Abstract
Introduction:
Recent studies have shown 11-oxygenated androgens (11oAs) are the dominant androgens in premature adrenarche (PA). Our objective was to compare 11oAs and conventional androgens in a well-defined cohort of children with PA or premature pubarche (PP), and correlate these androgens with metabolic markers.
Methods:
A prospective cross-sectional study was conducted at a university hospital. Fasting early morning serum steroids (including 11oAs) and metabolic biomarkers were compared and their correlations determined in children ages 3 – 8 years (F) or 3 – 9 years (M) with PA or PP (5 M, 15 F) and healthy controls (3 M, 8 F).
Results:
There were no differences between PA, PP, and controls or between PA and PP subgroups for sex, BMI z-score, or criteria for childhood metabolic syndrome (MetS). DHEAS was elevated only in the PA subgroup, as defined. 11oAs were elevated versus controls in PA and PP, although no differences in 11oAs were noted between PA and PP. Within the case cohort, there was high correlation of T and A4 with 11-ketotestosterone and 11β-hydroxyandrostenedione. While lipids did not differ, median insulin and HOMA-IR were higher but not statistically different in PA and PP.
Conclusions:
PA and PP differ only by DHEAS and not by 11oAs or insulin sensitivity, consistent with 11oAs – rather than DHEAS – mediating the phenotypic changes of pubarche. Case correlations suggest association of 11oAs with T and A4. These data are the first to report the early morning steroid profiles including 11oAs in a well-defined group of PA, PP, and healthy children.
Keywords: 11-oxygenated steroids, adrenarche, androgen, insulin resistance, pediatrics
Introduction
Adrenarche is the pubertal maturation of the adrenal zona reticularis in both boys and girls. Premature adrenarche (PA) is the most common cause of premature pubarche (PP), or the early onset of pubic hair and or axillary hair/odor before the age of 8 in girls and 9 in boys. PA is associated with elevated adrenal androgens and occurs in the absence of gonadotropin-dependent puberty. Other causes of hyperandrogenism such as congenital adrenal hyperplasia, androgen-secreting tumors, or exogenous androgen exposure must be excluded in order to make the diagnosis of PA [1]. Pubic hair growth in PA is a physical manifestation that correlates with increased production of dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) from the adrenal zona reticularis, as well as 5-androstenediol sulfate and androstenedione (A4). Laboratory data typically demonstrate increased DHEA, DHEAS, and A4 levels that correlate with stage of pubic hair development. Cases of PP, however, have been documented in the absence of DHEAS levels above the adrenarchal range of 50 μg/dL [1–3]. To explain this paradox of PP in the absence of PA, it has been speculated that these children have an increased receptor sensitivity to low circulating levels of androgens [4].
Though previously thought to be a benign normal variant of puberty, PA has been associated with obesity, development of metabolic syndrome (MetS), and polycystic ovary syndrome (PCOS) [5–7]. Hyperinsulinemia, dyslipidemia, insulin resistance, and increased free insulin-like growth factor-1 (IGF-1) have been reported in PA [8–12]. Both PCOS and PA are associated with decreased insulin-like growth factor binding protein-1 (IGFBP-1) concentrations, which are inversely correlated with fasting insulin levels and adrenocorticotropin hormone (ACTH)-stimulated adrenal steroid levels [1, 10].
Recent studies have shown that the human adrenals are also the source of abundant 11-oxygenated 19-carbon (C19) steroids (11-oxygenated androgens, 11oAs), including 11β-hydroxyandrostenedione (11OHA4), 11β-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), and 11-ketodihydrotestosterone (11KDHT). The androgenic activity of 11KT and 11KDHT shows comparable potency and efficacy to testosterone (T) and dihydrotestosterone (DHT), respectively [13, 14]. Measurement of 11oAs in various disease states of androgen excess have been explored including castration-resistant prostate cancer [15], use as biomarkers for disease severity and treatment response in 21-hydroxylase deficiency [16–18], and potential use for biomarkers of PCOS as well as differentiation of its subtypes [13, 19]. The 11oAs appear to significantly contribute to the clinical signs observed in adrenarche [13–15, 20], including PA [21]. These findings suggest that elevated 11oAs – rather than DHEA, DHEAS, and A4 – may be of particular diagnostic and clinical significance in cases of PP without the biochemical diagnosis of PA. These findings could potentially also be broadly applied to other disease states of androgen excess.
A recent study showed that 11oAs correlate with surrogate markers of metabolic risk in a large cohort of women with PCOS [19], but this relationship has not been explored in PA. In our study, our primary aim was to assess the 11oAs as biomarkers of androgen excess in PA and/or PP compared to healthy controls of a clinically well-defined cohort. We further sought to correlate the 11oAs and traditional androgens in a PA and PP cohort with surrogate markers of metabolic risk, including the criteria for childhood MetS.
Materials and Methods
Study design and participants
This was a cross-sectional analysis of baseline data collected prospectively from a cohort of pediatric subjects (cases and controls) in a university hospital in a tertiary medical center. Approval was obtained from the Institutional Review Board at Columbia University Irving Medical Center (CUIMC). Over a 21-month period, 31 prepubertal children ages 3 to 8 (girls) and 3 to 9 (boys) were recruited to be cases or controls from the pediatric endocrinology practices of CUIMC, affiliated general pediatric practices, and in response to community flyers and the CUIMC RecruitMe website. Inclusion criteria for the PA and PP groups (referred to as cases) were clinical signs of adrenarche including axillary odor, axillary hair, and/or pubic hair, in the absence of true puberty (Tanner I breast in girls and testicular volume ≤ 3 mL in boys). Subjects were excluded for the following criteria: a history of chronic illness or other known endocrinopathies, evidence of adrenal enzyme defect, elevated inflammatory marker (C-reactive protein), a history of hormone exposure, chronic glucocorticoid use, psychostimulant use, or use of antibiotics in the prior 3 months or probiotics in the last 1 month. Study subjects were subdivided into PA and PP groups based on the traditional cutoff of serum DHEAS level of 50 μg/dL (1.3 μmol/L) or greater for PA [22].
