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BackgroundAbdominal fat distribution is associated with the development of cardio-metabolic disease independently of body mass index (BMI). We assessed anthropometry, serum adipokines, and DXA as markers of abdominal subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) using magnetic resonance imaging (MRI).MethodsWe performed a cross-sectional study that included 197 healthy adolescents (114 boys) aged 10-15 years nested within a longitudinal population-based cohort. Clinical examination, blood sampling, DXA, and abdominal MRI were performed. SAT% and VAT% were adjusted to total abdominal volume.ResultsGirls had a higher SAT% than did boys in early and late puberty (16 vs. 13%, P

Context: Detailed evaluation of pubertal progression in girls from longitudinal studies is sparse, and the phenomenon of transient thelarche (TT), defined as the appearance, regression, and subsequent reappearance of breast buds, in healthy girls remains undescribed. Objective: To describe TT in terms of pubertal progression, growth, genotypes, and reproductive hormones and to apply new puberty nomograms for breast stages, pubic hair, and menarche. Design: A prospective, longitudinal population-based study. Patients or Other Participants: Ninety-eight healthy Danish schoolchildren (Caucasian girls) followed longitudinally as part of the COPENHAGEN Puberty Study were included in the evaluation of TT. A total of 1466 girls from 2 cross-sectional studies were included in the creation of the puberty nomograms. Intervention(s): None. Main Outcome Measure(s): Pubertal progression, specifically thelarche, reproductive hormones, genotype, and growth. Results: Twelve of 98 (12%) girls experienced TT. A larger proportion of girls with TT entered puberty by the pubarche pathway (50%) compared with girls with normal progression (15.4%), P = 0.014. Girls with TT progressed through puberty normally when evaluated using puberty nomograms. Reproductive hormones and growth velocity were lower at the first (transient) thelarche than the second (permanent) thelarche. Conclusion: TT is a frequent phenomenon that appears to be a peripheral occurrence independent of central puberty. It does not appear to affect subsequent pubertal progression as evaluated by our new puberty nomograms.

Appearance of glandular breast tissue may be difficult to distinguish from fat tissue by palpation, especially in obese girls. To our knowledge, validation of the clinical assessment of pubertal breast stages by magnetic resonance imaging (MRI) has never been performed. Our objective was to report normative data of glandular breast tissue volume and validate the clinical evaluation of pubertal breast staging by MRI of breast tissue and to evaluate circulating reproductive hormone levels and estrogen-dependent transabdominal ultrasound (TAUS) parameters as markers of glandular breast tissue.Glandular breast tissue volume quantified by MRI and breast stage evaluation was performed in 100 healthy peripubertal girls. Circulating levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin B, and estradiol were measured by immunoassays. Ovarian volume, uterine volume, and endometrial thickness were assessed by TAUS.Glandular breast tissue volume was positively associated with Tanner stages (r = 0.858, P0.001). The sensitivity and specificity of breast palpation to detect presence of glandular breast tissue using MRI as gold standard were 96 and 95%, respectively. The best parameters to distinguish prepubertal girls from girls with breast development were: LH (area under the curve (AUC) by receiver operating characteristic analysis = 0.871), inhibin B (AUC = 0.847) and estradiol (AUC = 0.830).Clinical palpation reliably detects the presence of glandular breast tissue.

Adrenarche is characterized by steadily rising levels of adrenal androgen metabolites from 4–6 years of age. We recently described marked gender-specific differences in circulating ratios between selected adrenal androgen metabolites in a cross-sectional study. This may suggest gender differences in steroidogenic enzyme activities. We therefore aimed at verifying these findings in a prospective, longitudinal study of healthy boys and girls who were examined during pubertal transition.A longitudinal study of 20 healthy children from the COPENHAGEN Puberty Study, followed every 6 months for 5 years. Clinical examinations were conducted and serum concentrations of Androstenedione (Adione), 17-hydroxyprogesterone (17-OHP), testosterone (T), dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) were quantified by a newly developed LC-MS/MS method. DHEA, DHEAS, Adione, 17-OHP and T increase with age. Boys had higher levels of DHEAS from 10.5 years of age, whereas girls had higher levels of Adione from 13 years of age compared to boys. Interestingly, we observed significantly higher ratios of DHEAS/DHEA (sulfotransferase activity) in boys before and after pubertal onset compared to girls, whereas Adione/17-OHP (CYP 17 activity) appeared to increase more in pubertal girls compared to boys. This suggests that adrenal steroidogenic enzyme activities show developmental as well as gender-specific changes in healthy children.

