Menstrual Cycle Phases Influence on Cardiorespiratory Response to Exercise in Endurance-Trained Females

this study was to analyse the impact of sex hormone uctuations throughout the menstrual cycle on cardiorespiratory response to high-intensity interval exercise in athletes. Twenty-one eumenorrheic endurance-trained females performed an interval running protocol in three menstrual cycle phases: early-follicular phase (EFP), late-follicular phase (LFP) and mid-luteal phase (MLP). It consisted of 8 3-min bouts at 85% of their maximal aerobic speed with 90-s recovery at 30% of their maximal aerobic speed. To verify menstrual cycle phase, we applied a three-step method: calendar-based counting, urinary luteinizing hormone

endurance-trained females performed an interval running protocol in three menstrual cycle phases: early-follicular phase (EFP), late-follicular phase (LFP) and mid-luteal phase (MLP). It consisted of 8 3-min bouts at 85% of their maximal aerobic speed with 90-s recovery at 30% of their maximal aerobic speed. To verify menstrual cycle phase, we applied a three-step method: calendar-based counting, urinary luteinizing hormone measurement and serum hormone analysis. Mixed-linear model for repeated measures showed menstrual cycle impact on ventilatory (EFP: 78.61 11.09; LFP: 76.45 11.37; MLP: 78.59 13.43) and heart rate (EFP: 167.29 11.44; LFP: 169.89 10.62; MLP: 169.89 11.35) response to high-intensity interval exercise (F 2.59= 4.300; p= 0.018 and F 2.61= 4.648;p= 0.013, respectively). Oxygen consumption, carbon dioxide production, respiratory exchange ratio, breathing frequency, energy expenditure, relative perceived exertion and perceived readiness were unaltered by menstrual cycle phase. Most of the cardiorespiratory variables measured appear to be impassive by menstrual cycle phases throughout a high-intensity interval exercise in endurance-trained athletes. It seems that sex hormone uctuations throughout the menstrual cycle are not high enough to disrupt tissues' adjustments caused by the high-intensity exercise. Nevertheless, HR based training programs should consider menstrual cycle phase. Keywords: sex hormones; estradiol; progesterone; eumenorrheic; high intensity interval exercise; athletes 1. Introduction The natural menstrual cycle is perhaps the second most important biological rhythm, next to the circadian one [1], and it is regulated by the hypothalamic-pituitary-ovarian axis and all hormones involved in it (predominantly, follicle-stimulating hormone [FSH], luteinizing hormone [LH], 17 -estradiol [E2] and progesterone). Despite individual vari- ations, female sex hormones uctuate fairly predictably over 23–38 days [2], giving rise to the different phases of the menstrual cycle. The rst one is the early-follicular phase (EFP), characterised by low concentrations of sex hormones, which starts at the onset of menstruation. Then, E2 starts to rise throughout the mid-follicular phase, reaching its peak in the late-follicular phase (LFP), followed by the peak in LH and FSH, just prior to ovulation. These hormones drastically decrease after ovulation whereas progesterone Int. J. Environ. Res. Public Health2021,18, 860.

at the onset of menstruation. Then, E2 starts to rise throughout the mid-follicular phase, reaching its peak in the late-follicular phase (LFP), followed by the peak in LH and FSH, just prior to ovulation. These hormones drastically decrease after ovulation whereas progesterone Int. J. Environ. Res. Public Health2021,18, 860.

Int. J. Environ. Res. Public Health2021,18, 860 2 of 12 starts to increase, achieving its peak in the mid-luteal phase (MLP), coinciding with high levels of E2 as well. Finally, during the late luteal phase all sex hormones drop, starting the cycle again [2,3]. Female sex hormones, specially E2 and progesterone, have receptors in several tissues of the body. Thereby, other than reproductive functions, these hormones may in uence many other physiological systems such as hypothalamus, cardiovascular system, kidney tubules, liver, skeletal muscle and adipose tissue [1,3–5], which may have an impact on females' exercise performance. In this sense, an increase in ventilation (Ve) has been reported in sedentary [6] and active females [7,8] as well as an increase in heart rate (HR) in both, sedentary [9] and trained females [10], during the luteal phase. In addition, higher fat utilisation in the luteal phase has been observed in active females, resulting in a lower respiratory exchange ratio (RER) during this phase [4]. However, other studies concluded no impact of menstrual cycle on maximal oxygen consumption (VO2max), Ve, RER, lactate and HR in physically active females [11–13]. These con icting ndings may be explained by methodological shortcomings, mainly the measurements trials carried out in different moments of the menstrual cycle since it has been divided into two [12,13], three [8,10] or four [6,11] phases. An additional limitation is the menstrual cycle veri cation, as studies often rely on calendar counting [4,11,12] or measuring body basal temperature [4], and it is well known than these methods are not accurate enough and should be accompanied by urinary LH tests and serum sex hormone veri cation, as a recent review concluded [3]. Therefore, the aim of this investigation was to assess the in uence of sex hormone uctuations throughout the menstrual cycle on cardiorespiratory response to high intensity interval exercise. Based on previous literature, we hypothesis that cardiorespiratory response to exercise is altered by sex hormones uctuations over the menstrual cycle in endurance-trained females. 2. Material and Methods 2.1. Participants A total of twenty-one eumenorrheic females (age: 30.5 6.5 years; height: 163.1 6.4 cm;

