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The aim of this study was to investigate the impact of sex and puberty stage on circadian changes in sodium excretion, sodium-regulating hormones, and hemodynamics. Thirty-nine healthy volunteers (9 prepuberty boys, 10 prepuberty girls, 10 puberty boys, and 10 puberty girls) were included. They all underwent a 24-h circadian in-patient study under standardized conditions regarding activity, diet, and fluid intake. Blood samples were drawn every 4 h, and the urine was collected in fractions. Blood pressure and heart rate were noninvasively monitored. Atrial natriuretic peptide (ANP), angiotensin II, aldosterone, and renin were measured in blood. Children in puberty had lower plasma levels of renin (P < 0.05) and angiotensin II (P < 0.05) and a 26% reduction in filtered sodium without changes in sodium excretion compared with prepuberty children. A circadian rhythm in sodium excretion, the renin-angiotensin system, ANP, and blood pressure was found with a midnight ANP peak (P < 0.001), a nighttime decrease in hemodynamic parameters (P < 0.001), an increase in plasma renin (P < 0.001) and angiotensin II (P < 0.001), and a decrease in sodium excretion (P < 0.001) mainly on the basis of increased sodium reabsorption (P < 0.001). The timing of the changes did not depend on sex or puberty group. There is a circadian rhythm of sodium excretion and sodium regulation in 7- to 15-yr-old children. This rhythm is similar in boys and girls. As an important new finding, puberty changes the plasma levels of renin and angiotensin II without changing the amount of sodium excreted or the day to night sodium excretion ratio. [ABSTRACT FROM AUTHOR]

We investigated the influence of sex and puberty stage on circadian urine production and levels of antidiuretic hormone [arginine vasopressin (AVP)] in healthy children. Thirty-nine volunteers (9 prepuberty boys, 10 prepuberty girls, 10 midpuberty boys, and 10 midpuberty girls) were included. All participants underwent a 24-h circadian inpatient study under standardized conditions regarding Na+ and fluid intake. Blood samples were drawn every 4 h for measurements of plasma AVP, serum 17-β-estradiol, and testosterone, and urine was fractionally collected for measurements of electrolytes, aquaporin (AQP)2, and PGE2.We found a marked nighttime decrease in diuresis (from 1.69 ± 0.08 to 0.86 ± 0.06 ml·kg-1·h-1, P < 0.001) caused by a significant nighttime increase in solute-free water reabsorption (TcH2O; day-to-night ratio: 0.64 ± 0.07, P < 0.001) concurrent with a significant decrease in osmotic excretion (day-to-night ratio: 1.23 ± 0.06, P < 0.001). Plasma AVP expressed a circadian rhythm (P < 0.01) with a nighttime increase and peak levels at midnight (0.49 ± 0.05 pg/ml). The circadian plasma AVP rhythm was not influenced by sex (P = 0.56) or puberty stage (P = 0.73). There was significantly higher nighttime TcH2O in prepuberty children. This concurred with increased nighttime urinary AQP2 excretion in prepuberty children. Urinary PGE2 exhibited a circadian rhythm independent of sex or puberty stage. Levels of serum 17β-estradiol and testosterone were as expected for sex and puberty stage, and no effect on the AVP-AQP2- TcH2O axis was observed. This study found a circadian rhythm of plasma AVP independent of sex and puberty stage, although nighttime TcH2O was higher and AQP2 excretion was more pronounced in prepuberty children, suggesting higher prepuberty renal AVP sensitivity. [ABSTRACT FROM AUTHOR]

Abstract Background The role of estrogens in male physiology has become evident. However, clinically useful normative data for estradiol secretion in boys has not previously been established due to the insensitivity of current methods used in clinical routine. By use of a validated ultra-sensitive extraction RIA, our aim was to establish normative data from a group consisting of healthy boys in prepuberty and during pubertal development. Methods Sixty-two 24-hours serum profiles (6 samples/24 hours) were obtained from 44 healthy boys (ages; 7.2–18.6 years) during their pubertal development, classified into five stages: prepuberty (testis, 1–2 mL), early (testis, 3–6 mL), mid (testis, 8–12 mL), late-1 (testis,15–25 mL, not reached final height) and late-2 (testis,15–25 mL, reached final height). Serum estradiol was determined by an ultra- sensitive extraction radioimmunoassay with detection limit 4 pmol/L and functional sensitivity 6 pmol/L. Results Mean estradiol concentrations during 24-hours secretion increased from prepuberty (median: Conclusion With the use of an ultra-sensitive extraction RIA, we have provided clinically useful normative data for estradiol secretion in boys.

Aim: We aimed to evaluate aspects of pubertal development to identify the most reliable clinical sign of pubertal onset in males., Methods: We performed a mini review of the literature., Results: In 1951 Reynolds and Wines categorised pubic hair growth and genital development in five stages by visual inspection. Today the Tanner scale is used to assess the five stages of pubertal development, The second genital stage, characterised by enlargement of the scrotum defines pubertal onset in males. Testicular volume may be evaluated by using a calliper or by ultrasound scan. The Prader orchidometer, described in 1966, offers a method for evaluating testicular growth by palpation. Pubertal onset is commonly defined as testicular volume >3 or ≥4 mL. The development of sensitive laboratory methods has enabled studies analysing hormonal activity in the hypothalamus-pituitary-gonadal axis. We review the relationships between physical and hormonal signs of puberty. We also discuss the results of studies assessing different aspects of pubertal development with a focus on identifying the most reliable clinical sign of pubertal onset in males., Conclusion: A substantial amount of evidence supports testicular volume of 3 mL as the most reliable clinical sign of male pubertal onset., (© 2023 The Authors. Acta Paediatrica published by John Wiley & Sons Ltd on behalf of Foundation Acta Paediatrica.)

