Introduction
Cytokines as paracrine regulators of spermatogenesis and steroidogenesis have been reported to play a key role in the normal testis (14 11 11 40 2 2 29 2
Increasing evidence, from a growing number of research groups, substantiates the modulating effect of physical exercise as a type of physiological stress on proinflammatory cytokines and redox status in several organs and tissues (15,16 10 15,16,48 15,16,21 12 34 36 23,24,26,50 23,24,26,50
Methods
Experimental Approach to the Problem
Building on the Compendium of Physical Activities, a questionnaire was used to classify the level of physical activity (3 15–17,23,24,26,50 n = 283) provided written informed consent, entered the study, and were randomly assigned to 1 of 4 groups: MI (n = 71), RT (n = 71), CT (n = 71), and nonexercise (NON-EX, n = 70) groups (Table 1 ) (Figure 1 ). Randomization was performed by random number generation, and group assignment was placed in a sealed envelope, which was opened by the study coordinator at the time of randomization. Twenty-seven subjects (MI, n = 6; RT, n = 9; CT, n = 7; and NON-EX, n = 5) could not complete the study protocol and were excluded from the study. Thus, 256 subjects remained in the analysis. Subjects had anthropometric characteristics (body mass, body mass index [BMI], body fat, and waist circumference), aerobic capacity (V̇o 2 max), seminal markers of inflammation (IL-β, IL-6, IL-8, and TNF-α), oxidative stress (ROS, malondialdehyde [MDA], and 8-isoprostane), antioxidants (superoxide dismutase [SOD] activity, catalase activity, and total antioxidant capacity [TAC]), semen parameters and sperm DNA damage measured at baseline (T1 ), the end of week 12 (T2 ), the end of week 24 (T3 ), and 7 (T4 ), and 30 days (T5 ) after the intervention. The data analysts and the assessor who collected the data were blinded to the group allocation. We used markers of male reproductive function including semen quality parameters and sperm DNA integrity as the primary outcomes to calculate the sample size and considered progressive motility as the largest sample. We considered the power of 80% to detect the actual differences and set type 1 error to 0.05. Moreover, the clinical difference was determined based on an expert opinion so that the mean values for the 4 study groups (MI, RT, CT, and NON-EX) were considered 50, 52, 60, and 52, respectively. The commonly predicted SD was set at 18. According to the computations run using PASS, version 11, and considering approximately 40% loss in the follow-up, it was made apparent that 60 participants were required per group (i.e., a total of 240 participants for the entire study groups). We sampled 376 individuals so that at the end of the follow-up, we met the minimum sample size required.
Table 1.: Demographic and physiological parameters at baseline.*± SD
Figure 1.: Follow-up diagram. MI = moderate intensity treadmill exercise training; RT = resistance training; CT = combined treadmill + resistance training; NON-EX = nonexercise.
Subjects
A total of 376 healthy male volunteers were recruited for the study. The study enrolled sedentary healthy married men aged 25–40 years who did not have a history of chronic illness, serious systemic diseases, testicular varicocele, and genital infection. The research protocol was approved by the Human Subject Internal Review Board committee of Urmia University of Iran. As mentioned in the previous section, all subjects provided written informed consent.
Procedures
Trained dietitians collected dietary data at baseline and 30 days post-training using a validated semiquantitative food frequency questionnaire (15–17,20,25,26
Exercise Protocols
Before the initiation of the study protocol, each subject performed a Bruce treadmill test (Ergo XELG90 Spezial; Woodway, Weil am Rhein, Germany) to determine the maximal oxygen uptake (V̇o 2 max) (7 o 2 ) and carbon dioxide production (V̇co 2 ) were measured using an automated breath-by-breath system (CPX; Medical Graphics, Saint Paul, MN, USA). Employing the Borg scale, rating of perceived exertion (RPE) was recorded in the last 10 seconds of each stage. Two of the following 4 criteria were required for a test to be considered maximal (1 o 2 despite increasing workload (2 3 4 28
Moderate Intensity Treadmill Exercise Training Protocol
The subjects participated in 25–30 minutes of moderate intensity aerobic exercise on treadmill 3 times a week for 24 weeks. The exercise training commenced with 50% of V̇o 2 max, and the intensity of the training increased by 5% after every 3 training sessions, which reached 70% of V̇o 2 max by the end of the fourth week. The subjects were re-evaluated every 4 weeks (at the end of each 12 session of training) and were exercised at 70% of new V̇o 2 max for the upcoming 4 weeks. Exercise adherence was documented using Polar heart rate monitors, and subjects received feedback to adjust themselves to the prescribed intensity.
