The study population consisted of 25 Caucasian children (11 males), aged 9.8 ± 3.5 years (range 4.2-18.7) with untreated idiopathic SH. The diagnosis of idiopathic SH was made on the basis of the following criteria: serum TSH concentration between 4.5 mU/l and the conventional limit of 10 mU/l with a serum free thyroxine (FT4) concentration within the reference range, along with the absence of anti-thyroglobulin (Tg-Ab) and anti-thyroperoxidase antibodies (TPO-Ab), normal echogenicity of the parenchyma on thyroid ultrasound and adequate urinary iodine excretion. The criteria for enrollment also included the persistence of a similar biochemical pattern of SH for at least 2 years prior to entering into the study and the absence of palpable goiter or symptoms related to hypothyroidism.
In 13/25 SH children, thyroid function measurement had been made for short stature (5 cases), obesity (1 case) or familial history of thyroid diseases (7 cases). In the remaining 12 cases, TSH measurement had been performed as part of a check-up. From the first finding of SH until the time of this study, all children were periodically followed at our center. At enrollment in the study, the duration of SH was 4.6 ± 2.8 (range 2.0-13.3) years. Six/25 subjects were pubertal. None of them was ever been treated with L-thyroxine. Twenty-five healthy euthyroid children (14 males), matched for age, sex, and pubertal status, who had undergone a check-up including also TSH measurement participated in the study as controls.
At the entry into the study, all subjects underwent to clinical examination, anthropometric measurements, determination of urinary iodine, fasting blood sample for calcium (Ca), phosphorus (P), alkaline phosphates (ALP), parathyroid hormone (PTH), TSH, FT4, thyroglobuline (Tg), Tg-Ab, TPO-Ab, insulin-like growth factor 1 (IGF1), bone age (BA) assessment and bone status evaluation. Bone maturation was evaluated using the Greulich and Pyle method and was expressed as BA/chronological age (CA) ratio. Moreover, all participants answered questionnaires on lifestyle and health related topics including questions on physical activity, smoking habits, intake of calcium, former or current diseases and use of medications.
The study protocol was approved by the Local Ethical Committee and written informed consent to participate into the study was obtained from the all subjects’ parents when the chronological age was lower than 18 and directly from each older subject.
Standing height was measured using a mechanical stadiometer to the nearest 0.1 cm and body weight was measured with a mechanical balance to the nearest 0.1 kg. The body mass index (BMI) was calculated as weight/height2. Parental height was measured and target height (TH) was calculated according to Tanner et al. . Height, TH and BMI were also expressed as Z-score, adjusted for age and gender in accordance with Italian standards .
A venous blood sample was drawn from all participants at 8.00 in the morning after a 12-hour fast. TSH, FT4, Tg, Tg-Ab, TPO-Ab and PTH serum concentrations were measured by electrochemiluminescence immunoassay (ECLIA) using a commercial kit (Elecsys ecobas e. Roche Diagnostics) (reference ranges: TSH, 0.3–4.2 mU/l; FT4, 0.9–1.7 ng/dl; Tg, 0–50 ng/ml; Tg-Ab, 0–115 mIU/l; TPO-Ab, 0–34 mIU/l; PTH, 10–75 pg/ml). Serum IGF1 levels were measured using a two-site IRMA kit (Diagnostics System Laboratories, Inc., Webster, TX, USA). The IGF1 intra- and inter-assay coefficients of variation (CV) were 3.4 and 8.2% respectively. The values obtained were standardized by age and sex and expressed in SDS. Urine iodine levels were analyzed with an automated system (Autoanalyzer 3 system, Bran CLuebbe GmbH, Nordestedt, Germany) (reference range: 100–200 mg/l). The other parameters were measured by a standardized automatic colorimetric method using the Cobas Integra 400 Analyzer (USA).
In all subjects bone status was evaluated by DXA, the most widespread diagnostic tool to assess bone health, and by QUS. Bone mineral density was measured by DXA at the lumbar spine from the first to the fourth lumbar vertebra (L1–L4), using a Hologic QDR 1000 densitometer (Hologic, Waltham, MA, USA). Daily calibrations of the densitometer performed with a phantom during a 1-year period had provided a coefficient of variation of 0.56%. All examinations were carried out in the continuous presence of trained technicians. BMD results were expressed as g/cm2 and as BMD Z-score calculated on the basis of the normal reference values for age and sex provided by the DXA system manufacturer. Bone quality was assessed in each subject by QUS measurements performed with a DBM Sonic 1200 bone profiler (Igea S.r.l., Carpi, MO, Italy) employing a sound frequency of 1.25 MHz. QUS is an easy, cheap and radiation-free technique to evaluate bone mineral status at peripheral skeleton through amplitude-dependent speed of sound (Ad-SoS), that expresses the ultrasound velocity inside the bone, and bone transmission time (BTT), reflecting the bone characteristics without the interference of the soft tissue. QUS was performed on the second to the fifth proximal phalanges of the non-dominant hand and the mean value per person was calculated. Measurements were performed by the same operator and the coefficient of variation was 0.73%, determined by repeated measurements in a subgroup of 12 subjects (three measurements per person on three different days). Ad-SoS and BTT results were expressed as Z-scores calculated on the basis of the normal values for age and sex obtained in a large Italian population sample .
In agreement with the International Society for Clinical Densitometry , BMD was considered to be normal when values were above −2 Z-score , a similar cut-off was used for Ad-SoS and BTT.
The statistical analyses were conducted using SPSS for Windows, version 15.0 (SPSS Inc., Chicago, IL, USA). All data are expressed as mean ± S.D. Comparison between patients and controls for all variables was performed by paired Student’s t-test or Wilcoxon matched pairs test as appropriate. Pearson’s correlation coefficient was used to evaluate the relationship between variables. Multiple stepwise regression analysis was used to evaluate the effect of age, sex, BMI, thyroid hormone status and duration of SH on BMD, Ad-SoS and BTT. Differences were considered statistically significant when P <0.05.