Objectives: In laboratory medicine, external quality assessment (EQA) schemes have become versatile tools for detecting analytical flaws. However, EQA schemes are lacking for pediatric sex steroid levels. We aimed to investigate the suitability of different estradiol and testosterone immunoassays in a pediatric setting in comparison with clinical liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays., Methods: The study was conducted by staff and the advisory group on endocrinology at Equalis, the Swedish provider of EQA schemes for laboratory medicine. The test material consisted of five pooled serum samples from children who were either prepubertal or in puberty. Clinical laboratories enrolled in Equalis EQA schemes for estradiol and testosterone were invited to participate, as were clinical laboratories using LC-MS/MS-assays. Samples were analyzed by either routine immunoassays (n=18) or in-house LC-MS/MS assays (n=3)., Results: For estradiol, LC-MS/MS assays showed a high degree of conformity with interlaboratory coefficients of variation (CV) below 24.2 %. Reported levels were between 4.9 ± 1.2 and 33.9 ± 1.6 pmol/L (group mean ± standard deviation). The direct immunoassays had lower precision; their CVs were up to 81.4 %. Reported concentrations were between 25.3 ± 18.1 and 45.7 ± 19.4 pmol/L, an overestimation compared to LC-MS/MS. Testosterone LC-MS/MS also showed a high degree of conformity, CVs were below 13.4 %, and reported concentrations were from 0.06 ± 0.00 to 1.00 ± 0.11 nmol/L. The direct immunoassays had a larger discrepancy between results; CVs were up to 95.8 %. Concentrations were between 0.12 ± 0.11 and 0.85 ± 0.23 nmol/L., Conclusions: For the safe diagnosis and determination of sex steroids in children, analysis with mass spectrometry-based methods is recommended., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Objective: To study the impact of GH dose and age at GH start in girls with Turner syndrome (TS), aiming for normal height and age at pubertal onset (PO) and at adult height (AH). However, age at diagnosis will limit treatment possibilities., Methods: National multicenter investigator-initiated studies (TNR 87-052-01 and TNR 88-072) in girls with TS, age 3-16 years at GH start during year 1987-1998, with AH in 2003-2011. Of the 144 prepubertal girls with TS, 132 girls were followed to AH (intention to treat), while 43 girls reduced dose or stopped treatment prematurely, making n=89 for Per Protocol population. Age at GH start was 3-9 years (young; n=79) or 9-16 years (old; n=53). Treatment given were recombinant human (rh)GH (Genotropin ® Kabi Peptide Hormones, Sweden) 33 or 67 µg/kg/day, oral ethinyl-estradiol (2/3) or transdermal 17β-estradiol (1/3), and, after age 11 years, mostly oxandrolone. Gain in height SDS , AH SDS , and age at PO and at AH were evaluated., Results: At GH start, height SDS was -2.8 (versus non-TS girls) for all subgroups and mean age for young was 5.7 years and that of old was 11.6 years. There was a clear dose-response in both young and old TS girls; the mean difference was (95%CI) 0.66 (-0.91 to -0.26) and 0.57 (-1.0 to -0.13), respectively. The prepubertal gain SDS (1.3-2.1) was partly lost during puberty (-0.4 to -2.1). Age/height SDS at PO ranged from 13 years/-0.42 for GH 67young to 15.2 years/-1.47 for GH 33old . At AH, GH 67old group became tallest (17.2 years; 159.9 cm; -1.27 SDS; total gain SDS , 1.55) compared to GH 67young group being least delayed (16.1 years; 157.1 cm; -1.73 SDS; total, 1.08). The shortest was the GH 33young group (17.3 years; 153.7 cm: -2.28 SDS; total gain SDS , 0.53), and the most delayed was the GH 33old group, (18.5 years; 156.5 cm; -1.82 SDS; total gain SDS , 0.98)., Conclusion: For both young and old TS girls, there was a GH-dose growth response, and for the young, there was less delayed age at PO and at AH. All four groups reached an AH within normal range, despite partly losing the prepubertal gain during puberty. Depending on age at diagnosis, low age at start with higher GH dose resulted in greater prepubertal height gain, permitting estrogen to start earlier at normal age and attaining normal AH at normal age, favoring physiological treatment and possibly also bone health, hearing, uterine growth and fertility, psychosocial wellbeing during adolescence, and the transition to adulthood., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kriström, Ankarberg-Lindgren, Barrenäs, Nilsson and Albertsson-Wikland.)

