Influence of Oral Contraceptive Use on Adaptations to Resistance Training

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Influence of Oral Contraceptive Use on Adaptations to Resistance Training © 2019 The Authors Published version Dalgaard, Line B.; Dalgas, Ulrik; Andersen, Jesper L.; Rossen, Nicklas B.; Møller, Andreas Buch; Stødkilde-Jørgensen, Hans; Jørgensen, Jens Otto; Kovanen, Vuokko; Couppé, Christian; Langberg, Henning; Kjær, Michael; Hansen, Mette Dalgaard, L. B., Dalgas, U., Andersen, J. L., Rossen, N. B., Møller, A. B., Stødkilde-Jørgensen, H., Jørgensen, J. O., Kovanen, V., Couppé, C., Langberg, H., Kjær, M., & Hansen, M. (2019). Influence of Oral Contraceptive Use on Adaptations to Resistance Training. Frontiers in Physiology, 10, Article 824. https://doi.org/10.3389/fphys.2019.00824 2019 fphys-10-00824 June 28, 2019 Time: 15:13 # 1 ORIGINAL RESEARCH published: 02 July 2019 doi: 10.3389/fphys.2019.00824 Edited by: Hassane Zouhal, University of Rennes 2 – Upper Brittany, France Reviewed by: Michael D. Roberts, Auburn University, United States Aaron Petersen, Victoria University, Australia Nathalie Boisseau, University of Auvergne, France *Correspondence: Mette Hansen [email protected] Specialty section: This article was submitted to Exercise Physiology, a section of the journal Frontiers in Physiology Received: 28 February 2019 Accepted: 12 June 2019 Published: 02 July 2019 Citation: Dalgaard LB, Dalgas U, Andersen JL, Rossen NB, Møller AB, Stødkilde-Jørgensen H, Jørgensen JO, Kovanen V, Couppé C, Langberg H, Kjær M and Hansen M (2019) Influence of Oral Contraceptive Use on Adaptations to Resistance Training. Front. Physiol. 10:824. doi: 10.3389/fphys.2019.00824 Influence of Oral Contraceptive Use on Adaptations to Resistance Training Line B. Dalgaard1, Ulrik Dalgas1, Jesper L. Andersen2, Nicklas B. Rossen3,4, Andreas Buch Møller3, Hans Stødkilde-Jørgensen5, Jens Otto Jørgensen3,4, Vuokko Kovanen6, Christian Couppé2, Henning Langberg2,7, Michael Kjær2and Mette Hansen1,2* 1Section of Sport Science, Department of Public Health, Aarhus University, Aarhus, Denmark, 2Department of Orthopedic Surgery M, Bispebjerg Hospital and Center for Healthy Aging, Institute of Sports Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark, 3Steno Diabetes Center Aarhus, Aarhus University Hospital, Aarhus, Denmark, 4Department of Endocrinology and Internal Medicine, Medical Research Laboratories, Aarhus University Hospital, Aarhus, Denmark, 5Center of Magnetic Resonance, Aarhus University Hospital, Skejby, Denmark, 6Faculty of Health Sciences, University of Jyväskylä, Jyväskylä, Finland, 7CopenRehab, Department of Public Health, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Introduction: The majority of young women use oral contraceptives (OCs). Use of OCs has been associated with lower myofibrillar protein and tendon collagen synthesis rates, but it is unknown whether OCs will limit the adaptive response of myotendinous tissue to resistance training. Design and Methods: Fourteen healthy untrained young regular OC users (24 ±1 years, fat% 32 ±1, 35 ±2 ml·min−1·kg−1) and 14 NOC users (nonOC, controls) (24 ±1 years, fat% 32 ±2, 34 ±2 ml·min−1·kg−1) performed a 10-week supervised lower extremity progressive resistance training program. Before and after the intervention biopsies from the vastus lateralis muscle and the patellar tendon were obtained. Muscle (quadriceps) and tendon cross-sectional area (CSA) was determined by magnetic resonance imaging (MRI) scans, and muscle fiber CSA was determined by histochemistry. Maximal isometric knee extension strength was assessed by dynamometry while 1 repetition maximum (RM) was determined during knee extension. Results: Training enhanced CSA in both muscle (p<0.001) and tendon (p<0.01). A trend toward a greater increase in muscle CSA was observed for OC (11%) compared to NOC (8%) (interaction p= 0.06). Analysis of mean muscle fiber type CSA showed a trend toward an increase in type II muscle fiber area in both groups (p= 0.11, interaction p= 0.98), whereas type I muscle fiber CSA increased in the OC group (n= 9, 3821 ±197 to 4490 ±313 mm2,p<0.05), but not in NOC (n= 7, 4020 ±348 to 3777 ±354 mm2,p= 0.40) (interaction p<0.05). Post hoc analyses indicated that the effect of OCs on muscle mass increase was induced by the OC-users (n= 7), who used OCs containing 30 µg ethinyl estradiol (EE), whereas the response in users taking OCs with 20 µg EE (n= 7) did not differ from NOC. Both the OC and NOC group experienced an increase in maximal knee strength (p<0.001) and 1RM leg extension (p<0.001) after the training period with no difference between groups. Frontiers in Physiology | www.frontiersin.org 1July 2019 | Volume 10 | Article 824 fphys-10-00824 June 28, 2019 Time: 15:13 # 2 Dalgaard et al. OC Effect on Training Adaptations Conclusion: Use of OCs during a 10-week supervised progressive resistance training program was associated with a trend toward a greater increase in muscle mass and a significantly greater increase in type I muscle fiber area compared