Utility of Antioxidants in the Treatment of Male Infertility: Clinical Guidelines Based on a Systematic Review and Analysis of Evidence

Review Article Male reproductive health and infertility pISSN: 2287-4208 / eISSN: 2287-4690 World J Mens Health Published online Jan 15, 2021 https://doi.org/10.5534/wjmh.200196

Ashok Agarwal1 , Kristian Leisegang2 , Ahmad Majzoub1,3,4 , Ralf Henkel1,5,6 , Renata Finelli1 , Manesh Kumar Panner Selvam1 , Nicholas Tadros7 , Neel Parekh8 , Edmund Y. Ko9 , Chak-Lam Cho10,11 , Mohamed Arafa1,3,12 , Marco G. Alves13 , Pedro Fontes Oliveira14 , Juan G. Alvarez15 , Rupin Shah16 1American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA, 2School of Natural Medicine, Faculty of Community and Health Sciences, University of the Western Cape, Bellville, South Africa, 3Department of Urology, Hamad Medical Corporation, 4Department of Urology, Weill Cornell Medicine - Qatar, Doha, Qatar, 5Department of Metabolism, Digestion and Reproduction, Imperial College London, London, UK, 6Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa, 7Division of Urology, Southern Illinois University School of Medicine, Springfield, IL, 8Department of Urology, Cleveland Clinic, Cleveland, OH, 9Department of Urology, Loma Linda University, Loma Linda, CA, USA, 10Department of Surgery, Union Hospital, 11S. H. Ho Urology Centre, Department of Surgery, The Chinese University of Hong Kong, Hong Kong, 12Andrology Department, Cairo University, Giza, Egypt, 13Department of Anatomy & Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, 14QOPNA & LAQV, Department of Chemistry, University of Aveiro, Aveiro, Portugal, 15Centro Androgen, La Coruña, Spain and Harvard Medical School, Boston, MA, USA, 16Department of Urology, Lilavati Hospital and Research Centre, Mumbai, India

It is widely accepted that oxidative stress plays an important role in the pathophysiology of male infertility and that antioxidants could have a significant role in the treatment of male infertility. The main objectives of this study are: 1) to systematically review the current evidence for the utility of antioxidants in the treatment of male infertility; and 2) propose evidence-based clinical guidelines for the use of antioxidants in the treatment of male infertility. A systematic review of the available clinical evidence was performed, with articles published on Scopus being manually screened. Data extracted included the type of antioxidant used, the clinical conditions under investigation, the evaluation of semen parameters and reproductive outcomes. The adherence to the Cambridge Quality Checklist, Cochrane Risk of Bias for randomized controlled trials (RCTs), CONSORT guidelines and JADAD score were analyzed for each included study. Further, we provided a Strength Weakness Opportunity Threat (SWOT) analysis to analyze the current and future value of antioxidants in male infertility. Of the 1,978 articles identified, 97 articles were included in the study. Of these, 52 (53.6%) were uncontrolled (open label), 12 (12.4%) unblinded RCTs, and 33 (34.0%) blinded RCTs, whereas 44 (45.4%) articles tested individual antioxidants, 31 (32.0%) a combination of several products in variable dosages, and 22 (22.6%) registered antioxidant products. Based on the published evidence, we 1) critically examined the necessity of additional double-blind, randomized, placebo-controlled trials, and 2) proposed updated evidence-based clinical guidelines for antioxidant therapy in male infertility. The current systematic review on antioxidants and male infertility clearly shows that antioxidant supplementation improves semen parameters. In addition, it provides the indications for antioxidant treatment in specific clinical conditions, including varicocele, unexplained and idiopathic male infertility, as well as in cases of altered semen quality.

Keywords: Antioxidants; Oxidative stress; Practice guideline; Semen analysis; Sperm maturation

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Nov 22, 2020 Revised: Dec 8, 2020 Accepted: Dec 16, 2020 Published online Jan 15, 2021 Correspondence to: Ashok Agarwal https://orcid.org/0000-0003-0585-1026 Andrology Center and American Center for Reproductive Medicine, Cleveland Clinic, Mail Code X-11, 10681 Carnegie Avenue, Cleveland, OH 44195, USA. Tel: +1-216-444-9485, Fax: +1-216-445-6049, E-mail: [email protected], Website: www.Clevelandclinic.org/ReproductiveResearchCenter

INTRODUCTION been reported [8-11]. The topic was first summarized by a Cochrane meta-analysis in 2011, with updated Infertility affects approximately 15% of couples glo reviews in 2014 and 2019 [8,9,11]. These reviews inves bally, with 2.5%–12% believed to be solely due to male tigated the therapeutic benefit of male antioxidant factors. The incidence of male factor infertility varies treatment for couples undergoing assisted reproduc according to the geographical location, ranging from tive technology (ART). Based on limited randomized 20% to 70% [1]. The causes of male infertility are diverse, controlled trials (RCTs), the reviews concluded that low including genetic causes, varicocele, reproductive tract level evidence supports antioxidant therapy in infer infections, obstructive or non-obstructive azoospermia, tile males to increase pregnancy and live birth rates, male hypogonadism, and anti-sperm antibodies. How with no evidence for increased risk of miscarriage ever, a large proportion of cases remain unexplained [8,9,11]. Majzoub and Agarwal (2018) [10] conducted a (unexplained male infertility, UMI) (±15%) or idiopathic systematic review on antioxidant treatment in infer (idiopathic male infertility, IMI) (30%–50%) in the ab tile men, concluding that antioxidants have a positive sence of identifiable female factors [2,3]. In addition, effect on male fertility, including semen parameters numerous environmental and lifestyle factors have been and advanced sperm function, ART outcomes and live associated with poor reproductive outcomes in males [4]. birth rates. Antioxidants that are commonly used clini Importantly, oxidative stress has been established as a cally and investigated scientifically as either an indi significant mediator in many known causes and risk vidual application or in combination include vitamin factors of male infertility, and has further been associ A, vitamin C, vitamin E, carnitine, N-acetyl cysteine, ated with 30% to 80% of IMI cases in a condition termed coenzyme Q10 and lycopene, along with important male oxidative stress infertility (MOSI) [2,5]. Therefore, antioxidant co-factors zinc, selenium, and folic acid, as the use of antioxidants to reduce oxidative stress across these compounds are significantly involved in essential a range of etiologies and risk factors of male infertility sperm functions (Fig. 1) [12-15]. However, the outcomes has gained increasing attention. This is supported by the of clinical trials included in the systematic reviews are wide availability of oral antioxidants, excellent safety not consistent, ranging from clear benefit to no clinical and bioavailability profiles, and that antioxidants are effect of the treatment, or even having significant de considered relatively cost effective [5,6]. Therefore, there trimental effects [16-20]. The reasons for this inconsis is a growing trend of prescribing antioxidants to all tency are multifactorial and include: small numbers of males with infertility, even without complete evaluation participants in the studies, variable treatment regimens, or relevant guidelines [7]. dosages, treatment duration, and the lack of placebo- Exogenous administration of antioxidants has been controlled studies. In addition, many of the trials did not explored for decades, and the effects of several antioxi evaluate final reproductive outcomes, such as live birth dants on male fertility have been extensively reported. rate, but only certain specific aspects such as seminal Numerous trials of different qualities using various volume, sperm concentration and motility, morpho antioxidants as mono- or poly-formulations, which may logy, seminal levels of reactive oxygen species (ROS) or include pharmacologically-active herbal extracts, have oxidative stress. Furthermore, these antioxidants were

L-Arginine L-carnitine CoQ10

Vitamin C V Molybdenum itality Vitamin E Sperm Thiamin Concentratio

Motility n

Lipid peroxidation Lycopene damage DNA & Sperm motility +morphology

damage Motility

Protamination damage Zinc Fig. 1. Individual antioxidant com- DNA Chromatin selenium pounds that have significant effects on N-Acetyl-L-cysteine DNA Folic acid Acetyl-L-carnitine sperm functions. CoQ: coenzyme Q.