Objective: The relationship between sex hormone concentrations during childhood and birth weight (BW) is poorly understood. We aimed to investigate this relationship and the associations with anthropometric data at 5, 6, 7, 8, and 10 years of age in preterm boys., Design: A prospective longitudinal single-centre study, including 58 boys with a BW of 1325-3320 g and gestational age (GA) of 32 + 2 to 36 + 6 weeks., Patients and Measurements: Data on GA, BW and anthropometric data between 5 and 10 years of age were recorded. Testicular development was assessed at 8 and 10 years of age. Serum concentrations of sex steroids were analysed with gas chromatography-tandem mass spectrometry at 5-10 years and luteinizing hormone (LH) and follicle-stimulating hormone (FSH) with immunoassays at 10 years of age., Results: At 8 years of age, testosterone and estrone correlated negatively with BW, (ρ = -0.35, p = .021) and (ρ = -0.34, p = .024), respectively. At 10 years of age, testosterone, dihydrotestosterone, estrone and estradiol correlated negatively with BW (ρ = -0.39, p = .010), (ρ = -0.38, p = .013), (ρ = -0.44, p = .003) and (ρ = -0.36, p = .019), respectively. Weight gain from birth correlated with testosterone at 5 years (ρ = 0.40, p = .002), 7 years (ρ = 0.30, p = .040), 8 years (ρ = 0.44, p = .003) and 10 years (ρ = 0.40, p = .008) of age. At 10 years of age, testosterone correlated with LH (ρ = 0.42, p = .006) and FSH (ρ = 0.33, p = .033) but not with testicular volume., Conclusions: Lower BW was associated with increased sex steroid concentrations from 8 years of age, independently of clinical signs of puberty., (© 2022 The Authors. Clinical Endocrinology published by John Wiley & Sons Ltd.)

Context: There is a lack of knowledge on longitudinal sex steroid patterns during infancy, especially for boys born preterm or with low birth weight (LBW)., Objective: To find out whether LBW boys have a disturbed sex steroid profile during infancy., Design and Setting: Population-based longitudinal study performed at Sahlgrenska University Hospital, Gothenburg, Sweden., Participants: Ninety-eight singleton boys (47 LBW) born at gestational age 32.0 to 36.9 weeks were included. Because of dropout, 83 of the boys were still in the study at 10 months' corrected age., Main Outcome Measures: Serum androgen and estrogen concentrations were analyzed by gas chromatography-tandem mass spectrometry and IGF-I was determined with radioimmunoassay in umbilical cord and at 0, 2, 5, and 10 months' corrected age., Results: Serum levels of androstenedione, estrone, and estradiol declined gradually from birth to 10 months corrected age. In both LBW boys and their counterparts, a surge was seen at 2 months' corrected age (3 months' chronological age) for testosterone, median (range) 6.5 (2.0-18.9) nmol/L, and in dihydrotestosterone 1.2 (0.4-4.3) nmol/L. At birth, LBW boys had higher median testosterone (0.7 vs 0.4 nmol/L, P = 0.019), and at 0 months' corrected age, both had higher testosterone (5.7 vs 3.5 nmol/L, P = 0.003) and dihydrotestosterone (1.2 vs 0.9 nmol/L, P = 0.006) than their counterparts. At 10 months' corrected age, catch-up in weight SD score from birth correlated with testosterone (rho = 0.27, P = 0.044) and androstenedione (rho = 0.29, P = 0.027)., Conclusions: Moderately to late preterm LBW boys showed a disturbed sex hormone profile, with elevated concentrations of androgens in early infancy., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aim: We investigated longitudinal adrenal androgen concentrations and any relationship between gestational age, birth size, anthropometric parameters and adrenal androgen concentrations during childhood in boys born moderate to late preterm., Methods: This longitudinal, prospective study included 58 boys born at 32+0 to 36+6 weeks of gestation. Dehydroepiandrosterone sulphate and androstenedione were analysed by liquid chromatography-tandem mass spectrometry, and anthropometric data were recorded from 5 to 10 years of age., Results: Dehydroepiandrosterone sulphate concentrations correlated with weight standard deviations scores (SDS) from 7 to 10 years of age and waist-to-height ratios at seven and 10 years of age. Androstenedione correlated with weight SDS from 7 to 10 years of age and waist-to-height ratios at 10 years of age. Longitudinal analysis showed a relationship between weight SDS and waist-to-height SDS and dehydroepiandrosterone sulphate (p < 0.001 and p < 0.001, respectively) and androstenedione (p = 0.002 and p = 0.003, respectively), independently of age., Conclusion: The trajectories of anthropometric parameters and adrenal androgen secretion were consistent from 5 to 10 years of age in this cohort. The body composition reflected by current weight and the waist-to-height ratio, rather than gestational age and birth size, was associated with adrenal androgen secretion., (© 2021 The Authors. Acta Paediatrica published by John Wiley & Sons Ltd on behalf of Foundation Acta Paediatrica.)

Quantitation of endogenous steroids and their precursors is essential for diagnosis of a wide range of endocrine disorders. Usually, these analyses have been carried out using immunoassays. However, immunoassays often overestimate concentrations due to assay interference by other endogenous steroids, especially for low concentrations. Mass spectrometry based methods offer superior specificity, accuracy, and sensitivity. We therefore present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with automated sample preparation for determination of 17α-hydroxyprogesterone (17OHP), cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), androstenedione (A 4 ), testosterone (T), and estrone sulfate (E 1 S). Samples were prepared using protein precipitation and 96-well filter plates, fully automated in a pipetting robot and analyzed by LC-MS/MS. Serum samples from 187 healthy children and adolescents aged 5-18 years were used to study hormone changes in relation to sex and pubertal stage. Lower limit of quantification for 17OHP was 0.7 nmol/L, for cortisol 11 nmol/L, for cortisone 2 nmol/L, for DHEAS 0.1 µmol/L, and for A 4 , T, and E 1 S, 0.2 nmol/L. This study showed a general increase in 17OHP, DHEAS, A 4 , T and E 1 S in both genders during puberty. In boys, A 4 and T increased significantly throughout pubertal development. Girls had significantly higher A 4 and E 1 S concentrations, while boys had higher T concentrations. No sex- or puberty-specific differences were seen in cortisol or cortisone concentrations. To the best of our knowledge, this is the first presentation of changes in serum E 1 S concentrations during pubertal development in healthy children.