Resistance Training Protocol
The subjects participated in RT intervention 3 times a week for 24 weeks. The intervention started with 50% of 1RM and intensity of the training was increased by 5% after every 3 training session, which reached 70% of 1RM by the end of the fourth week. The subjects were re-evaluated every 4 weeks (at the end of each 12 session of training) and were exercised at 70% of new 1RM for the next 4 weeks. Furthermore, 1RM measures were not taken on the identical days that the subjects completed their main training program sessions. To learn the correct form of each exercise, 2 sets of 12 repetitions, separated by 30-second active recovery, were performed in the first 4 weeks. From the fifth week on, 3 sets of 12 repetitions were performed. Recovery was held constant at 30 seconds in the following weeks as well. Resistance training included exercises for all major muscle groups. Exercises to strengthen the upper body included bench press (pectoralis), chest cross (horizontal flexion of the shoulder joint), shoulder press (trapezius and latissimus dorsi), pull downs (back muscles), biceps curls, triceps extension, upright row, trunk extension, and exercises for abdominal muscles (sit-ups). Lower-body exercises included leg press (quadriceps femoris), calf raises, hip extensions (biceps femoris), hamstring curls using quadriceps table, hip abduction, and hip adduction. The test of 1RM was performed for the upper body by bench press, chest cross, shoulder press, pull downs, biceps curls, triceps extension, and upright row exercises and for the lower body by leg press, hip extensions, hamstring curls, hip abduction, and hip adduction exercises with free weights. After the warm-up (8–10 repetitions using a light weight), and 3–5 repetitions using a moderate weight, participants were tested for 1RM strength (1–3 repetitions using a heavy weight) by increasing the load on subsequent attempts until the participants were no longer able to do the entire movement. To allow for recovery, an interval of 3–5 minutes was given between the attempts (20,22,43
Combined Treadmill + Resistance Training Protocol
The subjects participated in CT intervention 3 times a week for 24 weeks. The main program included aerobic exercise followed by the RT. The training progression was similar to that described in the sections on the aerobic and RT program (20
All training sessions were performed between 5.00 and 7.00 pm , and instructions in correct exercise techniques and supervision of the participants throughout the entire training period were performed by a professional instructor and an experienced physician. The training session consisted of a 10-minute warm-up period, which included walking and jogging and muscle stretches. At the end of each training session, there was a 10-minute cool-down period, which involved slow walking and gentle muscle stretches. To be consistent and compliant in this study, the subjects had to complete 90% or more of possible exercise sessions. None of the participants incurred an injury as a consequence of the training program. The NON-EX group subjects were requested to maintain their normal daily activities and not to modify their lifestyles during the intervention period other than to comply with the requirements of the study. The subjects also were asked to refrain from exercising during the recovery period.
Anthropometric Characteristics
The height and body mass were measured to the nearest 0.25 cm and nearest 0.1 kg, respectively, using a floor model physician's scale/stadiometer. Body mass index was calculated (kg·m−2 ). Using the bioelectrical impedance method, percent body fat was measured by a body fat analyzer (Omron HBF 306, Omron, Kyoto, Japan). Waist circumference was measured in standing position to the nearest 0.1 cm at a midpoint between the lowest rib and the iliac crest using a stretch resistant tape.
Semen Sampling and Assays
The study subjects were presented with clear instructions on how to collect their semen samples by masturbation on site. Each subject ejaculated into a sterile collection container (26 35 g for 10 minutes. The supernatant seminal plasma was then frozen at −80° C until examination. Semen evaluations were then performed for the measurement of sperm DNA damage, seminal markers of oxidative stress, and inflammation.
