Moderate aerobic exercise training for improving reproductive function in infertile patients: A randomized controlled trial

Cytokine 92 (2017) 55–67

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Moderate aerobic exercise training for improving reproductive function in infertile patients: A randomized controlled trial Behzad Hajizadeh Maleki a,⇑, Bakhtyar Tartibian b a b

Department of Sports Medicine, Justus-Liebig-University, Giessen, Germany Department of Sport Injuries, Faculty of Physical Education and Sport Sciences, Allameh Tabataba’i University, Tehran, Iran

a r t i c l e

i n f o

Article history: Received 6 October 2016 Received in revised form 3 January 2017 Accepted 9 January 2017

Keywords: Exercise intervention Infertility Proinflammatory cytokines Randomized controlled trial Redox status

a b s t r a c t This study investigated for the first time the changes in seminal markers of inflammation, oxidative stress status, semen parameters, sperm DNA integrity as well as pregnancy rate following 24 weeks of moderate aerobic exercise in infertile patients. A total of 1026 sedentary men (aged 25–40 years) attending the infertility clinic with history of more than one year of infertility, were screened and 419 were randomized to either exercise (EX, n = 210) or non-exercise (NON-EX, n = 209) groups. Exercise training favorably attenuated seminal markers of both inflammation (IL-1b, IL-6, IL-8, and TNF-a) and oxidative stress (ROS, MDA, 8-Isoprostane) as well as enhanced antioxidant defense system (SOD, catalase and TAC) (P < 0.05). These changes correlate with favorable improvements in semen parameters, sperm DNA integrity and pregnancy rate (P < 0.05). The results provide information about the effectiveness of moderate aerobic exercise training as a treatment option for male factor infertility. The 4-week detraining period was not enough to reverse all benefits promoted by exercise intervention. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Infertility is characterized by the inability of a couple to achieve a clinical pregnancy within one year or more of regular, unprotected and well-timed intercourse [1]. It is a worldwide problem and affects 15% of all couples of reproductive age, with about 50% being associated with impairments in the process of spermatogenesis and sperm function, called male factor infertility [2]. In fact, spermatogenesis and sperm function can be compromised owing to a number of factors such as high level of reactive oxygen species (ROS) and oxidative stress [3]. ROS-induced oxidative stress has already been correlated with negative changes in semen parameters [4] and DNA fragmentation [5], leading to poor semen quality and is the cause of infertility in men [3]. A number of authors further showed oxidative stress in the seminal plasma can be related to inflammatory conditions therein [6]; and men with an excessive production of ROS by sperm demonstrated to have elevated levels of proinflammatory cytokines and leukocytes infiltration in their semen [7]. Positive correlations also were demonstrated between proinflammatory cytokines IL-6 or IL-8 with lipid peroxidation as well as sperm DNA fragmentation [8] ⇑ Corresponding author at: Department of Sports Medicine, Justus-Liebig-University, 35394 Giessen, Germany. E-mail addresses: [email protected], behzad.hajizadeh-maleki@ sport.uni-giessen.de (B. Hajizadeh Maleki), [email protected] (B. Tartibian). http://dx.doi.org/10.1016/j.cyto.2017.01.007 1043-4666/Ó 2017 Elsevier Ltd. All rights reserved.

in male genital tract inflammation. Proinflammatory cytokines in seminal plasma furthermore have been reported to negatively influence standard semen quality parameters [9,10], as well as were found to damage sperm membranes and induce a significant loss of genomic integrity [11], all of which may have serious consequences for spermatogenesis and eventually male fertility. Data from recent studies consistently show an association between physical activity and male reproduction [12–18]. For instance, a more recent randomized controlled trial conducted by our research laboratory [19] found that, in healthy human subjects, moderate intensity aerobic exercise training can induce significant improvements in semen parameters and sperm DNA integrity mainly through adaptations in the seminal antioxidant defense system and attenuating seminal markers of inflammation. To date; however, there are no published reports on the effects of exercise training on reproductive function in men with impaired fertility. Therefore, taking into account the role of markers of inflammation and oxidative stress in male reproductive function and potential effects of exercise training on seminal inflammatory mediators and redox homeostasis, we hypothesized that, in infertile men, the moderate aerobic exercise training, as a novel treatment option, would be successful in reducing chronic inflammation and oxidative stress in seminal plasma and those changes would be correlated to improvements in spermatogenesis and eventually male reproductive function. This randomized controlled trial, thus,

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was conducted to evaluate the effects of 24 weeks of moderate aerobic exercise training on markers of male reproduction including seminal markers of oxidative stress and inflammation, semen quality parameters, sperm DNA integrity and pregnancy rate in infertile patients. To the best of our knowledge, ours is the first study to address this issue.

2. Materials and methods

This randomized controlled trial conducted in Dr. Bakhtyar Tartibian’s Exercise Physiology Laboratory of the Urmia University (Iran) fromMarch 2014 to September 2014. The data analyses were also conducted in UrmiaUniversity of Iran. The research protocol was approved by the Human Subject Internal Review Board Committee of the Urmia University (Iran). Thirty-three patients (asthenozoospermic, n = 8; asthenoteratozoospermic, n = 8; oligoasthenozoospermic, n = 4; oligospermic, n = 11; and oligoasthenoteratozoospermic, n = 2) could not complete the study protocol, the remaining 386 patients are included in the analysis (Fig. 1).