Study visit
Each subject underwent a one-day early morning study visit beginning at 0800 h at the Clinical Research Resource (CRR) at Columbia Irving Institute for Clinical and Translational Research (UL1TR001873) after obtaining appropriate parental consent and subject assent (7 years of age and older). For each study subject and control, a complete medical history was obtained using a standardized history template, in the language each participant/parent preferred (English or Spanish). A complete physical examination by a pediatric endocrinologist was performed including Tanner staging of puberty and measurements of blood pressure (BP), height, weight, and waist circumference (WC). Fasting early morning blood samples were obtained, centrifuged for 20 minutes at 3000 rpm, and frozen at −80°C until analysis. A bone age study was performed only for PA and PP cases and interpreted using the method of Greulich and Pyle [23].
Gestational size was calculated using the PediTools Electronic Growth Chart Calculator [24]; small for gestational age (SGA) status was defined as birth weight less than 10th percentile for gestational age. Body mass index (BMI) was calculated from the weight and height measurements. Subjects were classified as underweight (< 5th percentile) normal weight (5th to < 85th percentile), overweight (85th to < 95th percentile), and obese (≥ 95th percentile), using cutoff criteria based on the sex-specific 2000 Centers for Disease Control and Prevention (CDC) BMI-for-age growth curves. Height age was defined as the age at which the measured height plots at the 50th percentile on the CDC stature-for-age sex-specific growth curves. Age-, sex-, and height-specific blood pressure percentiles and z-scores were determined using a pediatric blood pressure reference chart (Children’s Nutrition Research Center, Baylor College of Medicine) [25]. Age- and sex-specific WC percentiles were determined using data from the Third National Health and Nutrition Examination Survey (NHANES III) [26]. Subjects were classified as having childhood metabolic syndrome (MetS) using de Ferranti et al. [27] definition of three or more of the following criteria: WC > 75th percentile for age and sex, fasting blood glucose (BG) ≥ 110 mg/dL (≥ 6.1 mmol/L), triglycerides (TG) ≥ 100 mg/dL (≥ 1.3 mmol/L), high-density lipoprotein cholesterol (HDL-C) ≤ 40 mg/dL (1.03 mmol/L), and BP ≥ 90th percentile for age, sex, and height.
Assays
The following serum analytes were assessed in the Biomarkers Core Laboratory of the Irving Institute for Clinical and Translational Research (Columbia University, New York, NY): IGF-1, insulin (Immulite 1000, Siemens); BG, hemoglobin A1c (HbA1c), total cholesterol, TG, HDL-C, high-sensitivity C-reactive protein (Cobas Integra 400, Roche Diagnostics), and low density lipoprotein cholesterol (LDL-C) (calculated from total cholesterol, HDL-C, and TG using the Friedewald formula). Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the formula (fasting glucose [mmol/L] × fasting insulin [mU/L] / 22.5). Intra-assay coefficients of variability were all less than 6%.
The following serum analytes were assessed by commercial laboratory (Esoterix, Inc, Calabasas Hills, CA) using these respective assay methods: DHEA, DHEAS, A4, total T, cortisol, 17-hydroxyprogesterone (17OH-Prog), 11-deoxycortisol, deoxycorticosterone (DOC), estradiol (E2), and estrone (E1) by high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS); free T by equilibrium dialysis; luteinizing hormone (LH), follicle-stimulating hormone (FSH), and ACTH by immunochemiluminometric assay (ICMA).
Sera for 11OHA4, 11OHT, 11KA4, and 11KT were analyzed by LC-MS/MS as previously described by Wright et al. [28] (University of Michigan, Ann Arbor, MI). The lower limit of quantification (LLOQ), defined as the minimum concentration achieving an extrapolated signal-to-noise ratio of 3, ranged from 3 ng/dL (0.1 nmol/L) (11OHT, 11KA4, 11KT) to 10 ng/dL (0.3 nmol/L) (11OHA4). Intra-assay coefficients of variability were all less than 12%.
Statistical methods
Laboratory results that were below the LLOQ for the assay were recorded as the LLOQ cut-off value for the respective assay. Descriptive statistics were used to summarize all variables of interest. Continuous data were reported as median [interquartile range (IQR)] unless otherwise stated. Categorical data are reported as frequencies and percentages. Statistical significance was determined by nonparametric Mann Whitney U test for continuous data and Fischer’s exact test for categorical data. A P value of < 0.05 was considered to indicate statistical significance. A stepdown bootstrap with replacement adjustment was used for multiple comparisons of between-group differences for variables of interest within metabolic data and steroid data [29]. Relationships between metabolic biomarkers and steroids were analyzed by Spearman correlation analysis, and the Hommel method was used for correction for multiple comparisons of Spearman correlations [30]. Data analyses were conducted using SAS software (version 9.4, Cary, NC, USA).
Results
Characteristics of subjects
The baseline characteristics for subjects with PA (n=11), PP (n=9), and controls (n=11) who completed the study are shown in Table 1. Case subjects (PA and PP subjects) were older than controls (P = 0.016); however, there was no difference in height age to chronological age (HA/CA) ratio for cases versus controls (P = 0.112), nor for PA and PP subgroups (P = 0.110). Sex composition did not differ between case and control groups, and there were no differences in race or Hispanic ethnicity for case and control groups.
Table 1.