2 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility often given in non-proven dosages, thus neglecting the ducted on July 15th, 2020 to identify studies investi fine bodily redox balance that is necessary for normal gating the use of antioxidants in the treatment of male physiology, including reproductive functions [21]. If the infertility, with no restriction on publication date. dosage is too low, the treatment might be ineffective; if The following keyword strings and Boolean Opera it is too high, it can result in an excess of antioxidants tors were used: TITLE-ABS-KEY (“antioxidant*”) causing ‘reductive stress’, which is as detrimental as AND TITLE-ABS-KEY (“male infertil*” OR “infertile oxidative stress [22,23]. Reductive stress due to inappro male*” OR “infertile men” OR “male subfertil*” OR priate antioxidant dosage may lead to infertility [23,24]. “male steril*” OR “sperm*” OR “seminal” OR “semen”). In this regard, high dosages of vitamin E have been Further specifications of the search are presented in shown to have adverse effects [20]. Recently more ba Supplementary Table 1. Automatic filters were used in lanced antioxidant formulations have shown promising the database to specifically include only English origi results whereby seminal oxidative stress was reduced, nal articles and exclude other types of publication such sperm function improved and pregnancies achieved as book chapters, conference papers, editorials, notes, [25,26]. letters, short surveys, erratum, and books. With the current rationale and increased use of anti The articles identified through the keyword search oxidants to counteract male infertility, and the hetero were subsequently screened manually by title, key geneous and inconsistent data currently available, this words and abstract for eligibility. This screening was study aims to: 1) systematically review the current evi independently done by three researchers (RF; KL; dence for antioxidant use to ameliorate male infertility; MKPS), and the number of articles excluded through and 2) propose updated evidence-based clinical guide screening was recorded. Full text articles were then lines for the use of antioxidants in male infertility. reviewed for eligibility using the inclusion and exclu sion criteria provided in Table 1 , and the number of MATERIALS AND METHODS articles excluded based on these criteria was recorded. Data was subsequently extracted from the eligible 1. Literature search strategy articles, including the clinical trial design, the type of In order to support the development of clinical antioxidant or antioxidant formulation used, the clini guidelines for antioxidant use in male infertility, a sys cal condition under investigation, the evaluation of se tematic review of the available clinical evidence was men parameters and/or sperm function tests (i.e., sperm performed to systematically identify relevant clinical DNA fragmentation [SDF], oxidative stress markers, trials investigating the impact of antioxidant therapy capacitation/acrosome reaction, and zona binding test) on semen quality. A literature search was performed as well as reproductive outcomes (i.e., fertilization, according to the Preferred Reporting Items for System implantation, pregnancy, miscarriage, and live birth atic Reviews and Meta-Analyses (PRISMA) guidelines rates). [27]. The Scopus database was chosen as it currently in cludes over 1.4 billion cited references, with over 70,000 2. Evaluation of study quality indexed articles (https://www.elsevier.com/solutions/ The quality of all studies included was evaluated by scopus/how-scopus-works/content). The search was con applying the Cambridge Quality Checklist [28]. More

Table 1. Proposed inclusion and exclusion for article selection Inclusion Exclusion Human participants Animal and in vitro studies Antioxidants used as intervention individually or combined Intervention not clearly reported as an antioxidant Open or controlled clinical trials Abstracts only, conference abstracts, book chapters, case series, review articles At least one semen parameter (sperm concentration, motility, mor- Non-english studies phology) and/or sperm function parameters (sperm DNA fragmentation, seminal oxidative stress markers, mitochondrial membrane integrity) reported after antioxidant treatment

www.wjmh.org 3 https://doi.org/10.5534/wjmh.200196 over, the quality of RCT was further evaluated by using the Cochrane Risk of Bias [29] and the JADAD 1 evidence evidence Quality of score [30], as well as by evaluating the adherence to published CONSORT guidelines [31]. Based on a combination of these quality evaluation tools, the studies were catego 4 JADAD JADAD (Oxford (Oxford Quality) rized into “low” (0) and “high” (1) quality. All uncon of 5) (out trolled studies were considered “low-quality”, in com parison with controlled studies, which were evaluated 20 CONSORT CONSORT (out of 25) (out according to the criteria reported in Supplementary Guidelines - Table 2. We have created this scoring system, as no - such method was previously reported in the literature.

In addition, the most recent clinical trials reporting of bias for random sequence generation, allocation - conceal selec ment, reporttive other ing, and sources blinding (outcome assessment) Unclear risk Cochrane Risk Cochrane of Bias for RCTof Bias for the effect of antioxidant treatment on male infertility, published from January 2019 to July 2020, were further 7 Checklist (out of 7) (out

ranked based on study design, the sample size analysed, causal for risk factors the inclusion/exclusion criteria used for selecting the

population, the antioxidant regimen used, the length 3 factors for risk for Checklist of treatment, the assessment of oxidative stress mark of 3) (out ers, pregnancy and live birth rates. This range of time 2 for for was specifically selected to gain an understanding of QualityCambridge Checklist correlate correlate Checklist the most recent evidence on the antioxidant therapy of 5) (out and the quality of the studies currently conducted. The - selection of these criteria was achieved through a con sensus among the male infertility experts involved in treatment antioxidant antioxidant

this study. The system provides a total score of a maxi Reproductive men parameters men parameters and after before treatment ROS levels binding outcomes after outcomes No difference in se No difference mum of 12 points and a classification of the articles in No difference zona Improved in “low” (<6 points) and “high” (≥6 points) quality, as reported in Supplementary Table 3. Clinical recommendations were proposed based on the quality of the evidence, classified as A, B, C, D Study population Study 30 infertile men (Oxford Centre for Evidence-Based Medicine 2011 - Levels of Evidence; https://www.cebm.net/wp-content/ uploads/2014/06/CEBM-Levels-of-Evidence-Introduc

tion-2.1.pdf and https://www.cebm.net/wp-content/up Antioxidant formulation formulation of treatment etate (Ephynal, (Ephynal, etate Hoffman-La F. Roche ltd) for mg/daily 300 3 months -tocopheryl ac α loads/2014/06/CEBM-Levels-of-Evidence-2.1.pdf). dosage and length

