Abstract
Introduction
Although a well-known phenomenon, the hormonal mechanism of thelarche in the absence of gonadotropin activation remains unclear. To reveal the biological mechanisms of such thelarche, we assessed serum concentrations of several hormones potentially related to breast development, as well as clinical signs of androgen action.
Methods
A population-based sample of 175 girls aged 9–11 was divided into those without thelarche (Tanner breast stage 1) and those with thelarche (Tanner breast stage ≥2). The girls were further categorized based on basal serum luteinizing hormone (LH) using a cut-off of 0.3 IU/L. This resulted in three groups: 114 girls with low LH without thelarche, 32 girls with low LH and thelarche, and 29 girls with higher LH and thelarche. Serum concentrations of sex steroids and insulin-like growth factor-1 (IGF-1) and clinical signs of androgen action were assessed at ages 7–9 and 9–11 years.
Results
Girls with low LH and thelarche had higher levels of dehydroepiandrosterone sulfate (DHEAS) (0.80 vs. 0.60 µmol/L, p = 0.03 at 7–9 years; 0.97 vs. 0.72 µmol/L, p = 0.03 at 9–11 years), testosterone (0.18 vs. 0.14 nmol/L, p = 0.02; 0.27 vs. 0.22 nmol/L, p = 0.02), estrone (14.5 vs. 11.5 pmol/L, p = 0.02; 24.3 vs. 18.2 pmol/L, p = 0.002), and IGF-1 (28.6 vs. 23.6 nmol/L, p = 0.001; 35.9 vs. 31.1 nmol/L, p = 0.006) than girls with low LH without thelarche. Differences in DHEAS, testosterone, and IGF-1 diminished after further adjustment for body fat percentage. Girls with low LH and thelarche also had more clinical signs of androgen action at both ages than girls with low LH without thelarche. There were no statistically significant differences in hormonal concentrations at 7–9 years between girls with low LH and thelarche and those with higher LH and thelarche. However, at 7–9 years, girls with low LH and thelarche had more signs of androgen action than girls with higher LH and thelarche. At 9–11 years, girls with higher LH and thelarche had higher hormonal concentrations except for DHEAS than girls with low LH and thelarche.
Conclusion
Thelarche with low LH is associated with elevated DHEAS, testosterone, estrone, and IGF-1 concentrations, which is partly explained by BF%, suggesting that aromatization of adrenal androgens to estrogens in adipose tissue is one hormonal mechanism of thelarche in girls without gonadotropin activation.
Keywords: Thelarche, Isolated thelarche, Sex steroids, Adrenal androgens, Adrenarche
Introduction
Thelarche, the appearance of a breast bud, is typically the first clinical sign of central puberty in girls [1, 2]. When thelarche occurs without activation of the hypothalamic-pituitary-gonadal axis and other clinical signs of puberty, including pubic hair growth or increased growth velocity, it is called isolated thelarche [3]. Thelarche is considered premature if it occurs before the age of 8 years [3]. The prevalence of premature thelarche has been reported to be 5–9% in girls aged 2–8 years [4, 5], which makes it a common reason for visiting a physician and referral to a pediatrician. Interestingly, over the last decades, the average age at thelarche has declined more than that of menarche, and this temporal trend in thelarche age appears independent of changes in serum estradiol and gonadotropin levels [6].
Several reproductive and metabolic hormones regulate the growth and differentiation of breast tissue in females [7]. Estrogen promotes ductal growth, whereas progesterone stimulates alveolar development in the breast. Growth hormone and insulin-like growth factor-1 (IGF-1) further mediate and amplify the effects of estrogen and progesterone on breast maturation [7]. Importantly, sex steroids are synthetized not only in the gonads by also in the adrenal cortex, which serves as an additional source of steroid hormone production [8]. In adrenarche, the production of androgen precursors, mainly dehydroepiandrosterone (DHEA), its sulfate (DHEAS), and androstenedione, is increased owing to the developmental maturation of the inner layer of the adrenal cortex, zona reticularis [9]. These sex steroids are inactive or weak androgens that can be converted into biologically active androgens and estrogens in adipose and other peripheral tissues [9]. Consequently, adrenal androgens may play a role in breast development in females.
The hormonal mechanisms underlying isolated thelarche remain unclear, with the exception of thelarche in early infancy, which is attributed to transient activation of the hypothalamic-pituitary-gonadal axis [10]. This lack of understanding is largely due to the limited number of studies examining the roles of circulating reproductive and metabolic hormones in breast development among girls with premature or isolated thelarche [11–14]. To fill this knowledge gap, we compared serum concentrations of several reproductive and metabolic hormones, including DHEAS, androstenedione, testosterone, estrone, estradiol, progesterone, and IGF-1, as well as the occurrence of clinical signs of androgen action among 32 girls with serum luteinizing hormone (LH) <0.3 IU/L and thelarche and among 114 girls with serum LH <0.3 IU/L without thelarche. We hypothesized that thelarche in the absence of clear gonadotropin activation is a consequence of the conversion of adrenal androgens to estrogens in peripheral tissues, particularly adipose tissue.
