit (QIAGEN GmBH, Albertslund, Denmark). All patients and the 42 healthy females were tested for small deletions, insertions, or point mutations in all exons and flanking introns of the CYP21B. Proximal promoter sequences and 3' terminal regions of the CYP21 gene were not evaluated. The 210 blood donors were tested for mutations detected among patient samples. Specificity was achieved using the procedure described by Bobba et al. (24) generating three CYP21B-specific fragments covering all of CYP21B. These fragments were submitted to a second round of PCR using primers as described by Bobba et al. (24) modified to contain GC clamps using the MELT94/MELT87 software. Denaturing gradient gel electrophoresis was performed as originally described by Fischer and Lerman (25). The sensitivity of this method is more than 90% in detecting sequence variations (26). A 6% polyacrylamide gel containing 20–70% linearly increasing denaturant (100% = 7 M urea, 40% formamide) was cast using a gradient gel mixer (Hoeffer). A 12-µl PCR sample was loaded in each lane, and the gel was run at 60 C at 150 V for 6 h in 1x Tris-acetate-EDTA buffer using a modified PROTEAN II slab electrophoresis cell (Bio-Rad, Herlev, Denmark). After electrophoresis, the gels were stained with ethidium bromide and examined by UV transillumination using a GelDoc instrument (Bio-Rad).
Samples revealing abnormal migration patterns were analyzed on a MegaBACE DNA sequencer (Amersham Biotech A/S, Hoersholm, Denmark). Sequencing was performed on both strands.
Analysis for the following mutations was performed using the restriction enzymes as indicated in brackets: P31L (AciI), L99V (AluI), I172N (SfaN1), V281L (ApaLI), and G319X (PstI).
All restriction enzymes were purchased at New England Biolabs (Herefordshire, UK). The intron 2 splice site mutation A/C656-21G was detected by real-time PCR using CYP21inv-21F 5'-TCCCTCAGCTGCCTTCATC-3' as forward primer and CYP21inv-21R 5'-CGGGTAGTTCTTAGACACCTGCTT-3' as reverse primer. As wild-type probes, the CYP21-21A 5'-FAM-TCCAGCCCCCACCT-MGB-TAMRA-3', CYP21-21A 5'-FAM-TCCAGCCCCCAACT-MGB-TAMRA-3', or the CYP21-21C 5'-VIC-CCAGCCCCCAACT-MGB-TAMRA-3' were used. The probe CYP21-21C 5'-HEX-CCAGCCCCCAGCT-MGB-TAMRA-3' was used to detect the mutant allele. All probe sets were tested pair-wise. Primer concentrations were 200 mM, probes were used at 900 mM, and PCR chemistry and PCR conditions were established as described by Applied Biosystems in application note 790910-004. Large rearrangements were analyzed using quantitative real-time PCR (Brusgaard, K., L. S. Peterson, D. Glintborg, M. Anderson, and C. Hagen, unpublished data). Analyses were performed using an ABI7700 DNA sequence analysis machine (Applied Biosystems). To evaluate quantitative differences, the serum albumin gene (gene ID 213) was used as internal standard as described by Kristensen et al. (27). Quantitative differences were calculated using the Ct method as devised by Applied Biosystems note 777802-002.
We based the definition of polymorphisms in the CYP21 gene on a database search including already described nonfunctional polymorphisms (http://www.ncbi.nlm.nih.gov/SNP/). A number of newly described polymorphisms had a high prevalence in both the hirsute and the control group, suggesting a minor risk as being disease causing. The remaining polymorphisms did not account for any amino acid changes and were distributed in both groups at equal frequencies.
Calculations and statistical analysis
Hormonal parameters were log-Gaussian distributed in healthy women and in patients, and data were expressed as geometric mean (±2 SD). One-way ANOVA was used on log-values to detect significant differences between several groups before applying the Bonferroni test on subgroups.
Nonparametric tests were used for comparison of basal, clinical characteristics between groups, and data were expressed as median (25–75 quartile). The Kruskall-Wallis test was used to compare several groups before performing the Mann-Whitney test.
The 
2 test on contingency tables was used to analyze differences in PCO frequencies, CYP21 carrier status, and 17OHP. P < 0.05 was considered statistically significant.
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Results |
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Clinical and hormonal characteristics of patients and controls are shown in Table 1
. Patients were divided according to diagnosis: IH (n = 180) and PCOS (n = 157). Both patient subgroups had significantly higher body mass index (BMI) (P < 0.05) compared with controls. Patients with PCOS were significantly younger (P < 0.001) compared with patients with IH.
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B17OHP.
Patients with PCOS had significantly higher B17OHP compared with patients with IH (P < 0.001) and with controls (P < 0.05) (Table 1
). The cutoff limit for B17OHP was defined as the upper limit of the 95% CI for the 97.5 percentile in controls: 4.0 µg/liter (12 nmol/liter). Using this limit on patients with IH and PCOS, B17OHP levels were elevated in 2.4% (four of 164) of IH patients and 5.3% (eight of 149) of PCOS patients (not significant).