to controls. Yet, use of OCs did not influence the overall increase in muscle strength related to training. Keywords: exercise, estrogen, estradiol, women, muscle hypertrophy, tendon, muscle strength INTRODUCTION Worldwide, more than 100 million fertile young women use oral contraceptives (OCs) for menstrual regulation and contraception (Christin-Maitre, 2013). It has been suggested that OCs may affect sports performance through influence on steroid hormone receptors located in the peripheral tissues such as skeletal muscle and tendon (Liu et al., 1996;Wiik et al., 2005;Ekenros et al., 2017). Yet, knowledge on the effects of OC on long term adaptations to training is sparse. Most women use monophasic OC pill formulations containing synthetic estrogen, ethinyl estradiol (EE) and synthetic progesterone, progestin, in a constant dose during the pill cycle. Depending on brand name and OC “generation,” the formulation of EE typically varies between 20 and 50 µg daily dose, while the different types of progestins differ in dose depending on potency (Stanczyk, 2003;Burrows and Peters, 2007). In addition, progestins also differ in androgenocity introducing a potential influence on adaptations to training (Ruzic et al., 2003). Furthermore, use of OCs is associated with reduced endogenous levels of natural estrogen, progesterone, free testosterone and insulin-like growth factor I (IGF-I), but enhanced levels of cortisol (Hansen et al., 2011;Zimmerman et al., 2014;Eisenhofer et al., 2017), which may also affect the anabolic response to training. To study the effects of OC on exercise induced muscle growth, resistance training seems as the optimal exercise modality because of its well-known hypertrophic effects (Folland and Williams, 2007). Nonetheless, only a few studies have so far investigated the potential role of OCs in relation to adaptation to resistance training (Ruzic et al., 2003;Nichols et al., 2008). One study showed no difference in strength gain between users (n= 13) and non-users of OC (n= 18) following 12 weeks of resistance training (Nichols et al., 2008), but no information on OC type was provided. This may be of importance since a smaller strength gain has been reported following 16 weeks resistance training in users of anti-androgenic OCs (EE and cyproterone acetate, n= 26) compared to users of high androgenic OCs (EE and levonorgestrel, n= 24) (Ruzic et al., 2003). Unfortunately, no control group of non-OC users was included in the latter trial, making the interpretation of the results challenging. Changes in skeletal muscle mass are determined by the balance between myofibrillar protein synthesis and breakdown. In users of low androgenic OCs (EE and gestoden) myofibrillar fractional protein synthesis rate has been reported to be lower 24 h following one-legged kicking exercise compared to non-users of OC (Hansen et al., 2011). The influence of OCs on myofibrillar protein breakdown rate has not been clearly elucidated, and has only been studied indirectly by analyzing interstitial fluid obtained from the skeletal muscle for 3-methyl-histidine, which did not show a difference between OC users and non-users (Hansen et al., 2011). Based on the sparse existing data, we therefore hypothesized, that OC use may negatively influence the gain in skeletal muscle mass and strength in response to resistance training compared to non-users of OC, but the effect may depend on the type of OC (content of EE and type of progestin). The biomechanical properties of tendon and ligaments improve in response to regular training (Svensson et al., 1985), and regular resistance training has been shown to induce tendon hypertrophy in groups of men (Kongsgaard et al., 2007) and in a mixed group of men and women (Arampatzis et al., 2007). However, cross-sectional data in runners suggests that the ability to adapt to training is reduced in women (Westh et al., 2008). Since tendon adaptations have important perspectives related to lowering of the risk of injury in tendon and ligaments, the effect of regular resistance training as a function of OC use must be clarified. It has been suggested that circulating estrogen (endogenous or exogenous) alters the risk of injuries by changing the structural composition and adaptations to exercise, and thereby also the mechanical properties ofthe tendon (Magnusson et al., 2007;Hansen and Kjaer, 2016). In support, estradiol increases tendon collagen synthesis and the overall tendon collagen turnover, while it reduces tendon and ligament stiffness (Lee et al., 2015). In contrast, use of OCs containing EE is associated with a lower tendon collagen synthesis rate in the patellar tendon compared to non-users both at rest and after acute knee-extensor exercise (Hansen et al., 2008, 2009a). Yet, no previous studies have looked into how OC use influences the response to regular resistance training. The overarching aim of the study was therefore to compare adaptations in skeletal muscle and tendon to 10 weeks of resistance training in users of low-androgenic OCs vs. NOC. We hypothesized that