Methods
Study Design and Participants
The present analyses are based on data from the Physical Activity and Nutrition in Children (PANIC) study (ClinicalTrials.gov NCT01803776), which is a long-term lifestyle intervention study in a representative population sample of primary-school children from the city of Kuopio, Finland [15]. We invited 736 children aged 7–9 years who started the first grade of primary school between 2007 and 2009 to participate in the study. Altogether, 512 children (248 girls) aged 7–9 years, who represented 70% of those invited, accepted the invitation and attended the baseline examinations between 2007 and 2009. The participants did not differ in terms of sex, age, height standard deviation score (SDS), or body mass index (BMI) SDS from all children who started the first grade in the city of Kuopio between 2007 and 2009.
Altogether, 438 children (218 girls) aged 9–11 years participated in the 2-year follow-up examinations between 2009 and 2012. The median (interquartile range) follow-up time was 2.1 (2.1–2.2) years. Of the 218 girls aged 9–11 years who attended the 2-year follow-up examinations, 39 were excluded because the use of inhaled or oral corticosteroids may affect adrenal function and thereby confound the associations studied. The remaining 179 girls aged 9–11 years were categorized into those with low serum LH (<0.3 IU/L) and thelarche, low serum LH but no thelarche, and higher serum LH (≥0.3 IU/L) and thelarche (Fig. 1). We used the serum LH of 0.3 IU/L as a cut-off as it has been shown to have a specificity of 98% to separate prepubertal and pubertal girls with Tanner B2 [16]. We did not include four girls who had higher LH but no thelarche, leaving 175 girls for the statistical analyses.
Fig. 1.
Flow diagram for study participants.
Assessment of Body Size and Composition
Body height was measured three times using a calibrated wall-mounted stadiometer, the children standing in the Frankfurt plane without shoes to an accuracy of 0.1 cm. The mean of the nearest two height values was used in the statistical analyses. Body weight was measured twice using a scale incorporated into the InBody® 720 bioelectrical impedance device, (Biospace, Seoul, South Korea) the children having fasted for 12 h, having emptied the bladder, and wearing light underwear to an accuracy of 0.1 kg. The mean of the two weight values was used in the statistical analyses. BMI was calculated by dividing weight (in kg) by the square of height (in m). Height SDS and BMI-SDS were assessed using the Finnish growth reference data [17]. Body fat percentage (BF%) was measured using the Lunar® dual-energy X-ray absorptiometry device (Lunar Prodigy Advance, GE Medical Systems, Madison, WI, USA), the children being in the non-fasting state, having emptied the bladder, and being in light clothing with all metal objects removed. Information on gestational weeks at birth and birth size was obtained from the register of Kuopio University Hospital. Birth length SDS and birth weight SDS were calculated using Finnish growth references for newborns [18].
Assessment of Secondary Sexual Characteristics and Clinical Signs of Androgen Action
Physicians specifically trained to assess secondary sexual characteristics evaluated breast development by inspection and palpation (Tanner stages B1-5) and pubic hair growth by inspection (Tanner stages P1-5) [19]. Thelarche was defined as breast development stage ≥ B2. The same trained physicians also assessed clinical signs of androgen action (adult type body odor, oily hair, acne, comedones, and axillary or pubic hair). Additionally, the caregivers filled out a questionnaire to provide information on their children’s body odor and oily hair.