Puberty is initiated by a complex interaction of suppressing and stimulating factors. Genetic studies of familial central precocious puberty have suggested makorin ring finger protein 3 (MKRN3) as a major inhibitor of GnRH secretion during childhood. Furthermore, genetic variation near MKRN3 (rs12148769) affects age at menarche in healthy girls.The purpose of this study was to evaluate whether serum levels of MKRN3 declined before pubertal onset in healthy girls.This was a population-based longitudinal study of healthy Danish girls and a cohort study of early maturing girls.The study was performed in the general community and in a tertiary referral center for pediatric endocrinology.Healthy girls (n = 38) aged 9.3 years (range, 5.9-11.3 years) at baseline and followed for 6.0 years (2.7-7.6 years) (2006-2014) with blood sampling every 6 months and early maturing girls (n = 13) with breast development ay8.3 years of age were included.Serum levels of MKRN3 were measured in 354 samples (median, 9 per girl; range, 2-14 per girl), and genotyping of variants near MKRN3 (rs12148769 and rs12439354) was performed.MKRN3 concentrations declined preceding pubertal onset; the geometric mean (95% confidence interval) 3 years before pubertal onset vs the last visit before pubertal onset was 304 pg/mL (264-350 pg/mL) vs 257 pg/mL (243-273 pg/mL), corresponding to a reduction of 15% (1-27%) (P = .033). In prepubertal girls, circulating MKRN3 correlated negatively with gonadotropin levels: for FSH, r = -0.262 (P = .015) and for LH, r = -0.226 (P = .037). After adjustment, MKRN3 levels were lower in early maturing girls than in age-matched prepubertal girls: 171 pg/mL (25-333 pg/mL) vs 262 pg/mL (94-624 pg/mL) (P = .051). Genetic variants near MKRN3 did not correlate with serum levels of MKRN3.Declining levels of circulating MKRN3 preceded pubertal onset. The negative correlation between MKRN3 and gonadotropins further supports MKRN3 as a major regulator of hypothalamic GnRH secretion during childhood. Undetectable or low MKRN3 levels were observed in a subgroup of patients with early onset of puberty.

In adult women, Anti-Müllerian hormone (AMH) is produced by small growing follicles, and circulating levels of AMH reflect the number of antral follicles as well as primordial follicles. Whether AMH reflects follicle numbers in healthy girls remains to be elucidated.This study aimed to evaluate whether serum levels of AMH reflects ovarian morphology in healthy girls.This was a population-based cohort study involving the general community.Included in the study were 121 healthy girls 9.8-14.7 years of age.Clinical examination, including pubertal breast stage (Tanner's classification B1-5), ovarian volume, as well as the number and size of antral follicles were assessed by two independent modalities: magnetic resonance imaging (MRI), Ellipsoid volume, follicles ≥2 mm; and Transabdominal ultrasound, Ellipsoid and 3D volume, follicles ≥1 mm. Circulating levels of AMH, inhibin B, estradiol, FSH, and LH were assessed by immunoassays; T and androstenedione were assessed by liquid chromatography-tandem mass spectrometry.AMH reflected the number of small (MRI 2-3 mm) and medium (4-6 mm) follicles (Pearson's Rho [r] = 0.531 and r = 0.512, P.001) but not large follicles (≥7 mm) (r = 0.109, P = .323). In multiple regression analysis, small and medium follicles (MRI ≤ 6 mm) remained the main contributors to circulating AMH (β, 0.501; P.001) whereas the correlation between AMH and estradiol was negative (β, -0.318; P = .005). In early puberty (B1-B3), the number of AMH-producing follicles (2-6 mm) correlated positively with pubertal stages (r = 0.453, P = .001), whereas AMH levels were unaffected (-0.183, P = .118).Similarly to adult women, small and medium antral follicles (≤6 mm) were the main contributors to circulating levels of AMH in girls.

Insulin-like factor 3 (INSL3) is a promising marker of Leydig cell function with potentially high clinical relevance. Limited data of INSL3 levels in relation to other reproductive hormones in healthy pubertal boys exist. In this study, we aimed to evaluate longitudinal serum changes in INSL3 compared with LH, FSH, testosterone, inhibin B, and anti-Müllerian hormone (AMH) during puberty in healthy boys. Ten boys were included from the longitudinal part of the COPENHAGEN Puberty Study. Pubertal evaluation, including testicular volume, was performed and blood samples were drawn every 6 months for 5 years. Serum concentrations of testosterone were determined by a newly developed LC–MS/MS method, and serum concentrations of INSL3, AMH, inhibin B, FSH, and LH respectively were determined by validated immunoassays. The results showed that serum INSL3 levels increased progressively with increasing age, pubertal onset, and testicular volume. In six of the ten boys, LH increased before the first observed increase in INSL3. In the remaining four boys, the increase in LH and INSL3 was observed at the same examination. The increases in serum concentrations of LH, testosterone, and INSL3 were not parallel or in ordered succession and varied interindividually. We demonstrated that INSL3 concentrations were tightly associated with pubertal onset and increasing testicular volume. However, the pubertal increases in LH, INSL3, and testosterone concentrations were not entirely parallel, suggesting that INSL3 and testosterone may be regulated differently. Thus, we speculate that INSL3 provides additional information on Leydig cell differentiation and function during puberty compared with traditional markers of testicular function.

