Iron and Muscular Damage: FEmale Metabolism and Menstrual Cycle During Exercise

Study Overview
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Brief Summary
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This project is an observational controlled randomized counterbalance study. One hundred and three physically active and healthy women were selected to participate in the IronFEMME Study, of which 57 were eumenorrheic, 30 were oral contraceptive users (OCP) and 16 were postmenopausal women. The project consisted on two sections carrying out at the same time: Iron metabolism (Study I) and Muscle damage (Study II). For the study I, the exercise protocol consisted on an interval running test (8 bouts of 3 min at 85% of the maximal aerobic speed), whereas the study II protocol was based on an eccentric-based resistance exercise protocol (10 sets of 10 repetitions of plate-loaded barbell parallel back squats at 60% of their 1RM with 2 min of rest between sets). In both studies, eumenorrheic participants were evaluated at three specific moments of the menstrual cycle: Early-follicular phase, late-follicular phase and mid-luteal phase; OCP performed the trial at two moments: Withdrawal phase and active pill phase. Lastly, postmenopausal women were tested only once, since their hormonal status does not fluctuate. The three-step method was used to verify the menstrual cycle phase: calendar counting, blood analyses confirmation and urine-based ovulation kits. Blood samples were obtained to measure sexual hormones (e.g., 17β-Estradiol, Progesterone), iron metabolism parameters (e.g., Hepcidin, Iron, Ferritin, Transferrin) and muscle damage related markers (e.g., Creatine Kinase, Myoglobin, Lactate Dehydrogenase).

Official Title
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Iron and Muscular Damage: FEmale Metabolism and Menstrual Cycle During Exercise

Conditions
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Iron-deficiency, Inflammation, Exercise Induced Iron-deficiency, Exercise Induced Muscle Damage, Regulation of Sex Hormones on Hepcidin, Regulation of Sex Hormones on Muscle Damage, Iron Metabolism Disorders

Intervention / Treatment
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* Procedure: Interval running protocol / eccentric-based resistance exercise protocol


