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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 8 2724-2728
Copyright © 1999 by The Endocrine Society

Original Studies

Profiling Steroid Hormones in Amniotic Fluid of Midpregnancy by Routine Stable Isotope Dilution/Gas Chromatography-Mass Spectrometry: Reference Values and Concentrations in Fetuses at Risk for 21-Hydroxylase Deficiency1

Stefan A. Wudy, Helmuth G. Dörr, Claudia Solleder, Mahmoud Djalali and János Homoki

Steroid Laboratory, Department of Pediatrics (S.A.W., C.S., J.H.), and Department of Medical Genetics (M.D.), University of Ulm, D-89070 Ulm/Donau; and the Department of Pediatric Endocrinology, University of Erlangen (H.G.D.), D-91054 Erlangen, Germany

Address all correspondence and requests for reprints to: PD Dr. Stefan A. Wudy, Steroid Laboratory, Department of Pediatrics, University of Ulm, D-89070 Ulm, Germany. E-mail: stefan.wudy{at}medizin


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Using routine stable isotope dilution/gas chromatography-mass spectrometry, 17-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, androstanediol, and 5{alpha}-dihydrotestosterone have been profiled in amniotic fluid of midgestation in 77 normal fetuses and 38 untreated or dexamethasone-treated fetuses at risk for 21-hydroxylase deficiency. Dexamethasone was suspended 5–7 days before amniocentesis. In normal fetuses, amniotic fluid concentrations (median, range; nanograms per mL) of 17-hydroxyprogesterone did not reveal a sex difference (1.48, 0.21–4.96), whereas those of androstenedione were lower in females (0.53, 0.00–2.71) than in males (0.93, 0.29–1.98). Testosterone levels were higher in males (0.24, 0.00–0.50) than in females (0.00, 0.00–0.27). No sex difference was found for dehydroepiandrosterone (0.47, 0.19–1.77). Levels of androstanediol and 5{alpha}-dihydrotestosterone were below the detection limit of our method in most cases. Regarding prenatal diagnosis of 21-hydroxylase deficiency, 17-hydroxyprogesterone and androstenedione presented the diagnostically most valuable steroids and were of equal diagnostic potential. They permitted successful diagnosis in 36 of 37 fetuses at risk: 12 were untreated and unaffected, 13 were treated and unaffected, 4 were untreated and affected (3 salt wasters and 1 simple virilizer), and 8 were treated and affected (5 salt wasters and 3 simple virilizers). In the latter group, one simple virilizer revealed normal steroid concentrations. Isotope dilution/gas chromatography-mass spectrometry, providing the highest specificity in steroid analysis, is proposed for routine use in clinical steroid analysis whenever maximal reliability is requested. Our study provides the first mass spectrometric reference data on amniotic fluid steroid concentrations and underscores the high accuracy of prenatal hormonal diagnosis of 21-hydroxylase deficiency.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT HAS RECENTLY been demonstrated that prenatal diagnosis of 21-hydroxylase deficiency by hormonal analysis in amniotic fluid of midgestation had a high accuracy rate of 95%, which was at least equivalent to other diagnostic procedures, such as human leukocyte antigen typing or DNA analysis (1). While currently routine determination of steroid hormones is almost exclusively based on immunoassays, application of the most specific technique of steroid analysis, stable isotope dilution/mass spectrometry (ID/MS), to amniotic fluid steroid determination has been lacking to date.

Therefore, the purpose of this study has been 2-fold. Firstly we aimed at obtaining first mass spectrometric data on the concentrations of six potentially diagnostic steroid hormones in amniotic fluid of normal fetuses and of untreated and dexamethasone-treated fetuses at risk for 21-hydroxylase deficiency. Secondly, we intended to demonstrate the applicability of a current microanalytical technique such as stable isotope dilution/mass spectrometry to clinical steroid analysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
We analyzed amniotic fluid specimens obtained by transabdominal amniocentesis from 115 pregnant women between the 13th and 21st weeks of gestation. Gestational age was assessed both from the time since the last menstrual period and by fetal ultrasound. Fetal sex was determined by karyotyping amniotic fluid cells. None of the samples was visually contaminated with blood or meconium. All samples were immediately frozen at -20 C until analysis. Amniotic fluid samples collected between the 13th and 21st weeks of gestation (median, 15 weeks) from 77 pregnancies at risk for fetal chromosomal abnormalities but with normal outcome served as controls. From 38 fetuses at risk for 21-hydroxylase deficiency (index case present in each family) amniotic fluid samples were obtained between the 13th and 18th weeks of gestation (median, 16 weeks). In 21 mothers dexamethasone (1.0–1.5 mg/day) was started before the 10th week of gestation and was stopped 5–7 days before amniocentesis. Therapy was continued until term in female affected fetuses only. The prenatal diagnoses of 26 unaffected and 12 affected fetuses were confirmed postnatally. The study was approved by the local ethical committee, and informed consent was obtained from the pregnant women.