Biochemical Measurements
Venous blood samples were taken between 8.30 and 9.30 a.m. following a 12-h overnight fast. Blood was centrifuged, and separated serum was stored at −75°C until biochemical analyses. Serum LH concentrations were analyzed using an electrochemiluminescence immunoassay (Roche Diagnostics GmbH, Mannheim, Germany) with the intra- and inter-assay coefficients of variation (CVs) of 1.6–2.2% and 0.7–1.2%, respectively, and the detection limit of 0.1 IU/L. Serum DHEAS concentrations were analyzed using an enzyme-linked immunosorbent assay (Alpha Diagnostic International Inc., San Antonio, TX, USA) with the intra- and inter-assay CVs of 7.5–11.5% and 7.0–11.0%, respectively, and the detection limit of 0.014 µmol/L. Biochemical adrenarche was defined as the serum DHEAS concentration of ≥1 µmol/L [9]. Serum progesterone, androstenedione, testosterone, estrone, and estradiol concentrations were analyzed by the ultrasensitive liquid chromatography – tandem mass spectrometry (LC-MS/MS) method [20]. The calibration range was 0.013–13.322 nmol/L for progesterone, 0.013–33.182 nmol/L for androstenedione, 0.003–13.300 nmol/L for testosterone, 1.33–6,591.00 pmol/L for estrone, and 6.68–13,362.00 pmol/L for estradiol. The detection limit of serum estradiol was 6.68 pmol/L. If the measured serum estradiol concentration was below this limit, the value of 6.68 pmol/L was used in the statistical analyses. Serum IGF-1 concentrations were determined using an enzyme-linked immunosorbent assay (Mediagnost, Reutlingen, Germany) with the intra- and inter-assay CVs of 5.1–6.6% and 7.7–9.2%, respectively.
Statistical Methods
Serum progesterone, DHEAS, androstenedione, testosterone, estrone, and estradiol concentrations had skewed distributions and therefore natural logarithmic transformation was used in the statistical analyses. The statistical analyses were conducted using the SPSS software, Version 27 (IBM Corp., Armonk, NY, USA). Differences in basic characteristics between the groups were studied by the T-test for independent samples for continuous variables and by the Chi-square test for categorical variables except that for Tanner stage ≥ B2 at age 7–9 years where Fisher’s test was used. Differences in serum hormone concentrations were analyzed using General Linear Models. To account for possible confounding due to age and body fat content, we adjusted the data for age (model 1) and additionally for BF% at the same age (model 2). Differences between the groups were considered statistically significant if the p value <0.05.
Results
Clinical Characteristics of Girls
Of all 175 girls included in the statistical analyses, 146 (83%) had serum basal LH <0.3 IU/L and 29 (17%) had serum basal LH ≥0.3 IU/L at age 9–11 (Table 1, Fig. 1). Of the 146 girls with low LH, 114 (78%) had no thelarche and 32 (22%) had thelarche at age 9–11 (Table 1; Fig. 1). Girls with low LH and thelarche were slightly older, taller, and heavier and had higher BF% at ages 7–9 and 9–11 than girls with no thelarche (Table 1). Four (14%) girls with low LH and thelarche had Tanner breast stage ≥ B2 already at age 7–9, whereas only one (1%) girl with no thelarche had Tanner breast stage ≥ B2 already at age 7–9 indicating transient thelarche. Girls with low LH and thelarche were also more likely to have pubic hair at age 9–11 and other clinical signs of androgen action at ages 7–9 and 9–11 than girls with no thelarche.
Table 1.
Clinical characteristics of the study groups
| | Girls with LH <0.3 IU/L without thelarche (n = 114) | Girls with LH <0.3 IU/L and thelarche (n = 32) | p value1 | Girls with LH ≥0.3 IU/L (n = 29) | p value2 |
|---|---|---|---|---|---|
| Birth weight SDS | 0.0 (1.1) | 0.1 (1.0) | 0.58 | 0.0 (1.0) | 0.66 |