2.1. Experimental design and patients 2.2. Exercise protocol A total of 1026 sedentary men (aged 25–40 years) attending the infertility clinic with a history of infertility longer than 12 months, were considered for inclusion in the study. All the patients had a history of infertility with no indication of hormonal, infective or physical causes. To be qualified to take part in the study, they had to be married men 25–40 years of age; not participating in a regular exercise program or accumulating 25 min or more of moderate physical exercise on 3 or more days a week; in good health, as ascertained through a routine physical examination and laboratory tests over the past 12 months; with no history of chronic illnesses, serious systemic diseases, testicular varicocele, genital infection and leukocytospermia; with no history of use of antioxidants as supplements like vitamins and medications known to alter the hypothalamic-pituitary-gonadal (HPG) axis, like anabolic steroids; with no history of cigarette use and alcohol consumption over the past 6 months; with no history of depression and eating disorders; with normal physical development and sexual maturation; not involving in occupations where the activity might influence fertilizing capacity; and had not undergone vasectomy reversal or varicocele removal surgeries. To be included in the study, patients had to be able to increase their level of physical activity as well. All patients were also needed to have stopped all medical therapy P12 weeks before study initiation [12,13,15,16,19]. Each patient provided 3 semen samples at 3-week intervals. All semen samples were evaluated in same laboratory and, on the basis of the fifth edition of World Health Organization (WHO) laboratory manual for the examination and processing of human semen [20], were classified into groups of asthenozoospermic (progressive sperm motility <32% motile), asthenoteratozoospermic (progressive sperm motility <32% motile, sperm morphology <4% normal), oligoasthenozoospermic (sperm concentration <15
106 sperm/mL, progressive sperm motility <32% motile), oligospermic (sperm concentration <15
106 sperm/mL), and oligoasthenoteratozoospermic (sperm concentration <15
106 sperm/mL, progressive sperm motility <32% motile, sperm morphology <4% normal) to give a total of five infertile subgroups. Patients were excluded for having azoospermia, leukocytospermia (leukocyte concentration >106/ml of ejaculate) and semen hyperviscosity. Each patient was evaluated by a full review of their clinical history, physical examination and routine biochemistry analysis, with any clinically significant abnormalities in any of these tests leading to exclusion from the study. A certificate attesting to the patient’s ability to participate in the study protocol was provided by the physician, and served as the final screen for participation in the study. Once they met the inclusion criteria, eligible patients (asthenozoospermic, n = 83; asthenoteratozoospermic, n = 84; oligoasthenozoospermic, n = 80; oligospermic, n = 88; and oligoasthenoteratozoospermic, n = 84) were further randomized to either exercise (EX) or non-exercise (NON-EX) groups, then provided written informed consent and entered the study (Table 1). With an a = 0.05, an effect size = 0.95 and a power of 0.94, a sample size of 40 was recommended. Randomization using random number generation was used to assign patients to intervention groups.

Baseline testing included a maximal oxygen uptake (VO2max) with the use of an automated breath-by-breath system (CPX, Medical Graphics, St. Paul, MN, USA). Exercise sessions began between 5 and 7 pm. Moderate aerobic exercise protocol included walking or jogging on a treadmill supervised through certified personal trainers. During the first 12 weeks of the study, the EX groups exercised (25–30 min/day, 3–4 days/week) at 45–55% of their VO2max and then the volume and the intensity of exercise sessions were increased during the final 12 weeks (40–45 min/day, 4–6 days/ week, and 56–69% of VO2max). Adherence to the exercise was acknowledged via Polar heart rate monitors, and patients received immediate feedback to adjust to the prescribed intensity [19,21,22]. Patients with training adherence less than 95% were excluded. The NON-EX group patients were requested to maintain their current physical activities and not to modify their lifestyles during the 24-week intervention period. 2.3. Dietary and medication intake measures Trained dietitians collected dietary data at baseline and 30 days post training using a validated semi-quantitative food frequency questionnaire (FFQ) [12,13,15–17,19]. Patients were asked to maintain their normal diet during the study period and were encouraged to eat a similar diet as far as possible in each sampling days. Patients were required to avoid any prescriptive or over the counter medications/supplements and foods that may impact the reproductive function one week before and during the study. Standard and self-reported questionnaires were also used to obtain information on use of medications/supplements during the study period. 2.4. Sampling Patients reported to the lab on sampling days after complete sexual abstinence of at least 3–4 days. Patients were given clear instructions about the procedure of semen collection by masturbation into a sterile wide-mouth and metal-free plastic container at site. The initial semen sample was draw 24 h before training session (baseline). Additional samples were collected 24 h after the last training sessions in weeks 12 and 24; as well as 7 and 30 days during recovery. Patients were asked not to exercise during the recovery period. 2.5. Analysis and measurements Semen analysis was performed, following liquefaction (at least 30 min), to assess semen volume, sperm motility, sperm morphology, sperm concentration, and number of spermatozoa according to WHO guidelines for the examination of human semen [20]. The supernatant seminal plasma was frozen after a 10 min centrifugation (10,000g) at 80 °C until examination [23]. The same experienced technician performed semen evaluations during the study for the assessment of percentage of TUNEL positive sperma-

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B. Hajizadeh Maleki, B. Tartibian / Cytokine 92 (2017) 55–67 Table 1 Individual characteristics of infertile patients. Asthenozoospermic