OC use reduces the anabolic effect of resistance training in the skeletal muscle and tendon. MATERIALS AND METHODS Subjects Thirty young, healthy women already using 3rd generation OCs or using no OCs were recruited through local newspapers and included in the study. All participants were non-smokers, nonusers of medication, and without orthopedic and metabolic disorders that would influence their participation in the training intervention. Exclusion criteria were: resistance training more than once every month within the last 6 months, other types of Frontiers in Physiology | www.frontiersin.org 2July 2019 | Volume 10 | Article 824 fphys-10-00824 June 28, 2019 Time: 15:13 # 3 Dalgaard et al. OC Effect on Training Adaptations physical training more than 2 h per week, bike transportation more than 20 km per day, and being on a hypocaloric diet to reduce weight. The 14 OC users had used OCs for 6.1 ±5.0 years. Third generation OCs are characterized as being low-androgenic (Burrows and Peters, 2007). The brand names of the OCs were Lindynette (30 µg EE and 75 µg gestoden per day, n= 7), Gestonette (20 µg EE and 75 µg gestoden per day, n= 5), or Novynette (20 µg EE and 150 µg desogestrel per day, n= 2). The 14 NOCs had regular menstrual cycles for at least 1 year (within the range of 24–35 days). The subjects were informed about the risks and benefits associated with the study and provided written informed consent. The protocol followed the Declaration of Helsinki and was approved by the Central Denmark Region Committees on Health Research Ethics (Journal No.: M-20100187). Design The study compared adaptations in skeletal muscle and tendon after 10 weeks of resistance training in OC and NOC users (Figure 1). Each participant visited the laboratory on three separate occasions within 1–2 weeks prior to the start of training period (baseline). At the first visit height, weight, body composition [Dual-Energy x-ray absorptiometry (DXA)], aerobic fitness (VO2max/kg) determined indirectly by a submaximal bike test (Åstrand and Rodahl, 1986), and physical activity (MET/24 h) level determined by questionnaire (Aadahl and Jorgensen, 2003). At the same day, the participants were familiarized with the testing procedures of the 1 repetition maximum (RM) and isometric dynamometer measurements to minimize any learning effects. At the second visit magnetic resonance imaging (MRI) of the thigh muscle and patellar tendon were collected. MRI was performed on average 5.9 days [range 2–13 days] after the initial visit and before collection of strength measures and tissue samples to avoid edema. At the third visit, blood, tendon and muscle tissue samples were collected in the morning after an overnight fast, and isometric muscle strength was determined by dynamometry. Muscle and tendon samples were collected in either the dominant or non-dominant leg as determined by randomization. The isometric muscle strength was determined in the leg that had not been biopsied. All tests were repeated after the resistance training intervention period (post-training). The isometric dynamometer strength was performed on the day before the last training session. MRI data were collected before the blood, muscle and tendon samples, at 48 or 72 h after the last training session. In addition, 4 days of self-reported dietary intake were recorded in week 1 and 10 of the intervention period (Vitakost, Kolding, Denmark) and recreational physical activity logs were collected during the training intervention. The participants were asked to remain weight-stable during the intervention. Resistance Training Protocol Test and training sessions were completed at the Section for Sports Science at Aarhus University. The exercise protocol consisted of 10 weeks of progressive resistance training performed 3 times per week supervised by physical therapists. Training intensities were estimated from the 1 RM test. The exercises consisted of seated knee extensions and inclined leg press performed in a progressive manner; week 1: 3x 12 repetitions of 15 RM; week 2–3: 3x 12 repetitions of 12 RM; week 4–5: 3x 10 repetitions of 10 RM; week 6–10: 4x 10 repetitions of 10 RM. Each training session was initiated with a 10 min warm-up on a stationary bike. The 1 RM test of knee extension strength was performed at the fourth training session (baseline test), and was repeated at every third training session as well as during the last training session of the intervention period (post-test). All subjects were provided with a standardized amount of protein (Maxim Pink protein bar, 10.6 g protein) after each training session. 