3. Statistical analysis

Statistical analysis was performed using MedCalc design Clinical trial

statistical software version 19.5.3. (MedCalc Software RCT blinded bv, Ostend, Belgium). Chi-square test was used to eval uate the association between the quality of the study and the outcomes (positive or no/negative effect) due Reference al (1995) [19]

to antioxidant treatment on semen parameters and et Kessopoulou 1

sperm function such as oxidative stress and SDF are SN presented in Table 2 [25,26,32-121]. A p-value <0.05 was considered statistically significant. When the p-value was ≥0.05, a sample size calculation was carried out to Clinical condition Infertile men

predict the required sample size to attain a statistical outcomes on reproductive treatment the impact 2. Articles of antioxidant investigating Table

4 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 0 0 0 0 0 evidence evidence Quality of published 3 3 N/A N/A N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 19 12 N/A N/A N/A CONSORT CONSORT (out of 25) (out Guidelines N/A N/A N/A identified identified No risk of bias No risk of bias Cochrane Risk Cochrane of Bias for RCTof Bias for 3 3 3 7 4 Checklist (out of 7) (out for causal for risk factors 3 3 3 3 3 of 3) for risk for Checklist factors (out 0 2 3 0 1 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - - treatment antioxidant antioxidant Reproductive Reproductive higher sperm decondensation men parameters binding capacity men parameters and apoptotic markers protamination between treated treated between and placebo fertilization, for implantation, and pregnancy, miscarriage rates higher in rate patients treated motility levels outcomes after outcomes No difference in se No difference No difference in se No difference Reduced SDF but Reduced Improved sperm Improved Reduced SDF, ROS SDF, Reduced DNA Improved No differences No differences pregnancy Live Improved sperm Improved MDA Reduced with high OS encing 2 previous encing 2 previous of IVF or failures ICSI, and DFI - decon chromatin densation>15% dence of seminal dence stress oxidative and SDF>25% TUNEL. The by number of total is not patients clearly reported. Study population Study 19 infertile men 50 infertile men - experi 58 patients Men with evi - 54 infertile men - - Antioxidant Antioxidant formulation formulation of treatment search, Geneve, Geneve, search, Switzerland) 200 mg daily for 3 months (Horphag Re Australia Ltd, Ltd, Australia - Austra Sydney, lia) 1 capsule/daily for 3 months for a maximum of 3 months (400 mg each), ß-carotene (18 mg), zinc (500 µmoL), selenium 3 (1 µmoL) for months mg) (Ephynal mg) (Ephynal 2 tab 100 mg, - Austra Sydney, lia) 1 capsule/day 3 months for lets) or selenium 3 (225 µg) for months Pycnogenol Pycnogenol Menevit (Bayer Menevit (Bayer Vitamins C and E Vitamins Vitamin E (400 Vitamin Menevit (Bayer, dosage and length design Clinical trial (open label) (open (open label) (open (open label) (open Uncontrolled Uncontrolled Uncontrolled Uncontrolled Uncontrolled Uncontrolled RCT unblinded RCT blinded Reference [32] (2009) [34] (2007) [20] et al (2003) [18] (2007) [33] Roseff (2002) Tunc et al Tunc Ménézo et al Ménézo Keskes-Ammar Keskes-Ammar et al Tremellen 2 6 5 3 4 SN Clinical condition Continued 1 2. Continued Table

www.wjmh.org 5 https://doi.org/10.5534/wjmh.200196 0 0 1 0 0 evidence evidence Quality of published 5 N/A N/A N/A N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 15 N/A N/A N/A N/A CONSORT CONSORT (out of 25) (out Guidelines N/A N/A N/A N/A identified No risk of bias Cochrane Risk Cochrane of Bias for RCTof Bias for 6 3 3 3 7 Checklist (out of 7) (out for causal for risk factors 3 3 3 3 3 of 3) for risk for Checklist factors (out 1 3 0 0 3 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - - treatment antioxidant antioxidant Reproductive Reproductive count and count seminal motility, plasma lipid levels, peroxide catalase, SOD, GSH and ascorbic acid parameters, uri - parameters, nary and semen TAC quality men parameters reduced TAC, DNA damage motility, count, TAC levels men parameters outcomes after outcomes Improved sperm Improved No difference in se No difference Improved semen Improved SDF Reduced embryo Improved No changes in se Increased seminal Increased sperm Increased ROS Reduced Study population Study 120 infertile men 70 infertile men 30 infertile men 57 infertile men 115 infertile men - - - Antioxidant Antioxidant formulation formulation of treatment seed powder 3 5 g/daily for months 5 mg/daily for 3 5 mg/daily for months docosahexae 6 mg/daily for 6 mg/daily for 45 days noic acid oil/ 10 daily for weeks tose, and citric tose, not acid (dosage 6 reported) for months Acetyl-L-Car CoQ10, nitine, Lycopene, acid, Folic Zinc, B12, Vitamin Selenium, Fruc Mucuna pruriens Folic acid Folic 1.5 g capsules of Melatonin L-Carnitine, L-Carnitine, dosage and length design Clinical trial (open label) (open (open label) (open (open label) (open (open label) (open RCT blinded Uncontrolled Uncontrolled Uncontrolled Uncontrolled Uncontrolled Uncontrolled Uncontrolled Reference (2013) [17] (2010) [35] et al (2016) [37] (2014) [36] et al (2016) [16] da Silva et al da Silva Shukla et al Martínez-Soto Bejarano et al Chattopadhyay Chattopadhyay 8 7 9 10 11 SN Clinical condition Continued 2 2. Continued Table

6 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 1 1 0 0 evidence evidence Quality of published 5 5 4 N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 23 19 19 N/A CONSORT CONSORT (out of 25) (out Guidelines N/A of bias other for sources identified of bias other for sources Unclear risk No risk of bias Unclear risk Cochrane Risk Cochrane of Bias for RCTof Bias for 5 7 7 7 Checklist (out of 7) (out for causal for risk factors 3 3 3 3 of 3) for risk for Checklist factors (out 0 2 4 0 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - - treatment antioxidant antioxidant Reproductive Reproductive men parameters - sperm concen count, tration, motility levels concentration, concentration, no change in DNA damage - concen count, mo total tration, tility and vitality, TAC inflammatory biomarkers outcomes after outcomes No difference in se No difference Decreased SDF Decreased Improved volume, volume, Improved MDA Reduced Improved sperm Improved Increased sperm Increased and MDA Reduced SDF≥15% with DFI ≥25% Study population Study 100 patients with 100 patients 60 infertile men 77 infertile men 60 infertile men - - Antioxidant Antioxidant formulation formulation of treatment 250 mg/daily for for mg/daily 250 3 months seed powder 3 5 g/daily for months mg), vitamin E (5 mg), vitamin B12 (0.5 ug), l- carnitine (750 mg), coenzyme Q10 (10 mg), acid (100 folic ug), zinc (5 mg), selenium (25 ug) with malto dextrin, calcium citric carbonate, ste acid, viol glycoside, beta- flavours, silicon carotene, for dioxide/daily 6 months 80 mg/daily for 10 weeks Ginger powder Mucuna pruriens Vitamin C (30 Vitamin Curcumin dosage and length design Clinical trial (open label) (open RCT blinded RCT blinded Uncontrolled RCT blinded Reference (2016) [38] (2018) [39] (2008) [40] (2018) [41] Hosseini et al Stenqvist et al Stenqvist et al Ahmad et al Alizadeh 12 13 14 15 SN Clinical condition Continued 3 2. Continued Table