of sex hormone uctuations throughout the menstrual cycle on cardiorespiratory response to high intensity interval exercise. Based on previous literature, we hypothesis that cardiorespiratory response to exercise is altered by sex hormones uctuations over the menstrual cycle in endurance-trained females. 2. Material and Methods 2.1. Participants A total of twenty-one eumenorrheic females (age: 30.5 6.5 years; height: 163.1 6.4 cm; body weight: 58.4 8.7 kg; body fat percentage: 25.2% 6.7%; lean mass, considering it as body weight minus fat mass and minus bone mineral content: 70.38% 6.51%; peak oxygen consumption [VO 2preak]: 48.4 4.4 mL min 1 kg 1 ) participated in this study. They had regular menstrual cycle, occurring from 23 to 38 days in length during the six months prior the study [2]. Concretely, volunteers´menstrual cycle ranged from 28 2 to 31 2 days in length. All of them were healthy and well-trained (7.4 5.3 years of endurance training experience with a training volume of 295.9 183.6 min per week during the 6 months prior to recruitment), in endurance activities such as running, obstacle races, triathlon and cycling. Participants were required to meet the following criteria: (a) healthy adult females between 18 and 40 years old; (b) presenting with healthy iron parameters (serum ferritin > 20 g/L, haemoglobin > 115 g/L and transferrin saturation > 16%); (c) performing endurance training between 3 and 12 h per week. Exclusion criteria included: (a) irregular menstrual cycles; (b) oral contraceptive use; (c) menopause; (d) smoking; (e) metabolic or hormonal disorder; (f) medication or dietary supplements that alter vascular function (e.g., tricyclic antidepressants, -blockers, -blockers, etc.); (g) any surgical interventions (e.g., ovariectomy); (h) pregnancies in the year preceding; (i) any musculoskeletal injury in the last six months. At the start of the data collection, all participants conducted a questionnaire gathering information about training experience, health status, dietary supplements and menstrual cycle aspects. All participants were informed about the procedures and risks involved and informed consent was provided by each participant. The experimental protocol was approved by the Institutional Ethics Committee and is in accordance with The Code of Ethics of the World

data collection, all participants conducted a questionnaire gathering information about training experience, health status, dietary supplements and menstrual cycle aspects. All participants were informed about the procedures and risks involved and informed consent was provided by each participant. The experimental protocol was approved by the Institutional Ethics Committee and is in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki).

Int. J. Environ. Res. Public Health2021,18, 860 3 of 12 2.2. Study Design The present work is part of the IronFEMME study, an observational cross-sectional study performed by physically active and healthy women. The project consisted on two sections carried out at the same time: iron metabolism (Study I, which exercise protocol was an interval running test) and muscle damage (Study II, which protocol was based on a resistance exercise trial). Concretely, the present work shows data from Study I. Participants came to our laboratory on four occasions. The initial screening visit was conducted during the EFP (i.e., between 2nd and 5th day of the menstrual cycle with day 1 being onset of menstrual bleeding). Volunteers came to our laboratory between 8 and 10 a.m. in a rested and overnight fasted state. Volunteers did not perform moderate or vigorous physical activity and did not take caffeine, alcohol or any supplementation 24 h prior to the screening day. Firstly, they signed all the informed consents and participant´s weight and height were recorded. Then, baseline blood samples were collected, for a complete blood count, genetic testing, biochemistry and hormonal analyses. Subsequently, an absorptiometry by dual-energy X-ray (DXA) was done. This screening session was completed with a maximal aerobic ramp test on a computerized treadmill (H/P/COSMOS 3PW 4.0, H/P/Cosmos Sports & Medical, Nussdorf-Traunstein, Germany) to determine their VO2peak. Expired gases were measured breath-by-breath with the gas analyser Jaeger Oxycon Pro (Erich Jaeger, Viasys Healthcare, Friedberg, Germany) for which validity and reliability have been previously demonstrated [14,15]. Heart response was continuously monitored with a 12-lead ECG. Participants began with a warm-up of 3 min at 6 km/h. Once the warm-up nished, the speed was set at 8 km/h and then increased by 0.2 km/h every 12 s until exhaustion. A slope of 1% was set throughout the test to simulate air resistance [8]. The maximal aerobic speed was considered as the minimum speed required to elicit the VO2peak [16]. To verify that VO2peak was reached, a con rmatory test was carried out as suggested in previous studies [17,18] after a 5 min recovery of