1RM Strength Test Participants warmed-up with 10 min ergometer cycling (60 W) followed by six repetitions at 60% of the 1 RM predicted from the familiarization session. The estimated starting weight was 85% of the previous 1 RM. The load was increased or reduced until a true 1 RM using a full range of motion was reached. Three to five trials separated by 5 min rest was allowed. One participant from each group was unable to perform the 1RM test after the training, reducing n to 13 in each group. Isometric Dynamometer Strength Participants were seated in an isometric dynamometer (Humac Norm, CSMi, Stoughton, MA, United States) with a hip angle of 90◦and strapped by the hip. The non-working leg was FIGURE 1 | Study overview. Frontiers in Physiology | www.frontiersin.org 3July 2019 | Volume 10 | Article 824 fphys-10-00824 June 28, 2019 Time: 15:13 # 4 Dalgaard et al. OC Effect on Training Adaptations positioned behind a stabilizing bar and participants were asked to keep their arms crossed over the chest. The rotational axis of the dynamometer was aligned with the transverse knee-joint axis of the working leg and attached to the lower leg by a length adjustable lever arm 3 cm proximal to malleolus medialis. After a standardized instruction (to contract as strongly and fast as possible) and two familiarization attempts, the subjects performed three to five (depending on the consistency of the first trials) maximal voluntary isometric contractions (MVIC) for the knee extensors at a knee angle of 70◦. The subjects received visual feedback and verbal encouragement during the maximal contractions. All contractions were separated by at least 1 min. of rest. The attempt with the highest MVIC, defined as peak torque measurement, was used for further analysis. All isometric strength data were sampled and exported using TeleMyo Direct Transmission System and MyoResearch Software (Noraxon USA, Scottsdale, AZ, United States). A sampling frequency of 1500 Hz was applied and the final analyses were performed using custom-made software. To normalize data, all strength measurements were scaled to body-weight (Jaric, 2003; Aagaard et al., 2007). Muscle and Tendon Cross-Sectional Area (CSA) Magnetic resonance imaging of muscle and tendon CSA was performed at the MR Centre at Aarhus University Hospital, Aarhus, Denmark. No strenuous exercise was performed within 48 h before the baseline-MRI and the post-MRI was performed 48–72 h after the last training session. This was done to minimize the risk of an exercise induced increase in muscle water content (Kristiansen et al., 2014). Participants were placed in a supine position with fully extended, relaxed legs. Muscle CSA was measured 20 cm proximal to the tibia plateau (TP), using MR-images (Siemens Magnetom Avanto 1.5 Tesla, Siemens AG, Munich, Germany, T1 weighted) from two lower extremity coils. An initial image was used to determine the proximal, lateral plateau of tibia and this point was then used as the reference position. The muscle CSA of vastus lateralis was manually outlined using the software program OsiriX DICOM Viewer (version 7, OsiriX HD, 2011 Pixmeo SARL, Bernex, Switzerland). The Patellar tendon CSA was measured in the same scanner in three anatomical regions directly beneath the patellar insertion (proximal) by determining the first picture on which the patella was not present, just above the tibia insertion (distal) by determining the first picture on which Hoffa’s fat pat was no longer seen, and midway between these two regions (central), using MR-images with the following parameters: repetition time (TR)/echo time (TE) 400/15 ms, field of view (FOV) 160, matrix 256 ×256, slice thickness 3 mm (Hansen et al., 2013). The MRIs of the Patellar tendon were performed using a special knee coil placing the knee in a fixed angle of ∼120◦. The perpendicular angle of the tendon was set using a localizer, and axial tendon slices were obtained. Patellar tendon CSA was manually outlined using the software program OSIRIX 7. For both muscle and tendon data analyses were performed by a single technician in a blinded fashion. A mean value of three measurements of the same image was calculated and used for analysis (Supplementary Figure S1). The coefficient of variation was found to be less than 5%. One participant from the NOC group did not have an MRI scan due to claustrophobia. Collection and Analyses of Muscle Tissue Participants were asked to avoid physical activity 48 h before biopsy collection. Muscle tissue samples were collected from m. vastus lateralis of the dominant or the non-dominant leg determined in a random fashion using a 5 mm Bergström needle under local anesthetic (1% lidocaine) after an overnight fast. Muscle samples of ∼100 mg were dissected free of fat and connective tissue, and one part was embedded in TissueTek and then frozen in pre-cooled isopentane, and the other part was snap frozen in liquid nitrogen. All samples were stored at −80◦C until analysis. Standard ATPase staining performed on 7 µm serial sections was used to evaluate fiber type distribution and muscle fiber CSA (Brooke and Kaiser, 1970). The serial sections were visualized and analyzed using a Leica DM2000 microscope (Leica, Stockholm, Sweden) and a Leica Hi-resolution Color DFC camera (Leica, Stockholm, Sweden) combined with imageanalysis software (Leica Qwin ver. 3, Leica, Stockholm, Sweden) essentially as previously described (Dalgas et al., 2010). Briefly, a fiber mask was automatically drawn by the software based on a