9 ± 0.5, 8.9 ± 0.5, 10.0 ± 0.5 [31], 12.4 ± 0.5 [32], and 16.4 ± 0.5 year [33]. During 1 year, between mean age of 7.9 and 8.9 years, half the cohort received a supplementation of calcium in a randomized, double-blind, placebo-controlled design, as previously reported [31]. Exclusion criteria at baseline were: ratio of weight/height <3rd or >97th percentile, physical signs of puberty, chronic disease,

malabsorption, bone disease, and regular use of medication as previously described [31]. The ethics committees of the Department of Pediatrics and the Department of Rehabilitation and Geriatrics of the University Hospitals of Geneva approved the protocol while informed consent was obtained from both EVP4593 parents and children [31]. All subjects were recruited within the Geneva area. Clinical assessment Body weight, standing height, and BMI (kg/m2) were retrospectively obtained at birth (n = 115) and 1 year of age (n = 96) through questionnaires sent to the parents and the pediatricians. These anthropometric

variables were then prospectively measured at each visit from 7.9 years of age on. At mean age (±SD) 7.9 ± 0.5 and 8.9 ± 0.5 year, pubertal stage was assessed by direct clinical examination made by a pediatrician–endocrinologist. At mean age of 10.0, 12.4, and 16.4 years pubertal maturation was assessed by a self-assessment questionnaire with drawings and written description of Tanner’s Ruboxistaurin datasheet breast and pubic hair. Silibinin At mean age 7.9 and 8.9 years, all girls were classified Tanner’s stage P1 while at mean age of 10.0 years, 38% of them had reached Tanner’s stage P2. Menarcheal age (MENA) was then assessed prospectively by direct interview at the second, third, fourth, and fifth visits, i.e., at the mean age of 8.9, 10.0, 12.4, and 16.4 years. MENA was within physiological range in all girls according to reference values established in the general population living in the same area [33]. Moreover,

there was no case of pathological delayed or precocious puberty. The use of contraceptive pill for more than 3 months was recorded as well as smoking expressed in yearly pack units. Calcium intake At each visit from 7.9 years, spontaneous, i.e., baseline calcium intake, as essentially assessed from dairy sources, was estimated by a frequency questionnaire [34]. Measurement of bone variables Areal bone mineral density (mg/cm2) was measured by dual-energy X-ray absorptiometry (DXA) at the level of the femoral neck (FN) with a Hologic QDR-4500 instrument (Waltham, MA, USA), as previously reported [33]. The coefficient of variation of repeated aBMD measurements varied between 1.0% and 1.6% [33].Volumetric bone density and microstructure were determined at the distal tibia by HR-pQCT on an XtremeCT instrument (Scanco medical AG®, Basserdorf, Switzerland), as previously described [35].