M17OHP and cortisol.
Patients with PCOS and IH had significantly higher mean M17OHP values compared with controls (P < 0.001). No significant differences in M17OHP levels were found between patients with PCOS and IH. 17OHP values were significantly higher in patients compared with controls (P < 0.001) (Table 1). Patients with IH had significantly higher mean 17OHP compared with patients with PCOS (P < 0.05). Two patients had elevated ACTH-stimulated 17OHP levels greater than 10 µg/liter (30 nmol/liter) suggestive of nonclassic adrenogenital syndrome (6). ACTH-stimulated 17OHP levels in these patients were 29.4 and 198.2 µg/liter (89.1 and 600 nmol/liter), respectively. In both patients, the diagnosis of nonclassic adrenogenital syndrome could be determined using basal 17OHP measurement.
The cutoff limit for M17OHP was defined as the upper limit of the 95% CI for the 97.5 percentile in controls. The cutoff limit was 5.3 µg/liter (16 nmol/liter). The fraction of patients with M17OHP responses above the cutoff limit was 20.5% (32 of 156) in IH and 20.8% (30 of 144) in PCOS (P = 1.0).
The 30-min ACTH-stimulated cortisol levels were significantly higher in hirsute patients compared with controls; the geometric mean (±2 SD) was 22.3 (15.5–32.1) µg/dl in hirsute patients vs. 20.6 (13.9–30.4) µg/dl in controls [615 (427–887) vs. 568 (384–839) nmol/liter; P < 0.05]. This remained significant when comparing only controls and patients with normal CYP21 genetics (P = 0.03). Patients with IH and PCOS were divided according to M17OHP levels (Table 2). IH and PCOS patients with M17OHP above the cutoff limit had significantly higher 30-min cortisol compared with controls (P < 0.05). No significant correlation was found between BMI and 30-min cortisol responses (r2 = 0.019). We found no significant differences in 30-min cortisol levels in overweight patients compared with cortisol levels in normal-weight patients (P = 0.5; data not shown).
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Genetic evaluation
The overall prevalence of CYP21 heterozygote carrier status was 8.6% (19 of 221) in patients and 6.3% (16 of 252) in healthy controls (P = 0.57) (Table 3). All detected mutations in hirsute patients and controls had previously been described in congenital adrenal hyperplasia. The mutation prevalence among patients with PCOS (eight of 93) was similar to patients with IH (11 of 128) (P = 0.99). We found significantly higher mean levels of M17OHP among carriers [7.0 (1.1–46.6) µg/liter; 21.2 (3.2–141.0) nmol/liter] compared with patients with no genetic defects [3.8 (1.9–7.6) µg/liter; 11.4 (5.7–22.9) nmol/liter] (P < 0.001). However, only nine of 19 heterozygote patients had M17OHP levels above the cutoff value. Of 41 patients with M17OHP levels above the cutoff limit of 5.3 µg/liter (16 nmol/liter), 32 had no mutations in the CYP21 gene. The most common mutation found in hirsute patients was in exon 7 (V281L); the prevalence was 5.9% in patients and 2.0% in controls (P = 0.03).
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Two controls used for establishment of B17OHP and M17OHP reference intervals were diagnosed with CYP21 mutations. No significant differences were found between clinical and paraclinical parameters in CYP21-positive healthy females compared with healthy females without mutations (data not shown).
Clinical and paraclinical data for CYP21 heterozygotes are shown in Table 4. A list of polymorphisms in hirsute patients and controls is given in Table 5.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We performed a thorough screening of the CYP21 gene and found a similar low prevalence of CYP21 carriers among hirsute patients and healthy females. No clinical or paraclinical characteristics were found in the mutation carriers. Although patients with CYP21 mutations had higher mean levels of M17OHP than patients with no mutations, a high M17OHP level could not predict CYP21 mutations in the individual patient. In 10 of 19 CYP21 carriers, M17OHP levels were below the cutoff limit. Our findings are in agreement with previous studies in which only minor clinical or paraclinical differences were found between CYP21 carriers and patients with no mutations (6, 7, 28, 29). A study evaluating obligate heterozygote females was not able to establish a higher risk of hyperandrogenemia or hirsutism in these females compared with healthy relatives (30).
Although we screened the whole CYP21 gene, we found a low prevalence of CYP21 carriers among hirsute patients in contrast to previous studies showing more than 20% of hirsute patients to be heterozygote CYP21 carriers (6, 7, 8, 29).
The most commonly observed mutation in our population was in exon 7 (V281L), seen in 13 patients; in six patients, it was seen in combination with exon 5. This mutation is usually associated with nonclassic adrenogenital syndrome and reduces 21-hydroxylase activity to 50% (31). In studies evaluating CYP21 carrier status in hyperandrogenic patients, this mutation has frequently been found (6, 8, 28). In our study, we were not able to confirm the findings of Escobar-Morreale et al. (6) and Witchel et al. (8) showing that the V281L mutation had the most prominent effect on clinical and paraclinical parameters.
