Reference intervals for serum 11-oxygenated androgens in children.

OBJECTIVE: Classic androgens such as dehydroepiandrosterone, androstenedione, and testosterone are generally measured for diagnosis and treatment monitoring in children and adolescents with hyperandrogenism, as can occur in congenital adrenal hyperplasia, premature pubarche, or polycystic ovarian syndrome. However, adrenally-derived 11-oxygenated androgens also contribute to the androgen pool and should therefore be considered in clinical management. Nevertheless, paediatric reference intervals are lacking. Therefore, we developed a serum assay to establish reference intervals for four 11-oxygenated androgens in addition to four classic androgens. DESIGN: Reference interval study for serum 11-oxygenated androgens in children. METHODS: We developed and validated a sensitive LC-MS/MS assay and quantified eight serum androgens, including four 11-oxygenated androgens, in serum of 256 healthy children (aged 0-17 years old). An age-dependency for all androgens was observed, and therefore we divided the cohort based on age (prepubertal (n=133; 94 boys, 39 girls) and pubertal (n=123; 52 boys, 71 girls)) to compute reference intervals (2.5th - 97.5th percentiles). RESULTS: In the prepubertal group, there was no significant sex-difference for any of the measured androgens. In the pubertal group, androstenedione, testosterone, and dihydrotestosterone showed a significant difference between boys and girls. In contrast, adrenal androgens dehydroepiandrosterone, 11-hydroxyandrostenedione, 11-ketoandrostenedione, 11-hydroxytestosterone, and 11-ketotestosterone did not. CONCLUSIONS: We developed and validated an assay for 11-oxygenated androgens, in addition to four classic androgens and established reference intervals. This enables a comprehensive evaluation of serum androgen status in children with clinical symptoms of hyperandrogenism.

Evaluation of Ex Vivo Adrenocorticotropic Hormone Responsiveness of Human Fetal Testis.

Testicular adrenal rest tumors (TARTs), commonly occurring in males with congenital adrenal hyperplasia, may arise from chronic stimulation of adrenocorticotropic hormone (ACTH)-sensitive cells in the testes. It is not yet established whether the human fetal testis (HFT) is responsive to ACTH. To investigate this, we cultured HFT tissue with and without ACTH for up to 5 days, and quantified adrenal steroid hormones and expression of adrenal steroidogenic enzymes. Fetal testis and adrenal tissue produced high levels of testosterone and cortisol, respectively, indicating viability. In contrast to fetal adrenal tissues, the expression of ACTH receptor MC2R was either absent or expressed at extremely low levels in ex vivo HFT tissue and no clear response to ACTH in gene expression or steroid hormone production was observed. Altogether, this study suggests that the HFT is unresponsive to ACTH, which would indicate that a TART does not arise from fetal testicular cells chronically exposed to ACTH in utero.

Hormonal control during infancy and testicular adrenal rest tumor development in males with congenital adrenal hyperplasia: a retrospective multicenter cohort study.

IMPORTANCE: Testicular adrenal rest tumors (TARTs), often found in male patients with congenital adrenal hyperplasia (CAH), are benign lesions causing testicular damage and infertility. We hypothesize that chronically elevated adrenocorticotropic hormone exposure during early life may promote TART development. OBJECTIVE: This study aimed to examine the association between commencing adequate glucocorticoid treatment early after birth and TART development. DESIGN AND PARTICIPANTS: This retrospective multicenter (n = 22) open cohort study collected longitudinal clinical and biochemical data of the first 4 years of life using the I-CAH registry and included 188 male patients (median age 13 years; interquartile range: 10-17) with 21-hydroxylase deficiency (n = 181) or 11-hydroxylase deficiency (n = 7). All patients underwent at least 1 testicular ultrasound. RESULTS: TART was detected in 72 (38%) of the patients. Prevalence varied between centers. When adjusted for CAH phenotype, a delayed CAH diagnosis of >1 year, compared with a diagnosis within 1 month of life, was associated with a 2.6 times higher risk of TART diagnosis. TART onset was not predicted by biochemical disease control or bone age advancement in the first 4 years of life, but increased height standard deviation scores at the end of the 4-year study period were associated with a 27% higher risk of TART diagnosis. CONCLUSIONS AND RELEVANCE: A delayed CAH diagnosis of >1 year vs CAH diagnosis within 1 month after birth was associated with a higher risk of TART development, which may be attributed to poor disease control in early life.