Characteristics of PA+PP Case and Control Subjects with Additional Comparison of PA and PP Subgroups
| PA+PP (n=20) | Control (n=11) | PA (n=11) | PP (n=9) | |
|---|---|---|---|---|
| Chronological age (CA), y * | 7.4 [6.5 – 7.8] | 6.3 [3.9 – 7.0] | 6.6 [5.4 – 7.5] | 7.7 [7.4 – 7.8] |
| Height age (HA), y ** | 7.8 [7.4 – 8.8] | 5.8 [4.2 – 7.4] | 7.6 [5.7 – 7.9] | 8.0 [7.7 – 9.0] |
| HA/CA ratio | 1.13 [1.05 – 1.19] | 1.04 [0.86 – 1.16] | 1.15 [1.10 – 1.21] | 1.08 [0.99 – 1.15] |
| Bone age, y † | 8.4 [7.5 – 9.1] | - | 8.1 [6.7 – 9.1] | 8.6 [7.8 – 9.4] |
| Sex, n | ||||
| Female | 15 (75) | 8 (73) | 9 (82) | 6 (67) |
| Male | 5 (25) | 3 (27) | 2 (18) | 3 (33) |
| Race, n | ||||
| African American | 5 (25) | 2 (18) | 3 (27) | 2 (22) |
| Asian | 2 (10) | 2 (18) | 0 (0) | 2 (22) |
| Caucasian | 12 (60) | 5 (45) | 7 (64) | 5 (55) |
| Other | 1 (5) | 2 (18) | 1 (9) | 0 (0) |
| Hispanic ethnicity, n *** | 7 (35) | 5 (45) | 10 (91) | 3 (33) |
| Age of onset, y | ||||
| Axillary odor (PA=10, PP=6) | - | - | 3.0 [3.0 – 4.8] | 7.0 [5.5 – 7.0] |
| Axillary hair (PA=8, PP=5) *** | - | - | 3.0 [2.8 – 4.3] | 7.0 [6.0 – 7.0] |
| Pubic hair (PA=10, PP=4) | - | - | 4.5 [4.0 – 5.8] | 5.5 [4.5 – 6.3] |
| Findings present by exam, n | ||||
| Axillary odor | - | - | 10 (91) | 7 (78) |
| Axillary hair | - | - | 8 (73) | 7 (78) |
| Pubic hair | - | - | 11(100) | 6 (67) |
| Family history, n | ||||
| Diabetes mellitus | 12 (60) | 7 (64) | 6 (55) | 6 (67) |
| Cardiovascular disease | 17 (85) | 9 (82) | 8 (73) | 9(100) |
| Infertility | 5 (25) | 1 (9) | 3 (27) | 2 (22) |
| Balding | 8 (40) | 6 (55) | 6 (55) | 2 (22) |
Abbreviations: PA, premature adrenarche; PP, premature pubarche.
Data are medians [IQR] or counts (percentage). Statistical significance was determined by nonparametric Mann Whitney U test for continuous data and Fischer’s exact test for categorical data. A P value of < 0.05 was considered to indicate statistical significance.
P < 0.05 for PA+PP vs. Controls
P < 0.01 for PA+PP vs. Controls
P < 0.05 for PA vs. PP
Based on available data from 10 PA and 8 PP
Preterm gestations were more prevalent in cases (7/20) vs. controls (0/11) (P = 0.033), but there was no difference between the groups for SGA status (0 of 20 cases and 2 of 11 controls; P = 0.120). Further, there were no differences between the groups for gestational diabetes, preeclampsia, or in vitro fertilization. Family history of diabetes, cardiovascular disease, infertility, and balding did not differ between cases and controls.
Between PA and PP subgroups, there were no differences in chronological age, sex composition, preterm gestations, gestational diabetes, in vitro fertilization, or pertinent family history. There were more subjects of Hispanic ethnicity in the PA subgroup (10/11) compared to PP (3/9) (P = 0.017). Parent-reported age of onset of developmental signs were earlier in PA compared to PP for axillary odor, axillary hair (P = 0.030), and pubic hair. There were no differences between PA and PP subgroups for birth history or family history.
Height age was greater in cases compared to controls (P = 0.002), but similar in PA and PP. Bone age data were available for 18 cases (10/11 PA and 8/9 PP); neither height age nor bone age differed between PA and PP subgroups. Bone age was slightly older than height age in both PA and PP subgroups. Bone age was advanced (greater than 1 year) in 8/10 children with PA and 4/8 children with PP, with a median difference of bone age and chronological age of 1.2 [1.1 – 1.9] years and 0.9 [0.0 – 1.6] years, respectively.
Metabolic profiles in PA and PP
The clinical and biochemical data related to subject metabolic profiles are summarized in Table 2. Case and control cohorts were similar in BMI percentiles and BMI z-scores. Criteria for childhood MetS were met in 3 cases (15%), including 1 subject with PA, 2 subjects with PP, and 1 control (9%). For PA and PP subgroups, there were no differences in any of the metabolic parameters assessed. None of the subjects had serum fasting BG ≥ 110 mg/dL; similarly, serum HbA1c was normal in all subjects. Median serum insulin was elevated in cases compared to controls (8.1 [3.6 – 10.0] μIU/mL vs. 2.0 [2.0 – 3.6] μIU/mL) as was median HOMA-IR (1.7 [0.8 – 2.1] vs. 0.4 [0.4 – 0.8]). However, these differences were not statistically significant after adjustment of metabolic data for multiple comparisons. Serum IGF-1 levels were higher in cases compared to controls (P = 0.001), but were similar in PA and PP.
Table 2.
