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Randomized, triple-blind, placebo-controlled clinical trial examining the effects of alpha-lipoic acid supplement on the spermatogram and seminal oxidative stress in infertile men

      Objective

      To evaluate effects of supplementation with alpha-lipoic acid (ALA) on the spermatogram and seminal oxidative stress biomarkers.

      Design

      Randomized, triple-blind, placebo-controlled clinical trial.

      Setting

      Infertility clinic.

      Patient(s)

      Infertile men.

      Intervention(s)

      ALA (600 mg) or placebo for 12 weeks.

      Main Outcome Measure(s)

      Semen analysis, anthropometric, dietary, and physical activity assessments, total antioxidant capacity, and malondialdehyde.

      Result(s)

      At the end of study, the total sperm count, sperm concentration, and motility in the intervention group were significantly higher than in the control group. In the ALA group, the total sperm count, sperm concentration, and motility levels were also significantly increased at the end of study compared with baseline values. However, there were no significant differences in ejaculate volume, normal morphology percentage, and live sperm between groups. ALA supplementation also resulted in a significant improvement in seminal levels of total antioxidant capacity (TAC) and malondialdehyde compared with the placebo.

      Conclusion(s)

      According to the results, medical therapy of asthenoteratospermia with ALA supplement could improve quality of semen parameters. However, further investigation is suggested in this regard.

      Clinical Trial Registration Number

      IRCT2013111010181N3.

      Key Words

      Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/haghighianh-lipoic-acid-spermatogram/
      Infertility is defined as conception failure after regular sexual activities in the absence of contraception for at least 1 year (
      • Gelbaya T.A.
      • Potdar N.
      • Jeve Y.B.
      • Nardo L.G.
      Definition and epidemiology of unexplained infertility.
      ). Infertility is a major clinical concern, affecting 15% of married couples (
      • Barnes A.
      • Riche D.
      • Mena L.
      • Sison T.
      • Barry L.
      • Reddy R.
      • et al.
      Efficacy and safety of intrauterine insemination and assisted reproductive technology in populations serodiscordant for human immunodeficiency virus: a systematic review and meta-analysis.
      ). Causes of infertility in couples are varied, but the main causes are attributable to the male partner (
      • Winters B.R.
      • Walsh T.J.
      The epidemiology of male infertility.
      ). Although certain cases of male infertility are due to anatomic abnormalities, such as varicocele, ductal obstructions, and ejaculatory disorders, an estimated 40%–90% of cases are due to deficient sperm production of unidentifiable origin (
      • Balercia G.
      • Regoli F.
      • Armeni T.
      • Koverech A.
      • Mantero F.
      • Boscaro M.
      Placebo-controlled double-blind randomized trial on the use of L-carnitine, L-acetylcarnitine, or combined L-carnitine and L-acetylcarnitine in men with idiopathic asthenozoospermia.
      ). Recently, a special concern has been raised about the low sperm concentration and poor quality of semen found in young men in some countries (
      • Jensen T.K.
      • Gottschau M.
      • Madsen J.O.
      • Andersson A.-M.
      • Lassen T.H.
      • Skakkebæk N.E.
      • et al.
      Habitual alcohol consumption associated with reduced semen quality and changes in reproductive hormones; a cross-sectional study among 1221 young Danish men.
      ). At the present time, the etiology of suboptimal semen quality is not well understood, and many physiologic, environmental, and genetic factors, such as oxidative stress, have been suggested (
      • Moslemi M.K.
      • Tavanbakhsh S.
      Selenium–vitamin E supplementation in infertile men: effects on semen parameters and pregnancy rate.
      ).
      Reactive oxygen species (ROS) can have beneficial or detrimental effects on sperm function, depending on the nature and the concentration of the ROS as well as the location and length of exposure to ROS (
      • Baumber J.
      • Ball B.A.
      • Gravance C.G.
      • Medina V.
      • Davies-Morel M.C.
      The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation.
      ). During epididymal transit, sperm acquire the ability to move progressively; however, they acquire the ability to fertilize in the female tract through a series of physiologic changes called “capacitation” (
      • Dacheux J.-L.
      • Dacheux F.
      New insights into epididymal function in relation to sperm maturation.
      ). Under physiologic states, spermatozoa make little amounts of ROS, which are needed for capacitation and acrosomal reaction. Superoxide anion appears to play a role in this process (
      • Wu J.
      • Wu S.
      • Xie Y.
      • Wang Z.
      • Wu R.
      • Cai J.
      • et al.
      Zinc protects sperm from being damaged by reactive oxygen species in assisted reproduction techniques.
      ). It is known that spermatozoa are susceptible to oxidative damage because their plasma membranes are rich in polyunsaturated fatty acids and have low concentrations of scavenging enzymes (
      • Linhartova P.
      • Gazo I.
      • Shaliutina-Kolesova A.
      • Hulak M.
      • Kaspar V.
      Effects of tetrabrombisphenol A on DNA integrity, oxidative stress, and sterlet (Acipenser ruthenus) spermatozoa quality variables.
      ). Studies have indicated that male germ cells at various stages of differentiation have the potential to generate ROS. Excessive generation of ROS in semen by leukocytes as well as by abnormal spermatozoa could be a cause of infertility (
      • Gharagozloo P.
      • Aitken R.J.
      The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy.
      ). It has been reported that moderately elevated concentrations of ROS do not affect sperm viability but cause sperm immobilization, mostly via depletion of intracellular adenosine triphosphate (ATP), and decreased phosphorylation of axonemal proteins (
      • Alvarez J.G.
      • Aitken R.J.
      Lipid peroxidation in human spermatozoa.
      ). High concentrations of hydrogen peroxide, the main ROS producer, also prompt lipid peroxidation and result in cell death (
      • Takei G.L.
      • Mukai C.
      • Okuno M.
      Transient Ca2+ mobilization caused by osmotic shock initiates salmonid fish sperm motility.
      ). In several studies, ROS levels were higher and levels of seminal antioxidants significantly lower in subfertile patients than in normal fertile men (
      • Atig F.
      • Raffa M.
      • Habib B.-A.
      • Kerkeni A.
      • Saad A.
      • Ajina M.
      Impact of seminal trace element and glutathione levels on semen quality of Tunisian infertile men.
      ). A simple tool to assay the effect of lipid peroxidation on the spermatozoa is the evaluation of seminal levels of malondialdehyde (MDA), which is a stable lipid peroxidation product (
      • Tavilani H.
      • Doosti M.
      • Saeidi H.
      Malondialdehyde levels in sperm and seminal plasma of asthenozoospermic and its relationship with semen parameters.
      ).
      Dietary antioxidants may also have a positive effect on semen quality (
      • Delimaris I.
      • Piperakis S.M.
      The importance of nutritional factors on human male fertility: a toxicological approach.
      ). Recently, Buhling et al. reported that a lower intake of some antioxidant nutrients, such as vitamins A, C, and E, carnitine, folate, zinc, and selenium, is associated with male infertility (
      • Buhling K.J.
      • Laakmann E.
      The effect of micronutrient supplements on male fertility.
      ). In a cross-sectional study of 97 healthy male subjects, a higher intake of vitamins C, E, and β-carotene was associated with a higher sperm count and motility (
      • Gaskins A.J.
      • Colaci D.S.
      • Mendiola J.
      • Swan S.H.
      • Chavarro J.E.
      Dietary patterns and semen quality in young men.
      ).
      Alpha-lipoic acid (ALA; also called thioctic acid) acts as the coenzyme for pyruvate dehydrogenase and α-ketoglutarate dehydrogenase in the mitochondria (
      • Bingham P.M.
      • Stuart S.D.
      • Zachar Z.
      Lipoic acid and lipoic acid analogs in cancer metabolism and chemotherapy.
      ). Exogenous ALA supplementation results in increased unbound ALA levels, which can act as a strong antioxidant and improve oxidative stress status both in vitro and in vivo. Inside cells and tissues, ALA is reduced to dihydrolipoic acid (DHLA), which is even more potent as an antioxidant (
      • Grasso S.
      • Bramanti V.
      • Tomassoni D.
      • Bronzi D.
      • Malfa G.
      • Traini E.
      • et al.
      Effect of lipoic acid and α-glyceryl-phosphoryl-choline on astroglial cell proliferation and differentiation in primary culture.
      ). ALA or its reduced form, DHLA, quenches a number of oxygen-free radical species in both lipid and aqueous phases, chelates transition metals, and prevents membrane lipid peroxidation and protein damage via interactions with glutathione (
      • Ali Y.F.
      • Desouky O.S.
      • Selim N.S.
      • Ereiba K.M.
      Assessment of the role of α-lipoic acid against the oxidative stress of induced iron overload.
      ). Recent studies results have also suggested that ALA is a main factor in the Krebs cycle and contributes to ATP biosynthesis, which is crucial for the sperm viability (
      • Ibrahim S.F.
      • Osman K.
      • Das S.
      • Othman A.M.
      • Majid N.A.
      • Rahman M.P.
      A study of the antioxidant effect of alpha lipoic acids on sperm quality.
      ).
      Based on the above facts, the present study was conducted to study the effect of daily oral supplementation of ALA on the quality of semen parameters and seminal markers of oxidative stress, including MDA, and total antioxidant capacity (TAC) levels in infertile men. As such, the results of this study may have wide clinical importance in fertility clinics and laboratories.