Background: Boys born small for gestational age (SGA) are at increased risk of testicular dysgenesis syndrome, and girls born SGA face the risk of polycystic ovary syndrome later in life. Our aim was to study whether neonates born SGA have an altered profile of steroid hormones at birth. Materials and methods: A total of 168 singletons (99 boys, 69 girls) born at 32.0-36.9 gestational weeks were recruited to a population-based, university hospital, single-center study. Of these, 31 infants (17 boys, 14 girls) were born SGA. The concentrations of dehydroepiandrosterone sulfate (DHEAS), androstenedione, testosterone, dihydrotestosterone, estrone, estradiol, cortisone, and cortisol were analyzed in umbilical cord serum with mass spectrometry. Results: Girls born SGA had higher levels of androstenedione than girls born appropriate for gestational age (AGA) (4.0 versus 2.6 nmol/L, p  = .002). Boys born SGA had lower levels of estrone than boys born AGA (33 822 versus 62 471 pmol/L, p  = .038). Infants born SGA had lower levels of cortisone than infants born AGA, both in girls (340 versus 579 nmol/L, p  = .010) and in boys (308 versus 521 nmol/L, p  = .045). Furthermore, boys born SGA had a higher cortisol/cortisone ratio than boys born AGA (0.41 versus 0.25, p  = .028). Gestational age correlated with DHEAS (boys r  = 0.48, p  = .000, girls r  = 0.35, p  = .013), and cortisol (boys r  = 0.48, p  = .000, girls r  = 0.29, p  = .039). Conclusions: In moderate-to-late preterm infants born SGA, we observed a different steroid hormone profile in cord serum. Girls born SGA show increased levels of androstenedione and boys born SGA show decreased levels of estrone in cord serum, which could be related to placental aromatase deficiency in intrauterine growth restriction.

Background: We have previously shown that adult height (AH) in males with Silver-Russell syndrome (SRS) correlated negatively with prepubertal estradiol concentrations. We aimed to identify the source of estradiol by analyzing androgen secretion profiles and measuring anti-Müllerian hormone (AMH) and inhibin B concentrations during childhood and puberty in this group of patients. Methods: In a retrospective longitudinal single-center study, 13 males with SRS were classified as non-responders (NRs = 8) or responders (Rs = 5), depending on the AH outcome. From 6 years of age, androgens were determined by mass spectrometry, and AMH, inhibin B and sex hormone-binding globulin concentrations were analyzed by immunoassays. Results: AH outcome correlated negatively with dehydroepiandrosterone-sulfate (DHEAS) at 8 ( r = -0.72), 10 ( r = -0.79), and 12 years ( r = -0.72); testosterone at 10 ( r = -0.94), 12 ( r = -0.70) and 14 years ( r = -0.64); dihydrotestosterone (DHT) at 10 ( r = -0.62) and 12 years; ( r = -0.57) and AMH at 12 years ( r = 0.62) of age. Compared with Rs, NRs had higher median concentrations of DHEAS (μmol/L) at 10 years (2.9 vs. 1.0); androstenedione (nmol/L) at 10 (1.1 vs. 0.6) and 12 years (1.7 vs. 0.8); testosterone (nmol/L) at 10 (0.3 vs. 0.1), 12 (7.8 vs. 0.2) and 14 years (15.6 vs. 10.4); and DHT (pmol/L) at 10 (122 vs. 28) and 12 years (652 vs. 59) of age. AMH (ng/mL) was lower in NRs than in Rs at 12 years of age (11 vs. 50). No significant differences were observed in the inhibin B concentrations at any age. Conclusions: The elevated androgen concentrations before and during puberty, originated from both adrenal and gonadal secretion and correlated negatively with AH outcomes in males with SRS., (Copyright © 2019 Kvernebo Sunnergren, Ankarberg-Lindgren and Dahlgren.)

Objective: Transdermal estradiol patches are primarily designed for adult women. No low-dose patches are licensed for pubertal induction in hypogonadal girls. Low doses can be achieved by cutting a matrix patch into smaller pieces. However, the manufacturers do not guarantee stability or utility of cut estradiol patches. The aim of the study was to assess 1-month stability of cut estradiol patches from four different manufacturers in the laboratory at room temperature (+21°C) and at an elevated temperature (+35°C)., Design and Methods: Estraderm MX 50 µg, Systen 50 µg and Oesclim 25 µg matrix patches were cut into eight pieces while Estradot 50 µg small patches were cut in half. The cut patches were stored in their respective pouches at +21°C or at +35°C for up to 1 month. The estradiol drug was extracted from the patch by ethyl acetate n-hexane and determined by radioimmunoassay., Results: Storage at +21°C or +35°C up to 1 month did not reduce the estradiol concentration in Estraderm MX, Systen and Oesclim patches. However, although the estradiol in Estradot patches was not affected by storage at +21°C, at +35°C, estradiol decreased by 57% (±1%) in cut pieces., Conclusions: Unused Estraderm MX, Systen and Oesclim patch pieces may be stored for at least 1 month at ≤+35°C. Where estradiol patches for children are not available, cut pieces of these or similar patches can be used for pubertal induction. The Estradot patch was too small to properly cut into low doses and not stable in elevated temperatures.