Asthenoterato zoospermic

Oligospermic

Oligoastheno zoospermic

Oligoasthenoterato zoospermic

P value

Age (years) EX NON-EX P value

33.7 ± 6.3 32.5 ± 7.4 0.083

32.1 ± 7.8 31.5 ± 8.4 0.081

34.2 ± 5.6 33.6 ± 6.3 0.108

33.3 ± 6.6 32.8 ± 7.0 0.094

32.8 ± 7.2 33.2 ± 6.8 0.072

0.084 0.112

Height (cm) EX NON-EX P value

175.3 ± 7.9 176.5 ± 8.3 0.077

174.4 ± 8.2 176.5 ± 6.9 0.091

176.1 ± 8.5 174.3 ± 7.8 0.156

175.2 ± 9.2 174.1 ± 9.3 0.201

176.1 ± 7.7 177.1 ± 5.7 0.080

0.091 0.242

Weight (kg) EX NON-EX P value

84.3 ± 11.1 83.4 ± 11.0 0.304

83.4 ± 11.5 83.0 ± 10.0 0.064

83.7 ± 10.2 83.9 ± 11.0 0.087

84.4 ± 12.6 83.0 ± 9.9 0.098

82.5 ± 7.3 83.7 ± 10.4 0.073

0.107 0.075

BMI (kg/m2) EX 27.5 ± 4.4 NON-EX 27.0 ± 3.6 P value 0.142

27.6 ± 5.0 26.9 ± 4.7 0.095

27.1 ± 5.9 27.5 ± 6.4 0.072

27.6 ± 5.6 27.4 ± 5.8 0.086

27.4 ± 5.6 26.9 ± 4.5 0.077

0.164 0.301

Fat (%) EX NON-EX P value

22.8 ± 9.9 22.1 ± 7.6 0.109

21.6 ± 8.1 22.3 ± 9.2 0.248

21.6 ± 8.8 22.8 ± 9.4 0.080

21.8 ± 10.0 21.7 ± 8.2 0.170

0.104 0.096

Waist circumference (cm) EX 125.6 ± 6.9 NON-EX 125.0 ± 6.7 P value 0.065

125.8 ± 7.2 126.5 ± 6.0 0.096

124.3 ± 6.4 124.8 ± 11.0 0.085

123.6 ± 8.1 124.2 ± 9.6 0.073

124.1 ± 11.4 124.6 ± 9.7 0.108

0.197 0.323

VO2max (ml kg1 min1) EX 36.0 ± 2.9 NON-EX 36.3 ± 3.0 P value 0.091

35.9 ± 4.5 36.1 ± 3.9 0.109

36.2 ± 3.9 36.3 ± 3.2 0.082

36.3 ± 6.2 36.1 ± 6.0 0.094

36.8 ± 6.3 37.1 ± 6.8 0.078

0.088 0.247

22.3 ± 8.1 23.1 ± 7.3 0.082

BMI = body mass index; VO2max = maximal oxygen uptake. P < 0.05, significant difference between subgroups. P < 0.05, significant difference between groups (EX vs. NON-EX groups). Values are mean ± SD. *

y

tozoa, seminal plasma oxidative stress biomarkers [ROS, malondialdehyde (MDA), and 8-Isoprostane], antioxidants [superoxide dismutase (SOD), catalase, and total antioxidant capacity (TAC)] as well as seminal cytokines IL-1b, IL-6, IL-8, and TNF-a. 2.5.1. 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). About 10000 spermatozoa were evaluated at a flow rate of <100 cells/s and the percentage of TUNEL-positive spermatozoa (TUNEL+ve) was calculated [24]. 2.5.2. ROS assay The formation of 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, Badwildbad, Germany) in the integrated mode for 15 min [25]. 2.5.3. Lipid peroxidation (LPO) 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 (BHT). The samples were immediately stirred and extracted with 5 mL of pentane after derivatisation with 2.4 dinitrophenylhydrazine. The samples then were dried using nitrogen and analyzed by high-performance liq-

uid chromatography (HPLC). For the MDA quantifications, we used a calibration curve with 0.5–10 nmol/mL of MDA. The MDA hydrazone quantification was performed at 307 nm using isocratic HPLC by a Waters 600 E System Controller (Milford, MA, USA) equipped with a Waters Dual k 2487 UV detector (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 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) [26].

2.5.4. 8-Isoprostane assay 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) according to the instructions provided by the manufacturer [27]. 2.5.5. TAC assay Initially 12 lL of seminal plasma was mixed with 1000 lL of the reconstituted chromogen, 2,20 -Azino-di-(3-ethylbenzthiazoline sulphonate) (ABTS)-metmyoglobin. Trolox (6-hydroxyl-2,5,7,8-tet ramethylchroman-2-carboxylic acid) at a concentration and volume of 1.71 mmol/L and 20 lL, respectively, was used as the standard. 1000 lL of chromogen was added to the Trolox and 20 lL of deionized water as a blank. The initial absorbance (A1) was read by spectrophotometer at 600 nm. Finally, 200 lL of H2O2 (250 lmol/ L) was added to standard, blank, and sample tubes, and final absorbance (A2) was read exactly after 3 min. The TAC of the sample was then calculated by the following formula:

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Fig. 1. Follow-up diagram.

TAC ¼ Concentration of the Standard
ðDA Blank  DA SampleÞ=ðDA Blank  DA StandardÞ: In this equation, A1 is the initial absorbance rate, A2 is the final absorbance rate, and DA is the difference between A2 and A1 [28]. 2.5.6. SOD activity assay SOD activity was measured by commercially available colorimetric method (Randox Laboratories Ltd, Antrim, UK). In this

method, the SOD activity is measured by the degree of inhibition of 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazoliumchlor ide (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

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of percentage inhibition for each standard against Log10 of standards [23].

change more than would be expected throughout the 24 weeks of the intervention (P > 0.05).

2.5.7. Catalase activity assay CAT activity was measured according to the method described earlier by Aebi [29]. The assay was based on the calculation of the rate constant of the H2O2 decomposition at 240 nm.

3.2. Physical characteristics

2.5.8. Cytokines assay Seminal IL-1b, IL-6, IL-8, and TNF-a were evaluated by the Predicta (Cambridge, MA) enzyme immunoassay kits. Briefly, a specimen containing control buffer or standard substance was added to each test well pre-coated 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 IL1b, IL-6, IL-8, or TNF-a. 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 then was 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 to quantitate cytokine concentrations a standard curve was constructed [6]. 2.5.9. Pregnancy and live birth rate Information about successful pregnancy during intervention and at 3 months afterwards was gathered through telephone interview. Live birth rate also was confirmed using phone interview and by medical record. 2.6. Statistical analysis Repeated measures analysis of variance (ANOVA) was employed to detect significant (P < 0.05) differences for study variables at 5 time periods among the groups. Post-hoc tests were done using Tukey adjustment method for differences between times periods. Also, differences among groups were detected through an analysis of covariance (ANCOVA) adjusting for baseline measurements. Further, Pearson correlation coefficients and mixed linear regression model were employed to evaluate the association between the quantitative variables studied. We also evaluated the odds ratio (OR) and 95% CI for rates of pregnancy and live birth using a firth penalized likelihood logistic regression model. The statistical software program STATA 14 (StataCorp, College Station, TX, USA) was used for data analysis. Statistical significance was set at P 6 0.05. 3. Results At baseline, EX and NON-EX groups were similar with respect to physical characteristics, body composition, semen quality, sperm DNA fragmentation, markers of inflammation and oxidative stress (P > 0.05). 3.1. Dietary and medication intake All foods consumed and their quality, quantity and frequency of consumption (including red meat, chicken, fish, eggs, vegetables, fruits and milky products) were similar in all groups, and the dietary intakes between the groups or within the groups did not