immunohistochemically staining of the basement membrane. This mask was fitted manually to the cell borders of the included muscle fibers. Only fibers cut perpendicularly to their longitudinal axis were used for the determination of fiber size. Images of the three ATPase stainings were then fitted with the fiber mask. Descriptive statistical analysis by the software allowed determination of the relative proportion of the various fiber types and fiber CSA. Blinded analyses were performed by trained laboratory personnel. The determination of muscle fiber CSA and typing of fibers were carried out essentially in accordance with the procedure described by Andersen et al. (Andersen and Aagaard, 2000). Calculation of muscle fiber distribution was performed for the three fiber types (I, IIa, and IIx) and muscle fiber CSA was performed for the two major fiber types (I and II). For the analysis of muscle fiber type distribution, n= 13 from each group before and after the intervention period (average number of fiber per subject 148 ±3). Several of the muscle biopsy slides for histochemical analysis were destroyed due to freeze damage and problems with the analysis procedure. Therefore, the analysis of changes in muscle fiber area, was performed on n= 9 for OC and n= 7 for NOC (average number of fibers per subject 123 ±6) and type IIx muscle fibers were not included in the analysis due to absence or very low numbers particularly at the post-time-point. Muscle Protein Expression Western blot analyses were used to assess protein expression and phosphorylation levels of muscle proteins. Muscle samples were Frontiers in Physiology | www.frontiersin.org 4July 2019 | Volume 10 | Article 824 fphys-10-00824 June 28, 2019 Time: 15:13 # 5 Dalgaard et al. OC Effect on Training Adaptations homogenized in an ice cold buffer containing (in mM) 50 HEPES, 137 NaCl, 10 Na4P2O7, 10 NaF, 2 EDTA, 1 MgCl2, 1 CaCl2, 2 Na3VO4, 1% (vol/vol) Nonidet P-40, 10% (vol/vol) glycerol, 2µg/ml aprotinin, 5 µg/ml leupeptin, 0.5 µg/ml pepstatin, 10 µg/ml antipain, 1.5 mg/ml benzamidine, and 100 µM 4-(-2-aminoethyl)-benzenesulfonyl fluoride, hydrochloride (pH 7.4), and rotated for 60 min at 4◦C. Subsequently, the samples were centrifugation at 14,000 ×gfor 20 min at 4◦C and the pellet discharged. With this method, we have previously been able to detect many skeletal muscle proteins, however, we might also lose proteins of interest (Murphy and Lamb, 2013). Protein concentration in the supernatant was determined using the Bradford assay. After separating the proteins by gel electrophoresis and blotting them on to a PVDF membrane, the membranes were incubated overnight with the following antibodies: from Cell Signaling Technology (MA, United States): mTOR (cat. no. 2972), p-mTOR Ser2448 (cat. no. 2971), p70S6K (cat. no. 9202), eIF4E (cat. no. 9742), AKT-pan (cat. no. 4691), p-AKT Ser473 (cat. no. 9271), 4EBP1 (cat. no. 9644), non-p-4EBP1 Thr46 (cat. no. 4923), and LC3B (cat. no. 3868); from Abcam (Cambridge, United Kingdom): Fbx32 (cat. no. ab92281) and MURF1 (cat. no. ab172479). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse IgG were used as secondary antibodies and visualized by BioWest enhanced chemiluminescence (Pierce). Quantification was performed using UVP Bio Imaging System software (UVP, Upland, CA, United States). Expression of both non-phosphoprotein and phospho-protein are presented as ratios of target protein to total protein load quantified using the Stain Free Technology. Due to difficulties in obtaining enough muscle tissue, the analysis was performed on n= 8 for OC and n= 12 for NOC. Tendon Samples Tendon biopsies were randomly obtained from the dominant or the non-dominant leg, according to procedures described in detail elsewhere (Hansen et al., 2009c;Kongsgaard et al., 2010). In brief, following sterilization of the insertion site, the skin was injected with local anesthetic (1% lidocaine), and a 3to 5-mm-long incision was made distally to the patella apex. A tendon biopsy was obtained by using a 16-G Monopty biopsy instrument (Bard, Covington, GA, United States) with a disposable core biopsy needle (14 gauge). The biopsy needle was inserted into the tendon surface at a ∼30◦angle and fired, securing a tissue sample of approximately 8–10 mg. The tissue sample was cleared of external adipose tissue and blood, snap-frozen in liquid nitrogen and stored at -80◦C for subsequent analysis. Biochemical analysis of collagen cross-links was performed as previously described in details (Hansen et al., 2013). Freeze-dried tendon samples were hydrolyzed and redissolved in H2O. The collagen-specific cross-linking compounds hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LyP), and pentosidine were analyzed spectrophotometrically by reversedphase high-performance liquid chromatography (HPLC) to quantify collagen protein (Creemers et al., 1997). Analyses of Blood Parameters All blood samples were taken in the morning after an overnight fast (<6 h) and from an antecubital vein. Serum albumin, estradiol, progesterone, testosterone, and sex hormone