17-Hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, 5{alpha}-dihydrotestosterone (17ß-hydroxy-5{alpha}-androstan-3-on), and androstanediol (5{alpha}-androstan-3{alpha},17ß-diol) were simultaneously determined in a single profile according to our own ID/gas chromatography (GC)-MS procedure (2, 3). Deuterium-labeled analogs of the analytes, [11,11,12,12-2H4]17-hydroxyprogesterone, [7,7-2H2]andro-stenedione, [16,16,17-2H3]testosterone, [7,7-2H2]dehydroepiandrosterone, [16,16,17-2H3]5{alpha}-dihydrotestosterone, and [16,16,17-2H3]androstanediol, served as internal standards (4). In brief, amniotic fluid (0.5–1 mL) was equilibrated with a cocktail containing 1 ng of each internal standard. After solvent extraction, the dried organic extract was purified on Sephadex LH-20 minicolumns. Then, derivatization with heptafluorobutyric anhydride followed, and a 4-µl portion of the total processed amniotic fluid extract (50 µl) was analyzed. GC was carried out on an OV-1 fused silica column (Macherey-Nagel, Düren, Germany; 25 m x 0.2 mm; film thickness, 0.1 µm) housed in a DANI 6500 gas chromatograph. The gas chromatograph was directly interfaced to a Hewlett-Packard Co. 5970B mass selective detector (Palo Alto, CA) operated in the selected ion-monitoring mode. Quantitation was computerized using the peak area ratios between the ion pairs of the analytes and their corresponding labeled analogs. The following ion pairs of analytes and corresponding internal standards were used: 17-hydroxyprogesterone, m/z 465; [11,11,12,12-2H4]17-hydroxyprogesterone m/z 469; androstenedione, m/z 482; [7,7-2H2]androstenedione, m/z 484; testosterone, m/z 680; [16,16,17-2H3]testosterone, m/z 683; dehydroepiandrosterone, m/z 270; [7,7-2H2]dehydroepiandrosterone, m/z 272; 5{alpha}-dihydrotestosterone, m/z 414; [16,16,17-2H3]5{alpha}-dihydrotestosterone, m/z 417; androstanediol, m/z 470; and [16,16,17-2H3]androstanediol, m/z 473. Calibration plots were prepared in amniotic fluid samples to which mixtures of analytes and corresponding internal standards were added, so that the amounts of analyte injected into the GC/MS covered the range of 16 pg to 1.6 ng with a fixed amount (80 pg) of internal standard, respectively. Standard plots were linear: 17-hydroxyprogesterone, y = 0.97x + 0.15, r = 1.000; 4-androstenedione, y = 1.00x + 0.29, r = 0.998; testosterone, y = 0.99x + 0.41, r = 0.998; dehydroepiandrosterone, y = 1.16x + 0.10, r = 1.000; androstanediol, y = 0.59x + 0.07, r = 1.000; and 5{alpha}-dihydrotestosterone, y = 0.66x + 0.59, r = 0.995. For the steroids studied, intra- and interassay coefficients of variation were between 2.7–6.0% and 2.2–5.4%, respectively. Sensitivity was lowest for testosterone, with a signal to noise ratio of 2.4 for 10 pg, and was highest for androstanediol with a signal to noise ratio of 17.0 for 10 pg.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Application of our assay in a clinical setting is demonstrated by the example of typical ion chromatograms. In Fig. 1Go, an amniotic fluid steroid profile shows exclusion of 21-hydroxylase deficiency in a midgestational amniotic fluid specimen.



