Abstract
Exercise-related menstrual dysfunction (ExMD) is associated with low energy availability (EA), decreased bone mineral density (BMD), and increased risk of musculoskeletal injury. We investigated whether a 6-month carbohydrate-protein (CHO-PRO) supplement (360 kcal/day, 54 g CHO/day, 20 g PRO/day) intervention would improve energy status and musculoskeletal health and restore menses in female athletes (n = 8) with ExMD. At pre/post-intervention, reproductive and thyroid hormones, bone health (BMD, bone mineral content, bone markers), muscle strength/power and protein metabolism markers, profile of mood state (POMS), and energy intake (EI)/energy expenditure (7 day food/activity records) were measured. Eumenorrheic athlete controls with normal menses (Eumen); n = 10) were measured at baseline. Multiple linear regressions were used to evaluate differences between groups and pre/post-intervention blocking on participants. Improvements in EI (+382 kcal/day; p = 0.12), EA (+417 kcal/day; p = 0.17) and energy balance (EB; +466 kcal/day; p = 0.14) were observed with the intervention but were not statistically significant. ExMD resumed menses (2.6 ± 2.2-months to first menses; 3.5 ± 1.9 cycles); one remaining anovulatory with menses. Female athletes with ExMD for >8 months took longer to resume menses/ovulation and had lower BMD (low spine (ExMD = 3; Eumen = 1); low hip (ExMD = 2)) than those with ExMD for <8 months;
intervention but were not statistically significant. ExMD resumed menses (2.6 ± 2.2-months to first menses; 3.5 ± 1.9 cycles); one remaining anovulatory with menses. Female athletes with ExMD for >8 months took longer to resume menses/ovulation and had lower BMD (low spine (ExMD = 3; Eumen = 1); low hip (ExMD = 2)) than those with ExMD for <8 months; for 2 ExMD the intervention improved spinal BMD. POMS fatigue scores were 15% lower in OPEN ACCESS
Nutrients 2014, 6 3019 ExMD vs. Eumen (p = 0.17); POMS depression scores improved by 8% in ExMD (p = 0.12). EI, EA, and EB were similar between groups, but the intervention (+360 kcal/day) improved energy status enough to reverse ExMD despite no statistically significant changes in EI. Similar baseline EA and EB between groups suggests that some ExMD athletes are more sensitive to EA and EB fluctuations. Keywords: amenorrhea; oligomenorrhea; energy intake; energy expenditure; exercise performance; bone health; protein synthesis; protein degradation; muscle strength 1. Introduction Optimal energy intake (EI) and nutrition can improve exercise performance and maintain overall health in physically active individuals [1]. Female athletes, however, can find it difficult to meet energy and nutrient needs while maintaining a low fat or body weight considered optimal for sports performance. Thus, they often restrict EI to make weight goals [1]. Low EI, combined with high levels of exercise, increases the risk of developing exercise-related menstrual dysfunction (ExMD) and poor bone health [2,3]. ExMD can be high in physically active women, ranging from 0% to 60% [4], and occurs across a continuum from mild disruptions in menses (no ovulation or luteal phase deficiency) to oligomenorrhea (cycles ≥ 35 day) and amenorrhea (no menses for >90 day [2]. Low energy availability (EA) (i.e., energy remaining for body functions after exercise training) may lead to menstrual dysfunction through a leptin-controlled pathway [2]. In ExMD, females have low leptin levels that contribute to the disruption of luteinizing hormone (LH) pulsatility via interference of gonadotropin-releasing hormone (GnRH) pulsatile [5]. Sequentially, the ovaries decrease production of estrogen and progesterone, the hormones responsible for triggering the lining and egg of the uterus to be shed (menstruation) resulting in abnormal menses [5]. ExMD is also associated with compromised bone health, including low bone mineral density (BMD, prevalence = 20%–50%), osteoporosis (prevalence = 10%–13%), and stress fractures [2,3,6]. For premenopausal women, low BMD is defined as 2.0–2.5 standard deviation (SD) below the mean BMD of young adults, and osteoporosis is defined as a BMD > 2.5 SD below the mean. For athletes, BMD is typically 5%–15% higher
with compromised bone health, including low bone mineral density (BMD, prevalence = 20%–50%), osteoporosis (prevalence = 10%–13%), and stress fractures [2,3,6]. For premenopausal women, low BMD is defined as 2.0–2.5 standard deviation (SD) below the mean BMD of young adults, and osteoporosis is defined as a BMD > 2.5 SD below the mean. For athletes, BMD is typically 5%–15% higher than non-athletes, thus, a BMD > 1.0 SD below the mean may indicate poor bone health [2]. In addition, ExMD has been linked to other health consequences, such as cardiovascular dysfunction, endothelial dysfunction, abnormal metabolic hormonal profile (decreased thyroid, leptin and IFG-1; increased cortisol) and muscle injuries [3,7]. The impact of ExMD on skeletal muscle strength and power, however, has not yet been examined. Chronically low estrogen observed in ExMD could potentially cause muscle damage and alter the muscle repair process, thus prohibiting an athlete from reaching their highest level of sports performance. Estrogen in the skeletal muscle of rodents decreases muscle damage and accelerates repair by possibly acting as antioxidant, cell membrane stabilizer, and/or satellite cells stimulator [8,9]. In young physically active women, the research findings are mixed. Lower estrogen status in women has been reported to be associated with greater