| Birth length SDS | −0.1 (1.0) | −0.1 (1.1) | 0.78 | −0.1 (0.8) | 0.76 |
| Gestational age at birth, wk | 40.0 (1.6) | 40.0 (1.9) | 0.95 | 40.7 (1.5) | 0.21 |
| Age | |||||
| 7–9 years | 7.5 (0.4) | 7.8 (0.3) | 0.001 | 7.9 (0.4) | 0.17 |
| 9–11 years | 9.6 (0.4) | 9.9 (0.4) | 0.001 | 10.0 (0.4) | 0.25 |
| Body weight, kg | |||||
| 7–9 years | 24.7 (3.8) | 31.6 (6.3) | <0.001 | 28.7 (4.3) | 0.04 |
| 9–11 years | 30.9 (5.1) | 42.0 (8.9) | <0.001 | 36.8 (5.3) | 0.009 |
| BMI-SDS | |||||
| 7–9 years | −0.5 (1.0) | 0.7 (1.0) | <0.001 | 0.0 (1.0) | 0.009 |
| 9–11 years | −0.4 (0·9) | 0.8 (0.9) | <0.001 | 0.0 (0.8) | <0.001 |
| Body fat percentage | |||||
| 7–9 years | 21.7 (7.0) | 32.5 (7.8) | <0.001 | 25.1 (8.6) | 0.001 |
| 9–11 years | 24.0 (7.3) | 36.7 (6.9) | <0.001 | 25.8 (8.8) | <0.001 |
| Body height, m | |||||
| 7–9 years | 1.26 (0.05) | 1.32 (0.05) | <0.001 | 1.32 (0.05) | 0.95 |
| 9–11 years | 1.37 (0.05) | 1.45 (0.06) | <0.001 | 1.45 (0.05) | 0.60 |
| Body height SDS | |||||
| 7–9 years | −0.1 (0.9) | 0.8 (1.0) | <0.001 | 0.6 (0.9) | 0.49 |
| 9–11 years | −0.2 (0.8) | 0.7 (0.9) | <0.001 | 0.7 (0.9) | 0.89 |
| Tanner stage ≥ B2 at age 7–9 years, n (%) | 1 (1) | 4 (13) | <0.007 | 3 (10) | 0.54 |
| Pubic hair, n (%) | |||||
| 7–9 years | 1 (1) | 1 (3) | 0.33 | 0 (0) | 0.34 |
| 9–11 years | 10 (9) | 8 (25) | 0.01 | 10 (34) | 0.42 |
| Clinical signs of androgen action3, n (%) | |||||
| 7–9 years | 26 (23) | 15 (47) | 0.007 | 5 (17) | 0.01 |
| 9–11 years | 66 (58) | 29 (91) | 0.001 | 26 (90) | 0.90 |
| Biochemical adrenarche (DHEAS ≥1 µmol/L), n (%) | |||||
| 7–9 years | 17 (15) | 9 (28) | 0.08 | 9 (31) | 0.67 |
| 9–11 years | 25 (22) | 12 (38) | 0.06 | 11 (38) | 0.95 |
Data are means (standard deviations) for continuous variables from the T test for independent samples and numbers (percentages) for categorical variables from the Chi-square test except that for Tanner stage ≥ B2 at age 7–9 years where Fisher’s test was used.
The classification into groups based on LH levels and thelarche was performed at the age of 9–11 years.
LH, luteinizing hormone; SDS, standard deviation score; BMI, body mass index; B2, beginning of breast bud formation and areola widening15; DHEAS, dehydroepiandosterone sulfate.
p value for differences in characteristics between girls with LH <0.3 IU/L without thelarche and girls with LH <0.3 IU/L and thelarche.
p value for differences in characteristics between girls with LH <0.3 and thelarche and girls with LH ≥0.3 IU/L and thelarche.
Adult-type body odor, oily hair, acne, comedones, and axillary or pubic hair.
Girls with low LH and thelarche were slightly heavier and had higher BF% at age 9–11 compared to girls with higher LH and thelarche (Table 1). They were also more likely to have clinical signs of androgen action at age 7–9. There were differences in serum reproductive and metabolic hormone concentrations between girls with low LH and thelarche and girls with no thelarche.
Girls with low LH and thelarche had higher age-adjusted serum DHEAS, testosterone, estrone, and IGF-1 at ages 7–9 and 9–11 and lower serum progesterone at age 9–11 compared to girls with no thelarche (Table 2). None of these differences, except that of serum estrone at age 9–11, between these two groups remained statistically significant after further adjustment for BF%. There was no statistically significant difference in serum androstenedione or estradiol at age 7–9 or 9–11 between girls with low LH and thelarche and those with no thelarche, adjusted for age. However, the difference in serum estradiol at age 9–11 between these two groups became stronger and statistically significant after additional adjustment for BF%.
Table 2.