      Material and methods

       Subjects

      This randomized, triple-blind, placebo-controlled clinical trial was conducted on 44 infertile men with idiopathic asthenozoospermia in the infertility clinic of Ahvaz Jundishapur University of Medical Sciences, Iran, in 2014. After laboratory investigations, if the mobility of sperm was <50% and rapid mobility in the direct path of sperm was <25%, the diagnosis was idiopathic asthenozoospermia (
      • Jungwirth A.
      • Giwercman A.
      • Tournaye H.
      • Diemer T.
      • Kopa Z.
      • Dohle G.
      • et al.
      European Association of Urology guidelines on male infertility: the 2012 update.
      ). Patients were recruited in the study after fulfilling certain criteria, including unwilling childlessness at least 24 months in duration with a female partner, no medical condition that could account for infertility, and a normal fertile female partner according to investigations. All patients were needed to have stopped all medical therapy ≥12 weeks before study initiation. Exclusion criteria included the history of epididymo-orchitis, prostatitis, genital trauma, testicular torsion, inguinal or genital surgery, urinary tract infection, or previous hormonal therapy; another genital disease (cryptorchidism, current genital inflammation or varicocele); severe general or central nervous system disease and endocrinopathy; use of cytotoxic drugs, immunosuppressants, anticonvulsants, androgens, or antiandrogens; and a recent history of sexually transmitted infection. Patients were also excluded from analysis if they had psychologic or physiologic abnormalities that would impair sexual performance or the ability to provide semen samples; drug or alcohol abuse; hepatobiliary disease; significant renal insufficiency; occupational and environmental subjections to possible reproductive toxins (
      • Safarinejad M.R.
      Efficacy of coenzyme Q10 on semen parameters, sperm function and reproductive hormones in infertile men.
      ); a body mass index of ≥30 kg/m2; participation in another investigational study; and unlikely availability for follow-up. The study was approved by Medical Ethical Committee of the Ahvaz Jundishapur University and recorded by the identification code of IRCT2013111010181N3 in the clinical trials registry of Iran. Written consent was obtained from each of the participants. The work was financially supported by a grant from the Vice-Chancellor for Research Affairs of Jundishapur University of Medical Sciences, Ahvaz, Iran.