At 12 and 24 weeks, weight, BMI, body fat and waist circumference had significantly decreased from baseline in the EX group (P < 0.05). Values remained significantly lower even after 30 days of detraining (P < 0.05). Moderate aerobic exercise training also induced significant increases in VO2max levels at 12 and 24 weeks (P < 0.05) and values remained significantly higher even after 30 days of detraining (P < 0.05) (Fig. 2). Similar changes were observed for weight, body fat, waist circumference and VO2max in the asthenozoospermic, asthenoteratozoospermic, oligospermic, oligoasthenozoospermic and oligoasthenoteratozoospermic groups, while BMI showed a post exercise decrease in the subgroups only at 24 week (P < 0.05) (Fig. 2). There were slight but non-significant changes in weight, BMI, body fat, waist circumference and VO2max at 24 wks in the NON-EX groups (P > 0.05). 3.3. Semen parameters At 12 and 24 weeks, progressive motility, sperm morphology, sperm concentration and number of spermatozoa had significantly increased from baseline in the EX group (P < 0.05). Values remained significantly higher even after 30 days of detraining (P < 0.05). Similar kinetics were observed for these levels in the asthenozoospermic, asthenoteratozoospermic, oligospermic, oligoasthenozoospermic and oligoasthenoteratozoospermic groups (P < 0.05). At 24 wks of the intervention, the mean values of semen volume were significantly increased from the baseline values in the EX group (P < 0.05) and remained significantly higher even after 30 days of detraining (P < 0.05). Similar changes were observed for semen volume in the asthenozoospermic, asthenoteratozoospermic, oligospermic, oligoasthenozoospermic and oligoasthenoteratozoospermic groups (P < 0.05) (Fig. 3). The NON-EX groups demonstrated no significant changes in progressive motility, sperm morphology, sperm concentration and number of spermatozoa in the 24 wks (P > 0.05) (Fig. 3). 3.4. Sperm DNA fragmentation At 12 and 24 weeks, sperm DNA fragmentation, as indicated by a decrease of percentage of TUNEL positive spermatozoa, had significantly improved from baseline in the EX group (P < 0.05). Values remained significantly lower even after 30 days of detraining (P < 0.05). Similar changes were observed for percentage of TUNEL positive spermatozoa in the asthenozoospermic, asthenoteratozoospermic, oligospermic, oligoasthenozoospermic and oligoasthenoteratozoospermic groups (P < 0.05) (Fig. 3). There were slight but non-significant increases in percentage of TUNEL positive spermatozoa at 24 wks in the NON-EX groups (P > 0.05) (Fig. 3). 3.5. Oxidants and antioxidants At 12 and 24 wks of the intervention, the mean values of ROS, MDA and 8-isoprostane were significantly decreased from the baseline values in the EX group (P < 0.05). Values remained significantly lower even after 30 days of detraining (P < 0.05). Similar changes were observed for ROS, MDA and 8-isoprostane in the asthenozoospermic, asthenoteratozoospermic, oligospermic, oligoasthenozoospermic and oligoasthenoteratozoospermic groups (P < 0.05). There were slight but non-significant increases in seminal ROS, MDA and 8-isoprostane at 24 wks in the NON-EX groups (P > 0.05) (Fig. 4).

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Fig. 2. Changes in body composition and VO2max in the EX and NON-EX groups during the course of the study. *: P < 0.05, significantly different from baseline values (within groups, baseline vs. week 12, week 24, 7 and 30 days during recovery).

At 12 and 24 weeks, SOD, catalase and TAC had significantly increased from baseline in the EX group (P < 0.05). Values remained significantly higher even after 30 days of detraining (P < 0.05). Similar changes were observed for SOD, catalase and TAC in the asthenozoospermic, asthenoteratozoospermic, oligospermic, oligoasthenozoospermic and oligoasthenoteratozoospermic groups (P < 0.05). TAC levels returned to baseline 30 days post exercise in the asthenoteratozoospermic, oligoasthenozoospermic and oligoasthenoteratozoospermic groups (P < 0.05). There were slight but non-significant decreases in these levels at 24 wks in the NON-EX groups (P > 0.05) (Fig. 4).

3.6. Cytokines At 12 and 24 weeks, IL-1b, IL-6, IL-8 and TNF-a had significantly decreased from baseline in the EX group (P < 0.05). There were slight but non-significant increases in these levels at 24 wks in the NON-EX groups (P > 0.05) (Fig. 5). IL-1b, IL-6, IL-8 and TNF-a values remained significantly lower even after 30 days of detraining (P < 0.05). Similar changes were observed for these levels in the asthenozoospermic, asthenoteratozoospermic, oligospermic,

oligoasthenozoospermic and oligoasthenoteratozoospermic groups (P < 0.05) (Fig. 5). 3.7. Correlations The association between body composition, maximal oxygen consumption, oxidants, antioxidants, cytokines, semen parameters and sperm DNA damage in all groups are shown in Table 2. Semen volume, progressive motility, sperm morphology, sperm concentration, number of spermatozoa and percentage of TUNEL positive spermatozoa were negatively related to weight (r = 0.342; P = 0.005, r = 0.515; P = 0.002, r = 0.486; P = 0.001, r = 0.502; P = 0.001, r = 0.396; P = 0.002, r = 0.557; P = 0.003, respectively), BMI (r = 0.321; P = 0.009, r = 0.442; P = 0.002, r = 0.489; P = 0.002, r = 0.369; P = 0.001, r = 0.458; P = 0.001, r = 0.562; P = 0.001, respectively), body fat (r = 0.334; P = 0.001, r = 0.473; P = 0.001, r = 0.429; P = 0.002, r = 0.371; P = 0.001, r = 0.349; P = 0.001, r = 0.524; P = 0.002, respectively), waist circumference (r = 0.355; P = 0.008, r = 0.502; P = 0.003, r = 0.426; P = 0.001, r = 0.452; P = 0.002, r = 0.329; P = 0.004, r = 0.603; P = 0.002, respectively), ROS (r = 0.419; P = 0.006, r = 0.623; P = 0.006, r = 0.634; P = 0.001, r = 0.579; P = 0.002,

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Fig. 3. Changes in semen quality parameters and sperm DNA damage in the EX and NON-EX groups during the course of the study. *: P < 0.05, significantly different from baseline values (within groups, baseline vs. week 12, week 24, 7 and 30 days during recovery).