every 12 s until exhaustion. A slope of 1% was set throughout the test to simulate air resistance [8]. The maximal aerobic speed was considered as the minimum speed required to elicit the VO2peak [16]. To verify that VO2peak was reached, a con rmatory test was carried out as suggested in previous studies [17,18] after a 5 min recovery of the maximal aerobic test [18]. The speed equivalent to 85% of the maximal aerobic speed was calculated to use in the interval running protocol. After this screening day, participants attended the laboratory to perform the interval running protocol in three different menstrual cycle phases: EFP (day 3.43 0.93), LFP (day 11.95 2.54), and MLP (day 21.86 3.05). In addition, the average day of the positive result in the LH test was 14.02 2.55. In order to avoid learning effects that could in uence our results, the order of these running protocols was randomized, and in no case, an order involved evaluating a volunteer in more than two cycles: EFP-LFP-MLP; LFP-MLP-EFP; MLP-EFP-LFP; LFP-EFP-MLP; EFP-MLP-LFP. 2.3. Interval Running Protocol To avoid diurnal variability [3], participants came to the laboratory between 8 and 10 a.m., after abstaining from alcohol or caffeine consumption and any intense physical activity or sport practice the 24 h prior the testing day. Nutritional recommendations were provided to the participants by a nutritionist in order to standardize the diet, and volunteers followed these 24 h prior to every test. In addition, participants replicated the same breakfast in each protocol performed in the different menstrual cycle phases.Figure shows the protocol of the testing procedure day. Firstly, a blood sample was collected to analyze sex hormones, followed by a standing blood pressure (BP) measurement, using the auscultatory method with a calibrated sphygmomanometer. Subsequently, participants started the interval running protocol consisting of a 5 min warm-up at 60% of their maximal aerobic speed followed by 8 bouts of 3 min at 85% of their maximal aerobic speed with 90-s recovery at 30% of their maximal aerobic speed between bouts. Finally, 5 min cool down was performed at 30% of their maximal aerobic

sphygmomanometer. Subsequently, participants started the interval running protocol consisting of a 5 min warm-up at 60% of their maximal aerobic speed followed by 8 bouts of 3 min at 85% of their maximal aerobic speed with 90-s recovery at 30% of their maximal aerobic speed between bouts. Finally, 5 min cool down was performed at 30% of their maximal aerobic speed. During this protocol, Ve, breathing frequency (BF), VO2, carbon dioxide production (VCO2), RER, HR and energy expenditure (EE) were continuously measured using the same apparatus as mentioned for the maximal aerobic test. Cardiorespiratory values were obtained as the mean of the 5 min warm-up, as well as the mean of the 5 min cool down. Likewise, values over the interval running

Int. J. Environ. Res. Public Health2021,18, 860 4 of 12 protocol were elicited as the mean of the 3 min high intensity intervals and the mean of the 90-s recovery intervals.Int. J. Environ. Res. Public Health 2021, 18, x FOR PEER REVIEW 13 of 13 tocol, Ve, breathing frequency (BF), VO 2, carbon dioxide production (VCO2), RER, HR and energy expenditure (EE) were continuously measured using the same apparatus as mentioned for the maximal aerobic test. Cardiorespiratory values were obtained as the mean of the 5 min warm-up, as well as the mean of the 5 min cool down. Likewise, values over the interval running protocol were elicited as the mean of the 3 min high intensity intervals and the mean of the 90-s recovery intervals. Additionally, rate of perceived exertion (RPE) and perceived readiness (PR) were respectively measured by RPE Borg 6–20 scale [19] and PR Nurmekivi 1–5 scale [20]. Participants were asked for RPE in the last 5 s of warm-up and every running bout, and at the end of the cool down. PR scale was applied in the last 5 s of warm-up and active recovery intervals from 1 to 7, and at the end of the cool down. Figure 1. Protocol of the testing procedure day. EFP: early-follicular phase; LFP: late-follicular phase; MLP: mid-luteal phase; v VO 2peak: maximal aerobic speed. 2.4. Menstrual Cycle Monitoring and Phase Determination Considering the first day of the cycle the onset of menstruation, the days of testing were: between the 2nd and the 5th day of the cycle for the EFP, between one and three days before the ovulation day for the LFP and between five and nine days following ov- ulation for the MLP. These three specific phases were selected in order to analyse dif- ferent hormonal environments as literature suggests [2,21]: low E2 and progesterone levels in the EFP, low progesterone but high E2 levels in the LFP and elevated levels of Figure 1. Protocol of the testing procedure day. EFP: early-follicular phase; LFP: late-follicular phase; MLP: mid-luteal phase; v VO 2peak: maximal aerobic speed. Additionally, rate of