The aim of the present study was to determine the frequency of CYP21 mutations in hirsute patients, and we did not address polymorphisms with no direct functional defects. It is therefore possible that some of the described polymorphisms could be of pathogenic importance in hirsutism, and future association studies are needed to determine this. We, however, found functional analysis beyond the scope of the present study.
Our data suggest that screening for CYP21 carrier status is not relevant in hirsute patients. Because of the need for genetic counseling in future pregnancy, we suggest, however, the determination of basal 17OHP levels to detect patients with nonclassic adrenogenital syndrome.
Our findings of higher B17OHP levels in PCOS may be a result of elevated basal adrenal or ovarian 17OHP secretion in these patients, as previously described (32, 33). In contrast, B17OHP levels in IH were comparable to those in healthy individuals.
Significantly higher 17OHP levels were found in IH compared with PCOS, but also patients with PCOS had significantly higher 17OHP levels than controls. In a study by Azziz et al. (34), the authors described normal 17OHP responses superimposed on high basal 17OHP levels in PCOS patients. However, more recent studies from this group have described elevated ACTH- or CRH-stimulated adrenal androgen levels in hyperandrogenic patients, thus supporting a hypothesis of adrenal hyperactivity (15, 35, 36).
The adrenal involvement in IH has been only sparsely examined (10, 11). In the present study, however, patients with IH had significantly higher M17OHP responses than controls during the ACTH test, and the mean M17OHP levels were comparable to patients with PCOS. In agreement with these findings, Rossi et al. (11) used a 5-h ACTH test in 48 patients with IH and found an elevated adrenal androgen production in 52% of the patients. Supporting the importance of adrenal hyperactivity in IH, we found significantly higher 30-min cortisol levels in the subgroup of patients with 17OHP responses greater than 5.3 µg/liter (16 nmol/liter) compared with patients with low 17OHP responses. To our knowledge, we are the first to report these findings in IH.
In our study, 20.5% of patients with PCOS had M17OHP levels above the cutoff limit. Previous studies have found elevated ACTH-stimulated 17OHP levels in 25–50% of patients with PCOS (15). Furthermore, ACTH- or CRH-stimulated cortisol levels in patients with PCOS were higher compared with controls (14, 16, 17, 37, 38, 39, 40).
Several authors have described dysregulation of the enzyme p450c17 (12, 13, 36), and Kelestimur et al. (14) proposed an alternate pathway of 17,20-lyase activity with enhanced conversion of 11-deoxycortisol to androstenedione in the adrenals in PCOS patients. Whether a specific enzymatic dysregulation or several mechanisms underlie the increased adrenal response in PCOS has not been clarified. Studies measuring 24-h cortisol profiles in PCOS have found increased cortisol levels or higher numbers of cortisol pulses in patients compared with controls (41, 42).
In the present study, lean and obese hirsute patients had comparable elevated cortisol levels in contrast to the nonhirsute controls. No correlation was found between BMI and cortisol in hirsute patients. These results exclude obesity as the only reason for elevated cortisol levels in hirsutism and are in contrast with observations from healthy individuals (42, 43, 44).
An exaggerated 17OHP response during the ACTH test would indicate a potential for steroid treatment in these patients. Previous studies using steroid treatment in hirsute patients showed improvement of menstrual cycles (45, 46, 47). However, because of side effects, the clinical relevance of these studies may be minor (48, 49). No studies used elevated 17OHP responses as inclusion criteria for initiation of steroid treatment. We would expect the best response to steroid treatment in the subgroup of patients with hyperactive adrenals. More studies, however, are needed to establish an indication for steroid treatment in these patients.
In conclusion, we found significantly higher ACTH-stimulated cortisol and 17OHP levels in hirsute patients, suggesting adrenal hyperresponsiveness in IH and PCOS. Our data indicated that CYP21 carrier status has only minor clinical relevance in hirsute patients.
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Footnotes |
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First Published Online December 14, 2004
Abbreviations: BMI, Body mass index; B17OHP, basal 17OHP; CI, confidence interval; IH, idiopathic hirsutism; M17OHP, ACTH-stimulated maximum 17OHP; 17OHP, 17-hydroxyprogesterone; PCOS, polycystic ovary syndrome; SHBG, sex hormone-binding globulin.
Received June 25, 2004.
Accepted December 7, 2004.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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. Fertil Steril 62:521–530[Medline]
-hydroxycorticosteroid intermediates response to adrenocorticotropin in hyperandrogenic women: apparent relative impairment of 17,20-lyase activity. J Clin Endocrinol Metab 81:881–886[Abstract]
dysregulation. Hum Reprod 12:910–913
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