www.wjmh.org 7 https://doi.org/10.5534/wjmh.200196 0 0 0 0 evidence evidence Quality of published 3 N/A N/A N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 15 N/A N/A N/A CONSORT CONSORT (out of 25) (out Guidelines N/A N/A N/A of bias for of bias for selective reporting and other sources; high risk of bias for random sequence generation, allocation - conceal and ment blinding (outcome assessment) Unclear risk Cochrane Risk Cochrane of Bias for RCTof Bias for 3 3 3 7 Checklist (out of 7) (out for causal for risk factors 3 3 3 3 of 3) for risk for Checklist factors (out 2 5 2 4 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - treatment antioxidant antioxidant Reproductive Reproductive sperm count, sperm count, and motility, normal morphol - ogy parameters damage rate=16.8% count, total mo total count, and normal tility, morphology FSH and LH increased levels, and testosterone, inhibin levels concentration, concentration, and motility, morphology 8-OHdG levels outcomes after outcomes Improved volume, volume, Improved Improved semen Improved DNA Reduced Pregnancy Improved sperm Improved In serum: reduced Improved sperm Improved and MDA Reduced with DFI>27% by with DFI>27% by SCSA Study population Study 24 infertile men 485 infertile men 58 infertile men 25 infertile men Antioxidant Antioxidant formulation formulation of treatment and CoQ10 and CoQ10 8 (200 mg) for months vitamin C (500 mg) and CoQ10 3 (100 mg) for months 3 2 g/daily for months 8 mg/daily for 1 8 mg/daily for month L-arginine (1 g) L-arginine Vitamin E (50 mg), Vitamin L-carnitine Astaxanthin dosage and length design Clinical trial (open label) (open (open label) (open label) (open Uncontrolled Uncontrolled RCT blinded Uncontrolled Uncontrolled Uncontrolled Reference (2019) [43] al (2020) [44] (2019) [42] (2020) [45] Hasoon et Nurmawati Salehi et al Hadi et al 17 18 16 19 SN Clinical condition Continued 4 2. Continued Table

8 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 0 0 evidence evidence Quality of published 3 N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 14 N/A CONSORT CONSORT (out of 25) (out Guidelines - N/A of bias for of bias for random sequence generation, allocation - conceal other ment, sources, selective re porting and blinding Unclear risk Cochrane Risk Cochrane of Bias for RCTof Bias for 3 7 Checklist (out of 7) (out for causal for risk factors 3 3 of 3) for risk for Checklist factors (out 2 2 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - - treatment antioxidant antioxidant -HCG–detected -HCG–detected Reproductive Reproductive concentration concentration and acrosome reaction and 8-OH- levels dG levels men parameters; men parameters; SDF improved assay; Comet by no significant in differences β clini - pregnancy, cal intrauterine ec pregnancy, pregnancy, topic pregnancy with multiple fetuses, birthlive rate outcomes after outcomes Improved sperm Improved ROS Reduced No changes in se - - tients 11 varicocele patients History of cryptor chidism (n=2), pa - with male tients accessory gland infection (n=7), immunological infertility (n=4), endocrine cause (n=1) of couples plan - of couples infer ning IVF for tility treatment Study population Study 7 idiopathic pa - 7 idiopathic 1,185 male partners - - -carotene β -tocopherol -tocopherol Antioxidant Antioxidant formulation formulation of treatment (600 mg) or cap (30 mg) and α (180 mg)/daily. In addition, - capsules con taining essential a fatty acids for of daily intake docosahexae sules providing sules providing a daily amount of noic acid (1 g), gammalinolenic acid (0.25 g) and acid arachidonic 6 (0.10 g) for months daily and 30 6 mg zinc for months Acetylcysteine Acetylcysteine Folic acid 5 mg/ Folic dosage and length design Clinical trial (open label) (open RCT blinded Uncontrolled Uncontrolled Reference al (2020) [46] (2000) [47] Schisterman et Schisterman Comhaire et al Comhaire 20 21 SN Clinical condition Varicocele Continued 5 2. Continued Table

www.wjmh.org 9 https://doi.org/10.5534/wjmh.200196 1 evidence evidence Quality of published 5 JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 16 CONSORT CONSORT (out of 25) (out Guidelines identified No risk of bias Cochrane Risk Cochrane of Bias for RCTof Bias for 7 Checklist (out of 7) (out for causal for risk factors 3 factors for risk for Checklist (out of 3) (out 3 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out treatment antioxidant antioxidant Reproductive Reproductive parameters pregnancy rate outcomes after outcomes Improved semen Improved No change in - patients with patients oligo persistent spermia 6 months after retrograde embolization Study population Study 42 varicocele 42 varicocele - Antioxidant Antioxidant formulation formulation of treatment preparation of:preparation (10 mg/kg/ NAC die), vitamin C vi - (3 mg/kg/die), tamin E (0.2 mg/ vitamin kg/die), A (0.06 IU/kg/ die), thiamine (0.4 mg/kg/ die), riboXavin (0.1 mg/kg/ die), piridoxin (0.2 mg/kg/die), nicotinamide (1 mg/kg/die), pantothenate (0.2 mg/kg/ die), biotin (0.04 mg/kg/die), cyanocobalamin (0.1 mg/kg/die), ergocalciferol cal - (8 IU/kg/die), cium (1 mg/kg/ die), magnesium (0.35 mg/kg/die), (0.45 phosphate iron mg/kg/die), die), (0.2 mg/kg/ manganese (0.01 cop mg/kg/die), per (0.02 mg/kg/ die), zinc (0.01 a for mg/kg/die) minimum of 90 days Ccommercial Ccommercial dosage and length design Clinical trial RCT unblinded Reference Galatioto et Galatioto al (2008) [48] Paradiso Paradiso 22 SN Clinical condition Continued 6 2. Continued Table

10 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 0 0 0 0 evidence evidence Quality of published 2 2 N/A N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 14 12 N/A N/A CONSORT CONSORT (out of 25) (out Guidelines - - N/A N/A of bias for of bias for random sequence generation, allocation - conceal and ment, incomplete outcome data of bias for of bias for random sequence generation, allocation - conceal selec ment, reporttive other ing, and sources, incomplete outcome data Unclear risk Unclear risk Cochrane Risk Cochrane of Bias for RCTof Bias for 4 3 7 7 Checklist (out of 7) (out for causal for risk factors 3 3 3 3 factors for risk for Checklist (out of 3) (out 0 2 0 0 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - - treatment antioxidant antioxidant Reproductive Reproductive tion, progressive tion, progressive motility, and TAC parameters men parameters, men parameters, SDF and prot amine damage assay TAC between the between TAC groups activity in group A and C outcomes after outcomes - Sperm concentra Improved semen Improved No changes in se No difference in No difference SOD Increased patients patients patients patients Study population Study 38 varicocele 38 varicocele 36 varicocele 36 varicocele 100 varicocele 100 varicocele 160 varicocele 160 varicocele Antioxidant Antioxidant formulation formulation of treatment 100 mg/daily for for mg/daily 100 3 months g), folic acid (5 g), folic mg) and zinc (66 mg) sulfate 3 months for 750 mg/daily for for mg/daily 750 6 months folic acid folic mg/daily) (66 mg/daily) Placebo D) CoQ10 Pentoxifylline (1.2 Pentoxifylline L-carnitine A) Zinc sulphate/ A) acid (5 B) Folic C) Zinc sulphate 6 months For dosage and length design Clinical trial (open label) (open (open label) (open RCT unblinded Uncontrolled Uncontrolled RCT unblinded Uncontrolled Uncontrolled Reference (2014) [51] (2014) [50] Mahani et al (2014) [52] (2009) [49] Pourmand et al Pourmand Festa et al Festa Nematollahi- Oliva et al Oliva 25 24 26 23 SN Clinical condition Continued 7 2. Continued Table