Challenges in treatment of patients with non-classic congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) due to 21α-hydroxylase deficiency (21OHD) or 11ß-hydroxylase deficiency (11OHD) are congenital conditions with affected adrenal steroidogenesis. Patients with classic 21OHD and 11OHD have a (nearly) complete enzyme deficiency resulting in impaired cortisol synthesis. Elevated precursor steroids are shunted into the unaffected adrenal androgen synthesis pathway leading to elevated adrenal androgen concentrations in these patients. Classic patients are treated with glucocorticoid substitution to compensate for the low cortisol levels and to decrease elevated adrenal androgens levels via negative feedback on the pituitary gland. On the contrary, non-classic CAH (NCCAH) patients have more residual enzymatic activity and do generally not suffer from clinically relevant glucocorticoid deficiency. However, these patients may develop symptoms due to elevated adrenal androgen levels, which are most often less elevated compared to classic patients. Although glucocorticoid treatment can lower adrenal androgen production, the supraphysiological dosages also may have a negative impact on the cardiovascular system and bone health. Therefore, the benefit of glucocorticoid treatment is questionable. An individualized treatment plan is desirable as patients can present with various symptoms or may be asymptomatic. In this review, we discuss the advantages and disadvantages of different treatment options used in patients with NCCAH due to 21OHD and 11OHD.

Transcriptional comparison of testicular adrenal rest tumors with fetal and adult tissues.

Background: Testicular adrenal rest tumors (TART) are a common complication of unknown cellular origin in patients with congenital adrenal hyperplasia (CAH). These benign tumors have both adrenal and testicular characteristics and are hypothesized to either derive from cells of adrenal origin from the fetal adrenogonadal primordium or by atypical differentiation of adult Leydig-progenitor cells. Objective: This study aims to unravel the identity and etiology of TART. Methods: Co-expression of adrenal-specific CYP11B1 and Leydig cell-specific HSD17B3 in TART was studied using immunohistochemistry. We studied the possibility of TART being derived from atypical differentiation of adult Leydig-progenitor cells by the quantification of adrenal-specific enzyme expression upon adrenocorticotrophic hormone (ACTH)-like stimulation of ex vivo cultured platelet-derived growth factor receptor alpha-positive cells. By comparing the transcriptome of TART (n = 16) with the transcriptome of fetal adrenal (n = 13), fetal testis (n = 5), adult adrenal (n = 11), and adult testis (n = 10) tissues, we explored the identity of TART. Results: We demonstrate co-expression of adrenal-specific CYP11B1 and testis-specific HSD17B3 in TART cells, indicating the existence of a distinct TART cell exhibiting both adrenal and testicular characteristics. Ex vivo cultured adult Leydig-progenitor cells did not express the ACTH-receptor MC2R but did express CYP11B1 upon stimulation. Unsupervised clustering of transcriptome data showed that TART was most similar to adult adrenal tissue, followed by adult testis tissue, and least similar to either fetal tissue. Conclusion: Our data suggest that TART is induced - most likely via activation of a cAMP/protein kinase A-dependent receptor - from a progenitor cell into a unique mature adrenal-like cell type, sometimes exhibiting both adrenal and testicular features.

Diurnal salivary androstenedione and 17-hydroxyprogesterone levels in healthy volunteers for monitoring treatment efficacy of patients with congenital adrenal hyperplasia.

OBJECTIVE: Treatment of congenital adrenal hyperplasia (CAH) patients with glucocorticoids is often challenging since there is a delicate balance between over- and undertreatment. Treatment can be monitored noninvasively by measuring salivary androstenedione (A4) and 17-hydroxyprogesterone (17-OHP). Optimal treatment monitoring requires the establishment of reference values in saliva. DESIGN: A descriptive study. PATIENTS: For this study saliva of 255 healthy paediatric and adult volunteers with an age range of 4-75 years old was used. MEASUREMENTS: We developed a sensitive liquid chromatography-tandem mass spectrometry method, assessed salivary A4 and 17-OHP stability, and measured A4 and 17-OHP concentrations in saliva collected in the morning, afternoon, and evening. RESULTS: We quantified A4 and 17-OHP concentrations in the morning, afternoon, and evening and demonstrated that there is a significant rhythm with the highest levels in the morning and decreasing levels over the day. A4 and 17-OHP concentrations display an age-dependent pattern. These steroids remain stable in saliva at ambient temperature for up to 5 days. CONCLUSIONS: Good stability of the steroids in saliva enables saliva collection by the patient at home. Since salivary A4 and 17-OHP display a diurnal rhythm and age-dependent pattern, we established reference values for both children and adults at three time points during the day. These reference values support treatment monitoring of children and adults with CAH.