binding globin (SHBG) were determined at baseline and after the training intervention (post) by standard procedures performed at the Department of Clinical Biochemistry at the Aarhus University Hospital. For each variable, all samples were measured in the same assay run. Statistical Analyses All data are reported as means ±SE unless stated otherwise. All data were tested for normal distribution by visual inspection and using a Shapiro-Wilk normality test. Statistical analyses for differences between OC and NOC in subject characteristics, serum hormones levels, fibril characteristics and tendon content of collagen and cross-links were performed using an unpaired t-test. A paired t-test was used to test for differences between baseline and post-in serum albumin, progesterone and testosterone. The log-transformed normal distributed data for FM, weight, PAL and BMI was used in an unpaired t-test since the data did not pass the normality test. Estradiol and SHBG did not pass normality tests even after log-transformation, thus a Wilcoxon Signed-Rank test was used to test for significance in the raw data between OC and NOC. Test for difference in muscle CSA, muscle fiber CSA, muscle strength, tendon CSA and tendon cross-links caused by training load or use of OC was performed using a two-way ANOVA with repeated measurements followed by a Holm-Sidak post hoc test. Data for tendon CSA at the mid and distal level only passed the normality test after log-transformation. Therefore, log-transformed data for these parameters were used in the analysis. Unpaired t-tests were performed to test for differences in response to training in muscle CSA between different types of OC (containing either 20 or 30 µg EE/pill) and NOC. A Spearman correlation was used to test the association between daily protein intake per kg bodyweight and change in muscle CSA after the intervention. The level of significance was set at p≤0.05, while p≤0.10 was designated as a trend. The following labels were used: ∗∗∗p<0.001, ∗∗p<0.01, and ∗p≤0.05 unless stated otherwise. The statistical analyses were performed using the statistical software package Sigma Plot version 13.0. RESULTS The OC and NOC groups were comparable regarding age, height, weight, body mass index (BMI), physical activity level (MET/24 h), aerobic fitness (VO2max/kg), and body composition (Table 1). The recreational activity logs were checked for changes in activity level during the intervention period, but none of the participants performed strenuous physical training more than 2 h per week and no additional resistance training was performed besides the intervention. Every one of the included participants completed the study. All participants followed and received individual supervision for each training session, and compliance Frontiers in Physiology | www.frontiersin.org 5July 2019 | Volume 10 | Article 824 fphys-10-00824 June 28, 2019 Time: 15:13 # 6 Dalgaard et al. OC Effect on Training Adaptations TABLE 1 | Subject characteristics. NOC (n= 14) OC (n= 14) Age (years) 24 ±1 24 ±1 Height (m) 1.67 ±0.02 1.68 ±0.02 Weight (kg) (Baseline) 66.7±2.1 65.6±3.6 Weight (kg) (Post) 67.3±2.1 66.6±4.1 BMI (kg·m−2) 24 ±1 23 ±2 Body Fat (%) 32 ±2 32 ±2 Aerobic fitness, VO2max (ml·min−1·kg−1) 32 ±2 35 ±3 Physical activity level (MET/24 h) 1.8±0.1 1.6±0.1 Use of OC (years) 0 ±0 6 ±1∗∗∗ BMI, body mass index; Aerobic fitness, maximal oxidative uptake of oxygen/min/kg body weight; OC, oral contraceptives; NOC, non-users of oral contraceptives. No significant differences existed between groups except for use of OC (∗∗∗ denotes p<0.001). Values are mean ±SE. with the training protocol was 100%, as any missed training session was performed another day within the same week. Fourteen OC and 14 NOC users were eligible to be included in the analysis. Two NOC were excluded before the analysis, since they had deliberately reduced their energy intake in the intervention period and lost 8.4 and 7.3% in body weight, respectively, which may have reduced the anabolic potential of resistance training on muscle growth. Muscle CSA and Muscle Fiber CSA Resistance training induced an increase in muscle CSA in both OC (10.8 ±1.3%, p<0.001) and NOC users (7.9 ±0.09%, p<0.001) (Figure 2A and Supplementary Figure S1). Furthermore, a trend toward interaction between OC status and training effect was observed (p= 0.06), which indicated a greater increase in muscle CSA in the OC group than in the NOC group. OC sub analysis indicated that the increased muscle mass was driven by the OC-users (n= 7), who used OCs containing 30 µg EE, whereas the response in users taking OCs with 20 µg EE (n= 7) did not differ from NOC (30 vs. 20 µg EE: 13.1 ±1.7% vs. 8.5 ±1.4%, p= 0.08. 30 µg EE vs. NOC: p= 0.01. 