www.wjmh.org 11 https://doi.org/10.5534/wjmh.200196 0 0 evidence evidence Quality of published 5 N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 18 N/A CONSORT CONSORT (out of 25) (out Guidelines - - N/A of bias for of bias for random sequence generation, allocation - conceal se ment, lective reporting, other sourc blinding es, (partici - and pants personnel, outcome assess - and ment), incomplete outcome data Unclear risk Cochrane Risk Cochrane of Bias for RCTof Bias for 3 6 Checklist (out of 7) (out for causal for risk factors 3 3 of 3) for risk for Checklist factors (out 2 2 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out treatment antioxidant antioxidant Reproductive Reproductive sperm count and sperm count SDF reduced parameters outcomes after outcomes Improved total total Improved Improved semen Improved patients patients Study population Study 20 varicocele 20 varicocele 115 varicocele 115 varicocele g) for 3 g) for g), zinc (10 μ μ g), vitamin B12 Antioxidant Antioxidant formulation formulation of treatment mg), selenium (50 months mg), vitamin C (60 mg), CoQ10 (20 mg), vitamin E (10 mg), vitamin B9 (200 μ (1 250 mg/daily for for mg/daily 250 3 months L-Carnitine (1,500 L-Carnitine Vitamin C Vitamin dosage and length design Clinical trial (open label) (open RCT blinded Uncontrolled Reference (2015) [53] (2015) [54] Cyrus et al Cyrus et al Gual-Frau 27 28 SN Clinical condition Continued 8 2. Continued Table

12 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 0 0 evidence evidence Quality of published 2 2 JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 12 19 CONSORT CONSORT (out of 25) (out Guidelines - - of bias for of bias for random sequence generation, allocation - conceal selec ment, reporttive other ing, and sources blinding (partici - and pants personnel as as well outcome assessment) bias for random sequence generation, allocation - conceal other ment, sources, blinding (partici - and pants personnel, as - outcome sessment), incomplete outcome data Unclear risk High risk of Cochrane Risk Cochrane of Bias for RCTof Bias for 4 7 Checklist (out of 7) (out for causal for risk factors 3 3 of 3) for risk for Checklist factors (out 1 3 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out

treatment antioxidant antioxidant Reproductive Reproductive - sperm concen motility, tration, % morphology, of ROS negative - sperm and inten sity ROS sperm of normal Improved protamine and DNA content integrity NAC rate: group=33.4%, control No group=10%. p-value reported parameters rate outcomes after outcomes No changes for No changes for Pregnancy semen Improved Higher pregnancy patients patients Study population Study 35 varicocele 35 varicocele 90 varicocele - Antioxidant Antioxidant formulation formulation of treatment ine (NAC) for mg/daily 200 3 months (2 g), fumarate carni - Acetyl-L- tine HCl (1 g), fructose (2 g), citric acid (100 mg), vitamin C (180 mg), zinc (20 mg), folic acid (400 mg), selenium (100 mg), coenzyme Q-10 (40 mg), vitamin B12 (3 6 mg)/daily for months N-acetyl-L-cyste L-carnitine dosage and length design Clinical trial RCT blinded RCT unblinded Reference (2016) [55] Altay (2019) [56] Barekat et al and Kızılay 29 30 SN Clinical condition Continued 9 2. Continued Table

www.wjmh.org 13 https://doi.org/10.5534/wjmh.200196 1 0 1 evidence evidence Quality of published 4 3 5 JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 24 12 21 CONSORT CONSORT (out of 25) (out Guidelines of bias for of bias for allocation - conceal other ment, sources; high risk of bias for blinding (partici - and pants personnel, outcome assessment) of bias for of bias for random sequence generation, allocation - conceal other ment, sources, blinding (partici - and pants personnel, as - outcome sessment), incomplete outcome data identified Unclear risk Unclear risk No risk of bias Cochrane Risk Cochrane of Bias for RCTof Bias for 7 7 7 Checklist (out of 7) (out for causal for risk factors 3 3 3 of 3) for risk for Checklist factors (out 2 4 0 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - treatment antioxidant antioxidant Reproductive Reproductive count and motil - count ity motility levels birthand live rates men parameters outcomes after outcomes Improved sperm Improved Improved sperm Improved MDA Reduced Higher pregnancy No changes in se ------patients (n=74), azoosper mic (n=38), asthe nospermic (n=94), oligospermic (n=30) patients High viscosity (n=22); oligosper mic with high viscosity (n=6); asthenosper mic with high viscosity (n=12); oligoastheno spermic with high viscosity (n=10) mic patients Study population Study 60 varicocele 60 varicocele Oligoastheno- 31 asthenozoosper Antioxidant Antioxidant formulation formulation of treatment Selenium (200 µg) and vitamin E (400 IU)/daily 6 months for for mg/daily 300 6 months mg) and Vitamin E (800 mg)/daily 56 days for Folic acid (5 mg), Folic E Vitamin Vitamin C (1,000 Vitamin dosage and length design Clinical trial RCT unblinded RCT blinded RCT blinded Reference Zadeh et al (2019) [57] (1996) [58] (1999) [59] Ardestani Ardestani Suleiman et al Rolf et al 31 32 33 SN - Clinical condition men quality Abnormal se Abnormal Continued 10 2. Continued Table

14 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 0 0 0 0 evidence evidence Quality of published N/A N/A N/A N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out N/A N/A N/A N/A CONSORT CONSORT (out of 25) (out Guidelines N/A N/A N/A N/A Cochrane Risk Cochrane of Bias for RCTof Bias for 3 4 3 6 Checklist (out of 7) (out for causal for risk factors 3 3 3 3 of 3) for risk for Checklist factors (out 4 3 3 1 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - - treatment antioxidant antioxidant Reproductive Reproductive sive motility after sive which treatment, after 6 reduced of wash- months out with rate=2.4%, 3 out of 22 pa - achieving tients a spontaneous pregnancy and parameters leukocytosper mia concentration, concentration, motilityand total activity progressive mo progressive tility and viability production rate outcomes after outcomes - progres Improved Pregnancy semen Improved SDF Reduced Improved sperm Improved No change in SOD Improved sperm Improved ROS Reduced Higher pregnancy - - - mic patients tozoospermic patients 47 non-nor mozoospermic patients with prostatovesiculo-epididy mitis Study population Study 22 asthenozoosper - 51 asthenotera 16 healthy men 16 healthy 54 OAT patients patients 54 OAT - - Antioxidant Antioxidant formulation formulation of treatment (Progine, Flor (Progine, maNord, Veyle, Veyle, maNord, Denmark) for mg/daily 400 6 months Italy) ence, for 2 tables/day 3 months 9.0 g/daily for 3 9.0 g/daily for months (Sigma-Tau, (Sigma-Tau, Pomezia-Rome, Italy) Twice/day months, for3 by followed a treatment- period of 3 free months CoQ10 (Phar CoQ10 M Fattore Sairei-to Sairei-to Carnitene® Carnitene® dosage and length design Clinical trial (open label) (open label) (open (open label) (open (open label) (open Uncontrolled Uncontrolled Uncontrolled Uncontrolled Uncontrolled Uncontrolled Uncontrolled Reference (2004) [62] (2008) [63] (2003) [61] Calogero Calogero (2001) [60] Balercia et al Balercia et al Piomboni Suzuki et al Vicari and Vicari 36 37 35 34 SN Clinical condition Continued 11 2. Continued Table