Serum reproductive hormones and IGF-1 concentrations of girls with LH <0.3 IU/L without thelarche and girls with LH <0.3 IU/L and thelarche adjusted for age (model 1) and age and body fat percentage (model 2)
| | Model 1 | Model 2 | ||||
|---|---|---|---|---|---|---|
| girls with lh <0.3 IU/L without thelarche (n = 114) | girls with lh <0.3 IU/L and thelarche (n = 32) | p value | girls with lh <0.3 IU/L without thelarche (n = 114) | girls with lh <0.3 IU/L and thelarche (n = 32) | p value | |
| DHEAS, µmol/L | ||||||
| 7–9 years | 0.60 (0.52–0.68) | 0.80 (0.66–0.95) | 0.03 | 0.63 (0.54–0.71) | 0.74 (0.57–0.91) | 0.46 |
| 9–11 years | 0.72 (0.62–0.82) | 0.97 (0.78–1.16) | 0.03 | 0.72 (0.62–0.83) | 0.98 (0.76–1.20) | 0.17 |
| Androstenedione, nmol/L | ||||||
| 7–9 years | 0.89 (0.77–1.00) | 1.12 (0.90–1.34) | 0.13 | 0.91 (0.78–1.03) | 1.04 (0.78–1.30) | 0.38 |
| 9–11 years | 1.31 (1.16–1.46) | 1.58 (1.28–1.87) | 0.08 | 1.29 (1.12–1.45) | 1.68 (1.33–2.03) | 0.08 |
| Testosterone, nmol/L | ||||||
| 7–9 years | 0.14 (0.13–0.15) | 0.18 (0.15–0.20) | 0.02 | 0.14 (0.13–0.16) | 0.16 (0.13–0.20) | 0.29 |
| 9–11 years | 0.22 (0.20–0.24) | 0.27 (0.23–0.31) | 0.02 | 0.22 (0.20–0.24) | 0.28 (0.23–0.33) | 0.07 |
| Estrone, pmol/L | ||||||
| 7–9 years | 11.5 (10.4–12.6) | 14.5 (12.4–16.5) | 0.02 | 11.5 (10.4–12.7) | 13.9 (11.5–16.3) | 0.14 |
| 9–11 years | 18.2 (16.7–19.8) | 24.3 (21.2–27.3) | 0.002 | 17.8 (16.1–19.5) | 25.6 (22.0–29.2) | 0.002 |
| Estradiol, pmol/L | ||||||
| 7–9 years | 7.7 (7.0–8.5) | 8.9 (7.5–10.3) | 0.11 | 7.7 (7.0–8.5) | 8.9 (7.5–10.3) | 0.22 |
| 9–11 years | 12.4 (10.6–14.1) | 15.6 (12.1–19.1) | 0.11 | 12.0 (10.0–13.9) | 17.0 (12.8–21.1) | 0.03 |
| IGF-1, nmol/L | ||||||
| 7–9 years | 23.6 (22.2–25.0) | 28.6 (26.0–31.2) | 0.001 | 24.1 (22.7–25.6) | 26.9 (24.0–29.9) | 0.12 |
| 9–11 years | 31.1 (29.5–32.6) | 35.9 (32.9–38.9) | 0.006 | 31.5 (29.8–33.1) | 34.6 (31.1–38.1) | 0.14 |
| Progesterone, nmol/L | ||||||
| 7–9 years | 0.15 (0.12–0.18) | 0.12 (0.06–0.18) | 0.30 | 0.15 (0.12–0.19) | 0.11 (0.04–0.18) | 0.23 |
| 9–11 years | 0.15 (0.12–0.19) | 0.12 (0.05–0.19) | 0.02 | 0.15 (0.12–0.19) | 0.13 (0.05–0.21) | 0.41 |
Data are means (95% confidence intervals) from general linear models adjusted for age (model 1) and additionally for body fat percentage at the same age (model 2).
The classification into groups based on LH levels and thelarche was performed at the age of 9–11 years.
BF%, body fat percentage; DHEAS, dehydroepiandrosterone sulfate; IGF-1, insulin-like growth factor 1.
Differences in Serum Reproductive and Metabolic Hormone Concentrations between Girls with Low LH and Thelarche and Girls with Higher LH and Thelarche
Girls with higher LH and thelarche had higher serum androstenedione, testosterone, estrone, estradiol, progesterone, and IGF-1 at age 9–11 compared to girls with low LH and thelarche adjusted for age (Table 3). All these differences in serum reproductive and metabolic hormones, except progesterone, between these two groups at age 9–11 remained statistically significant after further adjustment for BF%. There was no statistically significant difference in any serum hormone at age 7–9 or serum DHEAS at age 7–9 or 9–11 between girls with higher LH and thelarche and girls with low LH and thelarche adjusted for age or after additional adjustment for BF%.
Table 3.