Sperm DNA Fragmentation Assay
Sperm DNA fragmentation was performed by means of a terminal deoxynucleotidyl transferase–mediated fluorescein-dUTP nick end labeling (TUNEL) assay using Apo-Direct kit (Pharmingen, San Diego, CA, USA) and flow cytometry analysis. All fluorescence signals of labeled spermatozoa were analyzed by the flow cytometer FACScan (Becton Dickinson, San Jose, CA, USA). Approximately 10,000 spermatozoa were evaluated at a flow rate of <100 cells·s−1 , and the percentage of TUNEL-positive spermatozoa (TUNEL+ve) was calculated (19
Reactive Oxygen Species Assay
The formation ROS was evaluated by chemiluminescence assay by means of luminal (5-amino-2, 3 dihydro-1, 4 phtalazindione; Sigma Chemical Co., St. Louis, MO, USA) as the probe, using an Autolamat LB 935 Luminometer (Berthold Technologies, Bad Wildbad, Germany) in the integrated mode for 15 minutes (32
Lipid Peroxidation Assay
The LPO in seminal plasma was estimated by determining the MDA levels. Initially, 0.5 ml of seminal plasma was added to 0.5 ml of tris-hydrogen chloride (HCl) 0.04 M and acetonitrile containing 0.1% butylated hydroxytoluene. The samples were immediately stirred and extracted with 5 ml of pentane after derivatisation with 2.4 dinitrophenylhydrazine. The samples were then dried using nitrogen and analyzed by high-performance liquid chromatography (HPLC). For the MDA quantifications, we employed a calibration curve with 0.5–10 nmol·ml−1 of MDA. The MDA hydrazone quantification was performed at 307 nm using isocratic HPLC by a Waters 600 E System Controller (Waters 600 E System; Waters, Milford, MA, USA) equipped with a Waters Dual k 2487 UV detector (Waters Dual k 2487 UV detector; Waters, Milford, MA, USA). The hydrazone derivatisation was performed using a 5 L ultrasphere ODS column C18 (Beckman, San Ramon, CA, USA) at the flow rate of 0.8 ml·min−1 with the acetonitrile (45%)-HCl 0.01 N (55%) as mobile phase. The peak areas were used to calculate MDA concentrations by an Agilent 3395 integrator (Agilent Technologies, Santa Clara, CA, USA) (42
8-Isoprostane Assay
Drawing on the instructions provided by the manufacturer, the concentration of free 8-isoprostane in seminal plasma was measured by enzyme immunoassay (EIA) method using an EIA kit (Cayman Chemical, Ann Arbor, MI, USA) (33
Total Antioxidant Capacity Assay
Initially, 12 μL of seminal plasma was mixed with 1,000 μL of the reconstituted chromogen, 2, 2′-azino-di-(3-ethylbenzthiazoline sulphonate) (ABTS)-metmyoglobin. Trolox (6-hydroxyl-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid) at a concentration and volume of 1.71 mmol·L−1 and 20 µl, respectively, was used as the standard. Chromogen (1,000 μl) was added to the Trolox and 20 μl of deionized water as a blank. The initial absorbance (A1) was read by spectrophotometer at 600 nm. Finally, 200 µl of H2 O2 (250 μmol·L−1 ) was added to standard, blank, and sample tubes, and final absorbance (A2) was read exactly after 3 minutes. The TAC of the sample was then calculated by the following formula:
In this equation, A1 is the initial absorbance rate, A2 is the final absorbance rate, and ΔA is the difference between A2 and A1 (39
Superoxide Dismutase Activity Assay
Superoxide dismutase activity was measured by commercially available colorimetric method (Randox Laboratories Ltd., Antrim, United Kingdom). In this method, the SOD activity is measured by the degree of inhibition of 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazoliumchloride (I.N.T) reduction as a result of xanthine- and xanthine oxidase-generated superoxide radicals. After thawing, the seminal plasma was diluted 30-fold with 10 mM phosphate buffer at 37° C (pH 7.0) and then were mixed with xanthine oxidase and added into standards and sample tubes. The initial (A1) and final absorbance (A2) were then read by spectrophotometer at a wavelength of 505 nm. The SOD activity was measured using calibration curve of percentage inhibition for each standard against Log10 of standards (56
Catalase Activity Assay
Catalase activity was measured according to the method described earlier by Aebi (1 2 O2 decomposition at 240 nm. The intra-assay and interassay CVs for the catalase assay were 7.5 and 8.1%, respectively.