www.wjmh.org 15 https://doi.org/10.5534/wjmh.200196 0 0 1 evidence evidence Quality of published 4 4 N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 17 20 N/A CONSORT CONSORT (out of 25) (out Guidelines N/A of bias other for and sources blinding (partici - and pants personnel, outcome assessment) of bias for random sequence generation, allocation - conceal other ment, and sources blinding (partici - and pants personnel); high risk of bias for incomplete outcome data Unclear risk Unclear risk Cochrane Risk Cochrane of Bias for RCTof Bias for 4 7 7 Checklist (out of 7) (out for causal for risk factors 3 3 3 of 3) for risk for Checklist factors (out 2 4 0 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - treatment antioxidant antioxidant Reproductive Reproductive count and motil - count catalase, SOD, ity, and glutathione levels and Protein levels Carbonyl men parameters TAC improved concentration concentration and motility rate outcomes after outcomes Improved sperm Improved MDA Decreased No changes in se and MDA Reduced Increased sperm Increased Higher pregnancy - - astheno- (n=25) and normozoo spermic (n=25) patients nozoospermic nozoospermic patients Study population Study Oligo- (n=25), 60 OAT patients 60 OAT 60 oligoasthe Antioxidant Antioxidant formulation formulation of treatment nifera 3 5 g/daily for months (Nutraceutical (Nutraceutical InstiScience - NC, USA) tute, for mg/daily 200 3 months citrate (25 mg/ citrate and vitamin day) E (400 mg/day) 6 months for Withania som - Withania CoQ10 capsules CoQ10 Clomiphene dosage and length design Clinical trial (open label) (open RCT blinded RCT blinded Uncontrolled Reference al (2011) [66] (2010) [64] (2010) [65] Nadjarzadeh et Ghanem et al et al Ahmad 40 38 39 SN Clinical condition Continued 12 2. Continued Table

16 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 0 1 0 0 1 0 evidence evidence Quality of published 5 5 5 N/A N/A N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 21 19 20 N/A N/A N/A CONSORT CONSORT (out of 25) (out Guidelines N/A N/A N/A of bias other for and sources incomplete outcome data of bias other for and sources incomplete outcome data of bias for of bias for random sequence generation, selective reporting Unclear risk Unclear risk Unclear risk Cochrane Risk Cochrane of Bias for RCTof Bias for 4 3 4 7 7 7 Checklist (out of 7) (out for causal for risk factors 3 3 3 3 3 3 of 3) for risk for Checklist factors (out 2 1 2 3 3 2 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - treatment antioxidant antioxidant Reproductive Reproductive intracellular ROS intracellular and apoptosis; levels increased Zn2+, of Cu2+, and Au2+ Fe2+ parameters ous pregnancy parameters pregnancy and miscarriage rates sperm count, sperm count, concentration, normal motility, morphology, and seminal SOD, catalase - semen param seminal eters, catalase, SOD, and reproductive hormones - semen param SOD and eters, catalase-like LH, FSH, activity, PRL, TSH, - testicu lar volume outcomes after outcomes Decreased Decreased Improved semen Improved Higher spontane Improved semen Improved No change in Improved total total Improved No changes in No changes in - - astheno- (n=25) and normozoo spermic (n=25) patients spermic patients Study population Study Oligo- (n=25), 690 asthenoterato 287 OAT patients 287 OAT 211 OAT patients 211 OAT 278 OAT patients 278 OAT 260 OAT patients 260 OAT - Antioxidant Antioxidant formulation formulation of treatment nifera 3 5 g/daily for months µg), vitamin E (400 units)/daily 100 days for 300 mg/daily for for mg/daily 300 12 months (1.12 g) and docosahexae noic (0.72 g) acid/daily for 8 months 800 mg/daily for for mg/daily 800 6 months 60 mg/daily for 60 mg/daily for 26 weeks Withania som - Withania Selenium (200 CoQ10 Eicosapentanoic Eicosapentanoic Pentoxifylline sativus Crocus dosage and length design Clinical trial (open label) (open (open label) (open (open label) (open RCT blinded Uncontrolled Uncontrolled RCT blinded Uncontrolled Uncontrolled RCT blinded Uncontrolled Uncontrolled Reference (2011) [68] (2011) [67] (2011) [69] Tavanbakhsh Tavanbakhsh (2011) [70] al (2011) [71] (2012) [72] Safarinejad Shukla et al Safarinejad Moslemi and Safarinejad et Safarinejad 42 41 43 44 45 46 SN Clinical condition Continued 13 2. Continued Table

www.wjmh.org 17 https://doi.org/10.5534/wjmh.200196 0 0 evidence evidence Quality of published N/A N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out N/A N/A CONSORT CONSORT (out of 25) (out Guidelines N/A N/A Cochrane Risk Cochrane of Bias for RCTof Bias for 3 3 Checklist (out of 7) (out for causal for risk factors 3 3 of 3) for risk for Checklist factors (out 3 2 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out treatment antioxidant antioxidant Reproductive Reproductive parameters concentration, concentration, vital - motility, morphology, ity, DNA integrity rate=5% outcomes after outcomes Improved semen Improved Improved sperm Improved Pregnancy astheno- (n=33), (n=42) OAT patients men 65 healthy tozoospermic tozoospermic patients Study population Study Oligo- (n=20), - 20 asthenotera - g), n- μ Antioxidant Antioxidant formulation formulation of treatment nium (200 acetyl-L-cysteine (100 mg), L- carnitine (600 mg), citrulline (600 mg), gluta - (100 thione red. (8 mg), lycopene (30 mg), CoQ10 at mg)/daily for least 2 months mg), vitamin E (200 mg), folic acid (1 mg), zinc (50 mg), sele - Labora Pharma Alicante, tories, Spain) 1 capsule/daily 3 months for Vitamin C (200 Vitamin Androferti (Q Androferti (Q dosage and length design Clinical trial (open label) (open (open label) (open Uncontrolled Uncontrolled Uncontrolled Uncontrolled Reference (2013) [74] (2013) [73] Ajayi et al Ajayi Abad et al Abad 48 47 SN Clinical condition Continued 14 2. Continued Table

