Eighty-seven females followed by Meyer Children’s Hospital between October 2020 and March 2021 were enrolled. Of these, 54 children were evaluated for suspected precocious puberty (PP) and 33 children were enrolled as controls.
The study was conducted according to the Declaration of Helsinki and European Guidelines on Good Clinical Practice. Ethical approval was obtained from the Meyer Children’s University Hospital Ethics Committee (Paediatric Ethics Committee – Tuscany Region: number 228/2020 of 29 September 2020). Written informed consent was obtained from the parents of the participating patients after they had acknowledged their full understanding of the objectives of the research.
Subjects with melatonin-secreting tumors, genetic pathologies and/or established neurological or neuro-oncological pathologies were excluded from the study. We also excluded subjects under drug therapy with melatonin or drugs capable of interfering with the secretion or metabolism of melatonin.
Controls were selected and matched for age and sex from children investigated by the Allergology Unit of our Hospital. Only children who tested negative to tests for allergies were included.
Enrolled subjects were asked to take certain measures the day before the salivary test to avoid influencing melatonin levels. They were required to be in bed before 10.00 pm, not to use electronic devices (tablets, computers, cell phones, television, etc.) after 8 p.m and not to sleep with a light next to their bed or in the bedroom. All patients underwent an auxological evaluation and complete physical examination to record their height, weight, body mass index (BMI), growth velocity (HV), change in BMI in the last year, stage of pubertal development and rate of pubertal progression (according to Tanner's rating scale).
At the time of the visit a sample of 3 ml of saliva using a Salivette® cotton swab was collected. The sample was taken at least 30 minutes after a meal and the time was noted. Saliva samples were directly collected in clear sterile plastic tubes using sterile plastic straws. A 1-mL aliquot of each saliva sample was pipetted onto a Salivette® cotton swab (cotton saliva collection) and into clear sterile plastic tubes (passive saliva collection). All saliva samples were centrifuged at 1500× g for 5 min at room temperature and then frozen at -30°C until they were assayed.
Melatonin levels were analyzed in duplicate using commercially available ELISA kits (Direct Saliva Melatonin ELISA; Bühlmann Laboratories, Allschwil, Switzerland), and the mean values of the duplicates were used for analyzing the results. The kit sensitivity was 0.5 pg/mL. The intra- and inter-assay coefficients of variation were 12.6% and 22.9%, respectively.
Family and personal clinical data relevant to the study were collected (e.g., age at diagnosis of CPP, personal and family history for the presence of major diseases, family history for CPP).
Skeletal maturation (BA) was determined by radiographs of the left hand and wrist and then calculated according to the method of Greulich and Pyle.8 Pelvic ultrasound was also performed and the area (S) of the ovaries was calculated as follows: S = length x width × 0.8. Normal ovarian surface area is < 2 cm2 in prepubertal and 2 to 6 cm2 in pubertal subjects.9
All laboratory measurements were performed on blood samples collected after overnight fasting. Precocious puberty was defined as the development of pubertal changes before 8 years of age.10 We considered peak LH values of > 5 IU/L on GnRH in the presence of pubertal signs or a basal LH value of > 1.1 IU/L and a ratio of stimulated LH to stimulated FSH of > 1.0 combined with isolated and/or axillary hair growth accompanied by breast development 10, as indicative of activation of the hypothalamic GnRH pulse generator.
Statistical analysis
Data analysis was performed using the “Statistical Package for the Social Sciences for Windows” (SPSS, Inc, Chicago, IL, USA), version 13.0. Descriptive statistics are presented as numbers (percentages), median and range and mean ± SD values. Histograms and Shapiro-Wilk tests were used to verify the normality of continuous data. The variation of continuous variables at the beginning and end of observation was analysed by paired Student’s T for parametric data, while the Wilcoxon signed-rank test was used for nonparametric analysis. Categorical variables were compared by the χ2 test or Fisher exact test. The Pearson correlation test was used to determine correlation coefficients. All tests were two-sided, and values of p < 0.05 were considered statistically significant.