SummaryBackground Adrenal disorders such as congenital adrenal hyperplasia result in abnormal adrenal size and morphology, but little is known about the clinical value of magnetic resonance imaging (MRI) in determining adrenal volume. Objective To evaluate the potential usefulness of MR methodology, to estimate adrenal size in healthy children and to evaluate determinants of adrenal volume such as age, gender, body size, pubic hair stage and serum levels of adrenal androgens. Design Two hundred and thirty-five healthy children (116 girls and 119 boys) (age range 10·0–14·8 years) were examined by MRI. Clinical examinations (anthropometry and pubertal staging) were performed, and five androgen metabolites were measured in blood samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results It was possible to determine adrenal volume in 115/235 children using MRI. The adrenals were not measurable in 51% of children due to breathing and moving artefacts. The median volume of the right adrenal gland was 0·46 ml in girls and 0·46 ml in boys. The median volume of the left adrenal gland was 0·34 ml in girls and 0·40 ml in boys. Adrenal size was positively associated with body surface area (estimate B = 0·34 ml/year, P = 0·003), age (estimate B = 0·05 ml/year, P = 0·021) and pubic hair stage (estimate B = 0·05 ml/stage, P = 0·075). No associations between adrenal size and serum levels of adrenal androgens were observed. Conclusion It was possible to determine adrenal volume by MRI in only 50% of healthy children aged 10–15 years. Adrenal volume increased with age and Tanner stage of pubic hair. Future studies will unravel whether adrenal MRI is useful when evaluating children with adrenal diseases.

Aim To construct new Danish growth charts for 0- to 20-year-olds and to compare them with Danish references from 1982 and with World Health Organization (WHO) standards for children aged 0–5 years from 2006, by applying similar inclusion and exclusion criteria. Methods Anthropometric data from three contemporary Danish population-based studies were combined. References for height were based on healthy Caucasian children born at term. A total of 12 671 height measurements (8055 in boys and 4616 in girls) were included. Reference charts were developed using the generalised additive models for location, scale and shape. Results From prepubertal ages, a secular increase in height was observed for both genders. The differences were most pronounced in puberty, and final heights were increased by 1.4 cm in boys and 2.9 cm in girls compared to 1982 references. In boys, but not girls an upward shift in body mass index (BMI) above median levels was found. Reference curves for height were superimposable with standard curves based on the selective WHO criteria. Danish children were longer/taller and heavier and they had larger head circumferences than those reported in the recent multiethnic WHO standards. Conclusion We recommend national implementation of these contemporary 2014 Danish references for anthropometric measurements.

Longer androgen receptor gene CAG trinucleotide repeats, AR (CAG)n, have been associated with reduced sensitivity of the androgen receptor (AR) in vitro as well as in humans. Furthermore, short AR (CAG)n have been associated with premature adrenarche.The aim of the study was to evaluate associations between the AR (CAG)n polymorphism and development of pubic hair, levels of androgens, and body fat content in healthy boys.A longitudinal study of 78 healthy boys (age 6.2-12.4 years at inclusion) from the COPENHAGEN Puberty Study was conducted with clinical examinations and blood samples drawn every 6 months. The AR (CAG)n length was established by direct DNA sequencing and reproductive hormones were measured in serum by standardized analyses.Median AR (CAG)n length was 22 (range, 17-30). Before puberty (at 10 years of age), boys with long CAG repeats (CAG ≥ 24) had lower levels of SHBG (88 vs 125 nmol/L) (P.05) and a nonsignificant trend toward higher median skinfold thickness (41 vs 31 mm) (P = .06) compared with boys with an average number of CAG repeats (CAG 21-23). In contrast, the inverse association was observed at puberty (at 12 years of age) in boys with short CAG repeats (CAG 17-20) (P.05). Serum levels of LH and testosterone (at 12 years) were significantly higher in boys with long CAG repeats compared with boys with an average number of CAG repeats (P = .05).The observed associations between AR (CAG)n and peripubertal fat accumulation and serum SHBG concentrations indicate that this genetic polymorphism may influence the androgen-dependent fine-tuning of metabolic and reproductive factors at a young age.