r = 0.417; P = 0.007, r = 0.662; P = 0.003, respectively), MDA (r = 0.472; P = 0.003, r = 0.559; P = 0.001, r = 0.601; P = 0.001, r = 0.594; P = 0.002, r = 0.556; P = 0.001, r = 0.632; P = 0.002, respectively), 8-isoprostane (r = 0.297; P = 0.015, r = 0.433; P = 0.003, r = 0.478; P = 0.006, r = 0.491; P = 0.003, r = 0.428; P = 0.002, r = 0.519; P = 0.002, respectively), IL-1b (r = 0.327; P = 0.005, r = 0.466; P = 0.002, r = 0.451; P = 0.001, r = 0.429; P = 0.004, r = 0.407; P = 0.002, r = 0.494; P = 0.002, respectively), IL-6 (r = 0.453; P = 0.003, r = 0.582; P = 0.002, r = 0.601; P = 0.005, r = 0.612; P = 0.001, r = 0.573; P = 0.001, r = 0.607; P = 0.003, respectively), IL-8 (r = 0.462; P = 0.005, r = 0.513; P = 0.001, r = 0.539; P = 0.002, r = 0.587; P = 0.003, r = 0.564; P = 0.009, r = 0.588; P = 0.003, respectively) and TNF-a (r = 0.357; P = 0.017, r = 0.611; P = 0.001, r = 0.567; P = 0.010, r = 0.606; P = 0.001, r = 0.550; P = 0.002, r = 0.573; P = 0.001,

respectively) (Table 2). Results from the mixed model regression showed that for each unit (ml) increase in semen volume there was a decrease of 5.4 kg, 15.9 kg/m2, 6.1%, 1.4 cm, 0.09 RLU, 134.4 nmol/ml, 7.8 ng/ml, 6.8 pg/ml, 4.8 pg/ml, 0.1 pg/ml and 12.3 pg/ml, respectively in weight, BMI, body fat, waist circumference, ROS, MDA, 8-isoprostane, IL-1b, IL-6, IL-8 and TNF-a. Similarly, for each unit (%) increase in progressive motility there was a decrease of 1.6 kg, 4.6 kg/m2, 1.7%, 0.4 cm, 0.04 RLU, 34.4 nmol/ml, 2.0 ng/ml, 1.7 pg/ml, 1.3 pg/ml, 0.04 pg/ml and 3.2 pg/ml, respectively in weight, BMI, body fat, waist circumference, ROS, MDA, 8-isoprostane, IL-1b, IL-6, IL-8 and TNF-a. Furthermore, for each unit (%) increase in sperm morphology there was a decrease of 0.4 kg, 0.9 kg/m2, 0.4%, 0.08 cm, 0.01 RLU, 7.8 nmol/ ml, 0.5 ng/ml, 0.4 pg/ml, 0.3 pg/ml, 0.01 pg/ml and 0.7 pg/ml, respectively in weight, BMI, body fat, waist circumference, ROS,

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Fig. 4. Changes in markers of oxidative stress in the EX and NON-EX groups during the course of the study. *: P < 0.05, significantly different from baseline values (within groups, baseline vs. week 12, week 24, 7 and 30 days during recovery).

MDA, 8-isoprostane, IL-1b, IL-6, IL-8 and TNF-a. For each unit (
106/ml) increase in sperm concentration there was also a decrease of 1.1 kg, 3.0 kg/m2, 1.2%, 0.3 cm, 0.02 RLU, 25.1 nmol/ ml, 1.5 ng/ml, 1.3 pg/ml, 0.9 pg/ml, 0.03 pg/ml and 2.5 pg/ml, respectively in weight, BMI, body fat, waist circumference, ROS, MDA, 8-isoprostane, IL-1b, IL-6, IL-8 and TNF-a. Likewise, for each unit (106) increase in number of spermatozoa there was also a decrease of 4.1 kg, 11.9 kg/m2, 4.7%, 1.1 cm, 0.06 RLU, 99.7 nmol/ml, 5.8 ng/ml, 5.2 pg/ml, 3.7 pg/ml, 0.1 pg/ml and 9.2 pg/ml, respectively in weight, BMI, body fat, waist circumference, ROS, MDA, 8-isoprostane, IL-1b, IL-6, IL-8 and TNF-a. Additionally, for each unit (%) improvement in TUNEL positive spermatozoa there was also a decrease of 1.3 kg, 3.4 kg/m2, 1.4%, 0.3 cm, 0.2 RLU, 28.4 nmol/ml, 1.8 ng/ml, 1.5 pg/ml, 0.1 pg/ ml, 0.03 pg/ml and 2.7 pg/ml, respectively in weight, BMI, body

fat, waist circumference, ROS, MDA, 8-isoprostane, IL-1b, IL-6, IL8 and TNF-a (Table 2). Significant positive correlations were also observed between semen volume, progressive motility, sperm morphology, sperm concentration, number of spermatozoa and percentage of TUNEL positive spermatozoa with SOD (r = 0.431; P = 0.007, r = 0.594; P = 0.002, r = 0.617; P = 0.001, r = 0.598; P = 0.002, r = 0.447; P = 0.001, r = 0.624; P = 0.001, respectively), catalase (r = 0.319; P = 0.001, r = 0.376; P = 0.002, r = 0.421; P = 0.002, r = 0.452; P = 0.001, r = 0.264; P = 0.001, r = 0.485; P = 0.001, respectively), TAC (r = 0.336; P = 0.002, r = 0.397; P = 0.003, r = 0.416; P = 0.002, r = 0.404; P = 0.001, r = 0.347; P = 0.001, r = 0.429; P = 0.001, respectively) (Table 2). For each unit (ml) increase in semen volume there was an increase of 21.5 U/mL, 12.3 U/mL and 86.0 mM, respectively, in SOD, catalase and TAC. For each unit

B. Hajizadeh Maleki, B. Tartibian / Cytokine 92 (2017) 55–67

63

Fig. 5. Changes in proinflammatory cytokines in the EX and NON-EX groups during the course of the study. *: P < 0.05, significantly different from baseline values (within groups, baseline vs. week 12, week 24, 7 and 30 days during recovery).