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Participants were required to meet the following criteria: Healthy adult females between 18 and 40 years old for eumerroheic and oral contraceptive groups or under 60 years old for postmenopausal women. Presenting with healthy iron parameters (serum ferritin >20μg/l, haemoglobin >115 μg/l and transferrin saturation >16%). Performing endurance training between 5 and 12 h per week (study I) or experienced in resistance training performing at least 1 h session two times per week during a minimum of a year (study II). Exclusion Criteria: The exclusion criteria included: Irregular menstrual cycles. Any existing disease and/or metabolic or hormonal disorder. Any musculoskeletal injury in the last six months prior to the beginning of the project. Any surgery interventions (e.g. ovariectomy) or other medical conditions that would be exacerbated by an eccentric resistance exercise protocol. Regular use of medication or dietary supplements that could affect the results (e.g. nonsteroidal anti-inflammatory drugs). Taking medication that alters vascular function (e.g. tricyclic antidepressants, α-blockers, β-blockers, etc.). Pregnancies in the year preceding. Smoking.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 60 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Eumenorrheic women<br>The project consisted on two sections carrying out at the same time: Iron physiology (Study I) and Muscle damage (Study II). For the study I, the exercise protocol consisted on an interval running test. 5 min warm-up at 60% of the vVO2peak followed by 8 bouts of 3 min at 85% of the vVO2peak with 90 secs recovery at 30% of the vVO2peak between bouts. Finally, a 5 min cool down was performed at 30% of the vVO2peak. The study II protocol was based on an eccentric-based resistance exercise protocol consisted on 10 sets of 10 reps of plate-loaded parallel back squats at 60% of their previously calculated 1RM with 2 mins recoveries between sets. In both studies, eumenorrheic participants were evaluated at three specific moments of the menstrual cycle: Early-follicular phase (EFP), late-follicular phase (LFP) and mid-luteal phase (MLP); | Procedure: Interval running protocol / eccentric-based resistance exercise protocol<br> <br> |
| Oral contraceptive users<br>The project consisted on two sections carrying out at the same time: Iron physiology (Study I) and Muscle damage (Study II). For the study I, the exercise protocol consisted on an interval running test. 5 min warm-up at 60% of the vVO2peak followed by 8 bouts of 3 min at 85% of the vVO2peak with 90 secs recovery at 30% of the vVO2peak between bouts. Finally, a 5 min cool down was performed at 30% of the vVO2peak. The study II protocol was based on an eccentric-based resistance exercise protocol consisted on 10 sets of 10 reps of plate-loaded parallel back squats at 60% of their previously calculated 1RM with 2 mins recoveries between sets. Oral contraceptive users performed the trial at two moments: Withdrawal phase (WP) and active pill phase (APP). | Procedure: Interval running protocol / eccentric-based resistance exercise protocol<br> <br> |
| Postmenopausal women<br>he project consisted on two sections carrying out at the same time: Iron physiology (Study I) and Muscle damage (Study II). For the study I, the exercise protocol consisted on an interval running test. 5 min warm-up at 60% of the vVO2peak followed by 8 bouts of 3 min at 85% of the vVO2peak with 90 secs recovery at 30% of the vVO2peak between bouts. Finally, a 5 min cool down was performed at 30% of the vVO2peak. The study II protocol was based on an eccentric-based resistance exercise protocol consisted on 10 sets of 10 reps of plate-loaded parallel back squats at 60% of their previously calculated 1RM with 2 mins recoveries between sets. Postmenopausal women were tested only once, since their hormonal status does not fluctuate. | Procedure: Interval running protocol / eccentric-based resistance exercise protocol<br> <br> |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Hepcidin | Hepcidin is a protein that in humans is encoded by the HAMP gene. Hepcidin is a key regulator of the entry of iron into the circulation in mammals | pre-exercise | 
| Hepcidin | Hepcidin is a protein that in humans is encoded by the HAMP gene. Hepcidin is a key regulator of the entry of iron into the circulation in mammals | 0 hours post-exercise | 
| Hepcidin | Hepcidin is a protein that in humans is encoded by the HAMP gene. Hepcidin is a key regulator of the entry of iron into the circulation in mammals | 3 hours post-exercise | 
| Hepcidin | Hepcidin is a protein that in humans is encoded by the HAMP gene. Hepcidin is a key regulator of the entry of iron into the circulation in mammals | 24 hours post-exercise | 
| Creatine kinase | It is an enzyme that helps regulate the concentration of adenosine triphosphate within a cell. To do so, creatine kinase catalyzes the movement of a phosphate group from ATP to creatine, forming phosphocreatine. This molecules stores the phosphate group in a stable form, acting as an energy reservoir in cells. | pre-exercise | 
| Creatine kinase | It is an enzyme that helps regulate the concentration of adenosine triphosphate within a cell. To do so, creatine kinase catalyzes the movement of a phosphate group from ATP to creatine, forming phosphocreatine. This molecules stores the phosphate group in a stable form, acting as an energy reservoir in cells. | 2 hours post-exercise | 
| Creatine kinase | It is an enzyme that helps regulate the concentration of adenosine triphosphate within a cell. To do so, creatine kinase catalyzes the movement of a phosphate group from ATP to creatine, forming phosphocreatine. This molecules stores the phosphate group in a stable form, acting as an energy reservoir in cells. | 24 hours post-exercise | 
| Creatine kinase | It is an enzyme that helps regulate the concentration of adenosine triphosphate within a cell. To do so, creatine kinase catalyzes the movement of a phosphate group from ATP to creatine, forming phosphocreatine. This molecules stores the phosphate group in a stable form, acting as an energy reservoir in cells. | 48 hours post-exercise | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Iron |  | pre-exercise | 
| Iron |  | 0 hours post-exercise | 
| Iron |  | 3 hours post-exercise | 
| Iron |  | 24 hours post-exercise | 
| Transferrin |  | pre-exercise | 
| Transferrin |  | 0 hours post-exercise | 
| Transferrin |  | 3 hours post-exercise | 
| Transferrin |  | 24 hours post-exercise | 
| Ferritin |  | pre-exercise | 
| Ferritin |  | 0 hours post-exercise | 
| Ferritin |  | 3 hours post-exercise | 
| Ferritin |  | 24 hours post-exercise | 
| Mioglobin |  | pre-exercise | 
| Mioglobin |  | 2 hours post-exercise | 
| Mioglobin |  | 24 hours post-exercise | 
| Mioglobin |  | 48 hours post-exercise | 
| LDH | Lactate deshidrogenase | pre-exercise | 
| LDH | Lactate deshidrogenase | 2 hours post-exercise | 
| LDH | Lactate deshidrogenase | 24 hours post-exercise | 
| LDH | Lactate deshidrogenase | 48 hours post-exercise | 
| TNF-alfa |  | pre-exercise | 
| TNF-alfa |  | 2 hours post-exercise | 
| TNF-alfa |  | 24 hours post-exercise | 
| TNF-alfa |  | 48 hours post-exercise | 
| Interleukin-6 |  | pre-exercise | 
| Interleukin-6 |  | 0 hours post-exercise | 
| Interleukin-6 |  | 2 hours post-exercise | 
| Interleukin-6 |  | 24 hours post-exercise | 
| Interleukin-6 |  | 48 hours post-exercise | 
| CRP | C-reactive protein | pre-exercise | 
| CRP | C-reactive protein | 0 hours post-exercise | 
| CRP | C-reactive protein | 2 hours post-exercise | 
| CRP | C-reactive protein | 24 hours post-exercise | 
| CRP | C-reactive protein | 48 hours post-exercise | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Iron deficiency, Menstrual cycle, Muscle damage, Creatine kinase, Female, Hepcidin, Exercise, Estrogen, Progesterone, Oral contraceptives, Postmenopausal women