Nutrients 2014, 6 3020 muscle damage and oxidative stress after running [10,11] and slower muscle strength recovery after a stretch-shortening exercise load (i.e., 100 drops jumps from platform) [12] but not with exercise-induced muscle damage following either stretch-shortening exercise load [12] or eccentric exercise[13]. Thus, low estrogen status may negatively impact muscle health and strength, but more research in humans is needed [8]. In ExMD, low estrogen levels occur in conjunction with low EA, which potentially could lead to impairment of post-exercise protein metabolism. Miller et al. [14] compared myofibrillar protein synthesis in the follicular phase (i.e., low estrogen phase) and luteal phase (i.e., high estrogen phase) of the menstrual cycle in eumenorrheic physically active women and reported no difference in protein synthesis between phases. The women in this study, however, had normal cyclic variation in estrogens and not chronically low estrogen status as in ExMD. To our knowledge, protein metabolism in ExMD has not been examined and therefore, an aim of this study was to assess post-exercise indicators of protein metabolism (i.e., AMPK, p70S6K, and FOXO1). Oral contraceptives (OCs) are often prescribed to restore menses and prevent bone loss in ExMD, but their impact on bone health is equivocal [15]. In addition, OCs do not restore non-reproductive hormone levels (e.g., leptin, IFG-1, thyroid, cortisol) and can have undesirable side effects such as weight gain, fatigue, mood disorders, nausea, and headaches [16]. Currently there are no long-term interventions examining non-pharmacological treatments of active females with ExMD [17], thus, an alternative lifestyle approach, such as one that alters diet or physical activity is desirable. Based on our previous research [18], we hypothesized that an increase in EI (+360 kcal/day) would improve EB and bone health and restore reproductive function in ExMD. In addition, we hypothesized that muscle strength and power in the ExMD group would increase with the restoration of normal estrogen levels and EB improvements from the intervention. Therefore, the purpose of this 6-month intervention was to determine if increases in EI, using a daily carbohydrate-protein (CHO-PRO) supplement (360 kcal/day), would improve EB, bone health, muscle strength and power, and
In addition, we hypothesized that muscle strength and power in the ExMD group would increase with the restoration of normal estrogen levels and EB improvements from the intervention. Therefore, the purpose of this 6-month intervention was to determine if increases in EI, using a daily carbohydrate-protein (CHO-PRO) supplement (360 kcal/day), would improve EB, bone health, muscle strength and power, and mood state, and restore reproductive function in physically active women with ExMD. A secondary aim was to compare diet, indicators of protein metabolism, hormones, and bone biomarkers between women with ExMD and eumenorrheic (Eumen) physically active controls. 2. Experimental Section 2.1. Study Protocol and Participants Endurance trained women (12 amenorrheic/oligomenorrheic [ExMD]; 10 Eumen) were recruited. Overall, 8 of 12 women with ExMD completed the intervention; 4 women dropped out due to personal reasons. An ExMD control group was not included due to ethical reasons (i.e., no treatment for 6–12-months); the University Institution Review Board (IRB), which reviews and approves research involving human subjects, would only allow tracking ExMD participants that choose not to complete the intervention after the initial screening (none completed this option). After obtaining IRB approved informed consent, all participants completed baseline questionnaires to assess general health, exercise training, and menstrual/dietary history (0-month; Figure 1). Two subscales (Drive for Thinness and Body Dissatisfaction) of the Eating Disorder Inventory-2 (EDI-2) questionnaire [19] were also