       Study Design

      At the beginning of the study, patients were randomized to group 1, who received 600 mg ALA once daily, and group 2, who received matching placebo for 12 weeks. Each eligible patient received a randomization number which was determined by a computer-generated schedule. Then a randomization table was generated by the method of random permuted blocks. Persons who were operationally independent from the study investigator performed the study randomization. The investigator, clinician prescriber, and patients were blinded to the treatment condition. To maintain and guarantee blinding, ALA and placebo were identical in appearance. Patients' data collected during this trial were kept confidential and locked in a secure area. Randomization codes of the study were opened only after all participants had completed the study protocol.
      Demographic data, medical history, lifetime history of tobacco use, intake of multivitamin supplements, and lifestyle information were collected from each patient. Weight was measured with the use of digital scales (Soehne) with patients minimally clothed. Height was measured with the use of a fixed-to-wall nonstretch tape meter with patients in a standing position. The body mass index (BMI) was then calculated in kilograms per meters squared. Participants were interviewed face to face by trained professional nutritionists. After the baseline screening, data, including dietary habits, were collected from 44 asthenozoospermia patients.

       Preparation of Semen Samples

      Semen samples were obtained after 3 days of sexual abstinence at the urology unit of Imam Infertility Clinic, Ahvaz, Iran. All semen was held at 37°C to liquefy. After liquefaction, the samples were analyzed according to the World Health Organization (WHO) criteria (
      • Künzle R.
      • Mueller M.D.
      • Hänggi W.
      • Birkhäuser M.H.
      • Drescher H.
      • Bersinger N.A.
      Semen quality of male smokers and nonsmokers in infertile couples.
      ). Remnants of liquefied semen samples were immediately centrifuged at 300 rpm for 10 minutes. The seminal plasma was divided into several aliquot parts and kept frozen at −80°C for biochemical analysis.

       Assessment of Sperm Motility

      Motility assessment of sperm was performed according to WHO criteria (
      • Jensen T.K.
      • Swan S.
      • Jørgensen N.
      • Toppari J.
      • Redmon B.
      • Punab M.
      • et al.
      Alcohol and male reproductive health: a cross-sectional study of 8344 healthy men from Europe and the USA.
      ). Sperm were scored for motility evaluation expressed as grades a to d, and progressive motility rate was calculated as the percentage of a + b.

       Assessment of Seminal Total Antioxidant Capacity and Lipid Peroxidation

      Seminal malondialdehyde levels were detected by means of the tiobarbituric acid method (
      • Botsoglou N.A.
      • Fletouris D.J.
      • Papageorgiou G.E.
      • Vassilopoulos V.N.
      • Mantis A.J.
      • Trakatellis A.G.
      Rapid, sensitive, and specific thiobarbituric acid method for measuring lipid peroxidation in animal tissue, food, and feedstuff samples.
      ). Colorimetric method was used for analyzing seminal total antioxidant capacity (TAC; Randox Laboratories). This method has been completely described by Khosrowbeygi et al. (
      • Khosrowbeygi A.
      • Zarghami N.
      • Deldar Y.
      Correlation between sperm quality parameters and seminal plasma antioxidants status.
      ).

       Statistical Analysis

      All data were presented as mean ± SD. The distribution of the data was evaluated by means of the Kolmogorov-Smirnov test. Owing to normal distribution of variables, the independent-sample t test and the paired-sample t test were applied to analyze differences in semen variables between and within groups, respectively. To control confounding variables, analysis of covariance (ANCOVA) test were used to determine the differences between the two groups after intervention, adjusting for baseline measurements and covariates. Statistical computations were calculated with the use of SPSS 16 for Windows software. P<.05 was considered to be statistically significant.