ObjectiveGynaecomastia is a benign proliferation of glandular tissue of the breast; however, it is an important clinical observation because it can be the first symptom of an underlying disease. Some controversy exists concerning the clinical importance of an in-depth investigation of men who develop gynaecomastia. We hypothesise that a thorough work-up is required in adult men with gynaecomastia.DesignAll adult men (n = 818) referred to a secondary level andrological department at Rigshospitalet in Copenhagen, Denmark during a four-year period (2008–2011) under the diagnosis of gynaecomastia (ICD-10: N62) were included.MethodsThirty-two men who did not have gynaecomastia when examined were excluded; leaving 786 men for final analyses. They underwent an andrological examination, ultrasound of the testicles and analysis of endogenous serum hormones levels.ResultsIn 43% of men with adult onset of gynaecomastia (≥18 years) an underlying, and often treatable, cause could be detected. In men younger at onset an underlying cause for gynaecomastia could be detected in merely 7.7%. The study is limited by the fact that we did not have access to investigate men who were referred directly by their GP to private clinics for plastic surgery or who sought cosmetic correction without consulting their GP first.ConclusionsOur study demonstrates the importance of a thorough examination and provides a comprehensible examination strategy to disclose the underlying pathology leading to the development of gynaecomastia in adulthood.

Context: Epidemiological evidence on maternal and paternal heritability of the wide normal variation within pubertal timing is sparse. Objective: We aimed to estimate the impact of parental pubertal timing on the onset of puberty in boys and girls. Design: Annual pubertal examinations of healthy children in a longitudinal cohort study. Information on parental timing of puberty (earlier, comparable to, or later compared to peers) and menarche age was retrieved from questionnaires. Participants: A total of 672 girls and 846 boys. Main Outcome Measures: Age at onset of pubic hair (PH2+), breasts (B2+), and menarche in girls; and PH2+, genital stage (G2+), and testis >3 mL with orchidometer (Tvol3+) in boys. Results: In boys, pubertal onset was significantly associated with pubertal timing of both parents. PH2+ and Tvol3+ were earlier: −11.8 months (95% confidence interval, −16.8, −6.8)/−8.9 (−12.8, −4.9), and −9.5 (−13.9, −5.1)/−7.1 (−10.4, −3.7) if the father/mother, respectively, had early pubertal development compared to late. In girls, menarche was significantly associated with both parents' pubertal timing: −10.5 months (−15.9, −5.1)/−10.1 (−14.3, −6.0) if father/mother had early pubertal development compared to late. For the onset of PH2+ and B2+ in girls, estimates were −7.0 months (−12.6, −1.4) and −4.1 (−10.6, +2.4)/−6.7 (−11.0, −2.5), and −6.7 (−11.0, −2.0) for fathers/mothers, respectively. Maternal age of menarche was significantly associated with the onset of all pubertal milestones except PH2+ in girls. Conclusions: Maternal as well as paternal pubertal timing was a strong determinant of age at pubertal onset in both girls and boys. Age at breast and pubic hair development in girls, which has declined most during recent years, seemed to be least dependent on heritability.

Pubertal gynaecomastia is a clinical sign of an oestrogen-androgen imbalance, which occurs in 40-60% of adolescent Caucasian boys. In most cases no underlying endocrinopathy can be identified. A recent study reports higher plasma phthalate levels in Turkish boys with pubertal gynaecomastia. Therefore, we asked whether there was an association between concurrent measures of urinary phthalate metabolites and pubertal timing as well as the presence of gynaecomastia in otherwise healthy boys. We studied a total of 555 healthy boys (age 6.07-19.83 years) as part of the COPENHAGEN Puberty Study. Anthropometry and pubertal stages (PH1-6 and G1-5) were evaluated, and the presence of gynaecomastia was assessed. Non-fasting blood samples were analysed for serum testosterone and morning urine samples were analysed for the total content of 12 phthalate metabolites (MEP, MnBP, MiBP, MBzP, MEHP, MEHHP, MEOHP, MECPP, MiNP, MHiNP, MiONP and MCiOP) by LC-MS/MS. A statistically significant negative correlation was observed between chronological age and the urinary concentration of the sum of measured metabolites DEHP (∑DEHPm) (r = -0.164) and DiNP (∑DiNPm) (r = -0.224), respectively, and the sum of monobutyl phthalate (MBP) isomers (∑MBP((i+n))) (r = -0.139) (all with p < 0.01). In contrast urinary monoethyl phthalate concentration was positively correlated to age (r = 0.187, p < 0.01). The urinary levels of phthalate metabolites were not associated with age at pubertal onset, serum testosterone levels or presence of gynaecomastia. In conclusion, we did not find evidence of anti-androgenic effects of phthalates in our healthy boys. Thus, current phthalate exposure was not associated with pubertal timing, testosterone levels or with the presence of pubertal gynaecomastia in this cross-sectional study. However, longitudinal studies are needed to evaluate possible perinatal or long-term postnatal effects of phthalates on healthy boys.