Cytokines Assay
Seminal IL-1β, IL-6, IL-8, and TNF-α were evaluated by the Predicta (Cambridge, MA, USA) EIA kits. Briefly, a specimen containing control buffer or standard substance was added to each test well precoated with monoclonal antibody to a proper cytokine. After incubation, to capture proper cytokines by antibodies on the microtiter plate, the wells were washed and were mixed with a biotin-labeled polyclonal antibody to bind the captured IL-1β, IL-6, IL-8, or TNF-α. After the second wash, a peroxidase-labeled avidin reagent was introduced to the plates to attach the biotin. The wells were incubated and washed again, and then a peroxidase-labeled goat anti-rabbit immunoglobulin G was introduced to the plates to attach the polyclonal antibody. The plates then were washed again and a substrate buffer (peroxide) and chromogen (tetramethylbenzidine) were added to each well to produce a blue color in the presence of peroxidase. Sulfuric acid was then used for stopping the color reaction and converting the blue color to yellow. The absorbance was read at 450 nm with Multiscan Plus (Labsystems, Helsinki, Finland), and a standard curve was constructed to quantitate cytokine concentrations (40
Statistical Analyses
The statistical software program SPSS (version 23; SPSS Co., Chicago, IL, USA) for Windows was used for data analysis. All data are expressed as mean ± SD and checked for normality using a Kolmogorov-Smirnov and Q-Q plot. All statistical tests were performed and were considered statistically significant at p ≤ 0.05. Group differences were determined using a 3-factor analysis of variance for repeated measures and for continuous variables. If the main effects F -ratio was significant, differences among groups were subsequently identified using a Bonferroni post hoc analysis. Pearson and mixed model regression coefficients were used to evaluate the association between the variables studied.
Results
Recruitment to Trial
A total of 376 healthy sedentary male volunteers were screened for eligibility to the current trial. Of these, 94 subjects (25%) were excluded because they did not meet the inclusion criteria and the reasons for exclusion are given in Figure 1 . Also, 5 subjects were eligible for admission to the trial but declined to participate. Two hundred eighty-three (75%) subjects were finally included and of these 27 participants (9.5%) were excluded. The most common reasons for exclusion were that they could not adhere to dietary and exercise recommendations.
Dietary and Medication Intake
The subjects' dietary intakes were similar in all groups, and the dietary intakes between the groups or within the groups did not alter more than would be expected over the 24 weeks of the study (p > 0.05).
Muscular Strength
By 24 weeks, muscular strength improved in both RT and CT groups, ranging from 11.3 to 28.7% and from 8.9 to 25.6%, respectively (p ≤ 0.05). Total strength values, calculated by summing the 1RM values obtained in each of the 12 tested exercises, improved significantly from baseline to postexercise (p ≤ 0.05). These values were not different between the RT (20.2%) and CT (18.4) groups (p > 0.05).
Anthropometric Characteristics and Aerobic Capacity
All 3 MI, RT, and CT groups showed significant (p ≤ 0.05) improvements in body mass (12 weeks changes: MI −3.5% and CT −3.0%; 24 weeks changes: MI −7.0%, RT −2.6% and CT −6.0%), BMI (24 weeks changes: MI −6.9% and CT −6.0%), body fat percent (12 weeks changes: MI −12.0%, RT −12.1% and CT −14.2%; 24 weeks changes: MI −27.8%, RT −27.4% and CT −28.8%), waist circumference (12 weeks changes: MI −11.8% and CT −9.7%; 24 weeks changes: MI −21.6%, RT −10.2% and CT −21.1%), and V̇o 2 max (ml·kg−1 ·min−1 ) (12 weeks changes: MI +5.9%, RT +5.0% and CT +6.8%; 24 weeks changes: MI +9.5%, RT +14.0% and CT +15.9%). Training-induced changes in BMI, body fat percent, waist circumference, and V̇o 2 max sustained 30 days postexercise in the MI, RT, and CT groups. Body mass returned to baseline 7 and 30 days postexercise in the RT and CT groups, respectively; however, in the MI group, body mass remained significantly higher 30 days postexercise (p ≤ 0.05) (Figure 2 ). The NON-EX group demonstrated no significant changes in body mass, BMI, body fat percent, waist circumference, and V̇o 2 max in the 24 weeks (p > 0.05) (Figure 2 ). Results from the Bonferroni post hoc analysis showed that by 12 and 24 weeks and 7 and 30 days postexercise, MI and CT changes of body mass were greater than those observed in the RT group (p ≤ 0.05). By 12 and 24 weeks and 7 and 30 days postexercise, MI changes of BMI were greater than those observed in the RT and CT groups (p ≤ 0.05). No significant differences were observed between MI, RT, and CT groups during the course of the study in body fat percent (p ≤ 0.05). By 24 weeks and 7 days post exercise, MI and CT changes of waist circumference were greater than those observed in the RT group (p ≤ 0.05). At 30 days postexercise, the MI changes of waist circumference were greater than the other groups (p ≤ 0.05). At 24 weeks and 7 days postexercise, the MI changes of V̇o 2 max were greater than those observed in the RT and CT groups (p ≤ 0.05) (Figure 2 ).