      Results

      In this study, a total of 48 patients were recruited, but only 44 patients completed the whole study: 23 of 24 in the ALA group and 21 of 24 in the placebo group (Fig. 1). Table 1 lists characteristics of the study participants. There were no significant differences in baseline features of participants between the two groups. The mean age of all participants in the two groups was 33.56 ± 5.07 years. The mean age of subjects did not differ between the ALA and placebo groups (32.98 ± 5.35 vs. 34.12 ± 4.79 years). Also, the baseline weights (88.14 ± 9.51 vs. 89.51 ± 11.08 kg), BMI (28.04 ± 2.88 vs. 28.78 ± 3.39 kg/m2), and physical activity (31.79 ± 9.73 vs. 33.23 ± 10.69 MET-h/wk) did not vary significantly between the ALA and placebo groups (P>.05; Table 1). There were no significant changes in BMI, weight, and physical activity in the subjects after consuming of ALA and placebo (Table 1).
      Figure thumbnail gr1
      Figure 1Flowchart of patient recruitment for the triple-blind, placebo-controlled, randomized trial of alpha-lipoic acid supplementation in infertile men.
      Table 1Demographic and anthropometric characteristics of participants at baseline and end of the study.
      CharacteristicALA (n = 23)Placebo (n = 21)P value
      Age (y)32.98 ± 5.3534.12 ± 4.79.461
      Duration of marriage (y)4.08 ± 1.405.72 ± 2.35.02
      Smoking history
       Never15 (65.21%)14 (66.6%).813
       Current8 (34.79%)7 (33.3%)
      Education Status
       Less than high school8 (34.78%)6 (28.57%).937
       High school diploma10 (43.47%)10 (47.61%)
       Bachelor degree or higher5 (21.73%)5 (23.80%)
      Weight (kg)
       Baseline88.14 ± 9.5189.51 ± 11.08.537
       End88.58 ± 10.6290.01 ± 11.84.738
      Height (cm)177.23 ± 7.23176.35 ± 7.15.707
      Body mass index (kg/m2)
       Baseline28.04 ± 2.8828.78 ± 3.39.385
       End28.18 ± 3.2328.94 ± 3.62.313
      Physical activity (MET-h/wk)
       Baseline31.79 ± 9.7333.23 ± 10.69.572
       End31.83 ± 9.9332.44 ± 11.51.572
      Note: Data are expressed as mean ± SD or n (%), and were tested by means of independent-sample t test. ALA = alpha-lipoic acid.
      Sperm quality parameters of participants at baseline and end of the study are presented in Table 2. There were no significant differences in baseline levels of sperm concentration, sperm count, and sperm total motility between the two groups. However, ALA supplementation, compared with placebo, significantly increased sperm concentration, sperm count, and sperm total motility (P<.001).This effect remained significant even after adjusting for confounders. Within-group analyses indicated that the sperm concentration, sperm count, and sperm total motility significantly increased after intervention in the ALA-treated group (P<.05). Other sperm parameters, such as ejaculate volume and morphology, were not significantly different between the two groups (P>.05).
      Table 2Effects of ALA supplementation on sperm quality parameters in infertile men.
      VariableALA (n = 23)Placebo (n = 21)P value
      Independent-sample t test.
      P value
      Analysis of covariance in the adjusted models (adjusted for duration of marriage).
      Ejaculate volume
       Baseline3.59 ± 0.283.58 ± 0.31.990.990
       End3.58 ± 0.313.59 ± 0.31.991.991
      P value
      Paired-sample t test.
      .990.991
      Total sperm count (×106/ejaculate)
       Baseline77.88 ± 4.7677.43 ± 4.46.375.375
       End90.43 ± 6.2577.59 ± 4.56<.001<.001
      P value
      Paired-sample t test.
      <.001.133
      Sperm concentration (×106/mL)
       Baseline22.63 ± 1.9822.83 ± 2.69.666.666
       End26.35 ± 3.1722.89 ± 2.74<.001<.001
      P value
      Paired-sample t test.
      <.001.199
      Motility grade a + b (%)
       Baseline27.97 ± 2.9927.55 ± 2.40.662.662
       End33.48 ± 2.9127.14 ± 2.36<.001<.001
      P value
      Paired-sample t test.
      <.001.136
      Motility grade a (%)
       Baseline3.18 ± 1.222.88 ± 1.17.795.795
       End6.55 ± 2.282.78 ± 1.37<.001<.001
      P value
      Paired-sample t test.
      <.001.265
      Motility grade b (%)
       Baseline24.79 ± 2.7124.67 ± 2.32.511.511
       End26.93 ± 2.4624.36 ± 2.22.011.011
      P value
      Paired-sample t test.
      .008.121
      Motility grade c (%)
       Baseline7.13 ± 3.187.77 ± 3.01.167.167
       End7.17 ± 3.808.87 ± 3.20.122.122
      P value
      Paired-sample t test.
      0.1770.442
      Motility grade d (%)
       Baseline64.9 ± 3.4864.75 ± 3.67.161.161
       End59.35 ± 4.5163.99 ± 2.95.005.005
      P value
      Paired-sample t test.
      .006.185
      Motility grade a + b + c (%)
       Baseline35.1 ± 3.7835.32 ± 4.03.189.189
       End40.65 ± 4.9536.01 ± 3.16.004.004
      P value
      Paired-sample t test.
      .004.675
      Normal morphology (%)
       Baseline15.58 ± 3.614.64 ± 3.1.188.188
       End15.39 ± 3.613.80 ± 3.73.153.153
      P value
      Paired-sample t test.
      .929.171
      Live sperm (%)
       Baseline71.85 ± 3.7273.52 ± 4.2.686.686
       End71.46 ± 3.5872.8 ± 4.255.255
      P value
      Paired-sample t test.
      .164.295
      Note: Data are expressed as mean ± SD. ALA = alpha-lipoic acid.
      a Independent-sample t test.
      b Analysis of covariance in the adjusted models (adjusted for duration of marriage).
      c Paired-sample t test.
      The effect of ALA supplementation on seminal oxidative stress biomarkers in infertile men are summarized in Table 3. At baseline, there were no significant differences in seminal MDA and TAC levels between the two groups. However, ALA supplementation, compared with placebo, caused a significant increase in seminal TAC levels (1.13 ± 0.42 vs. 1.78 ± 0.40 μmol/L; P=.001). Seminal MDA levels were also affected by ALA supplementation: MDA levels significantly decreased in the ALA-treated group compared with the control group (P=.002).
      Table 3Effects of ALA supplementation on seminal oxidative stress biomarkers in infertile men.
      VariableALA (n = 23)Placebo (n = 21)P value
      Independent-sample t test.
      P value
      Analysis of covariance in the adjusted models (adjusted for duration of marriage).
      TAC (μmol/L)
       Baseline1.12 ± 0.431.14 ± 0.44.307.307
       End1.78 ± 0.401.13 ± 0.42.001.001
      P value
      Paired-sample t test.
      .001.153
      MDA (μmol/L)
       Baseline0.87 ± 0.30.96 ± 0.35.055.055
       End0.67 ± 0.240.98 ± 0.33.002.002
      P value
      Paired-sample t test.
      .003.129
      Note: Data are expressed as mean ± SD. ALA = alpha-lipoic acid; MDA = malondialdehyde; TAC = total antioxidant capacity.
      a Independent-sample t test.
      b Analysis of covariance in the adjusted models (adjusted for duration of marriage).
      c Paired-sample t test.

       Safety and Adverse Events

      No side effects due to the oral administration of ALA were observed in any participants. ALA resulted in no clinically significant changes in vital signs, urinalyses, serum chemical values, or hematologic values.