Background: Intrauterine growth retardation and short stature are common features in Silver-Russell syndrome (SRS). Despite recombinant growth hormone (rGH) treatment, poor pubertal height gain, affecting adult height (AH), is common. This study investigated whether growth patterns and estrogen concentrations are associated with AH outcome in rGH treated SRS males. Methods: In this retrospective longitudinal single-center study, 11 males with SRS were classified as non-responders ( NR = 6) or responders ( R = 5), depending on AH adjusted for midparental height. Epigenetic analysis and longitudinal growth measures, including bone age, rGH related parameters, pubertal development, gonadotropins and estrogen concentrations, were analyzed until AH. Results: Pubarche before 9 years was only observed in one NR. At 10 years of age, there was no difference in gonadotropins between NR and R. However, estradiol (E2) concentrations at 10 years of age showed a strong association to AH adjusted for MPH ( r = -0.78, p < 0.001). Serum E2 (pmol/L) was significantly higher in NR at ages 10 years [median (range) 2 (<2-5) vs. <2 (<2)], 12 years [23 (10-57) vs. 2 (<2-2)] and 14 years [77 (54-87) vs. 24 (<2-38)] but not at 16 years. Birth weight standard deviation score (SDS) was lower in NR [-4.1 (-4.7 to -2.1) vs. -2.7 (-3.3 to -1.7)]. Weight gain (SDS) until pubertal onset was greater in NR [2.4 (1.4-3.5) vs. 0.8 (-0.4 to 1.7)] and pubertal height gain (SDS) was lower in NR [-1.0 (-2.7-0.4) vs. 0.1 (-0.1 to 1.1)]. At AH, a number of NR and R had high E2 concentrations and small testes. Conclusion: Increased E2 concentrations at age 10, 12, and 14 years were associated to less pubertal height gain, thus affecting AH. Due to the small number of patients, the results need to be confirmed in larger cohorts. The finding of impaired testicular development stresses the need of hormonal evaluation as a complement to clinical and radiological assessment when predicting AH in males with SRS.

Background: Androgen and estrogen determinations serve as important diagnostic markers in a variety of clinical conditions. However, one challenge is to enhance assay sensitivity for determination in the lowest range, such as in prepubertal children. We here present a recently developed gas chromatography-tandem mass spectrometry (GC-MS/MS) method for determination of androstenedione (A 4 ), dihydrotestosterone (DHT), testosterone (T), estrone (E 1 ), and estradiol (E 2 ) in children, which we have compared with the sensitive radioimmunoassays; E 2 extraction-RIA and T-RIA., Methods: Steroids were extracted in ethyl acetate n-hexane solution from serum spiked with isotopically labeled internal standard and derivatized sequentially with pentafluorobenzyl bromide, pentafluorobenzyl hydroxylamine and pentafluoropropionic acid anhydride and analyzed by GC-MS/MS using a triple quadrupole mass spectrometer operated in negative chemical ionization mode. Leftover routine samples (n = 414) were used to evaluate the concordance between GC-MS/MS and RIAs and the validity of GC-MS/MS for pediatrics; of these samples, 101 were from seemingly healthy children. Pubertal stage was recorded for reference interval evaluation., Results: Lower limit of detection for A 4 , T, DHT, E 1 , and E 2 were 0.1 nmol/L, 0.1 nmol/L, 27 pmol/L, 9 pmol/L, and 2 pmol/L, respectively. Good agreement was found between GC-MS/MS and T-RIA (r = 0.98) as well as between GC-MS/MS and E 2 extraction-RIA (r = 0.98, for E 2 concentrations above 14 pmol/L). In boys, T and DHT increased significantly from prepuberty throughout pubertal development, and in girls the same increase was observed for E 1 and E 2 . The greatest increase in A 4 for both genders, as well as E 1 and E 2 in boys and T and DHT in girls, occurred in mid to late puberty., Conclusions: We report the development of a GC-MS/MS method sensitive enough to accurately determine serum levels of androgens and estrogens in children., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The overall goal of pubertal sex hormone replacement therapy (HRT) in girls is not only about development of secondary sexual characteristics, but also to establish an adult endocrine and metabolic milieu, as well as adult cognitive function. Estradiol (E2) is the first choice for HRT compared to ethinyl estradiol (EE2). E2 is the most potent endogenous estrogen in the circulation, with established levels during spontaneous puberty. Transdermal E2, compared to oral administration, is the first choice to start pubertal HRT. Transdermal application avoids liver exposure to supraphysiologic estrogen concentrations and provides a more physiologic mechanism for hormone delivery. By cutting E2 matrix patches in doses of 0.05-0.07 µg/kg or administrate E2 gel in doses of 0.1 mg/day, serum concentrations of E2 seen in early spontaneous puberty can be obtained. Patches can be removed in the morning and thereby mimic the normal circadian rhythm. For those clinics with access to sensitive E2 determinations methods (extraction followed by radioimmunoassay or mass spectrometry) monitoring the attained E2 serum levels is recommended in order to optimally mimic the levels seen in early puberty as well as growth velocity, breast and uterus development. Mid- and late pubertal HRT is obtained by increased doses of E2, adding cyclic oral or transdermal progestin, as well as testosterone gel over the pubic area if indicated., (© 2016 S. Karger AG, Basel.)