(%) increase in progressive motility there was also an increase of 5.5 U/mL, 3.2 U/mL and 22.1 mM, respectively, in SOD, catalase and TAC. Also, for each unit (%) increase in sperm morphology there was also an increase of 1.3 U/mL, 0.7 U/mL and 5.1 mM, respectively, in SOD, catalase and TAC. Likewise, for each unit (
106/ml) increase in sperm concentration there was also an increase of 4.0 U/mL, 2.3 U/mL and 16.9 mM, respectively, in SOD, catalase and TAC. Similarly, for each unit (
106) increase in number of spermatozoa there was also an increase of 16.2 U/mL, 9.4 U/mL and 64.5 mM, respectively, in SOD, catalase and TAC. Additionally, for each unit%) improvement in TUNEL positive spermatozoa there was also an increase of 4.6 U/mL, 2.8 U/mL and 17.7 mM, respectively, in SOD, catalase and TAC (Table 2). Percentages of TUNEL positive spermatozoa and VO2max levels were negatively correlated (r = 0.527; P = 0.001), while positive correlations between VO2max, semen volume, progressive motility, sperm morphology, sperm concentration and number of spermatozoa (r = 0.396; P = 0.012, r = 0.419; P = 0.002, r = 0.458; P = 0.001, r = 0.477; P = 0.001, r = 0.310; P = 0.002, respectively), were noted. The mixed model regression revealed that each unit increase in VO2max levels resulted in 1.9% decrease in percentages of TUNEL positive spermatozoa, and each unit increase in VO2max levels resulted in 2.2, 0.5, 1.6 and 6.5 unit increases in progressive motility, sperm morphology, sperm concentration and number of spermatozoa, respectively (Table 2). 3.8. Pregnancy rate and outcomes Intervention resulted in 70.5% (139 out of 197) partner pregnancies (OR, 80.0; 95% CI: 32.5 to 646.2), of which 31 (22.3%) were

spontaneous. However, 4 patients’ wives (2.9%) experienced miscarriage. As a result, the calculated live birth rate was 91.4% (OR, 197.0; 95% CI: 5.9–2149.6) (Table 3). As for subgroups, moderate aerobic exercise training was associated with elevated pregnancy rate in asthenozoospermic (OR, 186.7; 95% CI, 27.8– 1255.2), asthenoteratozoospermic (OR, 59.8; 95% CI, 10.2–351.4), oligospermic (OR, 108.7; 95% CI, 17.7–667.3), oligoasthenozoospermic (OR, 23.1; 95% CI, 4.0–132.7) and oligoasthenoteratozoospermic (OR, 41.7; 95% CI, 7.3–238.5) patients compared with NON-EX subgroups. 4. Discussion Perturbations in redox homeostasis [3–5] and overproduction of proinflamatory cytokines [9,10] in seminal plasma are known to be capable of significantly affecting sperm function and fertility. These support the notion that modulation of proinflammatory cytokines and redox imbalance in seminal plasma may be an early step toward improving fertility problems and reproductive outcomes in male infertility. In the present study, for the first time, we tested the effectiveness of moderate aerobic exercise training as a potential antioxidant and anti-inflammatory therapy on reproductive function in infertile men. The novel findings of this study are: (1) seminal proinflamatory cytokines, peroxidative biomarkers and DNA fragmentation index were attenuated with 24 weeks of moderate aerobic exercise, (2) aerobic exercise training improves standard semen quality parameters as well as increases enzymatic activities and concentration of seminal antioxidants, (3) changes in proinflamatory cytokines and redox homeostasis, in all groups combined, correlated with standard semen quality parameters

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Table 2 Correlation of body composition measures, VO2max, antioxidants, oxidants and proinflammatory cytokines with semen quality parameters and sperm DNA integrity in infertile patients. Semen volume (ml)

Progressive motility (%)

Sperm morphology (%)

Sperm concentration (
106/ml)

N of spermatozoa (
106)

TUNEL (%)

Correlation RC P<

0.342 5.4 0.005

0.515 1.6 0.002

0.486 0.4 0.001

0.502 1.1 0.001

0.369 4.1 0.002

0.557 1.3 0.003

Correlation RC * P<

0.321 15.9 0.009

0.442 4.6 0.002

0.489 0.9 0.002

0.369 3.0 0.001

0.458 11.9 0.001

0.562 3.4 0.001

Correlation RC P<

0.334 6.1 0.001

0.473 1.7 0.001

0.429 0.4 0.002

0.371 1.2 0.001

0.349 4.7 0.001

0.524 1.4 0.002

Correlation RC P<

0.355 1.4 0.008

0.502 0.4 0.003

0.426 0.08 0.001

0.452 0.3 0.002

0.329 1.1 0.004

0.603 0.3 0.002

Correlation RC * P<

0.396 8.6 0.012

0.419 2.2 0.002

0.458 0.5 0.001

0.477 1.6 0.001

0.310 6.5 0.002

0.527 1.9 0.001

Correlation RC P<

0.431 21.5 0.007

0.594 5.5 0.002

0.617 1.3 0.001

0.598 4.0 0.002

0.447 16.2 0.001

0.624 4.6 0.001

Catalase (U/mL)

Correlation RC * P<

0.319 12.3 0.001

0.376 3.2 0.002

0.421 0.7 0.002

0.452 2.3 0.001

0.264 9.4 0.001

0.485 2.8 0.001

TAC (mM)

Correlation RC * P<

0.336 86.0 0.002

0.397 22.1 0.003

0.416 5.1 0.002

0.404 16.9 0.001

0.347 64.5 0.001

0.429 17.7 0.001

Correlation RC P<

0.419 0.09 0.006

0.623 0.04 0.006

0.634 0.01 0.002

0.579 0.02 0.002

0.417 0.06 0.007

0.662 0.02 0.003

MDA (nmol/mL)

Correlation RC * P<

0.472 134.4 0.003

0.559 34.4 0.001

0.601 7.8 0.001

0.594 25.1 0.002

0.556 99.7 0.001

0.632 28.4 0.002

8-Isoprostane (ng/mL)