      Discussion

      During the past decade, understanding the reproductive performance of male in the incidence of infertility has been considered. Now many infertile men have disorders correctable with the use of medication, and if diagnosed and treated properly, natural fertilization can be attained.
      In the context of reproduction, a balance normally exists between ROS generation and antioxidant-scavenging activities conferred by seminal plasma, which contains enzymes that scavenge ROS, such as catalase and superoxide dismutase (
      • Agarwal A.
      • Saleh R.A.
      • Bedaiwy M.A.
      Role of reactive oxygen species in the pathophysiology of human reproduction.
      ). As a result of such balance, only minimal amounts of ROS remain. These metabolites usually enhance sperm function by stimulating DNA compaction and promotion of redox-regulated cyclic adenosine monophosphate–mediated pathways (
      • Ashour A.E.
      • Abdel-Hamied H.E.
      • Korashy H.M.
      • Al-Shabanah O.A.
      • Abd-Allah A.R.
      Alpha-lipoic acid rebalances redox and immune-testicular milieu in septic rats.
      ). That is central to the induction of sperm capacitation. However, the production of excessive amounts of ROS in semen can overwhelm the antioxidant defense mechanisms of spermatozoa and seminal plasma, stimulating DNA fragmentation and loss of sperm function that is associated with peroxidative damage to the sperm plasma membrane. This can eventually lead to loss of fertility (
      • Tremellen K.
      Oxidative stress and male infertility—a clinical perspective.
      ).
      ALA, and its reduced form, DHLA, is effective against conditions in which oxidative stress has a role (
      • Prahalathan C.
      • Selvakumar E.
      • Varalakshmi P.
      Lipoic acid modulates adriamycin-induced testicular toxicity.
      ). It shows beneficial effects in oxidative stress conditions because of its synergistic action with other antioxidants. ALA, which is a universal antioxidant, functions in both aqueous and membrane phases (
      • Valko M.
      • Rhodes C.
      • Moncol J.
      • Izakovic M.
      • Mazur M.
      Free radicals, metals and antioxidants in oxidative stress–induced cancer.
      ).
      The present study was an attempt to evaluate the efficacy of ALA for improving semen parameters and sperm function in a randomized controlled trial. Controlled studies are mandatory for assessing any clinical intervention for idiopathic oligoasthenoteratospermia. This study demonstrated that daily administration of 600 mg ALA for 12 weeks significantly improved semen parameters and sperm function.
      In the placebo group, ejaculate volume, sperm count, sperm concentration, and motility did not differ significantly after 12 weeks, whereas a statistically significant improvement in density, count, and motility was achieved in the ALA group.
      Statistically significant differences were found between the groups in 12th-week sperm count and motility values (P<.001). However, a significant difference was not found in sperm morphology.
      Until now, no studies have been conducted in humans on this topic with ALA. Studies have been conducted mainly with other antioxidants. For example, the administration of selenium to subfertile patients induced a statistically significant increase in sperm motility (
      • Moslemi M.K.
      • Tavanbakhsh S.
      Selenium–vitamin E supplementation in infertile men: effects on semen parameters and pregnancy rate.
      ). Antioxidants are essential for sperm function and male fertility. Antioxidant deficiency has been linked to reproductive dysfunctions in rats, mice, chickens, pigs, and cows, and supplementation with antioxidants has been reported to improve reproductive performance in sheep and mice (
      • Tremellen K.
      Oxidative stress and male infertility—a clinical perspective.
      ). Results of nonhuman animal studies showed that the percentage of motile sperm increased dramatically after ALA administration (
      • Ibrahim S.F.
      • Osman K.
      • Das S.
      • Othman A.M.
      • Majid N.A.
      • Rahman M.P.
      A study of the antioxidant effect of alpha lipoic acids on sperm quality.
      ).
      Regulation, structural integrity, and providing energy are three main factors that sperm mobility is largely dependent on. Movement is controlled at the midpiece, particularly the flagella and principal area. These locations handle a special function of sperm movement. The flagella midpiece controls the activation of motility, whereas the principal midpiece handles hyperactivation. ALA regulation of metabolism, increased availability of mitochondrial coenzymes, and improvement of protection from free radicals are thought to eventually lead to a reduced incidence of mitochondrial dysfunction, thus ensuring adequate ATP for sperm motility. In the present study, we observed that ALA administration improved sperm motility in the intervention group (
      • Liu J.
      The effects and mechanisms of mitochondrial nutrient α-lipoic acid on improving age-associated mitochondrial and cognitive dysfunction: an overview.
      ).
      Free radicals attack the unsaturated lipid and saturated protein channels in the midpiece in the sperm. ALA added into the extender media allows the antioxidant to protect these components by creating a shield surrounding the midpiece and within the structure itself. ALA creates a strong shield on the cell membrane, along with the liquid that encompasses the sperm indirectly, enhancing the ability of the sperm to tolerate higher volumes of free radical attack (
      • Azza M.G.
      The protective role of alpha lipoic acid against pesticides induced testicular toxicity—histopathological and histochemical studies.
      ). This ability will, in turn, indirectly reduce formation of deep pores and cracks on the sperm surface, thus protecting structural integrity. The rate of sperm motility is mainly dependent on the availability of its provided energy. Thus, normal-function sperm usually have very active functioning mitochondria, which in turn create high levels of free radicals (
      • Kaka A.
      • Wahid H.
      • Rosnina Y.
      • Yimer N.
      • Khumran A.
      • Behan A.
      • et al.
      Alpha-linolenic acid supplementation in Tris extender can improve frozen-thawed bull semen quality.
      ).
      To ensure constant yield of ATP, external and internal structural integrity of the organelle must be kept. Because the membrane wall and the various compartments of the organelle are high in lipid content, ALA addition would save these structures from the ever-increasing free radicals which are a by-product of the Krebs cycle (
      • Ibrahim S.F.
      • Osman K.
      • Das S.
      • Othman A.M.
      • Majid N.A.
      • Rahman M.P.
      A study of the antioxidant effect of alpha lipoic acids on sperm quality.
      ).
      Currently, sperm quality is determined through its morphology and motility (
      • Aitken R.
      • Finnie J.
      • Muscio L.
      • Whiting S.
      • Connaughton H.