Metabolic Data for PA+PP Case and Control Subjects with Additional Comparison of PA and PP Subgroups
| PA+PP (n=20) | Control (n=11) | PA (n=11) | PP (n=9) | |
|---|---|---|---|---|
| BMI percentile | 60 [31 – 89] | 71 [68 – 78] | 82 [37 – 91] | 48 [23 – 78] |
| BMI z-score | 0.25 [−0.51 – 1.22] | 0.55 [0.47 – 0.77] | 0.92 [−0.35 – 1.37] | −0.04 [−0.73 – 0.79] |
| BMI class * | ||||
| Underweight | 1 (5) | 0 (0) | 0 (0) | 1 (11) |
| Normal | 13 (65) | 9 (82) | 7 (64) | 6 (67) |
| Overweight | 3 (15) | 1 (9) | 2 (18) | 1 (11) |
| Obese | 3 (15) | 1 (9) | 2 (18) | 1 (11) |
| WC > 75th %ile, n † | 9 (45) | 4 (36) | 6 (55) | 3 (33) |
| Acanthosis nigricans, n | 8 (40) | 1 (9) | 4 (36) | 4 (44) |
| Systolic BP %ile ‡ | 74 [50 – 92] | 79 [60 – 80] | 70 [45 – 82] | 91 [67 – 93] |
| Systolic BP ≥ 90%ile, n | 6 (30) | 0 (0) | 1 (9) | 5 (56) |
| Childhood MetS, n § | 3 (15) | 1 (9) | 1 (9) | 2 (22) |
| Fasting BG, mg/dL | 88 [84 – 91] | 83 [80 – 88] | 84 [84 – 92] | 88 [87 – 89] |
| Insulin, μIU/mL | 8.1 [3.6 – 10.0] | 2.0 [2.0 – 3.6] | 8.1 [2.8 – 9.7] | 8.2 [7.5 – 10.1] |
| HOMA-IR | 1.7 [0.8 – 2.1] | 0.4 [0.4 – 0.8] | 1.7 [0.6 – 2.1] | 1.8 [1.6 – 2.2] |
| HbAlc, % | 5.3 [5.2 – 5.4] | 5.4 [5.2 – 5.5] | 5.3 [5.3 – 5.4] | 5.3 [5.0 – 5.4] |
| IGF-1, ng/mL ¶ | 179 [148 – 223] | 85 [61 – 137] | 178 [133 – 200] | 189 [149 – 233] |
| HDL-C, mg/dL | 59 [51 – 63] | 56 [50 – 60] | 57 [49 – 63] | 62 [57 – 64] |
| HDL ≤ 50 mg/dL, n | 5 (25) | 4 (36) | 3 (27) | 2 (22) |
| TG, mg/dL | 53 [41 – 57] | 51 [44 – 61] | 52 [41 – 55] | 53 [43 – 71] |
| TG ≥ 100 mg/dL, n | 2 (10) | 1 (9) | 0 (0) | 2 (22) |
Abbreviations: PA, premature adrenarche; PP, premature pubarche; BMI, body mass index; WC, waist circumference; BP, blood pressure; MetS, metabolic syndrome; BG, blood glucose; HOMA-IR, homeostasis model assessment of insulin resistance; HbA1c, hemoglobin A1c; IGF-1, insulin-like growth factor-1; HDL-C, high-density lipoprotein cholesterol; TG, triglycerides. Data are medians [IQR] or counts (percentage). Statistical significance was determined by nonparametric Mann Whitney U test for continuous data and Fischer’s exact test for categorical data. A stepdown bootstrap with replacement adjustment was used for multiple comparisons of between-group differences for the serum metabolic markers. A P value of < 0.05 was considered to indicate statistical significance.
Underweight: BMI ≤ 5th %ile; Normal: BMI 5 – < 85th %ile; Overweight: BMI 85 – < 95th %ile; Obese: BMI ≥ 95th %ile
Age- and sex- specific
Age-, sex-, and height-specific
Using de Ferranti et al. [24] criteria for childhood MetS
P = 0.001 for PA+PP vs. Control
11oA steroids in both PA and PP
Serum concentrations of adrenal steroids in PA, PP, and control subjects are shown in Figure 1. There were no differences in serum levels of T and A4 between PA and PP subgroups (Table 3). Although the serum levels of T and A4 were increased in cases compared to controls, these differences in laboratory values were not clinically significant and were in the expected range for the subjects’ Tanner stages for pubic hair. Serum levels of both DHEA and DHEAS were elevated in cases compared to controls (P = 0.002, P < 0.001), and as expected for the defined criteria for PA and PP, serum DHEAS was elevated in PA compared to PP (P < 0.001). Serum levels of 11OHT, 11KT, 11OHA4, and 11KA4 were all statistically higher in the PA and PP cases compared to controls (P < 0.001 for 11KT, 11OHA4, and 11KA4; P = 0.001 for 11OHT) but did not differ between PA and PP groups (Table 3). Importantly, 11KT values were 4-fold higher than those for T, indicating that 11KT is the major active androgen in the irculation for both PA and PP children. There were no differences between serum levels of cortisol, 17OH-Prog, ACTH, LH, FSH, and E2 between cases and controls or between PA and PP subgroups (Table 3). Serum E1 was higher in cases compared to controls, but all subjects had normal E1 levels. Differences in serum levels of DOC and 11-deoxycortisol between PA and PP subgroups reached statistical significance, attributable to one subject with PP, whose DOC, 11-deoxycortisol, and cortisol were all slightly above the normal range and were consistent with a stressful blood draw rather than an adrenal enzyme defect.
Fig. 1.
Serum concentrations of classic adrenal steroids (a-d) and 11oAs (e-h) in subjects with PA (n=11), PP (n=9), and healthy controls (n=11). Lower limit of quantification for each assay: 2.5 ng/dL for testosterone (T); 10 ng/dL for androstenedione (A4); 20 ng/dL for dehydroepiandrosterone (DHEA); 10 μg/dL for dehydroepiandrosterone sulfate (DHEAS); 3 ng/dL for 11β-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), and 11-ketotestosterone (11KT); and 10 ng/dL for 11β-hydroxyandrostenedione (11OHA4). To convert ng/dL to nmol/L, multiply by 0.035 for T, A4, and DHEA; 0.027 for DHEAS; and 0.033 for 11OHT, 11KT, 11OHA4, and 11KA4
Table 3.