Novel treatments for congenital adrenal hyperplasia.

Patients with classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHD) need life-long medical treatment to replace the lacking glucocorticoids and potentially lacking mineralocorticoids and to lower elevated adrenal androgens. Long-term complications are common, including gonadal dysfunction, infertility, and cardiovascular and metabolic co-morbidity with reduced quality of life. These complications can be attributed to the exposure of supraphysiological dosages of glucocorticoids and the longstanding exposure to elevated adrenal androgens. Development of novel therapies is necessary to address the chronic glucocorticoid overexposure, lack of circadian rhythm in glucocorticoid replacement, and inefficient glucocorticoid delivery with concomitant periods of hyperandrogenism. In this review we aim to give an overview about the current treatment regimens and its limitations and describe novel therapies especially evaluated for 21OHD patients.

Production of 11-Oxygenated Androgens by Testicular Adrenal Rest Tumors.

CONTEXT: Testicular adrenal rest tumors (TART) are a common complication in males with classic 21-hydroxylase deficiency (21OHD). TART are likely to contribute to the androgen excess in 21OHD patients, but a direct quantification of steroidogenesis from these tumors has not been yet done. OBJECTIVE: We aimed to define the production of 11-oxygenated 19-carbon (11oxC19) steroids by TART. METHODS: Using liquid chromatography-tandem mass spectrometry, steroids were measured in left (n = 7) and right (n = 4) spermatic vein and simultaneously drawn peripheral blood (n = 7) samples from 7 men with 21OHD and TART. For comparison, we also measured the peripheral steroid concentrations in 5 adrenalectomized patients and 12 age- and BMI-matched controls. Additionally, steroids were quantified in TART cell- and adrenal cell-conditioned medium, with and without adrenocorticotropic hormone (ACTH) stimulation. RESULTS: Compared with peripheral blood from 21OHD patients with TART, the spermatic vein samples displayed the highest gradient for 11ß-hydroxytestosterone (11OHT; 96-fold) of the 11oxC19 steroids, followed by 11-ketotestosterone (47-fold) and 11ß-hydroxyandrostenedione (11OHA4; 29-fold), suggesting production of these steroids in TART. TART cells produced higher levels of testosterone and lower levels of A4 and 11OHA4 after ACTH stimulation compared with adrenal cells, indicating ACTH-induced production of testosterone in TART. CONCLUSION: In patients with 21OHD, TART produce 11oxC19 steroids, but in different proportions than the adrenals. The very high ratio of 11OHT in spermatic vs peripheral vein blood suggests the 11-hydroxylation of testosterone by TART, and the in vitro results indicate that this metabolism is ACTH-sensitive.

Optimizing the Timing of Highest Hydrocortisone Dose in Children and Adolescents With 21-Hydroxylase Deficiency.

CONTEXT: Hydrocortisone treatment of young patients with 21-hydroxylase deficiency (21OHD) is given thrice daily, but there is debate about the optimal timing of the highest hydrocortisone dose, either mimicking the physiological diurnal rhythm (morning), or optimally suppressing androgen activity (evening). OBJECTIVE: We aimed to compare 2 standard hydrocortisone timing strategies, either highest dosage in the morning or evening, with respect to hormonal status throughout the day, nocturnal blood pressure (BP), and sleep and activity scores. METHODS: This 6-week crossover study included 39 patients (aged 4-19 years) with 21OHD. Patients were treated for 3 weeks with the highest hydrocortisone dose in the morning, followed by 3 weeks with the highest dose in the evening (n = 21), or vice versa (n = 18). Androstenedione (A4) and 17-hydroxyprogesterone (17OHP) levels were quantified in saliva collected at 5 am; 7 am; 3 pm; and 11 pm during the last 2 days of each treatment period. The main outcome measure was comparison of saliva 17OHP and A4 levels between the 2 treatment strategies. RESULTS: Administration of the highest dose in the evening resulted in significantly lower 17OHP levels at 5 am, whereas the highest dose in the morning resulted in significantly lower 17OHP and A4 levels in the afternoon. The 2 treatment dose regimens were comparable with respect to averaged daily hormone levels, nocturnal BP, and activity and sleep scores. CONCLUSION: No clear benefit for either treatment schedule was established. Given the variation in individual responses, we recommend individually optimizing dose distribution and monitoring disease control at multiple time points.