Written informed consent was obtained from all participants or their parents. The study was approved by the Poznań Medical Ethics Committee (no. 334/09). Menstrual status Each subject

completed a two-part medical questionnaire. The questions in the first part concerned menstruation: age at menarche, length of the menstrual cycles, and history of amenorrhea. Part two of the questionnaire referred to sport activities: age at the beginning of training, training period, number of training session per week, hours of training per day and per week. Primary amenorrhea was diagnosed where there was no onset of menses by 15 years, while secondary amenorrhea was diagnosed when there was no menstruation for 6 months, or for more than three times the previous cycle length. Menstrual VX-689 price periods that occurred more than 35 days apart

were described as oligomenorrhea [10]. Each participant underwent gynecological evaluation, including a pelvic ultrasound and measurements of luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P), 17β – estradiol (E2), prolactin (PRL), thyroid-stimulating hormone (TSH), testosterone (T), and sex-hormone-binding globulin (SHBG) serum concentration, in order to exclude independent causes of amenorrhea or oligomenorrhea (such as pregnancy, see more primary ovarian failure, hyperprolactinemia, thyroid dysfunction or polycystic ovary syndrome). Blood sampling and biochemical analyses Blood samples were obtained in menstruating subjects between days 2 and 5 of the menstrual cycle (in the early follicular phase), and at random in amenorrheic subjects. Blood serum samples were taken between 6.00 a.m. and 9.00 a.m. following overnight fasting and rest. The athletes were instructed to abstain from caffeine and alcohol for 24 hours prior to the blood sampling, and to refrain from performing strenuous

exercise on the day of sampling. Serum concentration of LH, FSH, E2, P, PRL, TSH, T and SHBG were measured by immunochemical methods using Chemiluminescent Microparticle Immunoassay (CMIA) and Casein kinase 1 Microparticle Chemiflex Flexible interassay protocols and making use of diagnostic sets and an ARCHITECT automatic analyzer. Serum leptin levels were estimated using Human Leptin Elisa by LINCO Research. All hormones concentrations were determined in duplicated. Body weight and body composition measurements In order to evaluate the nutritional status, the anthropometrical indices, height and weight were measured using an VX-680 manufacturer anthropometer coupled with a WPT 200 OC verified medical scale (Rad Wag). BMI (kg/m2) was calculated as body weight divided by squared body height. The participants were dressed in minimal clothing during the measurements, which were rounded to the nearest 0.5 kg and 0.5 cm.