20 µg EE vs. NOC: p= 0.73) (Supplementary Figure S2). For type I muscle fiber CSA, the two-way ANOVA demonstrated a significant interaction between OC status and time (p= 0.04) (Figure 2B). In OC users, type I muscle fiber CSA was significantly greater after the training period (3821 ±197 to 4490 ±313 µm2,p<0.05), whereas no significant change was observed in NOC users (NOC: 4020 ±348 to 3777 ±354 µm2, p= 0.40). Type II muscle fiber CSA showed a non-significant increase after the training period (p= 0.11) in both groups (OC: 3452 ±242 to 3891 ±387 µm2, NOC: 3239 ±344 to 3691 ±361 µm2), but no interaction between OC status and time was observed (p= 0.98) (Figure 2C). The training intervention induced a change in muscle fiber type distribution (Table 2). The training intervention reduced the relative number of type IIx muscle fibers (p<0.001), while the relative type IIa muscle fiber number increased (p<0.05). The changes in type IIx muscle fiber number (p= 0.05) was greater in OC than NOC. In contrast, type I muscle fiber relative number did not significantly change after the intervention. FIGURE 2 | Changes in muscle CSA and muscle fiber CSA. (A) Change in muscle cross-sectional area (mm2) in OC and NOC users (n= 14 and n= 13, respectively) from baseline to post-training. Change in cross-sectional area (µm2) of type I (B) and type II (C) muscle fibers in the OC and NOC group (n=9andn= 7, respectively) from baseline to post-training. OC is illustrated by closed circles, while NOC are shown by open circles. The horizontal line denotes mean ±SE. (∗) denotes a trend (p= 0.66), while a ∗denotes p<0.05. Muscle Strength Absolute muscle strength increased in both OC and NOC users following the training intervention as measured by 1RM test (OC: 18.2 ±3.6% vs. NOC: 15.4 ±1.6%; time p<0.001) and MVIC (OC: 17.3 ±3.6% vs. NOC: 13.8 ±3.5%; time p<0.001). Larger absolute increases were observed in the OC users, however, no significant difference between the two groups was observed in 1RM (interaction p= 0.46) and MVIC (interaction p= 0.36). Normalizing data to individual baseline Frontiers in Physiology | www.frontiersin.org 6July 2019 | Volume 10 | Article 824 fphys-10-00824 June 28, 2019 Time: 15:13 # 7 Dalgaard et al. OC Effect on Training Adaptations TABLE 2 | Muscle fiber type distribution. OC (n= 13) NOC (n= 13) Two-way ANOVA RM Fiber type% Baseline Post Baseline Post Training Group Interaction Type I 46.9 ±2.8 48.3 ±2.5 53.4 ±2.7 52.4 ±2.9 0.91 0.13 0.52 Type IIa 42.6 ±2.5 47.7 ±2.5 39.4 ±2.6 42.8 ±2.1 <0.05 0.16 0.64 Type IIx 10.5 ±2.2 3.9 ±1.5 7.1 ±2.1 4.8 ±1.2 <0.001 0.59 0.05 The relative distribution (%) of type I, type IIa, and type IIx muscle fibers in OC users and NOC users is shown. Data are reported as mean ±SE. and post-body weight (bw) did not influence the results (1RM; OC: 1.12 ±0.05 kg/kg bw (baseline) vs. 1.29 ±0.05 kg/kg bw (post); NOC: 1.05 ±0.09 kg/kg bw (baseline) vs. 1.20 ±0.10 kg/kg bw (post), p<0.001 for training effect, interaction p= 0.66. MVIC; OC: 3.0 ±0.14 Nm/kg bw (baseline) vs. 3.5 ±0.18 Nm/kg bw (post); NOC: 3.0 ±0.17 Nm/kg (baseline) vs. 3.3 ±0.19 Nm/kg (post), p= 0.01 for training effect, interaction p= 0.56) (Figure 3). Muscle Tissue Protein Expression The mTOR expression increased with time (p<0.05), while Murf-1 showed a trend toward a decrease at the post-time point (p= 0.08). eIF4E and p70S6K showed a positive interaction between time and OC-status (p<0.05) revealing an decreased expression in OC users and an increased expression in NOC users after the training period. In contrast, protein expression of mTORSer2448, Akt2, AktSer473, 4EBP1, 4EBP1Thr46(non−p), Atrogin-1, and the ratio of LC3B-II/LC3B-I were not different between OC and NOC groups (Supplementary Figure S3). Tendon Cross Sectional Area and Cross-Links Tendon CSA (proximal, mid, and distal) did not differ between the dominant and non-dominant leg and only average values for both legs are shown in Table 3. After the training period, tendon CSA at all three levels (proximal, mid, and distal) was significantly increased compared to baseline. However, no difference between groups was observed in the response to training. Analysis of tendon collagen content (dry weight), and cross-links (fractions of HP, LyP, and pentosidine) was performed in 14 OCs and 11 NOCs, since attempts to obtain a tendon biopsy were not successful in three NOCs (Table 3). A trend for higher concentration of HP in the OC than the NOC group was observed (p= 0.07), but otherwise no group difference between OC and NOC or difference in response to training were observed (Table 3). Blood Parameters At baseline and post-training, serum (s-) albumin and s-SHBG were higher in OC users compared to NOC, whereas s-estradiol, s-progesterone, and s-testosterone were lower in the OC compared to the NOC group (Table 4). S-estradiol was below the detection limit in 11 out of 14 OC-users at baseline and 8 out of 14 OC users after the intervention. The SHBG level in NOC users was higher post-training than at baseline (p<0.001). Similarly, NOC mean serum progesterone tended to be higher after the intervention vs. baseline (p= 0.06). Energy and Macronutrient Intakes Eating habits were not different at week 1 and 10 of the intervention. Therefore, only average energy and macronutrient intake from the two periods are included in Table 5. One subject from each group was excluded