18 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 0 1 0 evidence evidence Quality of published 3 4 N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 18 20 N/A CONSORT CONSORT (out of 25) (out Guidelines - - N/A of bias for of bias for random sequence generation, allocation - conceal se ment, lective reporting, other sourc blinding es, (participants and person - outcome nel, assess - and ment), incomplete outcome data of bias for of bias for allocation - conceal other ment, sources Unclear risk Unclear risk Cochrane Risk Cochrane of Bias for RCTof Bias for 7 3 7 Checklist (out of 7) (out for causal for risk factors 3 3 3 of 3) for risk for Checklist factors (out 4 0 2 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - - - treatment antioxidant antioxidant Reproductive Reproductive men parameters of CoQ10, level and SOD catalase activity; reduced of seminal level plasma 8-iso prostane concentration concentration and motility pregnancies of achieved; 16 were those, and spontaneous using ART 32 by men parameters - integ chromatin rity outcomes after outcomes No changes in se seminal Increased No difference in se No difference Improved sperm Improved 48 (28.4%) Increased sperm Increased Study population Study 60 OAT patients 60 OAT 83 OAT patients 83 OAT 169 OAT patients 169 OAT Antioxidant Antioxidant formulation formulation of treatment ceutical Science Science ceutical NC, Institute, USA) for mg/daily 200 3 months mg) and zinc (220 sulphate 4 mg)/daily for months vitamin C (80 mg), vitamin E (40 mg)/daily 6 months for - (Nutra CoQ10 Folic acid (5 Folic CoQ10 (120 mg), CoQ10 dosage and length design Clinical trial (open label) (open RCT blinded RCT blinded Uncontrolled Uncontrolled Reference al (2014) [75] (2014) [76] (2014) [77] Nadjarzadeh et Raigani et al Kobori et al Kobori 49 50 51 SN Clinical condition Continued 15 2. Continued Table

www.wjmh.org 19 https://doi.org/10.5534/wjmh.200196 0 0 0 0 evidence evidence Quality of published N/A N/A N/A N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out N/A N/A N/A N/A CONSORT CONSORT (out of 25) (out Guidelines N/A N/A N/A N/A Cochrane Risk Cochrane of Bias for RCTof Bias for 4 3 3 3 Checklist (out of 7) (out for causal for risk factors 3 3 3 3 of 3) for risk for Checklist factors (out 2 3 0 3 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - treatment antioxidant antioxidant Reproductive Reproductive motility and normal sperm morphology level progressive progressive total motility, and count, sperm activitycatalase concentration, concentration, and pro total motility gressive unchanged concentration outcomes after outcomes Improved sperm Improved MDA Decreased Improved volume, volume, Improved Improved sperm Improved Testosterone sperm Improved - - - - - oligoastheno- (n=7), (n=2), OAT (n=7), terato- asthenonecro- (n=2), asthe (n=20), noterato- asthenoteratone cro- (n=4), 0ligo asthenoteratone crozoospermic (n=2) patients mic patients men 60 healthy Study population Study Astheno- (n=1), 60 OAT patients 60 OAT patients 47 OAT 60 asthenozoosper - Antioxidant Antioxidant formulation formulation of treatment tablet 40 mg/daily for 3 months 150 mg/daily for for mg/daily 150 6 months - HI Pharmaceu Ltd, tical Co., Japan) 3 6 pills/daily for months for mg/daily 440 3 months Simvastation Simvastation Ubiquinol (KOBAYAS Edicare Zinc sulphate dosage and length design Clinical trial (open label) (open (open label) (open label) (open label) (open Uncontrolled Uncontrolled Uncontrolled Uncontrolled Uncontrolled Uncontrolled Reference (2009) [81] (2015) [78] (2015) [79] (2015) [80] Al-Hilli et al Thakur et al Thakur et al Kobori et al Hadwan 55 52 53 54 SN Clinical condition Continued 16 2. Continued Table

20 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 0 0 0 0 evidence evidence Quality of published 3 5 N/A N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 15 18 N/A N/A CONSORT CONSORT (out of 25) (out Guidelines N/A N/A of bias other for sources of bias for of bias for allocation - conceal other ment, sources; high risk of bias for incomplete outcome data Unclear risk Unclear risk Cochrane Risk Cochrane of Bias for RCTof Bias for 3 3 7 7 Checklist (out of 7) (out for causal for risk factors 3 3 3 3 of 3) for risk for Checklist factors (out 0 0 0 0 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out -tocopherol -tocopherol γ treatment antioxidant antioxidant -tocopherol -tocopherol Reproductive Reproductive tration, motility, motility, tration, normal morphol - ogy concentration of centrations α and in seminal fluid, TBARS, as as well of lipid a marker peroxidation of couples concentration concentration B and in group C, while total sperm motility in all improved groups concentration concentration and motility outcomes after outcomes - concen Improved Improved sperm Improved - con Reduced in 45% Pregnancy Improved sperm Improved Improved sperm Improved - - - - mic patients nozoospermic nozoospermic patients nozoospermic nozoospermic patients nozoospermic nozoospermic patients Study population Study 45 asthenozoosper 40 oligoasthe 90 oligoasthe 54 oligoasthe - Antioxidant Antioxidant formulation formulation of treatment Pharma s.r.l., s.r.l., Pharma Italy)Rome, 2 capsules/daily 3 months for (Tishcon Corp., (Tishcon Corp., NY, Westbury, USA) 2 softsules the first for 3 3 months, the softsules for last 3 months mg/daily) (25 mg/ citrate daily) E+clomiphene 6 for citrate months daily SG1002 (Nuevas Alternativas Ther Naturales mafat, S.A. de mafat, Monterrey, C.V., 750 mg/ Mexico) 75 days daily for (Andrositol, Lo.Li. Lo.Li. (Andrositol, Carni-Q-Nol A) Vitamin E (400 Vitamin A) B) Clomiphene C) Vitamin Resveratrol 25 mg/ Resveratrol dosage and length design Clinical trial (open label) (open (open label) (open RCT unblinded Uncontrolled RCT blinded Uncontrolled Reference (2015) [84] et al Oliva (2016) [85] (2015) [82] al (2015) [83] ElSheikh et al Montanino Martinez et al et Gvozdjáková 58 59 56 57 SN Clinical condition Continued 17 2. Continued Table

www.wjmh.org 21 https://doi.org/10.5534/wjmh.200196 0 0 0 0 evidence evidence Quality of published 2 N/A N/A N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 16 N/A N/A N/A CONSORT CONSORT (out of 25) (out Guidelines N/A N/A N/A of bias for of bias for allocation - conceal other ment, sources; high risk of bias for blinding (partici - and pants personnel, outcome assessment) Unclear risk Cochrane Risk Cochrane of Bias for RCTof Bias for 3 3 3 7 Checklist (out of 7) (out for causal for risk factors 3 3 3 3 of 3) for risk for Checklist factors (out 3 2 0 2 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - treatment antioxidant antioxidant Reproductive Reproductive count and motil - count glutathione ity, level concentration, concentration, and pro total motility gressive count, total and total count, progressive normal motility, morphology after treatment motility outcomes after outcomes Improved sperm Improved level MDA Reduced Improved sperm Improved Improved sperm Improved Improved sperm Improved - - - patients 31 oligoasthe nozoospermic patients patients 2 OAT nozoospermic nozoospermic patients nozoospermic nozoospermic patients Study population Study 7 oligozoospermic 7 oligozoospermic 32 oligoasthe 210 OAT patients 210 OAT 54 oligoasthe Antioxidant Antioxidant formulation formulation of treatment (Sun Pharma) 3 for Twice/day months (Hansal Pharm (Hansal Pharm GmbH, Germa - 3 months for ny) (Kagome Co., (Kagome Co., Japan) Ltd., Tablet nation (600 mg/ Shionogi day, - Pharmaceuti Japan), cal Co., N SoftJuvela (200 Capsule Tanabe mg/day, Seiyaku Hanbai Japan), and Co., Tablet Tathion (300 mg/day, Japan) Eisai Co., vitamin E (400 mg) and L- carnitine (2 6 g)/daily for months Tablet Fertisure M Fertisure Tablet Hansal A–Z Vital A) Natsushibori Natsushibori A) - B) CINAL Combi 3 months For Vitamin C (1 g), Vitamin dosage and length design Clinical trial (open label) (open (open label) (open (open label) (open Uncontrolled Uncontrolled RCT unblinded Uncontrolled Uncontrolled Uncontrolled Uncontrolled Reference (2016) [86] (2017) [88] (2017) [87] (2017) [89] Singh et al Yamamoto et al Yamamoto Alahmar Alahmar Magdi et al 60 62 61 63 SN Clinical condition Continued 18 2. Continued Table