Figure 2.: Baseline, week 12, week 24, and 7 and 30 days values of anthropometric characteristics and aerobic capacity in different groups of healthy male subjects. BMI = body mass index; MI = moderate intensity treadmill exercise training; RT = resistance training; CT = combined treadmill + resistance training; NON-EX = nonexercise. T1 : baseline (24 hours before training session). T2 : 24 hours after the last training session in week 12. T3 : 24 hours after the last training session in week 24. T4 : 7 days after the last training session in week 24. T5 : 30 days after the last training session in week 24. *p ≤ 0.05 significant difference between groups.
Semen Parameters
All 3 of MI, RT, and CT groups showed significant (p ≤ 0.05) improvements in semen volume (12 weeks changes: CT +8.1%; 24 weeks changes: MI +8.3% and CT +13.5%), progressive motility (12 weeks changes: MI +4.3%; 24 weeks changes: MI +10.6%, RT +2.6% and CT +9.1%), sperm morphology (12 weeks changes: MI +7.8% and CT +10.0%; 24 weeks changes: MI +17.1%, RT +11.2% and CT +19.2%), sperm concentration (12 weeks changes: MI +4.0% and CT +5.6%; 24 weeks changes: MI +9.1%, RT +4.7% and CT +11.0%), and number of spermatozoa (12 weeks changes: CT +14.2%; 24 weeks changes: MI +17.8% and CT +26.0%) (Figure 3 ). The NON-EX group demonstrated no significant changes in semen volume, progressive motility, sperm morphology, sperm concentration, and the number of spermatozoa in the 24 weeks (p > 0.05) (Figure 3 ). Training-induced changes in sperm concentration and number of spermatozoa returned to baseline 30 days postexercise in the MI, RT, and CT groups. Resistance training– and CT-induced changes in sperm morphology remained significantly higher after 30 days postexercise (p ≤ 0.05), whereas in the MI group, these changes returned to baseline after 30 days of detraining. Also, CT-induced changes in progressive motility returned to baseline 7 days postexercise, whereas these levels remained significantly elevated 30 days postexercise in the MI and RT groups (p ≤ 0.05) (Figure 3 ). The NON-EX group demonstrated no significant changes in semen parameters in the 24 weeks (p > 0.05) (Figure 3 ). Results from the Bonferroni post hoc analysis showed that at 12 and 24 weeks, the CT changes of semen volume were greater than those observed in the MI and RT groups (p ≤ 0.05). By 7 days postexercise, CT and MI changes of semen volume were greater than those observed in the RT group (p ≤ 0.05). By 12 and 24 weeks and 7 and 30 days postexercise, MI changes of progressive motility were greater than those observed in the RT and CT groups (p ≤ 0.05). By 30 days postexercise, CT changes of progressive motility were greater than those observed in the RT group (p ≤ 0.05). No significant differences were observed between MI, RT, and CT groups at 12 and 24 weeks and 7 days postexercise in sperm morphology (p ≤ 0.05). By 30 days postexercise, MI changes of sperm morphology were significantly greater than those observed in the RT and CT groups (p ≤ 0.05). At 24 weeks, the MI and CT changes of sperm concentration were significantly greater than those observed in the RT group (p ≤ 0.05). At 24 weeks, the MI changes of number of spermatozoa were greater than those observed in the RT and CT groups (p ≤ 0.05). Also, by 7 days postexercise, MI and CT changes of number of spermatozoa were greater than those observed in the RT group (p ≤ 0.05) (Figure 3 ). The CT changes of semen quality parameters further reveal that MI and RT interventions had a synergistic effect on the seminological parameters (Figure 3 ).