selected in order to analyse dif- ferent hormonal environments as literature suggests [2,21]: low E2 and progesterone levels in the EFP, low progesterone but high E2 levels in the LFP and elevated levels of Figure 1. Protocol of the testing procedure day. EFP: early-follicular phase; LFP: late-follicular phase; MLP: mid-luteal phase; v VO 2peak: maximal aerobic speed. Additionally, rate of perceived exertion (RPE) and perceived readiness (PR) were respectively measured by RPE Borg 6–20 scale [19] and PR Nurmekivi 1–5 scale [20]. Participants were asked for RPE in the last 5 s of warm-up and every running bout, and at the end of the cool down. PR scale was applied in the last 5 s of warm-up and active recovery intervals from 1 to 7, and at the end of the cool down. 2.4. Menstrual Cycle Monitoring and Phase Determination Considering the rst day of the cycle the onset of menstruation, the days of testing were: between the 2nd and the 5th day of the cycle for the EFP, between one and three days before the ovulation day for the LFP and between ve and nine days following ovulation for the MLP. These three speci c phases were selected in order to analyse different hormonal environments as literature suggests [2,21]: low E2 and progesterone levels in the EFP, low progesterone but high E2 levels in the LFP and elevated levels of both progesterone and E2 in the MLP. In order to meet this and based on the literature [2,3,21], we applied a three-step method: calendar-based counting, urinary LH measurement and serum hormone analysis.

Int. J. Environ. Res. Public Health2021,18, 860 5 of 12 Firstly, participants were asked to record information about the length of their last six menstrual cycles. These data were provided to a gynaecologist, who con rmed the menstrual cycles were regular and estimated the ovulation day (the middle day of the menstrual cycle 1) as well as the menstrual cycle phases. Then, a hormone ovulation predictor kit (Ellatest, Alicante, Spain) was used to identify the surge of LH in urine. Second morning mid-stream urine sample was collected day to day from three to ve days before LFP protocol until LH surge detection, which occurs 14–26 h before ovulation [2]. If LH surge was not detected or was detected more than 3 days after completion of LFP test, this test was discarded and the dates for the LFP test were recalculated to repeat it. Finally, serum sex hormones (LH, FSH, E2 and progesterone) were measured in each of the menstrual cycle phases selected for the study. Minimum progesterone was set at 16 nmol L 1 in the MLP as a reliable indicator of an ovulatory non luteal phase-de cient cycle [13,22,23]. 2.5. Blood Samples Analyses To avoid diurnal variability [3], blood samples were taken at the same time for all volunteers, between 8–10 a.m. They were obtained with venipuncture into a vacutainer containing clot activator. Following inversion and clotting, the whole blood was centrifuged (LMC-3000 version V.5AD, Biosan, Riga, Latvia) for ten minutes at 3000 rpm. After that, serum was transferred into eppendorf tubes and stored frozen at 80 C until further analysis. Within 1 to 15 days after testing, the serum samples were delivered to the clinical laboratory of the Spanish National Centre of Sport Medicine (Madrid, Spain) to determine sex hormones in order to verify hormonal pro les. Total E2, progesterone, FSH and LH were measured via ADVIA Centaur ® solid-phase competitive chemiluminescent enzymatic immunoassay (IMMULITE 1000 system; Siemens Healthineers AG, Munich, Germany). Inter- and intra-assay coef cients of variation (CV) reported by the laboratory for each variable were, respectively: 11.9% and 8.5% at 93.3 pg/mL and 6.8% and

This study examines how menstrual cycle phases affect exercise responses in trained female athletes.