Context: Physiological gynecomastia is common and affects a large proportion of otherwise healthy adolescent boys. It is thought to be caused by an imbalance between estrogen and testosterone, although this is rarely evident in analyses of serum. Objective: This study aimed to describe the frequency of physiological gynecomastia and to determine possible etiological factors (eg, auxology and serum hormone levels) in a longitudinal setup. Design, Settings, and Participants: A prospective cohort study of 106 healthy Danish boys (5.8–16.4 years) participated in the longitudinal part of the COPENHAGEN Puberty Study. The boys were examined every 6 months during an 8-year follow-up. Median number of examinations was 10 (2–15). Main outcome measurements: Blood samples were analyzed for FSH, LH, testosterone, estradiol, SHBG, inhibin B, anti-Müllerian hormone, IGF-1, and IGF binding protein-3 by immunoassays. Auxological parameters, pubertal development, and the presence of gynecomastia were evaluated at each visit. Results: Fifty-two of 106 boys (49%) developed gynecomastia, of which 10 (19%) presented with intermittent gynecomastia. Boys with physiological gynecomastia reached peak height velocity at a significantly younger age than boys who did not develop gynecomastia (13.5 versus 13.9 years, P = .027), and they had significantly higher serum levels of IGF-1 (P = .000), estradiol (P = .013), free testosterone (P < .001), and FSH (P = .030) during pubertal transition. However, no differences in serum LH or in the estradiol to testosterone ratio were found. Conclusions: Gynecomastia is frequent in pubertal boys. Increased IGF-1 levels and pubertal growth appear to be associated, whereas changes in estrogen to testosterone ratio seem negligible.

Objective To report normative data on uterine volume and endometrial thickness in girls, according to pubertal stages; to evaluate factors that affect uterine volume; and to compare transabdominal ultrasound (TAUS) and magnetic resonance imaging (MRI). Design Cross-sectional study of a nested cohort of girls participating in The Copenhagen Mother-Child Cohort. Setting General community. Patient(s) One hundred twenty-one healthy girls, aged 9.8–14.7 years. Intervention(s) None. Main Outcome Measure(s) Clinical examination, including pubertal breast stage (Tanner classification: B1–B5). Uterine volume: ellipsoid TAUS (n = 112) and 3-dimensional TAUS (n = 111); ellipsoid MRI (n = 61). Endometrial thickness: TAUS (n = 110) and MRI (n = 60). Result(s) Uterine volume and endometrial thickness were positively correlated with pubertal stages; e.g., ellipsoid TAUS: r = 0.753, and endometrium TAUS: 0.648. In multiple regression analyses, uterine volume was associated with the number of large follicles (TAUS >5 mm) (Beta 0.270); estradiol (E 2 ) (Beta 0.504); and height (Beta 0.341). Volumes from ellipsoid vs. 3-dimensional TAUS were strongly correlated ( r = 0.931), as were TAUS and MRI: ellipsoid volume ( r = 0.891) and endometrial thickness ( r = 0.540). Uterine volume was larger in TAUS compared with MRI; mean difference across the measured range: 7.7 (5.2–10.2) cm 3 . Agreement was best for small uteri. Conclusion(s) Uterine volume and endometrial thickness increased as puberty progressed. Circulating E 2 from large follicles was the main contributor to uterine and endometrial growth. The TAUS and MRI assessments of uterus and endometrium were strongly correlated.

Few data exist on the diagnostic criteria, and on the effects of puberty induction, in boys with constitutional delay in growth and puberty (CDGP).To develop puberty nomograms based on Danish boys with normal pubertal development. To evaluate the different diagnostic criteria and the effect of oral testosterone undecanoate (TU) in boys with CDGP.A cross-sectional and longitudinal study of Danish boys with normal pubertal development (COPENHAGEN puberty study). A retrospective observational study of 451 boys evaluated for delayed puberty between 1990 and 2013.Tertiary referral center for pediatric endocrinology.One hundred and sixty-four (36%) boys evaluated for CDGP were excluded due to missing data, reclassification, or associated comorbidities, yielding 287 (64%) eligible for analysis.The number of patients with CDGP classified by the puberty nomogram (genital stage-2 SD for age) versus the classical criteria (genital stage 1 at ≥ 14 years). The effect of one year of oral TU treatment on pubertal progression, circulating hormones, height, and predicted adult height (PAH).Seventy-eight (27%) of the 287 boys had delayed pubertal onset according to the classical criteria, whereas 173 (60%) of the 287 boys had impaired pubertal progression according to the puberty nomogram. Ninety-six (56%) of these 173 boys were treated with oral TU for 0.8 years (0.5; 1.3) [median (25th; 75th percentiles)], which resulted in beneficial effects on pubertal progression. Height increased from -1.9 SD (-2.5; -1.2) to -1.5 SD (-2.1; -0.7) (P.001), and PAH increased from 172.3 cm (170.3; 182.8) to 178.1 cm (171.4; 191.7) (P = .001) following one year of treatment.The puberty nomogram evaluates both delayed pubertal onset as well as delayed pubertal progression and allows separation of normal versus abnormal pubertal development. Oral TU treatment was followed by pubertal induction and progression and short-term growth without compromising final height.