Serum reproductive hormones and IGF-1 concentrations of girls with LH <0.3 IU/L and thelarche and girls with LH ≥0.3 IU/L and thelarche adjusted for age (model 1) and age and body fat percentage (model 2)
| | Model 1 | Model 2 | ||||
|---|---|---|---|---|---|---|
| girls with lh <0.3 IU/L and thelarche (n = 32) | girls with lh ≥0.3 IU/L and thelarche (n = 29) | p value | girls with lh <0.3 IU/L and thelarche (n = 32) | girls with lh ≥0.3 IU/L and thelarche (n = 29) | p value | |
| DHEAS, µmol/L | ||||||
| 7–9 years | 0.83 (0.64–1.02) | 0.86 (0.65–1.06) | 0.83 | 0.79 (0.29–0.99) | 0.90 (0.68–1.12) | 0.65 |
| 9–11 years | 0.96 (0.69–1.24) | 1.11 (0.83–1.40) | 0.63 | 0.89 (0.60–1.19) | 1.20 (0.88–1.52) | 0.52 |
| Androstenedione, nmol/L | ||||||
| 7–9 years | 1.17 (0.84–1.49) | 1.11 (0.77–1.45) | 0.43 | 1.06 (0.72–1.40) | 1.23 (0.87–1.59) | 0.87 |
| 9–11 years | 1.59 (1.25–1.92) | 2.66 (2.31–3.01) | <0.001 | 1.82 (1.45–2.18) | 2.41 (2.00–2.79) | 0.02 |
| Testosterone, nmol/L | ||||||
| 7–9 years | 0.18 (0.15–0.22) | 0.17 (0.13–0.20) | 0.17 | 0.17 (0.14–0.20) | 0.18 (0.15–0.22) | >0.99 |
| 9–11 years | 0.28 (0.22–0.34) | 0.47 (0.41–0.54) | <0.001 | 0.32 (0.26–0.39) | 0.43 (0.36–0.50) | 0.02 |
| Estrone, pmol/L | ||||||
| 7–9 years | 14.5 (12.2–16.9) | 13.4 (10.9–15.8) | 0.40 | 13.6 (11.2–16.0) | 14.4 (11.8–16.9) | 0.75 |
| 9–11 years | 25.2 (18.9–31.5) | 54.6 (48.1–61.1) | <0.001 | 28.5 (21.4–35.5) | 51.2 (43.8–58.5) | <0.001 |
| Estradiol, pmol/L | ||||||
| 7–9 years | 8.7 (6.9–10.5) | 10.1 (8.2–12.0) | 0.18 | 8.6 (6.7–10.5) | 10.2 (8.2–12.2) | 0.11 |
| 9–11 years | 17.4 (1.6–33.1) | 95.8 (79.2–112.4) | <0.001 | 22.1 (4.1–40.0) | 80.6 (71.5–109.7) | <0.001 |
| IGF-1, nmol/L | ||||||
| 7–9 years | 28.4 (25.5–31.2) | 26.7 (25.5–31.2) | 0.44 | 27.9 (24.9–30.9) | 27.3 (24.0–30.6) | 0.79 |
| 9–11 years | 36.1 (31.9–40.3) | 49.4 (45.0–53.8) | <0.001 | 36.6 (31.8–41.4) | 48.9 (43.9–53.9) | 0.002 |
| Progesterone, nmol/L | ||||||
| 7–9 years | 0.12 (0.02–0.22) | 0.18 (0.08–0.29) | 0.66 | 0.09 (−0.02–0.19) | 0.22 (0.11–0.33) | 0.27 |
| 9–11 years | 0.10 (0.06–0.13) | 0.15 (0.12–0.19) | 0.006 | 0.11 (0.07–0.15) | 0.14 (0.10–0.18) | 0.48 |
Data are means (95% confidence intervals) from general linear models adjusted for age (model 1) and additionally for body fat percentage at the same age (model 2).
The classification into groups based on LH levels and thelarche was performed at the age of 9–11 years.
LH, luteinizing hormone; BF%, body fat percentage; DHEAS, dehydroepiandrosterone sulfate; IGF-1, insulin-like growth factor 1.
Discussion
In the current study, girls with low LH and thelarche had higher serum DHEAS, testosterone, estrone, and IGF-1 concentrations at ages 7–9 and 9–11 years compared to girls with no thelarche. The differences in serum DHEAS, testosterone, and IGF-1 levels between these two groups were partly explained by BF%. Girls with low LH and thelarche were also more likely to present clinical signs of androgen action at ages 7–9 and 9–11 years than girls with no thelarche.
There are few previous studies on circulating androgen concentrations or adrenarche in relation to thelarche among prepubertal girls [11, 13, 14, 21]. The results of these studies are inconsistent due to differences in study designs, participant characteristics, and hormonal analyses. In a Croatian study among girls aged 14 months to 7 years, those with premature thelarche had higher serum levels of DHEA, but not androstenedione, than those without thelarche [11]. This suggests that DHEA, which is mainly produced by the adrenal cortex, may serve as a precursor for peripheral conversion of androgens to estrogens. A Chilean study reported higher plasma DHEAS levels among girls with thelarche occurring prematurely at the age of under 8 years than among girls with thelarche at age over 8 years [13]. Moreover, serum DHEAS and androstenedione levels were found to rise 1–2 years before thelarche among US girls [21]. Consistent with the results of these studies, we found that serum levels of DHEAS, a weak androgen that acts as a precursor for more potent androgens and is commonly used to assess biochemical adrenarche, were higher among girls with low LH and thelarche than among girls with no thelarche. This difference was partly explained by BF%, which is consistent with the results of previous studies linking childhood obesity to increased circulating DHEAS levels [9]. Indeed, there is a well-established positive correlation between serum DHEAS concentration and adiposity [22]. Moreover, we have previously shown that children with adiposity have increased likelihood of having clinical manifestations of adrenarche [23]. Our findings in the current study indeed reflect this dual role of adiposity: increased aromatization of adrenal androgens to estrogens and adiposity's effect on adrenocortical function. Serum levels of testosterone, but not androstenedione, were also higher among girls with low LH and thelarche than among girls with no thelarche, which was again partly explained by BF%. Consistent with these findings on DHEAS and testosterone, girls with low LH and thelarche were also more likely to present clinical signs of androgen action, which are typically seen in adrenarche. All these observations together suggest that adrenal androgens play an important role in thelarche without gonadotropin activation. It is important to note that, in adults, adipose tissue has been shown to possess a limited capacity for de novo androgen synthesis from cholesterol [24]. However, there are no corresponding data available for the pediatric population.