Correlation RC * P<

0.297 7.8 0.015

0.433 2.0 0.003

0.478 0.5 0.006

0.491 1.5 0.003

0.428 5.8 0.002

0.519 1.8 0.002

Correlation RC P<

0.327 6.8 0.005

0.466 1.7 0.002

0.451 0.4 0.001

0.429 1.3 0.004

0.407 5.2 0.002

0.495 1.5 0.002

Correlation RC * P<

0.453 4.8 0.003

0.582 1.3 0.002

0.601 0.3 0.005

0.612 0.9 0.001

0.573 3.7 0.001

0.607 0.1 0.003

Correlation RC P<

0.462 0.1 0.005

0.513 0.04 0.001

0.539 0.01 0.002

0.587 0.03 0.003

0.564 0.1 0.009

0.588 0.03 0.003

Correlation RC * P<

0.357 12.3 0.017

0.611 3.2 0.001

0.567 0.7 0.010

0.606 2.5 0.001

0.550 9.2 0.002

0.573 2.7 0.001

Weight (kg)

*

BMI (kg/m2)

Fat (%)

*

Waist circumference (cm)

*

VO2max (ml kg1 min1) SOD (U/mL)

*

ROS (RLU)

*

IL-1b (pg/mL)

*

IL-6 (pg/mL)

IL-8 (pg/mL)

*

TNF-a (pg/mL)

RC: Regression coefficient. P < 0.05, Adjusted for the group through the study based on mixed model.

*

and sperm DNA fragmentation index, and (4) alterations in semen parameters, sperm DNA integrity, inflammation and redox status in the seminal plasma following aerobic exercise training were coincide with pregnancy achievement in infertile couples. The effect of moderate aerobic exercise training on male factor infertility may therefore be positive. A close association between proinflammatory cytokines and male infertility has previously been postulated [9,10]. The elevated levels of seminal IL-6 and IL-8, for instance, are sensitive indicators of the early stage of the inflammatory process in the male genitourinary tract and augment the peroxidation process and influence sperm function, with consequent development of male infertility [6,30,31]. It is now widely accepted that the mediators of inflammation can also be a direct cause of peroxidative damage to the sperm plasma membrane, which can ultimately bring about

limited fertilizing abilities of the sperm cells [6,11,30,31]. Additionally, infertile men often demonstrate higher ROS and/or diminished antioxidant capacity within their seminal plasma and spermatozoa than do fertile men [32]. Enhanced levels of ROS and/or decreased levels of antioxidants in spermatozoa and seminal plasma produce a state known as oxidative stress that has long been considered as a major causative factor behind the pathogenesis of sperm dysfunction and sperm DNA fragmentation in male factor infertility [5,33]. In the present study, significantly lower seminal levels of proinflammatory cytokines IL-1b, IL-6, IL-8 and TNF-a have been observed after moderate aerobic exercise intervention in infertile patients. These findings parallel previous report from our laboratory that moderate aerobic exercise training for 24 weeks attenuates seminal proinflammatory cytokines in healthy human

65

B. Hajizadeh Maleki, B. Tartibian / Cytokine 92 (2017) 55–67 Table 3 Pregnancy and live birth rates during the course of the study. 0–12 wks (N)

13–24 wks (N)

25–36 wks (N)

Total pregnancies no./total (%)

Live births no./total (%)

Total pregnancies OR (95% CI)

Live births OR (95% CI)

5 No

19 1

11 No

35/39 (89.7) 1/36 (2.8)

34/35 (97.1) 0/1 (0.0)

186.7 (27.8–1255.2)

69.1 (1.9–2509.9)

Asthenoteratozoospermic EX (n = 38) 6 NON-EX (n = 38) No

14 No

7 1

27/38 (71.1) 1/38 (2.6)

25/27 (92.6) 0/1 (0.0)

59.8 (10.2–351.4)

30.6 (0.9–967.9)

Oligospermic EX (n = 40) NON-EX (n = 37)

16 No

9 No

33/40 (82.5) 1/37 (2.7)

31/33 (93.9) 0/1 (0.0)

108.7 (17.7–667.3)

37.8 (1.2–1190.7)

Oligoasthenozoospermic EX (n = 39) 3 NON-EX (n = 37) No

11 1

5 No

19/39 (48.7) 1/37 (2.7)

15/19 (78.9) 0/1 (0.0)

23.1 (4.0–132.7)

10.3 (0.4–299.9)

Oligoasthenoteratozoospermic EX (n = 41) 4 NON-EX (n = 41) 1

13 No

8 No

25/41 (61) 1/41 (2.4)

22/25 (88) 0/1 (0.0)

41.7 (7.3–238.5)

19.3 (0.6–573.8)

TOTAL EX(n = 197) NON-EX (n = 189)

73 2

40 1

139/197 (70.5) 5/189 (2.6)

127/139 (91.4) 0/5 (0.0)

80.0 (32.5–646.2)

197.0 (5.9–2149.6)

Asthenozoospermic EX (n = 39) NON-EX (n = 36)

8 1

26 2

OR: odds ratio. CI: confidence interval.

subject [19]. The mechanisms by which moderate aerobic exercise training attenuates seminal mediators of inflammation has not yet been fully understood; however, the anti-inflammatory effects of regular exercise training may partially explain by traininginduced alterations in the expressions of anti- and proinflammatory cytokines in favor of the former across body fluids, organs and tissues [34] as well as the training-induced reductions in monocyte toll-like receptor 4 (TLR4) which is responsible for activating the innate immune system [34]. These long-term adaptations may also be attributed to the anti-inflammatory response induced by every single session of exercise training, which is partially mediated through muscle-derived IL-6, as this cytokine provoke the release of the anti-inflammatory cytokines IL-1 receptor antagonist (IL-1ra) and IL-10 and inhibits the proinflammatory cytokine TNF-a production in body fluids, cells and/or tissues [35]. This study also demonstrated that moderate aerobic exercise training resulted in significant improvements in VO2max and body composition. Several studies have already shown an inverse relationship between markers of inflammation and maximal oxygen uptake [36]. Also, an inverse association has previously been reported between measures of body composition and circulating inflammatory biomarkers [37]. Attenuated inflammation in the present study is therefore likely related to the marked changes in VO2max and adiposity following participation in aerobic exercise training. Paralleling previous reports [12,17,19], our results showed a decrease in seminal oxidants, while radical depleting enzymes such as SOD, catalase and TAC were elevated after exercise training. These findings highlight the importance of regular physical activity in regulating the redox status of seminal plasma. With respect to physical activity, there is consistent proof that aerobic exercise training enhances antioxidant capacity through activation of redox-sensitive signaling pathways that lead to increased synthesis and expression of a number of enzymes and proteins that play important roles in maintenance of intracellular oxidantantioxidant homeostasis, which could result in an increased resistance against ROS induced lipid peroxidation as well as decreased accumulation of oxidative protein and DNA damage [38,39]. In this cohort of infertile patients, therefore post-exercise decreases in oxidative stress and lipid peroxidation biomarkers can be attributed to training-induced up-regulation of antioxidant

