Energy Availability in Female Athletes

The study population was recruited through the Danish and Swedish sport federations in weight-bearing endurance sports, and in local competitive endurance sports clubs, through flyers, webpage announcements and mailings. Interested athletes received the study protocol and a self-administered questionnaire, assessing age, sport, training regime, and self-assessed menstrual function. Athletes returning the questionnaire and who were interested in further participation were contacted by the research group. Before inclusion, all subjects were informed orally, and in writing, of all study procedures and signed an informed consent form. Subjects included and defined as elite endurance athletes were athletes at national team levels or competitive endurance athletes from regional sports clubs, 18-38 years of age, training a minimum of five times per week. Exclusion criteria included clinically verified menstrual dysfunction other than oligomenorrhea/functional hypothalamic amenorrhea; pregnancy; chronic illness; smoking; use of forms of contraceptives other than oral, e.g., hormonal coil/patches; inability or being unwilling to stop hormonal contraceptives for at least 6 weeks prior to investigation; or injuries preventing the athlete from training ≥ 2 weeks.

Data collection was performed on two consecutive days followed by a 7-day registration period in the athletes' normal environment. The first day comprised of examinations involving bone health, blood pressure and reproductive function. The second day included examinations of energy metabolism, aerobic capacity, as well as assessment of eating disorders. The subjects were instructed not to exercise for more than 30 min at low or moderate intensity on the day before, and on the first day of examination, and to arrive at the clinic in a fasted (from midnight) and rested state.

PROTOCOL OVERVIEW:

DAY 1: Start at 7:45 A.M.

1. Pragnancy test
2. Blood sampling
3. DXA scan
4. Blood pressure
5. Meal
6. Gynecological examination

DAY 2: Start at 7 A.M.

1. RMR
2. Blood sampling
3. Work efficiency
4. Meal
5. EDE interview
6. Incremental exercise test
7. EDI-questionnaire

7 DAY DIETARY AND ACTIVITY RECORD

The timing of the examination and registration period was planned individually for each subject in order to choose a period reflecting their habitual food habits and exercise regimes. Dietary intake and training intensity were recorded by the subjects for seven consecutive days to assess current EA. Energy intake was calculated from a prospective weighed (Exido 246030 Kitchen Scale, Gothenburg, Sweden) food record, using a nutrient analysis program, Dankost 2000 (Dankost, Copenhagen, Denmark) for Danish food items, and Dietist XP (Kost och Näringsdata AB, Bromma, Sweden) for Swedish food items. Subjects were given in-depth verbal and written instructions and a demonstration of how food and beverages should be weighed and registered. Subjects were instructed to maintain their normal food habits and eating patterns. Before entering data in the nutrient analysis program the same dietician reviewed all completed diet records and asked for supplementary information if needed, and the mean daily energy intake from the 7-day record was used in the analysis. In order to identify subjects who provided nutritional data of poor validity, the Goldberg cutoff was calculated using the equation described by Black (2000). Heart rate monitors (Polar RS400®, Stockholm, Sweden) and training logs were used to assess exercise energy expenditure. Subjects were instructed to maintain and to follow their normal training regime. They were, furthermore, instructed to describe each session in as much detail as possible, regarding type, duration, and intensity of exercise and to wear the heart rate monitor at all training sessions (except swimming) and during cycling (training as well as transportation).

Eating behavior was assessed using the Eating Disorder Inventory (EDI-3), a questionnaire assessing behavior and attitudes related to DE behavior, as well as to overt ED. Subjects were categorized as having DE behavior when the EDI risk subscale score for Drive for Thinness (DT) was ≥ 14, and/or a Body Dissatisfaction (BD) risk score ≥ 19 and without the presence of DSM-IV diagnosed ED. The Eating Disorder Examination (EDE-16) was used to determine whether subjects met the criteria for ED, according DSM-IVcriteria for anorexia nervosa, bulimia nervosa, and ED not otherwise specified (EDNOS). All interviews were performed by the same EDE-certified member of the research team.