Background: Immunoassays have been criticized for poor accuracy at low testosterone concentrations. Mass spectrometry (MS) has been proposed as the only reliable method for testosterone determination. The aim of this study was to compare a sensitive testosterone radioimmunoassay (RIA) with results from different MS., Methods: We compared testosterone concentrations determined by a sensitive testosterone RIA, lower limit of detection 0.03 nmol/L and limit of quantitation 0.1 nmol/L, with four tandem MS that were included in an international external quality assessment program for laboratory medicine. We also compared the morning concentrations of testosterone in girls and boys at different pubertal stages, using results from the RIA, with reported values determined by LC-MS/MS, developed for androgen determination in children., Results: The mean (SD), concentrations were similar between RIA and MS: 1.5 (0.3) and 1.4 (0.4) in the child/women range (0.8-2.6 nmol/L) and 16.0 (3.7) and 17.8 (4.5) nmol/L for the adult male range (10.1-30.0 nmol/L), respectively. The ratio between RIA and MS versus results from mean values of the four MS methods was 1.0 (0.18); 1.1 (0.18) for child/women concentrations and 0.9 (0.13) for male testosterone concentrations. Furthermore, compared to the pediatric reference values determined by LC-MS/MS, the sensitive testosterone RIA delivered similar testosterone values across the different pubertal stages., Conclusions: The comparison between different tandem MS methods and a sensitive testosterone RIA illustrates that there are immunoassays that deliver clinically useful information in prepubertal and pubertal children.

Background/aim: The goal of estrogen replacement therapy (ERT) in girls with hypogonadism is to achieve the endocrine milieu similar to natural puberty, where transdermal administration is the most physiological route. The aim of the study was to evaluate guidelines for the induction of puberty with transdermal estradiol (E2) patches in a large outpatient setting., Methods: In a retrospective study, serum E2 levels from 18 clinics were analyzed at the Göteborg Pediatric Growth Research Center laboratory, as part of the initiation of ERT in girls with hypogonadism. Exclusion criteria were pubertas tarda and pubertal arrest. Eighty-eight observations (50 with Turner syndrome, TS) were included. Serum E2 levels were determined by extraction + radioimmunoassay (detection limit 4 pmol/l) and analyzed in relation to the dose of Evorel(®) (25 µg/24 h, containing 1.60 mg estradiol hemihydrate; Janssen-Cilag Pharmaceutica N.V., Beerse, Belgium)., Results: There was a linear relationship between serum E2 and the weight-based dose, with r = 0.56, p < 0.0001 for all observations and r = 0.59, p < 0.0001 for the TS study group. Linear regression analysis for doses of 0.05-0.07 µg/kg resulted in serum levels of 17-23 pmol/l (TS 17-24 pmol/l) and doses of 0.08-0.12 µg/kg in 26-39 pmol/l (TS 27-39 pmol/l)., Conclusions: For the initiation of ERT with nocturnally administered E2 patches, we recommend reduced starting doses of 0.05-0.07 µg/kg, with the goal of mimicking E2 levels during gonadarche. In older girls, when breast development is of high priority, the starting dose can still be 0.08-0.12 µg/kg.

Background/aims: Growth Hormone (GH) dosage in childhood is adjusted for body size, but there is no consensus whether body weight (BW) or body surface area (BSA) should be used. We aimed at comparing the biological effect and cost-effectiveness of GH treatment dosed per m2 BSA in comparison with dosing per kg BW in girls with Turner syndrome (TS)., Methods: Serum IGF-I, GH dose, and adult height gain (AHG) from girls participating in two Dutch and five Swedish studies on the efficacy of GH were analyzed, and the cumulative GH dose and costs were calculated for both dose adjustment methods. Additional medication included estrogens (if no spontaneous puberty occurred) and oxandrolone in some studies., Results: At each GH dose, the serum IGF-I standard deviation score remained stable over time after an initial increase after the start of treatment. On a high dose (at 1 m2 equivalent to 0.056-0.067 mg/kg/day), AHG was at least equal on GH dosed per m2 BSA compared with dosing per kg BW. The cumulative dose and cost were significantly lower if the GH dose was adjusted for m2 BSA., Conclusion: Dosing GH per m2 BSA is at least as efficacious as dosing per kg BW, and is more cost-effective., (© 2014 S. Karger AG, Basel.)

Aging is associated with gradual decline of skeletal muscle strength and mass often leading to diminished muscle quality. This phenomenon is known as sarcopenia and affects about 30% of the over 60-year-old population. Androgens act as anabolic agents regulating muscle mass and improving muscle performance. The role of female sex steroids as well as the ability of skeletal muscle tissue to locally produce sex steroids has been less extensively studied. We show that despite the extensive systemic deficit of sex steroid hormones in postmenopausal compared to premenopausal women, the hormone content of skeletal muscle does not follow the same trend. In contrast to the systemic levels, muscle tissue of post- and premenopausal women had similar concentrations of dehydroepiandrosterone and androstenedione, while the concentrations of estradiol and testosterone were significantly higher in muscle of the postmenopausal women. The presence of steroidogenetic enzymes in muscle tissue indicates that the elevated postmenopausal steroid levels in skeletal muscle are because of local steroidogenesis. The circulating sex steroids were associated with better muscle quality while the muscle concentrations reflected the amount of infiltrated fat within muscle tissue. We conclude that systemically delivered and peripherally produced sex steroids have distinct roles in the regulation of neuromuscular characteristics during aging., (© 2011 The Authors. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

