P.-M. Holterhus

A Novel Ultrapressure Liquid Chromatography Tandem Mass Spectrometry Method for the Simultaneous Determination of Androstenedione, Testosterone, and Dihydrotestosterone in Pediatric Blood Samples: Age- and Sex-Specific Reference Data

CONTEXTnCurrent immunoassays for analysis of plasma androgens in children have several limitations due to antibody-specific variations of data and normal ranges. Mass spectrometry-based methods are available for individual steroids but need complex sample preparation and report only fragmentary reference data for the pediatric population.nnnOBJECTIVEnOur objective was to develop a state of the art sensitive and specific tandem mass spectrometry method for high-throughput simultaneous determination of plasma concentrations of androstenedione (A), testosterone (T), and dihydrotestosterone (DHT) and to report age-, sex-, and pubertal stage-specific reference levels for these steroids in children aged 0-18 yr.nnnSUBJECTS AND METHODSnPlasma (100 microl) was mixed with internal standard and extracted by solid-phase extraction. Androgens were measured by ultrapressure liquid chromatography tandem mass spectrometry. Samples of 138 boys and 131 girls with neither signs of endocrine nor systemic disease were considered for the generation of reference data. The following age groups were used: less than 1 wk, 2 wk to 2 months, 3-5 months, 6-11 months, 1-3 yr, 4-6 yr, 7-9 yr, 10-12 yr, 13-15 yr, and over 16 yr.nnnRESULTSnLower quantification limit was 2.9 ng/dl (0.1 nmol/liter) for A, T, and DHT. No relevant interference with other steroids was detected. Reference data for A, T, and DHT are reported as functions of age, sex, pubertal maturation, and testicular volume.nnnCONCLUSIONnSimplicity, velocity, sensitivity, specificity, and the availability of pediatric reference data allow application of our new method in clinical routine as well as in research settings.

Changes Over Time in Sex Assignment for Disorders of Sex Development

BACKGROUND AND OBJECTIVE: It is unclear whether the proportion of infants with a disorder of sex development who are raised as male or female has changed over time. The temporal trends in sex assignment of affected cases entered in the International Disorder of Sex Development (I-DSD) Registry were studied. METHODS: Cases of disorders of sex development reported as partial androgen insensitivity syndrome (PAIS; n = 118), disorder of gonadal development (DGD; n = 232), and disorder of androgen synthesis (DAS; n = 104) were divided into those who were born before 1990, 1990–1999, and after 1999. External appearance of the genitalia was described by the external masculinization score. RESULTS: The median (5th–95th percentile) external masculinization scores of those infants with PAIS, DGD, and DAS who were raised as boys were 6 (2–9), 6 (3–9), and 6 (1–12), respectively, and were significantly higher than in those raised as girls (2 [0–6], 2 [0–7], and 0 [0–5], respectively); this difference was maintained in the 3 temporal birth cohorts (P < .01). Of the 118 cases in the pre-1990 cohort, 41 (35%) were raised as boys; of the 148 cases in the 1990–1999 cohort, 60 (41%) were raised as boys; and of the 188 cases in the post-1999 cohort, 128 (68%) were raised as boys. CONCLUSIONS: Although there is an association between the external appearance of the genitalia and the choice of sex assignment, there are clear temporal trends in this practice pointing toward an increased likelihood of affected infants being raised as boys. The impact of this change in practice on long-term health outcomes requires additional focus.

Principles and clinical applications of liquid chromatography — tandem mass spectrometry for the determination of adrenal and gonadal steroid hormones

Liquid-chromatography — tandem mass spectrometry (LC-MS/MS) is becoming the method of choice for clinical steroid analysis. In most instances, it has the advantage of higher sensitivity, better reproducibility and greater specificity than commercial immunoassay techniques. The method requires only minimal sample preparation and a small sample volume. Furthermore, it has the potential to analyze multiple steroids simultaneously. Modern instruments guarantee high throughput, allowing an affordable price for the individual assay. All this makes LC-MS/MS an attractive method for use in a clinical setting. Reliable reference ranges for the detected analytes are the pre-requisite for their clinical use. If these are available, LC-MS/MS can find application in congenital disorders of steroid metabolism, such as congenital adrenal hyperplasia, disorders of sex development and disorders of salt homeostasis, as well as in acquired disorders of steroid metabolism, such as primary aldosteronism, Cushing’s disease, Addison’s disease, and hyperandrogenemia, as well as in psychiatric disease states such as depression or anxiety disorders. The principles of LC-MS/MS for steroid measurement, the pros and cons of LC-MS/MS compared with conventional immunoassays and the possible applications in clinical routine, with a special focus on pediatric endocrinology needs, are discussed here.