Nutrients 2014, 6 3021 administered to identify athletes with disordered eating [20]. At pre/post-intervention, mood state was assessed using a Profile of Mood State (POMS) questionnaire [21], which measures 6 different mood states (fatigue, anger, vigor, depression, confusion, anxiety). A total mood disturbance score (maximum = 60) was calculated to provide an overall measure of global effective state. Inclusion criteria included ≥7 h/week of exercise training for the last 2 years, a VO 2max > 38 mL/kg/min, no OCs or hormonal replacement therapy use for 6-months, EDI-2 subscale score <14, and no self-reported primary amenorrhea or non-exercise-related amenorrhea. Figure 1. Detailed Protocol of Study: Women with exercise-induced menstrual dysfunction (ExMD, n = 8) were assessed at pre (0-month)/post (6-months)-intervention; a mid-intervention (3-months) assessment was done to monitor compliance. ExMD were compared at pre/post-intervention to a group of Eumenorrheic athletes (n = 10), who were assessed at 0-month only. Participants were assigned to either the ExMD (n = 8) or Eumen (n = 10) group based on self-reported menstrual history and ovulation status (Clearblue ® Easy Fertility Monitor, Waltham, MA, USA), which the participants measured daily for ≥1 month. Participants who began menses during the intervention tested for ovulation each month until the study ended. Other types of menstrual dysfunction were eliminated based on assessments of LH, follicular stimulating hormone (FSH), prolactin levels, and LH/FSH ratio [2,22]. All participants had normal thyroid (T 3) levels. ExMD group was assessed at 0-month/6-months; Eumen group at 0-month only (Figure 1). 2.2. Maximal Aerobic Capacity Test (VO 2max) Participants completed a standardized treadmill VO 2max test using indirect calorimetry (ParvoMedics Metabolic Cart, Sandy, UT, USA) as previously reported [23]. 2.3. Blood Biochemistry Fasting blood was drawn for a general blood screen, including iron and vitamin (B-12, folate, 25-OH Vitamin D) status, T 3 and reproductive hormones (estradiol, LH, FSH, prolactin, progesterone) (Samaritan Health, Corvallis, OR, USA). The rationale for assessing the micronutrients and hormones
vitamin (B-12, folate, 25-OH Vitamin D) status, T 3 and reproductive hormones (estradiol, LH, FSH, prolactin, progesterone) (Samaritan Health, Corvallis, OR, USA). The rationale for assessing the micronutrients and hormones
Nutrients 2014, 6 3022 selected above are as follows: (1) Female athletes are at risk for deficiency of vitamins B12 and folate due to inadequate dietary intake and/or increased needs [24]; (2) Active women, especially endurance athletes, are at risk for low iron [1] and vitamin D status [25]; (3) With menstrual dysfunction, T 3 and reproductive hormones are typically evaluated [3]. In addition, serum osteocalcin, a bone-specific protein of osteoblasts, and Procollagen Type I Intact N-Terminal Propeptide (P1NP), an indicator of newly formed type I collagen, were assessed as indicators of bone formation [26]. Carboxyterminal telopeptide of type I collagen (CTX) was used as a marker of bone resorption [26]. These bone markers (osteocalcin, P1NP, and CTX) were measured using ELISA (CV: Osteocalcin = 2.5%; P1NP = 2.9%; CTX = 3.3%) (Immunodiagnostics Systems, Scottsdale, AZ, USA). 2.4. Energy Intake and Expenditure Measurement and Analysis 2.4.1. Measurements For 7-consecutive days participants complete weighed food records, physical activity (PA) logs and wore an accelerometer (ActiGraph LLC, Pensacola, FL, USA). To improve accuracy of energy expenditure (EE) and total energy expenditure (TEE) estimates, running energy expenditure (RunEE) and resting metabolic rate (RMR) were measured using indirect calorimetry [23]. Briefly, RunEE was measured as participants ran for 5-min at 4 self-selected speeds ranging from easy (warm-up) to fast (5 K race pace). RMR was measured on 2 separate days within a 7 day period (8-h fast; ~19 h since last exercise). 2.4.2. Analysis As previously reported [23], TEE was calculated as the sum of three components as outlined by Tomten and Hostmark [27]: RMR, all PA expenditures (activities of daily living plus EEE from 7 day activity logs), and the thermic effect of food. RunEE data was used to estimate each participant’s EEE during running. Diet and PA records were analyzed using a nutrient and activity analysis program (Food Processor SQL, ESHA Research, Salem, OR, USA). Under-reporters were identified using Goldberg et al. [28] criteria and one Eumen control was excluded from the dietary analysis. EB and EA were then calculated as follows: EB (kcal/day) = EI-TEE; EA (kcal/day) =EI-EEE. Exercise was defined
during running. Diet and PA records were analyzed using a nutrient and activity analysis program (Food Processor SQL, ESHA Research, Salem, OR, USA). Under-reporters were identified using Goldberg et al. [28] criteria and one Eumen control was excluded from the dietary analysis. EB and EA were then calculated as follows: EB (kcal/day) = EI-TEE; EA (kcal/day) =EI-EEE. Exercise was defined as PA > 4.0 metabolic equivalents (METs) to eliminate activity of daily living (i.e., bicycle commute) from EEE calculation. 2.5. Bone Density and Body Composition Dual-energy X-ray absorptiometry (DXA, Hologic QDR-4500 Elite A; Waltham, MA, USA) was used to measure whole body composition (bone, muscle, and fat mass) and areal BMD of the proximal femur (total hip, femoral neck, greater trochanter) and the lumbar spine (L1–L4). All scans were performed and analyzed by a trained technician with over 20 years of experience in bone density and body composition assessment (CV = 1.5% and 1.0% for whole body, and hip and lumbar spine BMD). Based on z-scores, BMD were classified as having normal BMD for z-score > −1.0, low BMD for z-scores between −1.0 and −2.5, and osteoporosis for z-scores < −2.5 [29].