Figure 3.: Baseline, week 12, week 24, and 7 and 30 days values of semen quality parameters and sperm DNA integrity in different groups of healthy male subjects. TUNEL = terminal deoxynucleotidyl transferase–mediated fluorescein-dUTP nick end labeling; MI = moderate intensity treadmill exercise training; RT = resistance training; CT = combined treadmill + resistance training; NON-EX = nonexercise. T1 : baseline (24 hours before training session). T2 : 24 hours after the last training session in week 12. T3 : 24 hours after the last training session in week 24. T4 : 7 days after the last training session in week 24. T5 : 30 days after the last training session in week 24. *p ≤ 0.05 significant difference between groups.
Sperm DNA Fragmentation
At 12 weeks of the intervention, values for TUNEL positive spermatozoa were not significantly different than baseline values and there were no difference between group values (Figure 3 ). However, by 24 weeks, all intervention groups showed a decrease in the percentage of TUNEL positive spermatozoa from baseline, and all had greater decreases than the NON-EX group (p ≤ 0.05). These values returned to baseline 30 days postexercise in the MI, RT, and CT groups. The NON-EX group demonstrated no significant changes in the percentage of TUNEL positive spermatozoa in the 24 weeks (p > 0.05) (Figure 3 ). No significant differences were observed between MI, RT, and CT groups during the course of the study in TUNEL positive spermatozoa (p > 0.05) (Figure 3 ).
Oxidants and Antioxidants
All 3 MI, RT, and CT groups demonstrated significant (p ≤ 0.05) improvements in SOD (24 weeks changes: MI +28.6% and CT +22.2%), catalase (12 weeks changes: MI +8.0%; 24 weeks changes: MI +32.5% and CT +22.5%), TAC (24 weeks changes: MI +38.8% and CT +66.7%), ROS (12 weeks changes: MI −15.1% and CT −12.1%; 24 weeks changes: MI −26.2%, RT −8.6% and CT −28.5%), MDA (12 weeks changes: MI −19.5%; 24 weeks changes: MI −39.0% and CT −38.6%), and 8-isoprostane (24 weeks changes: MI −15.5% and CT −18.3%) (Figure 4 ). The NON-EX group demonstrated no significant changes in seminal oxidants and antioxidants in the 24 weeks (p > 0.05) (Figure 4 ). Training-induced changes in SOD returned to baseline 30 days postexercise in the MI and CT groups. Moderate intensity treadmill exercise training– and CT-induced changes in catalase remained significantly elevated 30 days postexercise (p ≤ 0.05). Also, MI-induced changes in TAC returned to baseline 30 days postexercise in the CT group; however, these changes remained significantly higher even after 30 days postexercise (p ≤ 0.05) (Figure 4 ). Resistance training– and CT-induced changes in ROS returned to baseline 30 days postexercise in the MI group; however, these changes remained significantly higher even after 30 days postexercise (p ≤ 0.05). Malondialdehyde levels returned to baseline 7 and 30 days postexercise in the CT and MI groups. Moderate intensity treadmill exercise training– and CT-induced changes in 8-isoprostane levels also returned to baseline 30 days postexercise (Figure 4 ). The NON-EX group demonstrated no significant changes in the seminal markers of oxidative stress in the 24 weeks (p > 0.05) (Figure 4 ). Results from the Bonferroni post hoc analysis showed that at 12 weeks and 7 days post exercise, the CT changes of SOD were greater than those observed in the RT group (p ≤ 0.05). By 24 weeks, the MI, RT, and CT changes of SOD were greater than those observed in the NON-EX group (p ≤ 0.05). Also, the CT changes of SOD were greater than those observed in the RT group at 30 days postexercise (p ≤ 0.05). At 12 weeks, the MI and CT changes of catalase were greater than those observed in the RT group (p ≤ 0.05). No significant differences were observed between MI and CT groups during 12 weeks in catalase (p ≤ 0.05). By 12 and 24 weeks and 7 and 30 days postexercise, the MI and CT changes of catalase were greater than those observed in the RT group (p ≤ 0.05). At 24 weeks and 7 days postexercise, the MI and CT changes of TAC were significantly greater than those observed in the NON-EX group (p ≤ 0.05). By 12 and 24 weeks and 7 and 30 days postexercise, the RT changes of ROS were greater than those observed in the MI and CT groups (p ≤ 0.05). At 24 weeks, the MI changes of MDA were greater than those observed in the RT group (p ≤ 0.05). Also, at 7 days postexercise, the MI changes of 8-isoprostane were greater than those observed in the RT group (p ≤ 0.05) (Figure 4 ).