from the analysis due to unreliable energy intake registration based on cut-off values (Goldberg et al., 1991), despite that all subjects were weight-stable during the intervention. No significant difference in energy intake, fat, carbohydrate and protein intake was observed between groups, but the protein intake per kg body mass was higher in OC than NOC users. FIGURE 3 | Changes in muscle strength. (A) Difference in 1 repetition maximum (1RM) in OC and NOC users from baseline to post-training (kg) (n= 13 in each group). (B) Changes in maximum voluntary isometric strength (MVIC) in users of OC and NOC users from baseline to post-training (Nm/Kg Bw) (n= 14 in each group). Bw, body weight; OC, closed circles; NOC, open circles. The horizontal line denotes mean ±SE. Frontiers in Physiology | www.frontiersin.org 7July 2019 | Volume 10 | Article 824 fphys-10-00824 June 28, 2019 Time: 15:13 # 8 Dalgaard et al. OC Effect on Training Adaptations TABLE 3 | Tendon CSA, and concentrations of collagen and collagen cross-links. OC (n= 14) NOC (n= 14) Two-way ANOVA RM Baseline Post Baseline Post Training Group Interaction Tendon CSA proximal 81 ±6 87 ±5 77 ±3 85 ±5<0.05 0.71 0.70 Tendon CSA middle 78 ±4 90 ±4 80 ±3 97 ±7<0.001 0.37 0.57 Tendon CSA distal 95 ±5 101 ±5 100 ±5 109 ±5<0.05 0.31 0.57 OC (n= 14) NOC (n= 11) Baseline Post Baseline Post Training Group Interaction Collagen 0.62 ±0.02 0.64 ±0.02 0.61 ±0.03 0.62 ±0.04 0.61 0.66 0.72 HP/Collagen 0.73 ±0.06 0.80 ±0.05 0.63 ±0.06 0.65 ±0.06 0.39 0.07 0.56 LyP/Collagen 0.03 ±0.02 0.03 ±0.00 0.04 ±0.01 0.03 ±0.00 0.18 0.81 0.13 Pentosidine/Collagen 0.012 ±0.001 0.012 ±0.001 0.011 ±0.001 0.012 ±0.001 0.47 0.46 0.44 CSA (mm2) at the proximal, middle and distal part of the patella tendon in OC users and NOC users is reported along with collagen concentration (mg/mg d.w; dry weight) and collagen cross-links of tendon as fractions of HP, LyP, and pentosidine relative to collagen (pmol/pmol). Data are shown as mean ±SE. TABLE 4 | Blood parameters. NOC (n= 14) OC (n= 14) P-values Albumin (g/L) Baseline 42 ±1 46 ±1<0.01 Post 42 ±1 46 ±1<0.05 Estradiol (nmol/L) Baseline 0.45 ±0.06 11 out of 14 < 0.05 nmol/L <0.05 Post 0.72 ±0.17 8 out of 14 < 0.05 nmol/L <0.05 Progesterone (nmol/L) Baseline 15.2±4.2 1.1±0.1a<0.01 Post 27.2±8.8 1.1±0.2b<0.05 Testosterone (nmol/L) Baseline 1.18 ±0.14 0.76 ±0.07 <0.05 Post 1.35 ±0.12 0.90 ±0.09 <0.05 SHBG (nmol/L) Baseline 57 ±6 204 ±14 <0.01 Post 65 ±7#224 ±13 <0.01 Serum concentrations of albumin, estradiol, progesterone, testosterone, and sex hormone binding globin (SHBG) in OC and NOC at baseline and post-training is reported. Data are shown as mean ±SE. a2 out of 14 in OC had a progesterone level <0.03 nmol/L. bOne out of 14 in the OC group had a progesterone level <0.03 nmol/L. A#designate a significant difference between baseline and post-training values, p <0.05. P-values designate differences between OC and NOC. TABLE 5 | Energy and macronutrient intake. OC (n= 13) NOC (n= 13) P-values Energy intake (kJ/day) 8390 ±1960 8630 ±1716 0.74 Carbohydrates, E% 52 ±4 49 ±4 0.07 Fat, E% 27 ±4 30 ±6 0.17 Protein, E% 17 ±4 15 ±2 0.06 Protein, g/day 81 ±10 74 ±14 0.15 Protein g/kg bw/day 1.3 ±0.2 1.1 ±0.2 0.03 The average of two 4-day self-reported food records performed in week 1 and 10 of the intervention is shown for OC and NOC. E%, % of total daily energy intake; Bw, body weight. Data are shown as mean ±SE. DISCUSSION Studies investigating the influence of OCs on adaptation to resistance training are very few and conflicting. Here, we present the first data based on a controlled intervention focused on elucidating the effect of a single type of OC (3rd generation). Furthermore, the participants performed one-on-one supervised resistance training with a high level of compliance and a controlled progressive overload. These factors are important when interpreting the results since other studies have used a mix of OCs (Ruzic et al., 2003;Nichols et al., 2008;Ekenros et al., 2013;Wikstrom-Frisen et al., 2017) and failed to improve muscle strength following the resistance training intervention probably due to a less controlled (and intense) training intervention (Wikstrom-Frisen et al., 2017). Contrasting to our hypothesis, a trend toward a larger increase in muscle mass (MRI) (p= 0.06) was observed in OC compared to NOC users (11 vs. 8%). This contrasts our expectations based previous data showing a lower myofibrillar fractional protein synthesis rate in OC users of a similar type of low androgenic OC (EE and gestoden) compared to NOC (Hansen et al., 2011). Previous exercise studies in the field have failed to show any clear effect of OC on adaptation to resistance training. For example, one study by Nichols et al. (2008) showed no difference between OC and NOC in muscle strength gain after a 12-week strength period; however, included users of different types of OCs and included different types of athletes, who continued their discipline-specific training during Frontiers in Physiology | www.frontiersin.org 8July 2019 | Volume 10 | Article 824