Steroid hormone analysis in diagnosis and treatment of DSD: position paper of EU COST Action BM 1303 ‘DSDnet’

Disorders or differences in sex development (DSD) comprise a heterogeneous group of conditions with an atypical sex development. For optimal diagnosis, highly specialised laboratory analyses are required across European countries. Working group 3 of EU COST (European Cooperation in Science and Technology) Action BM 1303 ‘DSDnet’ ‘Harmonisation of Laboratory Assessment’ has developed recommendations on laboratory assessment for DSD regarding the use of technologies and analytes to be investigated. This position paper on steroid hormone analysis in diagnosis and treatment of DSD was compiled by a group of specialists in DSD and/or hormonal analysis, either from participating European countries or international partner countries. The topics discussed comprised analytical methods (immunoassay/mass spectrometry-based methods), matrices (urine/serum/saliva) and harmonisation of laboratory tests. The following positions were agreed upon: support of the appropriate use of immunoassay- and mass spectrometry-based methods for diagnosis and monitoring of DSD. Serum/plasma and urine are established matrices for analysis. Laboratories performing analyses for DSD need to operate within a quality framework and actively engage in harmonisation processes so that results and their interpretation are the same irrespective of the laboratory they are performed in. Participation in activities of peer comparison such as sample exchange or when available subscribing to a relevant external quality assurance program should be achieved. The ultimate aim of the guidelines is the implementation of clinical standards for diagnosis and appropriate treatment of DSD to achieve the best outcome for patients, no matter where patients are investigated or managed.

No Correlation between Androgen Receptor CAG and GGN Repeat Length and the Degree of Genital Virilization in Females with 21-Hydroxylase Deficiency

CONTEXTnIn 21-hydroxylase (CYP21A2) deficiency (21OHD), the level of in vitro enzymatic function allows for classification of mutation groups (null, A, B, C) and prediction of disease severity. However, genital virilization in affected females correlates only weakly with CYP21A2 mutation groups, suggesting the influence of genetic modifiers.nnnOBJECTIVEnThe objective of the study was to investigate the influence of the polymorphic CAG and GGn repeats of the androgen receptor (AR) gene on the degree of genital virilization in 21OHD females.nnnDESIGN AND PATIENTSnDesign of the study was the determination of CYP21A2 genotype, degree of genital virilization (Prader stage), and X-weighted biallelic mean of AR CAG and GGn repeat length in 205 females with 21OHD.nnnOUTCOME MEASUREMENTSnCorrelation of AR CAG and GGn repeat lengths with Prader stages using nested stepwise logistic regression analysis was measured.nnnRESULTSnCYP21A2 mutation groups null and A showed significantly higher levels of genital virilization than groups B and C (P < 0.01). However, Prader stages varied considerably within mutation groups: null, Prader I-V (median IV); A, Prader I-V (median IV); B, Prader I-V (median III); C, 0-III (median I). Mean GGn repeat length of patients was not significantly associated with Prader stages, classified as low (0-I), intermediate (II-III), or severe (IV-V) (odds ratio per repeat: 0.98, 95% confidence interval 0.71-1.35). In contrast, patients with Prader 0-I showed a trend toward longer CAG repeats without reaching statistical significance (P = 0.07, odds ratio per repeat: 0.82, 95% confidence interval 0.65-1.02).nnnCONCLUSIONnNeither CAG nor GGn repeat lengths are statistically significant modifiers of genital virilization in females with 21OHD.

Androgen receptor mutations are associated with altered epigenomic programming as evidenced by HOXA5 methylation.