Adrenal androgens can be converted to estrone and estradiol in adipose and other peripheral tissues [9]. Consistent with our findings, a study among US girls reported that serum levels of DHEAS, androstenedione, and estrone rose earlier than serum estradiol before the occurrence of thelarche [21]. In the Croatian study, there was no difference in serum estradiol or estrone levels between prepubertal girls with thelarche and those without it at age 14 months to 7 years [11]. However, in the Chilean study, girls with thelarche occurring prematurely at age below 8 years had twice the likelihood of having serum estradiol levels at least 5 pg/mL (18.3 pmol/L) at age 6.7 years compared to girls who experienced thelarche at age above 8 years [13]. We found higher serum estradiol levels among girls with low LH and thelarche than among girls with no thelarche at age 9–11 years after controlling for age and BF%. They also had higher serum estrone levels at ages 7–9 and 9–11 years. However, the difference in serum estrone levels at age 7–9 years but not at age 9–11 years between these two groups was partly explained by BF%. Peripheral estrogen production is known to favor estrone over estradiol. In the steroid synthesis pathway, androstenedione serves as the main precursor, which can be aromatized to testosterone or directly to estrone, whereas estradiol can be formed from testosterone and estrone [9, 25]. However, in this steroid synthesis pathway, minor amounts of estradiol are also produced. It is plausible that adipose tissue acts as a confounder that masks low-level gonadal estradiol production in girls with low LH and thelarche, explaining why estradiol levels became significantly higher among girls with low LH and thelarche than among girls with no thelarche only after accounting for BF%. In conclusion, our observations suggest that estrogens, particularly estrone, contribute to breast development during thelarche, prior to activation of the hypothalamus-pituitary-gonadal axis.
IGF-1 is known to regulate breast development by mediating the effects of estrogen and progesterone [7]. We have previously found higher serum IGF-1 levels among girls with premature adrenarche than among girls without it [26]. However, to our knowledge, there are no published reports comparing serum IGF-1 levels between prepubertal girls with thelarche and those without it. In the present study, we observed higher serum IGF-1 levels among girls with low LH and thelarche than among those with no thelarche at ages 7–9 and 9–11 years and that these differences were partly explained by BF%. These findings suggest that IGF-1 regulates breast development not only in puberty but also in isolated thelarche and this may be influenced by body fat content.
Progesterone, which is a metabolite in the steroid synthesis pathway and known to promote breast alveolar development, has not previously been found to be associated with premature thelarche or adrenarche [11, 27]. In our study, serum progesterone levels were slightly lower at age 9–11 years among girls with low LH and thelarche than among girls with no thelarche. This difference was partly explained by BF%. This finding may indicate that progesterone is more efficiently converted to adrenal androgens, leading to slightly lower serum levels of progesterone in girls with low LH and thelarche.
We found higher serum levels of several reproductive hormones, including androstenedione, testosterone, estrone, estradiol, and progesterone, among girls with higher LH and thelarche than among girls with low LH and thelarche at age 9–11 years. These differences are as expected, due to activation of the hypothalamus-pituitary-gonadal axis in girls with higher LH and thelarche. These findings in serum hormone levels also support our decision to use the basal serum LH cut-off 0.3 IU/L to distinguish prepubertal girls from pubertal ones. We also observed higher serum IGF-1 levels among girls with LH ≥0.3 IU/L and thelarche than among girls with LH <0.3 IU/L and thelarche at age 9–11 years. These differences in serum levels of reproductive and metabolic hormones, except progesterone, between girls with LH ≥0.3 IU/L and thelarche and girls with LH <0.3 IU/L and thelarche were independent of BF%. Interestingly, girls with LH <0.3 IU/L and thelarche were more likely to have clinical signs of androgen action at age 7–9 than girls with LH ≥0.3 IU/L and thelarche. This may be partly explained by the higher body fat content among girls with LH <0.3 IU/L and thelarche than among girls with LH ≥0.3 IU/L and thelarche. These findings suggest that isolated thelarche is associated with clinical signs of androgen action and provide further evidence that isolated thelarche is another presentation of adrenarche.