Steroid Data for PA+PP Case and Control Subjects with Additional Comparison of PA and PP Subgroups
| PA+PP (n=20) | Control (n=11) | P value | PA (n=11) | PP (n=9) | P value | |
|---|---|---|---|---|---|---|
| Steroids of Interest | ||||||
| DHEA, ng/dL | 128 [78 – 244] | 20 [20 – 31] | 0.002 | 204 [128 – 310] | 86 [65 – 129] | ns |
| DHEAS, μg/dL | 58 [44 – 94] | 10 [10 – 12] | < 0.001 | 92 [65 – 110] | 42 [36 – 46] | < 0.001 |
| T, ng/dL | 5.6 [4.2 – 7.2] | 2.5 [2.5 – 2.5] | < 0.001 | 5.2 [4.8 – 7.1] | 5.9 [4.0 – 7.3] | ns |
| A4, ng/dL | 22 [13 – 30] | 10 [10 – 10] | 0.002 | 21 [14 – 25] | 29 [13 – 38] | ns |
| 17OH-Prog, ng/dL | 14 [11 – 47] | 14.0 [13 – 19] | ns | 13 [10 – 20] | 47 [11 – 65] | ns |
| Cortisol, μg/dL | 6.6 [5.2 – 9.7] | 8.1 [4.9 – 10.5] | ns | 5.4 [5.1 – 7.5] | 9.4 [6.9 – 15.0] | ns |
| 11OHT, ng/dL | 5.7 [4.8 – 6.5] | 3.0 [3.0 – 3.8] | 0.001 | 5.1 [4.8 – 5.9] | 6.4 [5.0 – 6.9] | ns |
| 11KT, ng/dL | 24.2 [18.8 – 29.2] | 9.1 [5.8 – 10.4] | < 0.001 | 20.4 [18.8 – 27.2] | 25.5 [24.0 – 29.1] | ns |
| 11OHA4, ng/dL | 58.2 [51.4 – 94.4] | 16.1 [13.4 – 25.7] | < 0.001 | 57.5 [40.7 – 74.1] | 65.0 [52.8 – 110.0] | ns |
| 11KA4, ng/dL | 17.4 [14.0 – 20.2] | 5.8 [5.1 – 8.3] | < 0.001 | 17.3 [12.9 – 18.3] | 18.6 [14.5 – 20.7] | ns |
| Additional Assays | ||||||
| ACTH, ρg/mL* | 19 [11 – 25] | 15 [12 – 18] | ns | 18 [8 – 24] | 22 [19 – 31] | ns |
| LH, mlU/mL | 0.02 [0.02 – 0.05] | 0.04 [0.03 – 0.06] | ns | 0.02 [0.02 – 0.04] | 0.03 [0.02 – 0.06] | ns |
| FSH, mlU/mL | 1.15 [0.56 – 2.20] | 1.90 [0.69 – 3.20] | ns | 1.90 [1.10 – 3.15] | 0.60 [0.53 – 1.00] | ns |
| E2, pg/mL | 1.0 [1.0 – 1.7] | 1.3 [1.0 – 3.2] | ns | 1.0 [1.0 – 1.4] | 1.0 [1.0 – 2.0] | ns |
| E1, pg/mL | 3.1 [2.6 – 4.7] | 2.5 [2.5 – 3.0] | 0.033 | 3.1 [2.6 – 4.5] | 3.0 [3.0 – 5.9] | ns |
| 11-deoxycortisol, ng/dL | 23 [12 – 49] | 21 [15 – 32] | ns | 13 [10 – 26] | 41 [28 – 90] | 0.033 |
| DOC, ng/dL | 4.1 [2.7 – 5.6] | 5.6 [3.8 – 7.9] | ns | 3.3 [2.2 – 4.1] | 6.4 [4.5 – 9.9] | 0.008 |
Abbreviations: PA, premature adrenarche; PP, premature pubarche; DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate; T, testosterone; A4, androstenedione; 17OH-Prog, 17-hydroxyprogesterone; 11OHT, 11β-hydroxytestosterone; 11KT, 11-ketotestosterone; 11OHA4, 11β-hydroxyandrostenedione; 11-KA4, 11ketoandrostenedione; DOC, deoxycorticosterone; ACTH, adrenocorticotropin hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone; E2, estradiol; E1, estrone. Data are medians [IQR] or counts (percentage). Statistical significance was determined by nonparametric Mann Whitney U test for continuous data and Fischer’s exact test for categorical data. For Steroids of Interest, a stepdown bootstrap with replacement adjustment was used for multiple comparisons of between-group differences. A P value of < 0.05 was considered to indicate statistical significance.
Based on available data from 10 PA, 9 PP, and 10 controls
Correlations analyses
Spearman correlations with Hommel correction for multiple comparisons were conducted on the cohorts of cases (n = 20) and controls (n = 11) (Table 4). In the case cohort, serum T and serum A4 correlated strongly with serum 11KT and serum 11OHA4 (ρ = 0.70 – 0.78; P < 0.05); this relationship was not observed in the control cohort. There were no statistically significant correlations of serum insulin and HOMA-IR with serum levels of T, A4, DHEA, DHEAS, or the 11oAs within case or control cohorts.
Table 4.
Correlations of Androgens with Baseline Demographics and Metabolic Parameters for PA+PP Case and Control Cohorts
| Age | BMI-Z | IGF-1 | Insulin | HOMA-IR | DHEA | DHEAS | T | A4 | 11OHT | 11KT | 11OHA4 | 11KA4 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Age | −0.15 | 0.45 | 0.43 | 0.57 | 0.11 | −0.17 | 0.10 | 0.10 | 0.16 | 0.37 | −0.03 | 0.23 | |
| BMI-Z | −0.01 | 0.09 | 0.08 | 0.01 | 0.30 | 0.63 | 0.50 | 0.50 | 0.14 | 0.31 | −0.10 | 0.17 | |
| IGF-1 | 0.13 | 0.25 | 0.76 | 0.87 | 0.27 | 0.20 | 0.40 | 0.40 | −0.05 | 0.63 | 0.26 | 0.43 | |
| Insulin | 0.35 | 0.39 | 0.53 | 0.91 | 0.56 | 0.29 | 0.55 | 0.55 | 0.44 | 0.71 | 0.54 | 0.70 | |
| HOMA-IR | 0.35 | 0.33 | 0.51 | 0.99 | 0.50 | 0.29 | 0.50 | 0.50 | 0.37 | 0.77 | 0.45 | 0.60 | |
| DHEA | −0.28 | 0.01 | 0.17 | 0.01 | −0.06 | 0.86 | 0.68 | 0.63 | 0.73 | 0.82 | 0.84 | 0.84 | |
| DHEAS | −0.43 | 0.30 | −0.08 | 0.02 | −0.04 | 0.67 | 0.38 | 0.13 | 0.51 | 0.71 | 0.62 | 0.62 | |
| T | −0.30 | 0.08 | 0.18 | 0.05 | −0.02 | 0.55 | 0.64 | 1.00 | 0.38 | 0.50 | 0.40 | 0.40 | |
| A4 | −0.17 | 0.01 | 0.36 | 0.19 | 0.12 | 0.55 | 0.64 | 0.91 | 0.38 | 0.50 | 0.40 | 0.40 | |
| 11OHT | 0.14 | 0.07 | −0.16 | 0.34 | 0.31 | −0.01 | −0.03 | 0.26 | 0.31 | 0.61 | 0.50 | 0.50 | |
| 11KT | −0.17 | −0.03 | 0.33 | 0.13 | 0.05 | 0.44 | 0.12 | 0.70 | 0.78 | 0.46 | 0.63 | 0.75 | |
| 11OHA4 | −0.22 | −0.09 | −0.06 | −0.06 | −0.11 | 0.49 | 0.07 | 0.71 | 0.76 | 0.46 | 0.73 | 0.86 | |
| 11KA4 | −0.25 | −0.03 | 0.19 | 0.05 | 0.00 | 0.41 | 0.08 | 0.55 | 0.68 | 0.34 | 0.81 | 0.73 | |
Abbreviations: PA, premature adrenarche; PP, premature pubarche; BMI-Z, body mass index z-score; HOMA-IR, homeostatic model assessment of insulin resistance; DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate; T, testosterone; A4, androstenedione; 11OHT, 11β-hydroxytestosterone; 11KT, 11-ketotestosterone; 11OHA4, 11β-hydroxyandrostenedione; 11KA4, 11-ketoandrostenedione.