Objective: To characterise changes in testicular size and reproductive hormones and to investigate the aetiology of delayed puberty and impaired fertility in males with Noonan syndrome (NS)., Design: In this study, 12 males with NS were longitudinally followed from pre/early puberty until adulthood. Of the 12 males, ten had no medical history other than NS and were divided into two groups, undescended testes (UT), and descended testes (DT) and compared with a reference population., Methods: Hormone concentrations in serum were determined by immunoassays and testicular volume was measured using an orchidometer., Results: Before puberty, reproductive hormone levels were within the expected range in almost all cases. In some cases, LH, FSH and testosterone and oestradiol (E(2)) concentrations started to increase during puberty and inhibin B and anti-Müllerian hormone (AMH) declined to subnormal levels. Most of the boys studied had small testes that, in the majority of cases, progressed to normal size in adulthood. No difference in reproductive hormones was observed between the UT and DT groups either during puberty or at adulthood. However, as adults, males with NS had higher LH (5.7 vs 4.0 U/l, P<0.01), FSH (7.1 vs 2.5 U/l, P<0.001), testosterone (18.7 vs 15.6  nmol/l, P<0.01) and E(2) (66 vs 46  pmol/l, P<0.001) levels and lower AMH (33 vs 65  pmol/l, P<0.01) and inhibin B (median 108 vs 187  pg/ml, P<0.01) levels than the reference population., Conclusions: In NS males, both Sertoli and Leydig cell dysfunction is common with reproductive hormone levels deteriorating progressively to adulthood.

Although postmenopausal hormone-replacement therapy (HRT) is known to prevent fractures, knowledge on the influence of long-term HRT on bone strength and its determinants other than areal bone mineral density is scarce. This study used a genetically controlled design with 24 monozygotic female twin pairs aged 54 to 72 years in which one cotwin was using HRT (mean duration 8 years) and the other had never used HRT. Estimated bone strength, cross-sectional area, volumetric bone mineral density, bone mineral mass, and cross-sectional density and mass distributions were assessed in the tibial shaft, distal tibia, and distal radius with peripheral computed tomography (pQCT). In the tibial shaft, HRT users had 9% [95% confidence interval (CI) 3%-15%] higher estimated bending strength than their nonusing cotwins. Larger cortical area and higher cortical bone mineral density accounted for this difference. The cortex was larger in the HRT users in the endocortical region. In the distal tibia, estimated compressive strength was 24% (95% CI 9%-40%) higher and in the distal radius 26% (95% CI 11%-41%) higher in the HRT users than in their nonusing cotwins owing to higher volumetric bone mineral density. No difference between users and nonusers was observed in total bone cross-sectional area in any measured bone site. The added mineral mass in the HRT users was distributed evenly within and between bone sites. In postmenopausal women, long-term HRT preserves estimated bone strength systemically by preventing bone mineral loss similarly in body weight-loaded and non-weight-loaded bone., (Copyright © 2011 American Society for Bone and Mineral Research.)

Aim: To study the influence of growth hormone (GH) treatment on the initiation and progression of puberty in short children., Methods: This prospective, randomized, controlled study included 124 short children (33 girls) who received GH treatment (Genotropin®; Pfizer Inc.) from a mean age of 11 years until near adult height [intent-to-treat (ITT) population]. Children were randomized into three groups: controls (n = 33), GH 33 μg/kg/day (n = 34) or GH 67 μg/kg/day (n = 57). Prepubertal children at study start constituted the per-protocol (PP) population (n = 101). Auxological measurements were made and puberty was staged every 3 months. Serum sex-steroid concentrations were assessed every 6 months., Results: No significant differences were found between the groups, of both PP and ITT populations, in time elapsed from start of treatment until either onset of puberty, age at start of puberty or age at final pubertal maturation in either sex. In the ITT population, pubertal duration was significantly longer in GH-treated girls, and maximum mean testicular volume was significantly greater in GH-treated boys than controls, but there were no differences in testosterone levels between the groups., Conclusion: GH treatment did not influence age at onset of puberty and did not accelerate pubertal development. In boys, GH treatment appeared to increase testicular volume., (Copyright © 2011 S. Karger AG, Basel.)

We investigated whether long-term hormone replacement therapy (HRT) is associated with mobility and lower limb muscle performance and composition in postmenopausal women. Fifteen 54- to 62-yr-old monozygotic female twin pairs discordant for HRT were recruited from the Finnish Twin Cohort. Habitual (HWS) and maximal (MWS) walking speeds over 10 m, thigh muscle composition, lower body muscle power assessed as vertical jumping height, and maximal isometric hand grip and knee extension strengths were measured. Intrapair differences (IPD%) with 95% confidence intervals (CI) were calculated. The mean duration of HRT use was 6.9 +/- 4.1 yr. MWS was on average 7% (0.9 to 13.1%, P = 0.019) and muscle power 16% (-0.8 to 32.8%, P = 0.023) greater in HRT users than in their cotwins. Thigh muscle cross-sectional area tended to be larger (IPD% = 6%, 95% CI: -0.07 to 12.1%, P = 0.065), relative muscle area greater (IPD% = 8%, CI: 0.8 to 15.0%, P = 0.047), and relative fat area smaller (IPD% = -5%, CI: -11.3 to 1.2%, P = 0.047) in HRT users than in their sisters. There were no significant differences in maximal isometric strengths or HWS between users and nonusers. Subgroup analyses revealed that estrogen-containing therapies (11 pairs) significantly decreased total body and thigh fat content, whereas tibolone (4 pairs) tended to increase muscle cross-sectional area. This study showed that long-term HRT was associated with better mobility, greater muscle power, and favorable body and muscle composition among 54- to 62-yr-old women. The results indicate that HRT is a potential agent in preventing muscle weakness and mobility limitation in older women.