Male external genital differentiation is accompanied by implementation of a long-term, male-specific gene expression pattern indicating androgen programming in cultured genital fibroblasts. We hypothesized the existence of an epigenetic background contributing to this phenomenon. DNA methylation levels in 2 normal scrotal fibroblast strains from 46,XY males compared to 2 labia majora fibroblast strains from 46,XY females with complete androgen insensitivity syndrome (AIS) due to androgen receptor (AR) mutations were analyzed by Illumina GoldenGate methylation arrays®. Results were validated with pyrosequencing in labia majora fibroblast strains from fifteen 46,XY patients and compared to nine normal male scrotal fibroblast strains. HOXA5 showed a significantly higher methylation level in complete AIS. This finding was confirmed by bisulfite pyrosequencing of 14 CpG positions within the HOXA5 promoter in the same strains. Extension of the 2 groups revealed a constant low HOXA5 methylation pattern in the controls in contrast to a highly variable methylation pattern in the AIS patients. HOXA5 represents a candidate gene of androgen-mediated promoter methylation. The constantly low HOXA5 DNA methylation level of normal male scrotal fibroblast strains and the frequently high methylation levels in labia majora fibroblast strains in AIS indicate for the first time that androgen programming in sexual differentiation is not restricted to global gene transcription but also occurs at the epigenetic level.

Testosterone synthesis in patients with 17β-hydroxysteroid dehydrogenase 3 deficiency.

17β-hydroxysteroid dehydrogenase 3 (17β-HSD 3) deficiency is a rare cause of 46,XY disorders of sex development (DSD). At puberty, these patients experience a surge of androstenedione and also testosterone, leading to substantial virilization. The origin of testosterone synthesis in these patients remains elusive. We investigated the expression of the isoenzyme AKR1C3 (17β-HSD 5) in the testis and patient-derived genital skin fibroblasts (GSF) as well as the ability of GSF to synthesize testosterone. Supernatants of GSF cultures and serum samples of one patient before and after gonadectomy were analyzed by liquid and gas chromatography/mass spectrometry. The androgenic potential of GSF-derived supernatants was also assessed by androgen receptor-mediated transactivation of a reporter gene in transiently transfected Chinese hamster ovary cells. Although AKR1C3 is expressed both in the testes and in GSF, androstenedione is rapidly metabolized and is not synthesized to testosterone. The transactivation potential of GSF supernatants towards the androgen receptor is declining within 48 h. However, under testis-equivalent androstenedione concentration, testosterone can be synthesized in 17β-HSD 3-negative GSF. After gonadectomy, both androstenedione and testosterone decline rapidly in vivo. In 17β-HSD 3 deficiency, relevant amounts of testosterone are synthesized most probably through AKR1C3 in the testis and not peripherally in GSF.

LC–MS/MS based determination of basal- and ACTH-stimulated plasma concentrations of 11 steroid hormones: implications for detecting heterozygote CYP21A2 mutation carriers

OBJECTIVEnHeterozygosity in 21-hydroxylase deficiency (21OHD) has been associated with hyperandrogenemic symptoms in children and adults. Moreover, the carrier status is mandatory for genetic counseling. We aimed at defining a hormonal parameter for carrier detection by mass spectrometry.nnnDESIGNnEleven basal and ACTH-stimulated steroid hormones of heterozygous carriers of CYP21A2 mutations and control individuals were compared.nnnMETHODnHormones were determined in plasma samples by liquid chromatography tandem mass spectrometry (LC-MS/MS) in 58 carriers (35 males, 23 females, age range 6-78 years) and 44 random controls (25 males, 19 females, age range 8-58 years).nnnRESULTSnHeterozygotes could be identified best applying the 17-hydroxyprogesterone+21-deoxycortisol/cortisol×1000 ((17OHP+21S)/F×1000) equation 30u200a min after ACTH injection. An optimal cut-off value of 8.4 provided 89% sensitivity and specificity. Considering this data and a published frequency of heterozygotes of 1/50 to 1/61, the positive predictive value (PPV) of this cut-off is 12%. Of note, the negative predictive value (NPV) excluding heterozygosity in a given patient is 99.8%.nnnCONCLUSIONnConsidering only marginal biochemical effects anticipated from heterozygosity, the stimulated ((17OHP+21S)/F×1000) identifies and excludes heterozygotes remarkably well. Nevertheless, LC-MS/MS cannot replace genetic testing, since sensitivity and specificity did not reach 100%. However, due to the considerably high NPV of the optimal cut-off and to a specificity of even 100% applying a cut-off higher than 14.7, hormonal assessment of heterozygosity can be of significant aid in conditions with limited access to genetic testing, as in some health care systems. The ((17OHP+21S)/F×1000) equation can guide diagnostic considerations in the differential diagnosis of hyperandrogenism.