BACKGROUND AND OBJECTIVES:Studies of adolescents often use self-assessment of pubertal maturation, the reliability of which has shown conflicting results. We aimed to examine the reliability of child and parent assessments of healthy boys and girls.METHODS:A total of 898 children (418 girls, 480 boys, age 7.4–14.9 years) and 1173 parents (550 daughters, 623 sons, age 5.6–14.7 years) assessed onset of puberty or development of breasts, genitals, and pubic hair according to Tanner stages by use of a questionnaire and drawings. Physicians’ assessments were blinded and set as the gold standard. Percentage agreement, κ, and Kendall’s correlation were used to analyze the agreement rates.RESULTS:Breast stage was assessed correctly by 44.9% of the girls (κ = 0.28, r = 0.74, P < .001) and genital stage by 54.7% of the boys (κ = 0.33, r = 0.61, P < .001). For pubic hair stage 66.8% of girls (κ = 0.55, r = 0.80, P < .001) and 66.1% of boys (κ = 0.46, r = 0.70, P < .001) made correct assessments. Of the parents, 86.2% correctly assessed onset of puberty in girls (κ = 0.70, r = 0.71, P < .001) and 68.4% in boys (κ = 0.30, r = 0.37, P < .001). Children who underestimated were younger and children who overestimated older than their peers who made correct assessments. Girls and their parents tended to underestimate, whereas boys overestimated their pubertal stage.CONCLUSIONS:Pubertal assessment by the child or the parents is not a reliable measure of exact pubertal staging and should be augmented by a physical examination. However, for large epidemiologic studies self-assessment can be sufficiently accurate for a simple distinction between prepuberty and puberty.

Objective: To elucidate the natural course of circulating insulin-like peptide 3 (INSL3) levels according to puberty as well as its relation to other reproductive hormones. Design: Population-based cohort study. Setting: Not applicable. Patient(s): Healthy peripubertal girls (n¼10) examined every 6 months; total number of examinations was 84; median (range) per girl:9 (4–10), including staging of pubertal breast development and blood samples. Intervention(s): None. Main Outcome Measure(s): Serum levels of INSL3, inhibin B, E2, antim€ullerian hormone, LH, and FSH (validated immunoassays), and T and androstenedione (liquid chromatography–tandem mass spectrometry). Result(s): Serum levels of INSL3 varied considerably between girls (range, 0.01–0.27 ng/mL) and within each girl as puberty progressed; intraindividual variation, median (range) 102% (65%–143%). Insulin-like peptide 3 increased in late puberty (B1 to B4þB5); geometric mean 0.03 ng/mL to 0.15 ng/mL. Insulin-like peptide 3 levels reflected markers of large follicles (T, androstendione, inhibin B, and E2) better than markers of small follicles (antim€ullerian hormone), and INSL3 staining was localized in theca interna cells of antral follicles. Conclusion(s): Insulin-like peptide 3 increased in late puberty, albeit inter- and intraindividual variations were substantial. Immunohistochemistry and intraindividual variation, as well as relations to other ovarian hormones, reveal that INSL3 in girls is a unique and specific marker of theca cells surrounding antral follicles. The potential clinical use of INSL3 for evaluation of ovarian function in girls remains to be elucidated.

Age at pubertal onset varies substantially in healthy girls. Although genetic factors are responsible for more than half of the phenotypic variation, only a small part has been attributed to specific genetic polymorphisms identified so far. Follicle-stimulating hormone (FSH) stimulates ovarian follicle maturation and estradiol synthesis which is responsible for breast development. We assessed the effect of three polymorphisms influencing FSH action on age at breast deveopment in a population-based cohort of 964 healthy girls. Girls homozygous for FSHR -29AA (reduced FSH receptor expression) entered puberty 7.4 (2.5–12.4) months later than carriers of the common variants FSHR -29GG+GA, p = 0.003. To our knowledge, this is the strongest genetic effect on age at pubertal onset in girls published to date.