Spearman correlations in the combined PA+PP Case cohort (n=20) are shown below the diagonal and Spearman correlations within Control cohort (n=11) are shown above the diagonal in italics. Bolded correlation coefficients are statistically significant (P < 0.05) after Hommel correction for multiple comparisons.
Discussion
This is the first study to report the early morning serum 11oA levels of both male and female children with PA or PP, confirmed to be prepubertal on physical exam, with comparison to their healthy male and female counterparts Although chronological age was greater in the cases versus controls, HA/CA ratio was not significantly different between the groups (Table 1). Each PA and PP subject was confirmed to not have any undiagnosed endocrinopathies to explain their condition. There were early signs of metabolic dysfunction in prepubertal age seen in both cases and controls, including changes in serum HDL-C, serum TG, WC, serum insulin, and HOMA-IR; a total of four children met criteria for childhood metabolic syndrome (3 cases, 1 control).
We found that serum 11oAs were significantly increased in children with either PA or PP compared to controls, and comparison of children with PA or PP shows that beyond the Hispanic majority representation observed in the PA group, the only between-group differences exist in serum DHEAS levels. Circulating 11KT concentrations exceeded those of T by a factor of 4 in both PA and PP children. The findings that 11KT is the major circulating active androgen in both PA and PP children and that only DHEAS differed between the two groups suggest that pubarche is mediated by elevation of serum 11oAs—rather than by changes in serum DHEAS—and that PA and PP differ only by definition alone based on serum DHEAS cutoffs. While further study is warranted to explore whether this trend continues to be seen in other groups beyond this predominantly Hispanic sample size of children with PA, we propose measurement of serum 11oAs to be a more accurate way to screen and classify children with PA than serum DHEAS. That this inclusive definition can also capture the “PP” children is important; our study demonstrated that the PP subgroup had clinical and biochemical signs of elevated risk for metabolic disturbance that was no different from the PA subgroup.
While BMI did not differ between the PA and PP case groups and healthy controls, there were observable differences in serum insulin and HOMA-IR. A study of PCOS population by O’Reilly and colleagues [19] showed the circulating 11oAs 11OHA4, 11KA4, 11OHT, and 11KT were all higher in obese and non-obese subjects with PCOS compared to healthy controls, and serum 11OHA4 and serum 11KA4 correlated with markers of insulin resistance. The evolution of insulin resistance from childhood to adulthood in these patient populations with androgen excess requires further study to better delineate when the markers of metabolic risk become more evident and clinically relevant. A longitudinal follow-up to assess divergence between groups in a maturing population of children with history of PA or PP would offer additional insight into possible causal relationships between 11oAs and metabolic disturbance.
Decades ago, elevated IGF-1 levels were reported in children with PA, and their potential metabolic contributions were described [10, 11]. Both IGF-1 and insulin have been shown to induce steroidogenic enzymes in cultured human adrenocortical cells in vitro [31]; however, currently we do not know the stimulatory effects of IGF-1 on 11oAs. In this study, we observed moderate correlations of serum IGF-1 and HOMA-IR in children with PA or PP.
While many studies have reported decreased insulin sensitivity in children with PA compared to controls [12, 32–35], one study by Silfen and colleagues [10] did not detect a difference between their PA and control girls, possibly attributable in part to the prevalence of obesity in that study population. In our study, the PA and PP groups did not differ in insulin sensitivity from the control group. While our study subjects were recruited from the same geographic area as Silfen et al., ours were composed of mainly subjects of normal BMI z-scores. We hypothesize that there are likely both insulin-sensitive and insulin-resistant subtypes of PA and PP, which would be better studied as separate groups in order to better define the relationships of insulin sensitivity to the different phenotypes, by controlling for BMI.
The serum 11oA levels reported in this study cohort of PA and PP are similar to a cohort of girls with PA and age-matched controls described previously by Rege et al. [21]. In this present study, the degree of difference in 11oA levels between cases and controls is generally more pronounced and is likely in part due to the early morning collection of serum. Early morning collection of serum accounts for likely diurnal variation in 11oAs and ensures consistent reporting of values for all subjects.
Additionally, a prior report showed that urinary metabolites of DHEA and other C19 steroids increase exponentially starting at 3 years of age onwards [36]. Indeed, our youngest PA subject was 5.4 years of age, and our youngest PP subject was 7.4 years of age. Further, in a recent study by Janner et al., the 11β-hydroxy-metabolites as precursors to the 11-ketoandrogens were not higher in their PA subjects; however, this group did not directly measure the 11-ketoandrogens [37]. Biochemical adrenarche is likely a more gradual process than previously thought, and the present study further supports the reconsideration of the definition of adrenarche by an arbitrary cutoff of DHEAS or any other adrenal androgen. Future studies with more robust subject sample sizes will allow for subanalyses of these cohorts to answer further questions about the factors that regulate 11oAs production in obese and non-obese, or insulin-resistant and insulin-sensitive, groups. These current pilot findings in a unique well-described cohort demonstrate that serum 11oAs are elevated even in the absence of increased DHEAS, a traditional marker for androgen excess in adrenarche.