Birth weight in different etiologies of disorders of sex development

ContextnIt is well established that boys are heavier than girls at birth. Although the cause of birth weight (BW) difference is unknown, it has been proposed that it could be generated from prenatal androgen action.nnnObjectivenThe aim of the current study was to determine the BW of children with disorders of sex development (DSD) of different etiologies and to evaluate the effects of androgen action on BW.nnnMethodsnData regarding diagnosis, BW, gestational age, karyotype, and concomitant conditions were collected from the International Disorders of Sex Development (I-DSD) Registry (www.i-dsd). BW standard deviation score was calculated according to gestational age. Cases were evaluated according to disorder classification in I-DSD (i.e., disorders of gonadal development, androgen excess, androgen synthesis, androgen action, nonspecific disorder of undermasculinization groups, and Leydig cell defect).nnnResultsnA total of 533 cases were available; 400 (75%) cases were 46,XY, and 133 (25%) cases were 46,XX. Eighty cases (15%) were born small for gestational age (SGA). Frequency of SGA was higher in the 46,XY group (17.8%) than in the 46,XX (6.7%) group (P = 0.001). Mean BW standard deviation scores of cases with androgen excess and androgen deficiency [in disorders of gonadal development, androgen synthesis, and Leydig cell defect groups and androgen receptor gene (AR) mutation-positive cases in disorders of androgen action groups] were similar to normal children with the same karyotype. SGA birth frequency was higher in the AR mutation-negative cases in disorders of androgen action group and in the nonspecific disorders of the undermasculinization group.nnnConclusionsnBW dimorphism is unlikely to be explained by fetal androgen action per se. 46,XY DSDs due to nonspecific disorders of undermasculinization are more frequently associated with fetal growth restriction, SGA, and concomitant conditions.

Epigenetic Repression of Androgen Receptor Transcription in Mutation-Negative Androgen Insensitivity Syndrome (AIS Type II)

ContextnInactivating mutations within the AR gene are present in only ~40% of individuals with clinically and hormonally diagnosed androgen insensitivity syndrome (AIS). Previous studies revealed the existence of an AR gene mutation-negative group of patients with AIS who have compromised androgen receptor (AR) function (AIS type II).nnnObjectivenTo investigate whether AIS type II can be due to epigenetic repression of AR transcription.nnnDesignnQuantification of AR mRNA and AR proximal promoter CpG methylation levels in genital skin-derived fibroblasts (GFs) derived from patients with AIS type II and control individuals.nnnSettingnUniversity hospital endocrine research laboratory.nnnPatientsnGFs from control individuals (n = 11) and patients with AIS type II (n = 14).nnnMain Outcome Measure(s)nMeasurement of AR mRNA and AR promoter CpG methylation as well as activity of AR proximal promoter in vitro.nnnResultsnFifty-seven percent of individuals with AIS type II (n = 8) showed a reduced AR mRNA expression in their GFs. A significant inverse correlation was shown between AR mRNA abundance and methylation at two consecutive CpGs within the proximal AR promoter. Methylation of a 158-bp-long region containing these CpGs was sufficient to severely reduce reporter gene expression. This region was bound by the runt related transcription factor 1 (RUNX1). Ectopic expression of RUNX1 in HEK293T cells was able to inhibit reporter gene expression through this region.nnnConclusionsnAberrant CpGs methylation within the proximal AR promoter plays an important role in the control of AR gene expression and may result in AIS type II. We suggest that transcriptional modifiers, such as RUNX1, could play roles therein offering new perspectives for understanding androgen-mediated endocrine diseases.