ObjectivePubertal gynaecomastia is a frequent phenomenon occurring in 20–40% of otherwise healthy adolescent boys. Little is known about the aetiology of pubertal gynaecomastia. Markedly elevated thyroid hormone levels in adults with hyperthyroidism are associated with gynaecomastia.DesignA cross-sectional examination of 444 healthy boys with and without pubertal gynaecomastia.MethodsWe evaluated TSH, triiodothyronine (T3), thyroxine (T4), free T4 and free T3 in a cohort of healthy boys with and without pubertal gynaecomastia.ResultsBoys with gynaecomastia had significantly higher serum free T3, even after correction for age, BMI and pubertal stage. After inclusion of IGF1 in the model the differences disappeared. TSH, T4, free T4 and T3 did not differ between the groups.ConclusionsWe speculate that the GH/IGF1 axis and thyroid hormones interact and influence the development of pubertal gynaecomastia.

Background: Accurate and selective assessment of testosterone requires use of a sensitive LC-MS/MS method, especially at low levels as those seen in young children. Methods: The present longitudinal study of 20 healthy children from the Copenhagen Puberty Study followed every 6 months for 5 years evaluates the longitudinal increase in serum testosterone before, during and after pubertal onset quantified by a newly developed LC-MS/MS method in comparison with immunoassay. Testosterone concentrations in serum samples (n = 177) were determined by LC-MS/MS (detection limit 0.1 nmol/l) and by immunoassay (detection limit 0.23 nmol/l). Results: Serum concentrations of testosterone increased gradually with age by both methods. However, serum testosterone was quantifiable in 9/10 girls prior to pubic hair development measured with LC-MS/MS, and in 2/10 girls measured with immunoassay. In boys, testosterone was quantifiable in 10/10 boys 1 year prior to pubic hair development measured with LC-MS/MS, and only in 1/10 boys measured with immunoassay. Serum testosterone levels were quantifiable 1.5 years (range 0.5-2.5) earlier using LC-MS/MS. Conclusion: Assessment of longitudinal circulating levels of serum testosterone using a selective LC-MS/MS method proved to be more sensitive in predicting early peripubertal changes in healthy children compared to levels determined by immunoassay.

Objective Pubertal gynaecomastia is a very common condition. Although the underlying aetiology is poorly understood, it is generally accepted that excess of oestrogens and deficit of androgens are involved in the pathogenesis. Furthermore, adiposity as well as the GH/IGF-I axis may play a role. In this study, we elucidate the association of adiposity and levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), sex hormone-binding globulin (SHBG), testosterone, oestrogen, IGF-I and IGFBP-3 with the presence of pubertal gynaecomastia in a large cohort of healthy boys. Patients A total of 501 healthy Danish school boys (aged 6·1–19·8 year) from the COPENHAGEN Puberty Study. Measurements Anthropometry and pubertal stages (PH1-6 and G1-5) were evaluated, and the presence of gynaecomastia was assessed. Body fat percentage was calculated by means of four skin folds and impedance. Nonfasting blood samples were analysed for FSH, LH, testosterone, SHBG, oestradiol, IGF-I, IGFBP-3 and prolactin. Results We found that 23% (31/133) of all pubertal boys had gynaecomastia. More specifically, 63% (10/16) of boys in genital stage 4 had gynaecomastia. Boys with gynaecomastia had significantly higher IGF-I levels compared with controls (IGF-I SD-score 0·72 vs −0·037, P

Total body fat percentage (%BF) evaluated by dual energy X-ray absorptiometry (DXA) scans (DXA %BF) is widely recognized as a precise measure of fatness. We aimed to establish national reference curves for DXA %BF, %BF calculated from skinfolds (SF %BF) and waist circumference (WC) in healthy children, and to compare agreement between the different methods. Based on 11 481 physical examinations (anthropometry) and 1200 DXA scans from a longitudinal cohort of Danish children (n=2647), we established reference curves (LMS-method) for SF %BF, WC (birth to 14 years) and DXA %BF (8–14 years). Age- and sex-specific Z-scores for body mass index (BMI), WC and SF %BF were compared. Sensitivity and specificity were calculated for agreement of WC, SF %BF and BMI with DXA %BF to identify obese children (>+1 s.d.). %BF differed with age, sex, pubertal stage and social class. SF %BF correlated strongly with DXA %BF (r=0.86). BMI and WC also correlated positively with DXA %BF (Z-scores; r= 0.78 and 0.69). Sensitivity and specificity were 79.5 and 93.8 for SF %BF, 75.9 and 90.3 for BMI and 59.2 and 95.4 for WC. SF %BF showed the highest correlation and best agreement with DXA %BF in identifying children with excess fat (+1 s.d.).