Subjects were transported by car to the clinic on the morning of the second test day in order to minimize physical activity prior to the measurement. RMR was assessed between 7:00 and 8:30 h, after an overnight fast, using a ventilated open hood system (Oxycon Pro 4, Jeager, Germany). The system was automatically calibrated before each test, and again weekly, by an alcohol burning test with coefficients of variability (CV) of 0.7% for O2, 1.1% for CO2, 0.8%for the respiratory exchange ratio, and 2% for EE. After voiding, subjects laid down for 15 min before measurements of oxygen consumption (VO2) and carbon dioxide production (VCO2) over 35 min (the equation is defined under Calculations). Work efficiency was assessed by a standardized test protocol in the fasted state, initiated by the subject seated on the bicycle ergometer (Monark 939E, Monark Exercise AB, Vansbro, Sweden) for 6 min, followed by cycling for 6 min at 0W, 50W and 100W, respectively. An air-tight mask covering the mouth and nose was used in order to measure respiratory gas exchange (the equations for calculations are defined under Calculations). In order to calculate daily total EE, HR monitors (Polar RS400®) were used to assess EE during bicycle transportation, while actigraphy (ActiGraph GT3X®, Pensacola, FL, USA) and the data analysis software ActiLife 5 (ActiGraph) were used for assessment of NEAT. Subjects were instructed to wear an accelerometer on the wrist during sleep, and on the hip from getting up in the morning until bedtime, and only to take it off during showering, swimming, bicycle transportation, and training.

Two hours after a standardized breakfast, an incremental exercise test on the bicycle ergometer was performed, initiated by cycling for 6 min at 50W, followed by an increase in workload of 12-14 W/min until exhaustion. An air-tight mask covering the mouth and nose was used in order to measure VO2peak and respiratory exchange ratio, and HR (Polar RS400®) was measured.

Subjects using hormonal contraceptives were requested to stop for a minimum of 6 weeks prior to examination in order to secure a sufficientwashout period for exogenous estrogen and progesterone. Subjects not recovering their menstrual bleeding within the 6 weeks were contacted monthly by the research team during a follow-up period of a minimum of 3 months before gynecological assessment. Menstruating athletes were examined in the early follicular phase, on the third to fifth day of menstruation. A pregnancy test was performed on arrival at the hospital, and menstrual function was examined by an experienced gynecologist who performed a transvaginal ultrasound examination (Ultrasound Scanner, Class 1 type B, B-K Medical REF TYPE 2202, Bedfordshire, UK). The maximum number of ovarian follicles present in a single plane was counted, and total volume was assessed. Sex hormone status [estrogen, progesterone, LH, follicle stimulating hormone (FSH), sexual hormone binding globulin (SHBG), prolactin, dehydroepiandrosteron sulfate (DHEA-S), androstendion, and total testosterone] and anamnestic assessment, e.g., age of menarche, previous menstrual irregularities, use of hormonal contraceptives, and number of menstrual cycles during the last year, were recorded using the low EAin females questionnaire (LEAF-Q) (Melin et al., 2014). Subjects were then classified with eumenorrhea (menstrual cycles of 28 days ± 7 days and sex hormones within the normal range); oligomenorrhea (menstrual cycles > 35 days where other causes than hypothalamic suppression had been ruled out); FHA (either primary: no menarche after 15 years of age, or secondary: absence of at least three consecutive menstrual cycles where other causes than hypothalamic suppression had been ruled out) (Nattiv et al., 2007); other MDs (anatomic defects, hyperprolactinemia or other dysfunctional ovarian conditions); PCOS involving at least two of the following: (a) enlarged ovaries with a volume greater than 10 mL and/or ≥ one ovary demonstrating ≥ 12 follicles in one plane; (b) irregular or absence of bleeding; and (c) elevated androgen level, or otherwise androgen stigmatized.

Body weight was measured with an accuracy of 0.01 kg in underwear on an electronic scale (Lindeltronic 8000, Samhall Lavi AB, Kristianstad, Sweden). Height was measured without shoes and standing with legs together against a wall, using a fixed stadiometer (Hultafors AB, Hultafors, Sweden). After resting in a supine position for 7 min, HR and BP were measured three times (the mean was used) using an electronic sphygmomanometer (Microlife BP A100, Widnau, Switzerland). Hypotension was defined as a systolic BP < 90 mmHg and/or diastolic BP < 60 mmHg. Dual-energy X-ray absorptiometry (DXA) (Hologic, Model Discovery 2009, Hologic Inc.,Waltham, MA, USA) was used to determine fat-free, fat, and bone mass, respectively. BMD was determined for whole body, lumbar spine (L1-L4) and hip. All measurements and scans were performed in the fasted and resting state between 7:30 and 9:00 h, and were assessed by the same technician, and performed on the same scanner. Calibration of the Hologic Discovery 2009 was performed weekly, using a phantom provided by the manufacturer. Subjects were classified as having normal BMD: Z-scores > -1 in all measured sites, low BMD: Z-score -1 to -2 in at least one site, and osteoporosis: Z-score < -2 in at least one site together with minimum one secondary risk factor such as low EA, ED and oligomenorrhea/FHA.

Blood samples were drawn following an overnight fast from an antecubital vein on the first as well as on the second day, between 8:30 and 8:50 h, in a rested state.