      • Kuczera L.
      • et al.
      Potential importance of transition metals in the induction of DNA damage by sperm preparation media.
      ). Our study has shown that ALA, compared with placebo, increased motility.
      In Yeni et al.’s study from 2010, ALA administration effect was found to be significant (P<.05) in some reproductive tract measures—motility, membrane integrity, and abnormal rate of sperm—in adult male rats compared with the placebo group (
      • Yeni D.
      • Fidan A.
      • Ciğerci I.
      • Konuk M.
      • Avdatek F.
      • Gündoğan M.
      Effect ofα-lipoic acid on sperm quality, reproductive tract measures in thinner exposed rats.
      ).
      Ibrahim et al. added ALA into the semen of male goats and observed that ALA increased the sperm motility (
      • Ibrahim S.F.
      • Osman K.
      • Das S.
      • Othman A.M.
      • Majid N.A.
      • Rahman M.P.
      A study of the antioxidant effect of alpha lipoic acids on sperm quality.
      ). ALA is a thiol-consisting nucleophile, which acts in opposition to endogenous electrophiles, including free radicals and reactive drug metabolites. ALA is also reported to replenish the glutathione pool via reduction of oxidized glutathione. Consumption of ALA helps to overcome oxidative stress by increasing the reduce glutathione status, which results in increased free radical–scavenging activity (
      • Prahalathan C.
      • Selvakumar E.
      • Varalakshmi P.
      Modulatory role of lipoic acid on adriamycin-induced testicular injury.
      ).
      Results of the present study showed that seminal TAC and MDA levels were improved by ALA consumption compared with placebo. Although the effect of ALA on oxidative stress has been assessed among diabetic patients (
      • Borcea V.
      • Nourooz-Zadeh J.
      • Wolff S.P.
      • Klevesath M.
      • Hofmann M.
      • Urich H.
      • et al.
      α-Lipoic acid decreases oxidative stress even in diabetic patients with poor glycemic control and albuminuria.
      ,
      • Ziegler D.
      • Nowak H.
      • Kempler P.
      • Vargha P.
      • Low P.
      Treatment of symptomatic diabetic polyneuropathy with the antioxidant α-lipoic acid: a meta-analysis.
      ) and nonhuman animal models (
      • Hagen T.M.
      • Liu J.
      • Lykkesfeldt J.
      • Wehr C.M.
      • Ingersoll R.T.
      • Vinarsky V.
      • et al.
      Feeding acetyl-L-carnitine and lipoic acid to old rats significantly improves metabolic function while decreasing oxidative stress.
      ), as far as we could determine, this is the first study evaluating the effects of ALA on oxidative stress in infertile men.
      Studies on the peroxidation of phospholipids in mammalian sperm have suggested that peroxidation reaction causes membrane damage, which leads to loss of sperm motility (
      • Baumber J.
      • Ball B.A.
      • Gravance C.G.
      • Medina V.
      • Davies-Morel M.C.
      The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation.
      ). In the present study, ALA consumption improved the quality of sperm and increased the TAC. However, Suleiman et al. (
      • Suleiman S.A.
      • Ali M.E.
      • Zaki Z.
      • El-Malik E.
      • Nasr M.
      Lipid peroxidation and human sperm motility: protective role of vitamin E.
      ) showed no significant negative correlation between seminal plasma level of MDA and sperm count or motility, but they observed that spermatozoal MDA concentration was higher with decreased sperm motility. The results of the present study are compatible with earlier findings (
      • Colagar A.H.
      • Pouramir M.
      • Marzony E.T.
      • Jorsaraei S.G.
      Relationship between seminal malondialdehyde levels and sperm quality in fertile and infertile men.
      ,
      • Hsieh Y.-Y.
      • Chang C.-C.
      • Lin C.-S.
      Seminal malondialdehyde concentration but not glutathione peroxidase activity is negatively correlated with seminal concentration and motility.
      ).
      Bidmeshkipour et al.’s results (
      • Bidmeshkipour A.
      • Hosseinzadeh Colagar A.
      • Gholinezhad Chari M.
      • Biparva P.
      Seminal plasma total antioxidant capacity and vitamin-C levels in asthenozoospermia: a case-control study.
      ) indicated that TAC levels in the seminal plasma of asthenospermic men were significantly lower than in healthy men. In addition, they found a positive correlation between reduced TAC levels and low sperm motility.
      Our findings on the motility ratio were similar to other reports (
      • Prahalathan C.
      • Selvakumar E.
      • Varalakshmi P.
      Lipoic acid modulates adriamycin-induced testicular toxicity.
      ,
      • Ross C.
      • Morriss A.
      • Khairy M.
      • Khalaf Y.
      • Braude P.
      • Coomarasamy A.
      • et al.
      A systematic review of the effect of oral antioxidants on male infertility.
      ). Prahalathan et al. (
      • Azza M.G.
      The protective role of alpha lipoic acid against pesticides induced testicular toxicity—histopathological and histochemical studies.
      ) in 2006 declared that ALA could increase the count and motility and reduce the ratio of abnormal sperm. Their findings on count and motility ratio were similar to ours. Ross et al. (
      • Ross C.
      • Morriss A.
      • Khairy M.
      • Khalaf Y.
      • Braude P.
      • Coomarasamy A.
      • et al.
      A systematic review of the effect of oral antioxidants on male infertility.
      ), in a systematic review, evaluated 17 randomized trials of the effect of antioxidants supplementation, including vitamins C and E, zinc, selenium, and carnitine, on male infertility, considering a total of 1,665 men. Of the 17 trials, 14 (82%) showed an improvement in sperm quality after antioxidant therapy (
      • Ross C.
      • Morriss A.
      • Khairy M.
      • Khalaf Y.
      • Braude P.
      • Coomarasamy A.
      • et al.
      A systematic review of the effect of oral antioxidants on male infertility.
      ).
      The design of the present investigation, repeated assessment of diet and physical activity, exclusion of subjects using tobacco or with acute inflammatory disease, and control for covariates were strengths of our study. This study is the first that has investigated the effect of supplementation with ALA on infertile men, and the results have been statistically reported and discussed. Obviously, to clarify the clinical relevance of the data, more studies with larger sample sizes and longer durations are needed. Furthermore, owing to budget limitations we were not able to include healthy individuals in the study and measure ROS radicals and endogenous antioxidants such as glutathione, selenium, and vitamin E.
      In conclusion, ∼3 months of supplementation with ALA can improve sperm quality. After 12 weeks of treatment in oligoasthenoteratozoospermic men, mean count, concentration, and motility increased significantly compared with the placebo group. Based on our findings, medical therapy of asthenoteratospermia with oral antioxidants, such as ALA, can improve quality of semen parameters.