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  		Figure 1. Exclusion of 21-hydroxylase deficiency by hormonal prenatal ID/GC-MS analysis of a midgestational amniotic fluid specimen is demonstrated by the selected ion recordings of an aliquot (4 µl) of a processed extract (50 µl) of 0.5 mL amniotic fluid. Corresponding ion traces of analyte and deuterated (2H = d) internal standard were superimposed. The peaks of analytes and corresponding internal standards show practically identical retention times (tr) on the GC column. 17-Hydroxyprogesterone (17-OH-P; m/z 465; tr, 20.89 min; 2.81 ng/mL), [11,11,12,12-2H4]17-hydroxyprogesterone (d4-17-OH-P; m/z 469; tr, 20.86 min), androstenedione (4A; m/z 482; tr, 19.68 min; 1.24 ng/mL), [7,7-2H2]androstenedione (d2-4A; m/z 484; tr, 19.66 min), testosterone (T; m/z 680; tr, 19.66 min; 0.49 ng/mL), [16,16,17-2H3]testosterone (d3-T; m/z 683; tr, 19.65 min), dehydroepiandrosterone (DHEA; m/z 270; tr, 19.72 min; 0.56 ng/mL), [7,7-2H2]dehydroepiandrosterone (d2-DHEA; m/z 272; tr, 19.71 min), androstanediol (AD; m/z 470; no peak detected; 0.00 ng/mL), [16,16,17-2H3]androstanediol (d3-AD; m/z 473; tr, 18.59 min); 5{alpha}-dihydrotestosterone (DHT; m/z 414; no peak detected; 0.00 ng/mL), [16,16,17-2H3]5{alpha}-dihydrotestosterone (d3-DHT; m/z 417; tr, 20.28 min).

 
For all steroids no differences in concentrations were found between the first and second halves of the midgestational period studied. In normal pregnancies, 17-hydroxyprogesterone concentrations did not show a sex difference and therefore were combined (n = 77; median, 1.48 ng/mL; range, 0.21–4.96 ng/mL). Figure 2AGo depicts the concentrations of 17-hydroxyprogesterone in our 38 fetuses at risk for 21-hydroxylase deficiency. Choosing the upper limit of the normal range as threshold, all unaffected fetuses had lower 17-hydroxyprogesterone. Except for one patient with the simple virilizing form of congenital adrenal hyperplasia (CAH), all other cases of classical CAH had 17-hydroxyprogesterone values above the upper normal range.



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  		Figure 2. Amniotic fluid concentrations of 17-hydroxyprogesterone (A) and androstenedione (B) in 38 fetuses at risk for 21-hydroxylase deficiency. The horizontal dotted lines represent the cut-off levels (f, females; m, males) and correspond with the upper limits of the respective normal ranges. Simple virilizers are indicated by asterisks.

 
In normal subjects, amniotic fluid concentrations of androstenedione showed lower medians in females (n = 32; 0.53 ng/mL) than in males (n = 45; 0.93 ng/mL). Their ranges showed considerable overlap (females, 0.00–2.71 ng/mL; males, 0.29–1.98 ng/mL). The concentrations of androstenedione in the 38 fetuses at risk for 21-hydroxylase deficiency are shown in Fig. 2BGo. Choosing the upper limits of the normal ranges as cut-offs, all unaffected fetuses had lower concentrations of androstenedione. Except for one patient with the simple virilizing form of CAH (same patient who lacked an elevation of 17-hydroxyprogesterone), all other cases of classical CAH had androstenedione values above the upper limits of the ranges in normal fetuses.

Amniotic fluid testosterone levels were markedly higher in normal male fetuses (n = 45; median, 0.24 ng/mL; range, 0.00–0.50 ng/mL), but overlapped with the range in normal female fetuses (n = 32; median, 0.00; range, 0.00–0.27 ng/mL). In unaffected fetuses at risk, no elevations were noted, except for two females who showed slightly higher testosterone levels (0.32 and 0.30 ng/mL). In CAH fetuses, the only untreated female fetus showed a testosterone level (0.45 ng/mL) slightly below the upper range limit for normal males. In treated CAH females (n = 7), only two fetuses had elevated testosterone (0.32 and 0.39 ng/mL). One of three untreated male fetuses showed elevated testosterone (0.58 ng/mL). The only treated male fetus lay within the normal range.

For amniotic fluid dehydroepiandrosterone levels, no sex difference could be found in normal fetuses (n = 77; median, 0.47; range, 0.19–1.77 ng/mL). Fetuses at risk for but not affected with 21-hydroxylase deficiency showed concentrations between 0.13–2.32 ng/mL (median, 1.10 ng/mL). One of the untreated CAH fetuses had slightly elevated dehydroepiandrosterone (1.99 ng/mL). Two of the dexamethasone-treated CAH fetuses had elevated levels (4.42 ng/mL; 2.17 ng/mL).