Figure 4.: Baseline, week 12, week 24, and 7 and 30 days values of seminal oxidants and antioxidants in different groups of healthy male subjects. SOD = superoxide dismutase; TAC = total antioxidant capacity; ROS = reactive oxygen species; RLU = relative light units; MDA = malondialdehyde; MI = moderate intensity treadmill exercise training; RT = resistance training; CT = combined treadmill + resistance training; NON-EX = nonexercise. T1 : baseline (24 hours before training session). T2 : 24 hours after the last training session in week 12. T3 : 24 hours after the last training session in week 24. T4 : 7 days after the last training session in week 24. T5 : 30 days after the last training session in week 24. *p ≤ 0.05 significant difference between groups.
Cytokines
All 3 MI, RT, and CT groups revealed significant (p ≤ 0.05) improvements in IL-1β (12 weeks changes: MI −16.7%; 24 weeks changes: MI −33.3%, RT −14.7% and CT −45.0%), IL-6 (12 weeks changes: MI −19.0%; 24 weeks changes: MI −36.3%, RT −15.9% and CT −34.9%), IL-8 (12 weeks changes: MI −10.7%; 24 weeks changes: MI −16.9%, RT −8.6% and CT −18.2%), and TNF-α (12 weeks changes: MI −14.1%; 24 weeks changes: MI −23.4%, RT −19.7% and CT −26.2%) (Figure 5 ). The NON-EX group demonstrated no significant changes in seminal cytokines in the 24 weeks (p > 0.05) (Figure 5 ). Resistance training–induced changes in IL-1β, IL-6, and IL-8 levels returned to baseline 30 days postexercise; however, these levels remained significantly elevated 30 days postexercise in the MI and CT groups (p ≤ 0.05). Moderate intensity treadmill exercise training–, RT-, and CT-induced changes in TNF-α levels also remained significantly lower after 30 days postexercise (p ≤ 0.05) (Figure 5 ). Results from the Bonferroni post hoc analysis showed that by 12 and 24 weeks and 7 days postexercise, the MI and CT changes of IL-1β were significantly greater than those observed in the RT group (p ≤ 0.05). Also, at 7 days postexercise, the CT changes of IL-1β were significantly greater than those observed in the RT group (p ≤ 0.05). By 12 and 24 weeks and 7 and 30 days postexercise, the MI and CT changes of IL-6 and IL-8 were significantly greater than those observed in the RT group (p ≤ 0.05) (Figure 5 ).
Figure 5.: Baseline, week 12, week 24, and 7 and 30 days values of seminal cytokines in different groups of healthy male subjects. IL = interleukin; TNF = tumor necrosis factor; MI = moderate intensity treadmill exercise training; RT = resistance training; CT = combined treadmill + resistance training; NON-EX = nonexercise. T1 : baseline (24 hours before training session). T2 : 24 hours after the last training session in week 12. T3 : 24 hours after the last training session in week 24. T4 : 7 days after the last training session in week 24. T5 : 30 days after the last training session in week 24. *p ≤ 0.05 significant difference between groups.
Correlations
Each unit improvement in semen volume, sperm morphology, sperm concentration, number of spermatozoa, and percentage of TUNEL positive spermatozoa was associated with significant decreases in mass, BMI, body fat, waist circumference, ROS, MDA, 8-isoprostane, IL-1β, IL-6, IL-8, and TNF-α (p ≤ 0.05). Each unit improvement in semen volume, sperm morphology, sperm concentration, number of spermatozoa, and percentage of TUNEL positive spermatozoa was also associated with significant increases in SOD, catalase, and TAC (p ≤ 0.05). Furthermore, each unit increase in V̇o 2 max levels was associated with significant improvements in semen volume, sperm morphology, sperm concentration, number of spermatozoa, and percentage of TUNEL positive spermatozoa (p ≤ 0.05) (Table 2 ).