Acknowledgments
We thank the members of the Division of Pediatric Endocrinology, Diabetes and Metabolism at Columbia University Irving Medical Center (New York, NY) for their thoughtful referral of eligible subjects, as well as the subjects and their families for agreeing to participate. We thank Rachel Tao, B.A., for her initial help with patient recruitment and coordination of study visits. We also thank Ismael Castaneda, R.N., and the staff of the Clinical Research Resource (CRR) at Columbia Irving Institute for Clinical and Translational Research (UL1TR001873) for their valuable help in conducting the study visits. With gratitude we acknowledge Esoterix Laboratory and the Columbia Biomarkers Core Laboratory for performing laboratory measurements for this study.
Funding Sources
This work was supported in part by the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases Grant 5T32DK065522–14 (to BKW and SEO) and by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number UL1TR001873. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Footnotes
Statement of Ethics
Approval was obtained from the Institutional Review Board at Columbia University Irving Medical Center (CUIMC), reference number IRB-AAAR6323. Prior to any study procedures being conducted, written informed consent was obtained from the parents. Assent was obtained from the child when appropriate (7 years of age and older).
Conflict of Interest Statement
BKW, ACB, PO, AH, ABS, KMW, PMV, RN, DJM, RJA, and SEO have nothing to declare. IF is a consultant for Rhythm Pharmaceuticals and an Advisory Board Member for Ascendis Pharmaceuticals.
References
- 1.Oberfield SE, Tao RH, Witchel SF. Present Knowledge on the Etiology and Treatment of Adrenarche. Pediatr Endocrinol Rev. 2018;15(3):244–54. [DOI] [PubMed] [Google Scholar]
- 2.Paterson WF, Ahmed SF, Bath L, Donaldson MD, Fleming R, Greene SA, et al. Exaggerated adrenarche in a cohort of Scottish children: clinical features and biochemistry. Clin Endocrinol (Oxf). 2010;72(4):496–501. [DOI] [PubMed] [Google Scholar]
- 3.Utriainen P, Voutilainen R, Jaaskelainen J. Continuum of phenotypes and sympathoadrenal function in premature adrenarche. Eur J Endocrinol. 2009;160(4):657–65. [DOI] [PubMed] [Google Scholar]
- 4.Vottero A, Capelletti M, Giuliodori S, Viani I, Ziveri M, Neri TM, et al. Decreased androgen receptor gene methylation in premature pubarche: a novel pathogenetic mechanism? J Clin Endocrinol Metab. 2006;91(3):968–72. [DOI] [PubMed] [Google Scholar]
- 5.Friend A, Craig L, Turner S. The prevalence of metabolic syndrome in children: a systematic review of the literature. Metab Syndr Relat Disord. 2013;11(2):71–80. [DOI] [PubMed] [Google Scholar]
- 6.Idkowiak J, Lavery GG, Dhir V, Barrett TG, Stewart PM, Krone N, et al. Premature adrenarche: novel lessons from early onset androgen excess. Eur J Endocrinol. 2011;165(2):189–207. [DOI] [PubMed] [Google Scholar]
- 7.Williams RM, Ward CE, Hughes IA. Premature adrenarche. Arch Dis Child. 2012;97(3):250–4. [DOI] [PubMed] [Google Scholar]
- 8.Jean AM, Hassoun A, Hughes J, Pomeranz C, Fennoy I, McMahon DJ, et al. Utility of early insulin response and proinsulin to assess insulin resistance. J Pediatr. 2009;155(6):893–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Oberfield SE, Sopher AB, Gerken AT. Approach to the girl with early onset of pubic hair. J Clin Endocrinol Metab. 2011;96(6):1610–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Silfen ME, Manibo AM, Ferin M, McMahon DJ, Levine LS, Oberfield SE. Elevated free IGF-I levels in prepubertal Hispanic girls with premature adrenarche: relationship with hyperandrogenism and insulin sensitivity. J Clin Endocrinol Metab. 2002;87(1):398–403. [DOI] [PubMed] [Google Scholar]
- 11.Vuguin P, Linder B, Rosenfeld RG, Saenger P, DiMartino-Nardi J. The roles of insulin sensitivity, insulin-like growth factor I (IGF-I), and IGF-binding protein-1 and −3 in the hyperandrogenism of African-American and Caribbean Hispanic girls with premature adrenarche. J Clin Endocrinol Metab. 1999;84(6):2037–42. [DOI] [PubMed] [Google Scholar]
- 12.Denburg MR, Silfen ME, Manibo AM, Chin D, Levine LS, Ferin M, et al. Insulin sensitivity and the insulin-like growth factor system in prepubertal boys with premature adrenarche. J Clin Endocrinol Metab. 2002;87(12):5604–9. [DOI] [PubMed] [Google Scholar]
- 13.Pretorius E, Arlt W, Storbeck KH. A new dawn for androgens: Novel lessons from 11-oxygenated C19 steroids. Mol Cell Endocrinol. 2017;441:76–85. [DOI] [PubMed] [Google Scholar]
- 14.Rege J, Nakamura Y, Satoh F, Morimoto R, Kennedy MR, Layman LC, et al. Liquid chromatography-tandem mass spectrometry analysis of human adrenal vein 19-carbon steroids before and after ACTH stimulation. J Clin Endocrinol Metab. 2013;98(3):1182–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.du Toit T, Bloem LM, Quanson JL, Ehlers R, Serafin AM, Swart AC. Profiling adrenal 11beta-hydroxyandrostenedione metabolites in prostate cancer cells, tissue and plasma: UPC(2)-MS/MS quantification of 11beta-hydroxytestosterone, 11keto-testosterone and 11ketodihydrotestosterone. J Steroid Biochem Mol Biol. 2017;166:54–67. [DOI] [PubMed] [Google Scholar]