In both sexes, concentrations of androstanediol in amniotic fluid were very low, and in most cases lay below the detection limit of our method. In normal fetuses, the concentrations did not exceed 0.24 ng/mL in males and 0.10 ng/mL in females. Neither in unaffected fetuses at risk nor in CAH fetuses did the levels of androstanediol exceed those in normal fetuses. Likewise, 5{alpha}-dihydrotestosterone concentrations in amniotic fluid were mostly undetectable in normal subjects. In normal males their upper range limit was 0.19 ng/mL, and in females it was 0.10 ng/mL. With the exception of one untreated male fetus (0.17 ng/mL), the other three untreated CAH fetuses showed higher levels than normal subjects (males, 0.28 and 0.40 ng/mL; female, 0.19 ng/mL). In treated CAH fetuses, 5{alpha}-dihydrotestosterone concentrations were elevated in four of eight cases (male, 0.29; females, 0.26, 0.24, and 0.23 ng/mL).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The rising success of MS as a highly specific analytical technique in the biomedical sciences is due to its potential of supplying both qualitative and quantitative information on molecules based on their structural compositions. Although analytical techniques based on MS already constitute methods of choice in areas such as environmental studies, forensic chemistry, drug industry, or metabolic research, they have not yet found widespread use in clinical endocrinology. In the field of steroid analysis, ID/MS currently presents the most specific quantitative method for steroid determination, because, as a physicochemical method, it is independent of phenomena such as cross-reactivity or matrix effects (5, 6). We could previously demonstrate the applicability of bench-top GC-MS to clinical plasma steroid analysis, stressing its importance in difficult analytical situations, e.g. the neonatal period (3) or states of hyperandrogenism (7, 8).

The general principle of ID/MS consists of equilibrating internal standard with the endogenous analyte present in the sample. Stable isotope-labeled analogs are ideally suited as internal standards, because, due to similar chemical behavior of the analyte, they offer the advantage of compensation for losses of analyte during the work-up procedure and further avoid radioactive contamination of personnel and instrumentation (4). After work-up of the sample, MS-analysis follows. By monitoring specific ions of analyte and internal standard, the isotope ratio between both permits calculation of the concentration of the analyte. As GC bears the greatest potential in separating steroids (9), a hyphenated technique such as GC-MS permits simultaneous determination of multiple steroids in a single profile. Profiling is especially advantageous in pediatric endocrinology, because the amount of sample can be kept to a minimum.

Concerning amniotic fluid steroid analysis, GC-MS has to date only been used to characterize the major steroids of normal amniotic fluid in a nonselective approach (10). The concentrations of 17-hydroxyprogesterone in amniotic fluid of normal fetuses that have been published to date differ considerably. Direct RIAs (11, 12, 13), even in case solvent extraction was applied (14), overestimate 17-hydroxyprogesterone concentrations in amniotic fluid. Studies making use of solvent extraction and Celite chromatography showed the best agreement with our values (1, 15). In accordance with most investigations (11, 12, 13, 14), we could not find a significant sex difference (1, 16) for 17-hydroxyprogesterone. From 1975 onward, 17-hydroxyprogesterone was suggested as the most promising in utero predictor of 21-hydroxylase deficiency (17, 18, 19, 20, 21). In agreement with the literature, we found that 17-hydroxyprogesterone reliably predicted all cases of salt-wasting 21-hydroxylase deficiency (13, 22, 23). However, the parameter has not been found to be indicative of all cases of simple virilizers (1, 13, 22, 23). In our study 17-hydroxyprogesterone was elevated in all but one fetus with simple virilizing 21-hydroxylase deficiency.

Regarding androstenedione in amniotic fluid, immunoassays were in good agreement with our ID/GC-MS values (1, 24, 25, 26). Our data confirmed a sex difference, with slightly higher concentrations in males (1, 24, 26, 27, 28). In 1980, androstenedione was introduced as hormonal marker for the prenatal diagnosis of 21-hydroxylase deficiency (29). Although the parameter was not indicative of all simple virilizers in two studies (15, 22), another report demonstrated successful diagnosis of all cases of classical 21-hydroxylase (23). In our series of fetuses affected by 21-hydroxylase deficiency, one simple virilizer had normal 17-hydroxyprogesterone and showed a normal concentration of androstenedione, too. This finding might be due either to a still lasting suppressive effect of dexamethasone or to not sufficiently elevated amniotic fluid levels of 17-hydroxyprogesterone and androstenedione in mild forms of simple virilizing CAH.

