Energy Availability in Female Athletes
Condition: Female Athlete Triad
MeSH terms: Female Athlete Triad Syndrome
Study type: Observational

Summary: 
      The purpose of this project is to study the impact of energy availability (EA) on female
      endurance athletes and the adaptive consequences of insufficient EA with a special reference
      to the impact on energy metabolism, reproductive-, vascular- and bone health as well as
      exercise capacity, neuromuscular performance, ability to recover from intense exercise and
      the genetic interaction.

      Specific aims:

        -  To investigate the effects of EA on energy metabolism

        -  To investigate the effects of EA on reproductive, vascular- and bone health, and
           endocrine functions

        -  To investigate the effects of EA on exercise capacity

        -  To investigate the effects of EA on neuromuscular performance

        -  To investigate the effects of EA on recovery after exercise

        -  To identify potential dietary factors influencing EA, exercise capacity and
           neuromuscular performance

        -  To identify potential exercise factors influencing EA, exercise capacity and
           neuromuscular performance

        -  To identify potential psychological, behavoural and motivational aspects associated with
           insufficient EA

      The investigators hypothesize that female athletes with insufficient energy availability have
      attenuated energy metabolism, reduced BMD, impaired endothelial function and decreased
      ability to recover between exercise bouts compared to matched controls with sufficient energy
      and nutrient intake. The investigators also hypothesize that EA influences exercise capacity
      and neuromuscular performance.
    

Detailed description: 
      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.
    

Observational model: Cohort
Time perspective: Cross-Sectional
Sampling method: Non-Probability Sample
Study population: 
        Female endurance athletes exercising minimum 5 times a week will be recruited via the
        Danish and Swedish National Sport Federations (Team Danmark, Danmarks Idrætsforbund and
        Riksidrottsförbundet) and from competivite sport clubs.
      
Biospecimen description: 
      Whole Blood Serum Plasma
    
Keywords: Female endurance athletes; Energy Availability; Nutrient Availability; Bone Mineral Density; Menstrual dysfunction; Female athlete triad; Energy metabolism; Recovery after exercise; Endothelial function; Eating disorders; Performance