- 16.Turcu AF, Nanba AT, Chomic R, Upadhyay SK, Giordano TJ, Shields JJ, et al. Adrenal-derived 11-oxygenated 19-carbon steroids are the dominant androgens in classic 21-hydroxylase deficiency. Eur J Endocrinol. 2016;174(5):601–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Kamrath C, Wettstaedt L, Boettcher C, Hartmann MF, Wudy SA. Androgen excess is due to elevated 11-oxygenated androgens in treated children with congenital adrenal hyperplasia. J Steroid Biochem Mol Biol. 2018;178:221–228. doi: 10.1016/j.jsbmb.2017.12.016. [DOI] [PubMed] [Google Scholar]
- 18.Kamrath C, Wettstaedt L, Hartmann MF, Wudy SA. Height Velocity defined metabolic Control in Children with Congenital Adrenal Hyperplasia using urinary GC-MS Analysis [published online ahead of print, 2019 May 21]. J Clin Endocrinol Metab. 2019;jc.2019–00438. doi: 10.1210/jc.2019-00438. [DOI] [PubMed] [Google Scholar]
- 19.O’Reilly MW, Kempegowda P, Jenkinson C, Taylor AE, Quanson JL, Storbeck KH, et al. 11-Oxygenated C19 Steroids Are the Predominant Androgens in Polycystic Ovary Syndrome. J Clin Endocrinol Metab. 2017;102(3):840–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Turcu AF, Auchus RJ. Clinical significance of 11-oxygenated androgens. Curr Opin Endocrinol Diabetes Obes. 2017;24(3):252–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Rege J, Turcu AF, Kasa-Vubu JZ, Lerario AM, Auchus GC, Auchus RJ, et al. 11-Ketotestosterone Is the Dominant Circulating Bioactive Androgen During Normal and Premature Adrenarche. J Clin Endocrinol Metab. 2018;103(12):4589–98. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Korth-Schutz S, Levine LS, New MI. Serum Androgens in Normal Prepubertal and Pubertal Children and in Children with Precocious Adrenarche. J Clin Endocrinol Metab. 1976;42(1):117–24. [DOI] [PubMed] [Google Scholar]
- 23.Greulich WW, Pyle SI. Radiographic atlas of skeletal development of the hand and wrist. 2nd ed. Stanford, CA: Stanford University Press; 1959. [Google Scholar]
- 24.Chou JH, Roumiantsev S, Singh R. PediTools Electronic Growth Chart Calculators: Applications in Clinical Care, Research, and Quality Improvement. J Med Internet Res. 2020;22(1):e16204. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Shypailo RJ. Age-based Pediatric Blood Pressure Reference Charts [Internet]. Baylor College of Medicine, Children’s Nutrition Research Center, Body Composition Laboratory, Houston, Texas. [cited 2019 Nov 1]. Available from: https://www.bcm.edu/bodycomplab/BPappZjs/BPvAgeAPPz.html. [Google Scholar]
- 26.Fernández JR, Redden DT, Pietrobelli A, Allison DB. Waist circumference percentiles in nationally representative samples of African-American, European-American, and Mexican-American children and adolescents. J Pediatr. 2004;145(4):439–44. [DOI] [PubMed] [Google Scholar]
- 27.de Ferranti SD, Gauvreau K, Ludwig DS, Neufeld EJ, Newburger JW, Rifai N. Prevalence of the metabolic syndrome in American adolescents: findings from the Third National Health and Nutrition Examination Survey. Circulation. 2004;110(16):2494–7. [DOI] [PubMed] [Google Scholar]
- 28.Wright C, O’Day P, Alyamani M, Sharifi N, Auchus RJ. Abiraterone acetate treatment lowers 11-oxygenated androgens. Eur J Endocrinol. 2020;182(4):413–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Westfall PH, Young SS. Resampling-Based Multiple Testing : Examples and Methods for P-Value Adjustment. New York: John Wiley & Sons; 1993. [Google Scholar]
- 30.Hommel G. A Comparison of Two Modified Bonferroni Procedures. Biometrika. 1989;76(3):624–25. [Google Scholar]
- 31.Mesiano S, Katz SL, Lee JY, Jaffe RB. Insulin-like growth factors augment steroid production and expression of steroidogenic enzymes in human fetal adrenal cortical cells: implications for adrenal androgen regulation. J Clin Endocrinol Metab. 1997;82(5):1390–6. [DOI] [PubMed] [Google Scholar]
- 32.Ibanez L, Dimartino-Nardi J, Potau N, Saenger P. Premature adrenarche--normal variant or forerunner of adult disease? Endocr Rev. 2000;21(6):671–96. [DOI] [PubMed] [Google Scholar]
- 33.Ibanez L, Potau N, Zampolli M, Rique S, Saenger P, Carrascosa A. Hyperinsulinemia and decreased insulin-like growth factor-binding protein-1 are common features in prepubertal and pubertal girls with a history of premature pubarche. J Clin Endocrinol Metab. 1997;82(7):2283–8. [DOI] [PubMed] [Google Scholar]
- 34.Silfen ME, Manibo AM, McMahon DJ, Levine LS, Murphy AR, Oberfield SE. Comparison of simple measures of insulin sensitivity in young girls with premature adrenarche: the fasting glucose to insulin ratio may be a simple and useful measure. J Clin Endocrinol Metab. 2001;86(6):2863–8. [DOI] [PubMed] [Google Scholar]
- 35.Ibanez L, Potau N, Chacon P, Pascual C, Carrascosa A. Hyperinsulinaemia, dyslipaemia and cardiovascular risk in girls with a history of premature pubarche. Diabetologia. 1998;41(9):1057–63. [DOI] [PubMed] [Google Scholar]
- 36.Remer T, Boye KR, Hartmann MF, Wudy SA. Urinary markers of adrenarche: reference values in healthy subjects, aged 3–18 years. J Clin Endocrinol Metab. 2005;90(4):2015–21. [DOI] [PubMed] [Google Scholar]
- 37.Janner M, Sommer G, Groessl M, Flück CE. Premature Adrenarche in Girls Characterized By Enhanced 17,20-Lyase and 17β-Hydroxysteroid Dehydrogenase Activities. J Clin Endocrinol Metab. 2020;105(12):dgaa598. [DOI] [PubMed] [Google Scholar]