Background: The role of estrogens in male physiology has become evident. However, clinically useful normative data for estradiol secretion in boys has not previously been established due to the insensitivity of current methods used in clinical routine. By use of a validated ultra-sensitive extraction RIA, our aim was to establish normative data from a group consisting of healthy boys in prepuberty and during pubertal development., Methods: Sixty-two 24-hours serum profiles (6 samples/24 hours) were obtained from 44 healthy boys (ages; 7.2-18.6 years) during their pubertal development, classified into five stages: prepuberty (testis, 1-2 mL), early (testis, 3-6 mL), mid (testis, 8-12 mL), late-1 (testis,15-25 mL, not reached final height) and late-2 (testis,15-25 mL, reached final height). Serum estradiol was determined by an ultra- sensitive extraction radioimmunoassay with detection limit 4 pmol/L and functional sensitivity 6 pmol/L., Results: Mean estradiol concentrations during 24-hours secretion increased from prepuberty (median: <4 (5-95 percentiles: <4 - 7) pmol/L) to early puberty (6 (<4 - 12 pmol/L) but then remained relatively constant until a marked increase between mid-puberty (8 (4 - 17) pmol/L) and late-1 (21 (12 - 37) pmol/L) puberty, followed by a slower increase until late-2 puberty (32 (20 - 47) pmol/L). The diurnal rhythm of serum estradiol was non-measurable in pre- and early puberty, but discerned in mid-puberty, and become evident in late pubertal stages with peak values at 0600 to 1000 h., Conclusion: With the use of an ultra-sensitive extraction RIA, we have provided clinically useful normative data for estradiol secretion in boys.

Objective: To test the clinical usefulness of sensitive commercial immunoassays for determination of low 17beta-estradiol concentrations in children., Methods: The lower limit of detection and clinical usefulness (functional sensitivity) of three commercial estradiol immunoassays were validated by use of 500 sera from prepubertal and pubertal children and 55 pooled sera. The three immunoassays consisted of two modified direct immunoassays; one RIA (Spectria Estradiol RIA) and one time-resolved fluoroimmunoassay (AutoDELFIA Estradiol), both with increased serum volume in relation to antibody concentration and extended incubation time. In the third method, serum was purified and concentrated using diethyl ether extraction prior to measurement by the modified Spectria Estradiol RIA., Results: The lower limits of detection and clinical usefulness were 9 and 30 pmol/l for the direct RIA, 11 and 50 pmol/l for the AutoDELFIA, and 4 and 6 pmol/l for serum determined by extraction RIA. When measuring the serum pool originating from girls at breast stages 1-2, the direct RIA and AutoDELFIA resulted in significantly higher 17beta-estradiol concentrations when compared with the extraction RIA (+58 and +267%, P<0.001). We found a significant difference in 17beta-estradiol concentrations between girls at breast stages 1 (median 6 pmol/l) and 2 (median 16 pmol/l), when quantified by the extraction RIA (P<0.0001) but no difference when quantified with the direct RIA (median values 12 and 14 pmol/l respectively)., Conclusion: For determination of low serum 17beta-estradiol concentrations in children, an extraction step prior to commercial immunoassay is needed to achieve clinically useful results.

Objective: In males, the pubertal increase in sex hormone production has been associated with proatherogenic changes in lipid and carbohydrate metabolism. Aromatase inhibitors, a novel treatment modality for some growth disorders, may significantly influence these risk factors for cardiovascular disease by suppressing oestrogen biosynthesis and stimulating gonadal androgen production. In the current study, we explored the effects of aromatase inhibition on lipid metabolism, insulin sensitivity, body composition and serum adiponectin in peripubertal boys., Design: Prospective, double-blind, randomised, placebo-controlled clinical study., Methods: Thirty-one boys, aged 9.0-14.5 years, with idiopathic short stature were treated with the aromatase inhibitor letrozole (2.5 mg/day) or placebo for 2 years. During the treatment, the concentrations of sex hormones, IGF-I, lipids, lipoproteins and adiponectin were followed-up. The percentage of fat mass (FM) was assessed by skinfold measurements and insulin resistance by homeostasis model assessment (HOMA) index., Results: In pubertal boys, who received letrozole, high-density lipoprotein cholesterol (HDL-C) decreased by 0.47 mmol/l (P<0.01) during the study. Simultaneously, their percentage of FM decreased from 17.0 to 10.5 (P<0.001), in an inverse relationship with serum testosterone. The concentrations of low-density lipoprotein cholesterol, triglycerides and HOMA index remained at pretreatment level in both groups. Serum adiponectin decreased similarly in letrozole- and placebo-treated pubertal boys (2.9 and 3.3 mg/l respectively)., Conclusions: In males, aromatase inhibition reduces HDL-C and decreases relative FM after the start of puberty. The treatment does not adversely affect insulin sensitivity in lean subjects.