Objective To investigate whether genetic polymorphisms in the FSH pathway ( FSHB -211 G→T and FSHR 2039 A→G) affect serum levels of FSH, antimullerian hormone (AMH), and age at pubertal onset. FSH secretion and FSH signal transduction are enhanced in carriers of FSHB GG and FSHR AA, respectively. Furthermore, the combined genotype FSHB GG+ FSHR AA is the most favorable for male gonadal function, but the effect of this genotype has never been evaluated in peripubertal females. AMH is a marker of ovarian function and is negatively correlated with FSH in prepubertal girls. Design Secondary analyses of a prospective cohort study. Setting General community. Patient(s) We examined 78 healthy girls twice yearly for 6 years; the median age at baseline was 9.3 years. Intervention(s) None. Main Outcome Measure(s) Hormone levels were measured by immunoassays, and DNA was isolated from blood and genotyped by restriction fragment length polymorphism of polymerase chain reaction–amplified regions. Result(s) Carriers of FSHB GG+ FSHR AA had higher FSH before pubertal onset (median 2.2 vs. 1.5 IU/L) and lower AMH (13.8 vs. 19.4 pmol/L) compared with carriers of other genotypes. In crude analysis, girls with FSHB GG+ FSHR AA entered puberty earlier, 9.7 vs. 10.6 years. However, the difference was no longer statistically significant after including interval-, right-, and left-censored data in a probit analysis. Conclusion(s) The combined effect of FSHB GG+ FSHR AA may potentiate the FSH pathway, which increases serum levels of FSH and reduces AMH. Common variations in genes regulating follicle growth may affect AMH levels independently of the number of resting primordial follicles.

Pubertal gynaecomastia is a clinical sign of an oestrogen-androgen imbalance, which occurs in 40-60% of adolescent Caucasian boys. In most cases no underlying endocrinopathy can be identified. A recent study reports higher plasma phthalate levels in Turkish boys with pubertal gynaecomastia. Therefore, we asked whether there was an association between concurrent measures of urinary phthalate metabolites and pubertal timing as well as the presence of gynaecomastia in otherwise healthy boys. We studied a total of 555 healthy boys (age 6.07-19.83 years) as part of the COPENHAGEN Puberty Study. Anthropometry and pubertal stages (PH1-6 and G1-5) were evaluated, and the presence of gynaecomastia was assessed. Non-fasting blood samples were analysed for serum testosterone and morning urine samples were analysed for the total content of 12 phthalate metabolites (MEP, MnBP, MiBP, MBzP, MEHP, MEHHP, MEOHP, MECPP, MiNP, MHiNP, MiONP and MCiOP) by LC-MS/MS. A statistically significant negative correlation was observed between chronological age and the urinary concentration of the sum of measured metabolites DEHP (∑DEHPm) (r = -0.164) and DiNP (∑DiNPm) (r = -0.224), respectively, and the sum of monobutyl phthalate (MBP) isomers (∑MBP((i+n))) (r = -0.139) (all with p0.01). In contrast urinary monoethyl phthalate concentration was positively correlated to age (r = 0.187, p0.01). The urinary levels of phthalate metabolites were not associated with age at pubertal onset, serum testosterone levels or presence of gynaecomastia. In conclusion, we did not find evidence of anti-androgenic effects of phthalates in our healthy boys. Thus, current phthalate exposure was not associated with pubertal timing, testosterone levels or with the presence of pubertal gynaecomastia in this cross-sectional study. However, longitudinal studies are needed to evaluate possible perinatal or long-term postnatal effects of phthalates on healthy boys.

To describe available markers of male puberty, discuss associations between adiposity and pubertal timing and to review recent evidence of a possible secular trend in male pubertal timing.An expert panel reviewing existing American pubertal data from boys in 2005 could not confirm a secular trend in male pubertal timing. National Health and Nutrition Examination Survey III findings have been confirmed by the National Institute of Child Health and Human Development study reporting a mean age of 10.4 years for Caucasian boys entering Tanner stage G2. Furthermore, the Copenhagen Puberty Study reported a 3 months decline in pubertal onset during a 15-year period (from 11.92 years in 1991 to 11.66 years in 2008).A negative association between obesity and early puberty was found in the National Institute of Child Health and Human Development study, in contrast to the positive association found in a Danish study. Other studies have not been able to document an association between prepubertal BMI and age at pubertal onset.Evaluation of Tanner stage and especially assessment of testicular volume should both be used in epidemiological studies. We speculate that the association between fat mass and pubertal timing may be nonlinear and recent studies may indicate a small decline in age at pubertal onset in boys.