      References

        • Gelbaya T.A.
        • Potdar N.
        • Jeve Y.B.
        • Nardo L.G.
        Definition and epidemiology of unexplained infertility.
        Obstet Gynecol Surv. 2014; 69: 109-115
        • Barnes A.
        • Riche D.
        • Mena L.
        • Sison T.
        • Barry L.
        • Reddy R.
        • et al.
        Efficacy and safety of intrauterine insemination and assisted reproductive technology in populations serodiscordant for human immunodeficiency virus: a systematic review and meta-analysis.
        Fertil Steril. 2014; 102: 424-434
        • Winters B.R.
        • Walsh T.J.
        The epidemiology of male infertility.
        Urol Clin North Am. 2014; 41: 195-204
        • Balercia G.
        • Regoli F.
        • Armeni T.
        • Koverech A.
        • Mantero F.
        • Boscaro M.
        Placebo-controlled double-blind randomized trial on the use of L-carnitine, L-acetylcarnitine, or combined L-carnitine and L-acetylcarnitine in men with idiopathic asthenozoospermia.
        Fertil Steril. 2005; 84: 662-671
        • Jensen T.K.
        • Gottschau M.
        • Madsen J.O.
        • Andersson A.-M.
        • Lassen T.H.
        • Skakkebæk N.E.
        • et al.
        Habitual alcohol consumption associated with reduced semen quality and changes in reproductive hormones; a cross-sectional study among 1221 young Danish men.
        BMJ Open. 2014; 4: e005462
        • Moslemi M.K.
        • Tavanbakhsh S.
        Selenium–vitamin E supplementation in infertile men: effects on semen parameters and pregnancy rate.
        Int J Gen Med. 2011; 4: 99
        • Baumber J.
        • Ball B.A.
        • Gravance C.G.
        • Medina V.
        • Davies-Morel M.C.
        The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation.
        J Androl. 2000; 21: 895-902
        • Dacheux J.-L.
        • Dacheux F.
        New insights into epididymal function in relation to sperm maturation.
        Reproduction. 2014; 147: R27-42
        • Wu J.
        • Wu S.
        • Xie Y.
        • Wang Z.
        • Wu R.
        • Cai J.
        • et al.
        Zinc protects sperm from being damaged by reactive oxygen species in assisted reproduction techniques.
        Reprod Biomed Online. 2015; 30: 334-339
        • Linhartova P.
        • Gazo I.
        • Shaliutina-Kolesova A.
        • Hulak M.
        • Kaspar V.
        Effects of tetrabrombisphenol A on DNA integrity, oxidative stress, and sterlet (Acipenser ruthenus) spermatozoa quality variables.
        Environ Toxicol. 2014; 1: 1-11
        • Gharagozloo P.
        • Aitken R.J.
        The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy.
        Hum Reprod. 2011; 26: 1628-1640
        • Alvarez J.G.
        • Aitken R.J.
        Lipid peroxidation in human spermatozoa.
        in: Agarwal A. Aitken R.J. Alvarez J.G. Studies on men’s health and fertility. Humana Press, New York2012: 119-130
        • Takei G.L.
        • Mukai C.
        • Okuno M.
        Transient Ca2+ mobilization caused by osmotic shock initiates salmonid fish sperm motility.
        J Exp Biol. 2012; 215: 630-641
        • Atig F.
        • Raffa M.
        • Habib B.-A.
        • Kerkeni A.
        • Saad A.
        • Ajina M.
        Impact of seminal trace element and glutathione levels on semen quality of Tunisian infertile men.
        BMC Urol. 2012; 12: 6
        • Tavilani H.
        • Doosti M.
        • Saeidi H.
        Malondialdehyde levels in sperm and seminal plasma of asthenozoospermic and its relationship with semen parameters.
        Clin Chim Acta. 2005; 356: 199-203
        • Delimaris I.
        • Piperakis S.M.
        The importance of nutritional factors on human male fertility: a toxicological approach.
        J Transl Toxicol. 2014; 1: 52-59
        • Buhling K.J.
        • Laakmann E.
        The effect of micronutrient supplements on male fertility.
        Curr Opin Obstet Gynecol. 2014; 26: 199-209
        • Gaskins A.J.
        • Colaci D.S.
        • Mendiola J.
        • Swan S.H.
        • Chavarro J.E.
        Dietary patterns and semen quality in young men.
        Hum Reprod. 2012; 27: 2899-2907
        • Bingham P.M.
        • Stuart S.D.
        • Zachar Z.
        Lipoic acid and lipoic acid analogs in cancer metabolism and chemotherapy.
        Expert Rev Clin Pharmacol. 2014; 7: 837-846
        • Grasso S.
        • Bramanti V.
        • Tomassoni D.
        • Bronzi D.
        • Malfa G.
        • Traini E.
        • et al.
        Effect of lipoic acid and α-glyceryl-phosphoryl-choline on astroglial cell proliferation and differentiation in primary culture.
        J Neurosci Res. 2014; 92: 86-94
        • Ali Y.F.
        • Desouky O.S.
        • Selim N.S.
        • Ereiba K.M.
        Assessment of the role of α-lipoic acid against the oxidative stress of induced iron overload.
        J Radiat Res Appl Sci. 2015; 8: 26-35
        • Ibrahim S.F.
        • Osman K.
        • Das S.
        • Othman A.M.
        • Majid N.A.
        • Rahman M.P.
        A study of the antioxidant effect of alpha lipoic acids on sperm quality.
        Clinics. 2008; 63: 545-550
        • Jungwirth A.
        • Giwercman A.
        • Tournaye H.
        • Diemer T.
        • Kopa Z.
        • Dohle G.
        • et al.
        European Association of Urology guidelines on male infertility: the 2012 update.
        Eur Urol. 2012; 62: 324-332
        • Safarinejad M.R.
        Efficacy of coenzyme Q10 on semen parameters, sperm function and reproductive hormones in infertile men.
        J Urol. 2009; 182: 237-248
        • Künzle R.
        • Mueller M.D.