enzymes, reduced mitochondrial ROS production, and improvements in radical-scavenging capacity of body tissues and fluids [40,41]. Previous evidence likewise point out to an interaction between unbalanced cytokine release with ROS overproduction and subsequent oxidative stress in seminal plasma [6]. Thus, in our study, post exercise decreases in synthesis of proinflammatory cytokines also could be attributed to the attenuated levels of seminal oxidants and vice versa. In this study, moderate aerobic exercise training improved semen quality as demonstrated by significant improvements in progressive motility, sperm morphology, sperm concentration and number of spermatozoa. These findings parallel those of previous reports that have demonstrated the potential benefits of a physically active lifestyle [12,17] and moderate aerobic exercise intervention [19] on semen quality parameters. Consistent with our findings, our latest report has also suggested that moderate aerobic exercise training is capable of improving the sperm DNA integrity in healthy human subjects [19], and our data support this. The present study, the first of its kind, further found that the training-induced changes in semen quality parameters and sperm DNA integrity were coincide with pregnancy achievement in infertile couples. The results revealed total pregnancy rates of 70.5% and total live birth rates of 91.4% in infertile couples following the exercise intervention. The available data suggest that sperm DNA damage has a strong negative effect on male fertility potential [42] which might support the relevance of the current data for reproductive health of men with impaired fertility potential. A threshold value of 20% TUNEL-positive spermatozoa has earlier been demonstrated to differentiate between fertile and infertile men [43,44]. Likewise it has been reported that the miscarriage risk increased 4 times when the DNA fragmentation index exceeded 15% [45], while fewer than 15% sperm with fragmented DNA has been reported to be indicative of high fertility status [46]. The results of this study indicate that, in this cohort of infertile patients, such threshold levels have clearly been diminished by the actual training program. Therefore, in this cohort of infertile patients, the lower percentage of TUNEL-positive spermatozoa induced by a 24-week moderate aerobic exercise may contribute to improved reproductive outcomes. In the present study, semen quality parameters and sperm DNA integrity seem to be influenced by seminal markers of inflammation and redox status, as levels of

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oxidants and proinflammatory cytokines have been negatively correlated with semen quality parameters and percentage of TUNELpositive spermatozoa. Further, changes in the seminal levels of SOD, catalase and TAC were positively associated with alterations in semen quality parameters and sperm DNA integrity. Though, changes in seminal markers of inflammation and oxidative status alone cannot serve as indicators of an improved reproductive function in infertile patients, according to the WHO guidelines [20], it seems that positive changes in semen quality parameters and DNA integrity as well as marked improvement in pregnancy rate may serve as a predictor of an improved reproductive function among infertile men participated in aerobic exercise training. According to these findings, therefore, it can be speculated that post exercise alterations in seminal oxidative status and proinflammatory cytokines may prevent oxidative stress and inflammation in seminal plasma and thereby induce improvements in semen parameters and sperm DNA integrity, all of which may have positive consequences for spermatogenesis and eventually male fertility. Another sperm dysfunction and sperm DNA damage inductor is the unfavorable body composition [47], which is negatively correlated with the levels of physical fitness [48] and increase in the VO2max [49]. In the present study, exercise-induced changes in body composition and aerobic fitness were respectively negatively and positively correlated with improvements of semen quality parameters and sperm DNA integrity. It is likely that exercise training effectively improve semen quality parameters, sperm DNA integrity and subsequently reproductive outcomes, among others, in association with concomitant improvements in body composition and VO2max, as there were significant correlations between semen quality parameters, sperm DNA integrity, body composition and VO2max. In the present study, the magnitude of the changes in proinflammatory cytokines, oxidative status, semen quality parameters, sperm DNA integrity and reproductive outcomes increased by improvements in exercise intensity and duration in the second 12 weeks of the intervention, as training volume in the second 12 wks was greater than the first 12 wks, so it is possible that greater improvements in all studied variables in the second 12 wks of the intervention could be attributed to this. The results also suggest that the 4-week detraining period does not seem to be enough to reverse adaptations induced by moderate aerobic exercise training in infertile patients. Strengths of this study include randomization to treatment group, obtaining multiple samples during the course of the study, which enabled us to assess the chronic responses to aerobic exercise training in infertile patients, exclusion of physically active men, measurement of pregnancy rates, good adherence and retention rates assessed by comprehensive exercise monitoring, and prescription of a lengthy and supervised exercise intervention. In conclusion, these original findings provide compelling evidence that moderate aerobic exercise training may improve semen parameters, sperm DNA integrity and subsequently pregnancy rate in infertile couples. Our findings suggest that the beneficial effects of moderate aerobic exercise on male reproduction and fertility problems may primarily depend up on its anti-inflammatory and antioxidant effects. We recommend the moderate aerobic exercise training as a treatment option for male factor infertility. However, there is a need for future studies on physical activity in the infertile patients. References [1] S. Pfeifer, J. Goldberg, R. Lobo, M. Thomas, E. Widra, M. Licht, J. Collins, M. Cedars, M. Vernon, O. Davis, C. Gracia, W. Catherino, K. Thornton, R. Rebar, A. La Barbera, Definitions of infertility and recurrent pregnancy loss: a committee opinion, Fertil. Steril. 99 (1) (2013) 63. [2] R.I. McLachlan, D.M. de Kretser, Male infertility: the case for continued research, Med. J. Aust. 174 (3) (2001) 116–117.

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