Our ID/GC-MS data on amniotic fluid levels of testosterone in normal fetuses were in good agreement with RIA studies (24, 25, 26, 30, 31) and confirmed a sex difference. Due to overlapping ranges between sexes (25, 31), no reliance should be placed solely on amniotic fluid testosterone measurements for the determination of fetal sex (30). In accordance with previous studies amniotic fluid testosterone was of much less discriminatory value in the prenatal diagnosis of 21-hydroxylase deficiency than 17-hydroxyprogesterone or androstenedione (15, 25, 27, 32). Of all male CAH fetuses (n = 4), testosterone was only elevated in one of three untreated cases. For female CAH fetuses, amniotic fluid testosterone concentrations were reported not to show the usual sex difference and to lie in the normal male range (25, 27, 31). This applied to the only untreated female CAH fetus, but not to the majority (n = 5) of our dexamethasone-treated CAH females (n = 7), a finding most likely attributable to the lasting suppressive effect of dexamethasone.

Like other investigators (24), our findings did not confirm a sex difference (27) regarding dehydroepiandrosterone in amniotic fluid of normal subjects. Furthermore, the parameter hardly discriminated between CAH fetuses and normal fetuses. Both 5{alpha}-dihydrotestosterone and androstanediol represent end metabolites of androgen metabolism. To our knowledge, no data on the concentrations of these steroids in amniotic fluid of normal or CAH fetuses have yet been published. In normal fetuses, the concentrations of both steroids were generally below the limits of detection of our method. Males had higher upper range limits than females. In CAH fetuses, 5{alpha}-dihydrotestosterone was elevated more often in untreated (three of four fetuses) than in dexamethasone-treated cases (four of eight fetuses). Androstanediol was of no diagnostic value in untreated and treated CAH fetuses.

To conclude, we have provided the first mass spectrometric reference data on the concentrations of six steroid hormones in amniotic fluid of midgestation using routine ID/GC-MS, a microanalytical technique providing maximal reliability in steroid analysis. Regarding the prenatal diagnosis of 21-hydroxylase deficiency, 17-hydroxyprogesterone and androstenedione were diagnostically the most informative steroids. Midtrimester amniocentesis presents the safest invasive prenatal diagnostic technique (33). Our data support the finding (23) that reliable hormonal prenatal diagnosis is possible even in dexamethasone-treated fetuses, provided that dexamethasone therapy has been interrupted 5–7 days before amniocentesis. Our data exclude a rebound phenomenon, i.e. an excessive increase in steroid hormones after cessation of dexamethasone (26). Currently, chorionic villous sampling, which allows karyotyping and DNA analysis at the end of the first trimester of gestation, has become preferred method for prenatal diagnosis of 21-hydroxylase deficiency (34, 35). Whenever chorionic villous sampling will not be possible, amniocentesis, which permits karyotyping, DNA analysis, and hormonal amniotic fluid measurement, presents a highly accurate alternative diagnostic procedure.


   Acknowledgments
 
The authors appreciate the support of Prof. Vogel (Department of Clinical Genetics, University of Ulm), and thank Mrs. M. Hartmann (Steroid Laboratory, Department of Pediatrics, University of Ulm) for excellent technical assistance.


   Footnotes
 
1 Presented in part at the 10th International Congress on Hormonal Steroids, Quebec City, Canada, June 17–21, 1998. This work was supported by the Deutsche Forschungsgemeinschaft (Grant WU 148/3–2 to S.A.W.). Back

Received September 11, 1998.

Revised April 5, 1999.

Accepted April 14, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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P. C. White and P. W. Speiser
Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency
Endocr. Rev., June 1, 2000; 21(3): 245 - 291.
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 Arch. Dis. Child. Fetal Neonatal Ed.Home page
C G D Brook;, I. HUGHES;, and C. J H KELNAR
Antenatal treatment of a mother bearing a fetus with congenital adrenal hyperplasia
Arch. Dis. Child. Fetal Neonatal Ed., May 1, 2000; 82(3): 176F - 181.
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