The strengths of our study include the opportunity to examine a representative population sample of girls, the use of 2 time points for clinical and biochemical evaluation, the clinical assessment of breast development by physicians specifically trained to assess secondary sexual characteristics, and the use of the ultrasensitive LC-MS/MS method to measure serum androgen and estrogen concentrations. The main limitations of our study are the lack of precise information on the exact timing of thelarche and that girls with low LH without thelarche were younger than those with low LH and thelarche or those with higher LH and thelarche. We therefore controlled age in the statistical analyses. We used basal serum LH concentration (cut-off 0.3 IU/L) as a feasible and widely applied method to assess the onset of central puberty in larger study samples [28] instead of the gonadotropin-releasing hormone stimulation test. However, it should be acknowledged that the pubertal LH cut-offs used vary across studies. We did not study basal follicle-stimulating hormone (FSH) levels or the LH/FSH ratio, as these do not significantly improve sensitivity over basal LH alone in girls [29]. Ovarian ultrasound may have provided additional value to distinguish pubertal gonadal activation, but this was not included in the current study conducted in a population-based sample of children. Another limitation is that DHEAS was analyzed separately using an immunological method, as the LC-MS/MS method used for other steroids does not permit co-extraction of DHEAS. Therefore, DHEAS was analyzed separately using a well-established immunological method.
In conclusion, our findings suggest that girls with LH <0.3 IU/L and thelarche exhibit higher DHEAS production and more efficient conversion of DHEA to potent androgens and subsequently to estrogens, particularly estrone, compared to girls with LH <0.3 IU/L and no thelarche. These alterations in sex steroid metabolism appear to be partly mediated by body fat content, highlighting the role of adipose tissue in extra-gonadal steroidogenesis. These observations are consistent with a mechanism involving aromatization of adrenal-derived androgens to estrogens in adipose tissue, which could be one hormonal mechanism underlying isolated thelarche. However, further longitudinal studies are needed to confirm our findings and determine why some girls develop thelarche without gonadotropin activation while others do not. Subtle differences in estrogen receptor or aromatase enzyme function, for example, may explain this variation.
Acknowledgments
We are grateful to all children and their families for participating in the PANIC study and colleagues in our study group for their assistance during this study. We also thank Biocenter Finland and Biocenter Kuopio for supporting the mass spectrometry laboratory of the School of Pharmacy at the University of Eastern Finland.
Statement of Ethics
The study protocol was approved by the Research Ethics Committee of the Hospital District of Northern Savo in 2006 (Statement 69/2006). The children provided assent and their caregivers written informed consent to participate in the PANIC study. The study has been carried out in accordance with the principles of the Declaration of Helsinki as revised in 2008. The study participants provided assent and their caregivers written informed consent to participate in the PANIC study. The study has been carried out in accordance with the principles of the Declaration of Helsinki as revised in 2008.
Conflict of Interest Statement
Raimo Voutilainen was a member of the journal’s Editorial Board at the time of submission. The other authors have no conflicts of interest to declare.
Funding Sources
The Ministry of Education and Culture of Finland and the Research Council of Finland have been the most important funders for the PANIC study and the Doctoral School of the University of Eastern Finland for Saija Burtsov’s work. These funders had no role in the design, data collection, data analysis, and reporting of this study.
Author Contributions
S.B., A.V., S.S., M.H., S.A. R.V., J.J., and T.L. participated in substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data. S.B., A.V., S.S., M.H., S.A. A.M., R.V., J.J., and T.L participated in drafting the article or revising it critically for important intellectual content. All authors gave their final approval for this version to be published.
Funding Statement
The Ministry of Education and Culture of Finland and the Research Council of Finland have been the most important funders for the PANIC study and the Doctoral School of the University of Eastern Finland for Saija Burtsov’s work. These funders had no role in the design, data collection, data analysis, and reporting of this study.
Data Availability Statement
The PANIC study is still ongoing, and therefore the data are not fully anonymized and not openly available. Part of the data can be shared by request with researchers who provide a scientifically and ethically sound proposal for use. Such proposals should be sent to the Principal Investigator of the PANIC study (timo.lakka@uef.fi).
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The PANIC study is still ongoing, and therefore the data are not fully anonymized and not openly available. Part of the data can be shared by request with researchers who provide a scientifically and ethically sound proposal for use. Such proposals should be sent to the Principal Investigator of the PANIC study (timo.lakka@uef.fi).