        • Hänggi W.
        • Birkhäuser M.H.
        • Drescher H.
        • Bersinger N.A.
        Semen quality of male smokers and nonsmokers in infertile couples.
        Fertil Steril. 2003; 79: 287-291
        • Jensen T.K.
        • Swan S.
        • Jørgensen N.
        • Toppari J.
        • Redmon B.
        • Punab M.
        • et al.
        Alcohol and male reproductive health: a cross-sectional study of 8344 healthy men from Europe and the USA.
        Hum Reprod. 2014; 29: 1801-1809
        • Botsoglou N.A.
        • Fletouris D.J.
        • Papageorgiou G.E.
        • Vassilopoulos V.N.
        • Mantis A.J.
        • Trakatellis A.G.
        Rapid, sensitive, and specific thiobarbituric acid method for measuring lipid peroxidation in animal tissue, food, and feedstuff samples.
        J Agric Food Chem. 1994; 42: 1931-1937
        • Khosrowbeygi A.
        • Zarghami N.
        • Deldar Y.
        Correlation between sperm quality parameters and seminal plasma antioxidants status.
        Iran J Reprod Med. 2012; 2: 58-64
        • Agarwal A.
        • Saleh R.A.
        • Bedaiwy M.A.
        Role of reactive oxygen species in the pathophysiology of human reproduction.
        Fertil Steril. 2003; 79: 829-843
        • Ashour A.E.
        • Abdel-Hamied H.E.
        • Korashy H.M.
        • Al-Shabanah O.A.
        • Abd-Allah A.R.
        Alpha-lipoic acid rebalances redox and immune-testicular milieu in septic rats.
        Chem Biol Interact. 2011; 189: 198-205
        • Tremellen K.
        Oxidative stress and male infertility—a clinical perspective.
        Hum Reprod Update. 2008; 14: 243-258
        • Prahalathan C.
        • Selvakumar E.
        • Varalakshmi P.
        Lipoic acid modulates adriamycin-induced testicular toxicity.
        Reprod Toxicol. 2006; 21: 54-59
        • Valko M.
        • Rhodes C.
        • Moncol J.
        • Izakovic M.
        • Mazur M.
        Free radicals, metals and antioxidants in oxidative stress–induced cancer.
        Chem Biol Interact. 2006; 160: 1-40
        • Liu J.
        The effects and mechanisms of mitochondrial nutrient α-lipoic acid on improving age-associated mitochondrial and cognitive dysfunction: an overview.
        Neurochem Res. 2008; 33: 194-203
        • Azza M.G.
        The protective role of alpha lipoic acid against pesticides induced testicular toxicity—histopathological and histochemical studies.
        J Aquacult Res Dev. 2010; 1: 1-7
        • Kaka A.
        • Wahid H.
        • Rosnina Y.
        • Yimer N.
        • Khumran A.
        • Behan A.
        • et al.
        Alpha-linolenic acid supplementation in Tris extender can improve frozen-thawed bull semen quality.
        Reprod Domest Anim. 2015; 50: 29-33
        • Aitken R.
        • Finnie J.
        • Muscio L.
        • Whiting S.
        • Connaughton H.
        • Kuczera L.
        • et al.
        Potential importance of transition metals in the induction of DNA damage by sperm preparation media.
        Hum Reprod. 2014; 29: 2136-2147
        • Yeni D.
        • Fidan A.
        • Ciğerci I.
        • Konuk M.
        • Avdatek F.
        • Gündoğan M.
        Effect ofα-lipoic acid on sperm quality, reproductive tract measures in thinner exposed rats.
        Andrologia. 2012; 44: 74-80
        • Prahalathan C.
        • Selvakumar E.
        • Varalakshmi P.
        Modulatory role of lipoic acid on adriamycin-induced testicular injury.
        Chem Biol Interact. 2006; 160: 108-114
        • Borcea V.
        • Nourooz-Zadeh J.
        • Wolff S.P.
        • Klevesath M.
        • Hofmann M.
        • Urich H.
        • et al.
        α-Lipoic acid decreases oxidative stress even in diabetic patients with poor glycemic control and albuminuria.
        Free Rad Biol Med. 1999; 26: 1495-1500
        • Ziegler D.
        • Nowak H.
        • Kempler P.
        • Vargha P.
        • Low P.
        Treatment of symptomatic diabetic polyneuropathy with the antioxidant α-lipoic acid: a meta-analysis.
        Diabet Med. 2004; 21: 114-121
        • Hagen T.M.
        • Liu J.
        • Lykkesfeldt J.
        • Wehr C.M.
        • Ingersoll R.T.
        • Vinarsky V.
        • et al.
        Feeding acetyl-L-carnitine and lipoic acid to old rats significantly improves metabolic function while decreasing oxidative stress.
        Proc Natl Acad Sci U S A. 2002; 99: 1870-1875
        • Suleiman S.A.
        • Ali M.E.
        • Zaki Z.
        • El-Malik E.
        • Nasr M.
        Lipid peroxidation and human sperm motility: protective role of vitamin E.
        J Androl. 1996; 17: 530-537
        • Colagar A.H.
        • Pouramir M.
        • Marzony E.T.
        • Jorsaraei S.G.
        Relationship between seminal malondialdehyde levels and sperm quality in fertile and infertile men.
        Braz Arch Biol Technol. 2009; 52: 1387-1392
        • Hsieh Y.-Y.
        • Chang C.-C.
        • Lin C.-S.
        Seminal malondialdehyde concentration but not glutathione peroxidase activity is negatively correlated with seminal concentration and motility.
        Int J Biol Sci. 2006; 2: 23
        • Bidmeshkipour A.
        • Hosseinzadeh Colagar A.
        • Gholinezhad Chari M.
        • Biparva P.
        Seminal plasma total antioxidant capacity and vitamin-C levels in asthenozoospermia: a case-control study.
        Tehran Univ Med J. 2010; 67: 835-842
        • Ross C.
        • Morriss A.
        • Khairy M.
        • Khalaf Y.
        • Braude P.
        • Coomarasamy A.
        • et al.
        A systematic review of the effect of oral antioxidants on male infertility.
        Reprod Biomed Online. 2010; 20: 711-723