Nutrients 2014, 6 3023 2.6. Muscle Strength and Power Isokinetic concentric strength (peak power (watts) and torque (Nm)) of knee extension and flexion, and plantar and dorsiflexion were measured at speeds of 60, 90 and 120 degrees/s with gravity corrected using a validated and reliable mechanical system, the Biodex System 3 [30] (Biodex Medical Systems, Inc. Shirley, NY, USA). After being familiarized with the test, participants perform 10 trials of each exercise at low intensity to warm-up followed by three sets of 10 maximal efforts (one set at each speed) to determine peak torque (Nm) and power (watts). Each maximal effort was separated by ~60 s of rest. Strength assessment protocols were programmed into the dynamometer to set parameters for testing (i.e., start and stop angles and speed of contraction), and therefore ensured consistency. These protocols have good reliability within this population (CV = 4% to 8%). The dynamometer software (Biodex System 3 Advantage Software; Biodex Medical Systems Inc., Shirley, NY) generated peak power (watts) and torque (Nm) for each speed measured. The Bassey Power Rig (Medical Engineering Unit, University of Nottingham School of Biomedical Sciences, Nottingham, England) was used to assess explosive muscular power of the lower extremities as previously described [31]. Explosive power is a term used to describe the muscle’s ability to perform work in ≤0.5 s [31]. Participants performed a 5-min warm-up consisting of walking on the treadmill. The testing procedure required 10 maximal leg presses on each leg with their knee starting at a 90 degree angle. The participants were asked to press down on the push pedal one leg at a time to full extension, alternating right and left leg. The highest and lowest measurements were not used. The average of the remaining 8 measurements was used to calculate peak torque and force. Muscle strength and power assessments were conducted by trained researcher under supervision of exercise physiologist with over 20 years of experience in these measurements. 2.7. Submaximal Exercise Protocol and Skeletal Muscle Analysis 2.7.1. Submaximal Exercise and Post-Exercise Muscle Biopsy After an 8-h fast, the participants arrived to the laboratory and completed
measurements was used to calculate peak torque and force. Muscle strength and power assessments were conducted by trained researcher under supervision of exercise physiologist with over 20 years of experience in these measurements. 2.7. Submaximal Exercise Protocol and Skeletal Muscle Analysis 2.7.1. Submaximal Exercise and Post-Exercise Muscle Biopsy After an 8-h fast, the participants arrived to the laboratory and completed a 45-min run at 75% of VO 2max. After completing the run, the participants were provided a CHO-PRO nutrition shake (Gatorade ® Nutrition Drink; 325 mL; 360 kcal) to consume immediately after exercise. At 60-min post-exercise, a muscle biopsy was obtained from vastus lateralis by the percutaneous needle biopsy technique. First, the thigh was disinfected using an antiseptic/antimicrobial cleanser (chlorhexidine gluconate) and anesthetized with an injection of mercaine and lidocaine. A small incision, ~3/8 in long, was made through the skin and the fascia. Pressure was applied immediately to stop any bleeding prior to taking the biopsy. Suction was applied to maximize sample size. The biopsy was trimmed of adipose tissue and frozen in liquid nitrogen at −80°C for subsequent analysis. 2.7.2. Muscle Biopsy Preparation and Antibodies and Positive Controls Muscle samples (~40 mg) were homogenized in RIPA buffer (Cell Signaling, Beverly MA) and a DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA) was used to determine protein concentration. Monoclonal antibodies were used for phospho-p70S6k (Thr389), total p70S6k,
Description
This study investigates a dietary intervention's effect on restoring menses in female athletes with ExMD.