Table 2.: Correlation of body composition measures, V̇o 2 max, antioxidants, oxidants, and cytokines with semen quality parameters and sperm DNA integrity in healthy male subjects.*
Table 2-A.: Correlation of body composition measures, V̇o 2 max, antioxidants, oxidants, and cytokines with semen quality parameters and sperm DNA integrity in healthy male subjects.*
Discussion
The results showed that 24 weeks of MI, RT, and CT in men decreased the seminal markers of oxidative stress and inflammation and increased the seminal antioxidant capacity and these changes were correlated with the improvements in semen quality parameters and sperm DNA integrity in healthy human subjects. Moderate intensity treadmill exercise training and CT also induced significantly more profound effects on markers of male reproductive function than RT intervention. Furthermore, MI and RT acted synergistically to cause the improvements in several markers of male reproduction but it appears that the MI contributed more so to these improvements.
In this study, by 12 and 24 weeks, MI intervention resulted in significant decreases in IL-β, IL-6, IL-8, and TNF-α from the baseline values. Furthermore, at 24 weeks of the intervention, these proinflammatory cytokines decreased in the RT and CT groups. We could not find any randomized controlled trials in the literature that examined the effects of aerobic, resistance, and combined protocols on seminal markers of inflammation in healthy human subjects. However, the attenuating effects of exercise training on blood markers of inflammation have been observed by numerous researchers who used aerobic (5,52 45 4 13 13 18
Chronic moderate intensity (54 6,8 41 31,53 44 55 40
In this study, all 3 MI, RT, and CT groups showed significant improvements in semen parameters and sperm DNA integrity with different kinetics for the 3 types of exercise. The MI and CT changes of these parameters were significantly greater than observed in the RT group. Furthermore, at 12 weeks of the intervention, semen volume and number of spermatozoa had increased 8.1 and 14.2%, respectively, in the CT group, and this response seems to be a synergistic effect of the MI and RT interventions because there were no significant changes in either of these groups. These differences would be mediated, in part, by more profound training-induced improvements in seminal markers of inflammation and oxidative stress in the MI and CT groups than seen in the RT group. Recent studies published by our group demonstrated significantly better semen parameters and low percentage of TUNEL positive spermatozoa in recreationally active men compared with the competitive elite athletes and sedentary controls (27,49,51 38,47 23,26,27,49,50 24,50 16
In addition, in this study, the improvements in semen quality parameters and sperm DNA integrity coincide with the attenuations in seminal markers of oxidative stress and inflammation. Considering these, the significantly lower DNA damage and elevated semen quality parameters observed post-training in the MI, RT, and CT groups may be related to the increased antioxidant activity and reduced proinflammatory mediators in seminal plasma, as several studies have already implicated inflammation (11 2
Another sperm dysfunction and sperm DNA damage inductor is the unfavorable body composition (37 46 o 2 max (30 o 2 max. It is possible that exercise interventions effectively improve semen quality parameters and sperm DNA integrity, among others, in association with concomitant improvements in body composition and V̇o 2 max, as there were significant correlations between semen quality parameters and sperm DNA damage with body composition and V̇o 2 max. Several studies have also shown an inverse relationship between maximal oxygen uptake and markers of oxidative stress. According to Djordjevic et al. (9 o 2 max after participation in different exercise modalities. The findings might support the potential importance of exercise training for the prevention and treatment of male factor infertility. It is not known, however, to what degree training-induced changes in semen quality and sperm DNA integrity are capable of modifying the fertilizing potential of ejaculated human spermatozoa and the male reproductive function. These points are currently being addressed in our laboratory.
Practical Applications
In conclusion, we demonstrated that general features of the subjects were improved by all 3 MI, RT, and CT interventions. In the case of markers of male reproductive function, RT was not an optimal measure; however, MI and CT seem to be superior in terms of sperm quality. These results further indicate that, in respect to all the aspects studied, MI seems to be superior over the other interventions. With regard to the seminological parameters, however, CT had a synergistic effect in augmenting the semen quality parameters in healthy male subjects. The established effects can be attributed, at least in part, to training-induced improvements in antioxidant capacity, inflammation, and body composition. However, future studies may want to verify whether volume- and work-matched MI, RT, and CT and different training loads exhibit different reproductive responses.
Acknowledgments
The authors are grateful to the patients who participated in the study for without their dedication the study could not have been performed. This research did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector. The authors have no conflicts of interest to disclose.
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