22 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 0 1 0 evidence evidence Quality of published 4 5 N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 20 15 N/A CONSORT CONSORT (out of 25) (out Guidelines - - N/A of bias other for sources of bias for of bias for random sequence generation, allocation - conceal se ment, lective reporting, other sourc blinding es, (participants and person - outcome nel, assess - and ment), incomplete outcome data Unclear risk Unclear risk Cochrane Risk Cochrane of Bias for RCTof Bias for 6 7 6 Checklist (out of 7) (out for causal for risk factors 3 3 3 of 3) for risk for Checklist factors (out 3 0 1 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out treatment antioxidant antioxidant Reproductive Reproductive progressive progressive normal motility, morphology, - thiol con total total centration, disulfide linkage concentration, GPx levels parameters, parameters, sperm except morphology pregnancy in couples treated vs . 2 in the placebo parameters outcomes after outcomes Improved volume, volume, Improved Increased semen Increased 10 spontaneous semen Improved TAC Improved - - mic patients semen abnormali semen 52 those, ties (of with varicocele) patients Study population Study 60 asthenozoosper 104 patients with 104 patients 54 oligozoospermic Antioxidant Antioxidant formulation formulation of treatment 220 mg/daily for for mg/daily 220 3 months (Sigma-Tau (Sigma-Tau HealthScience, the Utrecht, Netherlands) 2 sachets/daily 6 months for for mg/daily 400 3 months Zinc Proxeed Plus Plus Proxeed Melatonin dosage and length design Clinical trial (open label) (open RCT blinded RCT blinded Uncontrolled Uncontrolled Reference 2018 [26] (2018) [91] (2018) [90] Busetto et al Busetto et al Lu Alsalman et al Alsalman 65 66 64 SN Clinical condition Continued 19 2. Continued Table

www.wjmh.org 23 https://doi.org/10.5534/wjmh.200196 0 0 0 evidence evidence Quality of published 3 N/A N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 16 N/A N/A CONSORT CONSORT (out of 25) (out Guidelines - N/A N/A of bias for of bias for selection, other sourc es of bias, blinding of participants and person - and nel, outcome assessment Unclear risk Cochrane Risk Cochrane of Bias for RCTof Bias for 3 2 7 Checklist (out of 7) (out for causal for risk factors 3 3 3 of 3) for risk for Checklist factors (out 0 3 3 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - treatment antioxidant antioxidant ‐glucosidase ‐glucosidase Reproductive Reproductive concentration, concentration, pro sperm count, motility, gressive normal morphol - ogy and vitality after therapy - seminal oxida after stress tive therapy tion check - sperm concen total tration, and progressive normal motility, morphology and protamine deficiency; TAC improved parameters; in - parameters; seminal creased carnitine and α activity; reduced SDF outcomes after outcomes Improved sperm Improved reduced Oxisperm; Unclear capacita - Improved volume, volume, Improved SDF MDA, Reduced Improved semen Improved - - mic patients nozoospermic nozoospermic patients Study population Study 32 OAT patients 32 OAT 50 asthenozoosper 175 oligoasthe g μ g folic g folic μ g selenium, μ Antioxidant Antioxidant formulation formulation of treatment CoQ10 (200 mg), (200 CoQ10 vitamin C (240 mg), vitamin B3 (27 mg), Tribulus (60 terrestris mg), ginseng (12 mg), inositol (100 mg), vita - min E (36 mg) 2 months for 600 mg/daily for for mg/daily 600 3 months sisting of 1 g LC, sisting of 1 g LC, 0.725 0.5 g ALC, 1 g g fumarate, 50 mg fructose, 10 citric acid, 20 mg mg zinc, coenzyme Q10, 50 90 mg vitamin C, 200 vitamin B12 for 6 months acid and 1.5 Arginine (3 g), Arginine N-acetylcysteine - con Plus, Proxeed dosage and length design Clinical trial (open label) (open (open label) (open Uncontrolled Uncontrolled RCT blinded Uncontrolled Uncontrolled Reference (2019) [93] (2019) [94] (2019) [92] Gambera et al Micic et al Jannatifar et al Jannatifar 68 69 67 SN Clinical condition Continued 20 2. Continued Table

24 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 0 0 1 evidence evidence Quality of published 4 5 N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 18 22 N/A CONSORT CONSORT (out of 25) (out Guidelines N/A identified of bias for of bias for allocation concealment, other sources No risk of bias Cochrane Risk Cochrane of Bias for RCTof Bias for Unclear risk 4 7 7 Checklist (out of 7) (out for causal for risk factors 2 3 3 of 3) for risk for Checklist factors (out 4 4 2 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - treatment antioxidant antioxidant Reproductive Reproductive concentration, concentration, and progressive motility, total TAC 10 level, CoQ and GPx and SDF levels sperm count, to sperm count, - tal and progres motility sive rate outcomes after outcomes total sperm count, sperm count, total concentration, total TAC motility, Improved sperm Improved ROS Reduced Improved total total Improved Higher pregnancy Improved volume, volume, Improved - nozoospermic nozoospermic patients altered semen altered Of those, quality. grade 52 showed I-III varicoceles patients Study population Study 65 oligoasthe 104 patients with 104 patients 44 oligozoospermic 44 oligozoospermic - g), g), μ μ g)/daily for 6 g)/daily for Antioxidant Antioxidant formulation formulation of treatment 200 mg/daily for for mg/daily 200 3 months vitamin B12 (1.5 μ months fumarate (725 fumarate mg), acetyl-L- carnitine (500 mg), fructose (1 g), citric acid (50 mg), sele nium (50 coenzyme Q10 (20 mg), vitamin C (90 mg), zinc (10 mg), folic acid (200 25 mg/daily for 3 25 mg/daily for months CoQ10 L-carnitine (1 g), L-carnitine dosage and length Lycopene design Clinical trial (open label) (open RCT blinded Uncontrolled RCT blinded Reference (2020) [96] (2020) [97] (2019) [95] Busetto et al Busetto et al Alahmar Nouri et al 70 71 72 SN Clinical condition Continued 21 2. Continued Table

www.wjmh.org 25 https://doi.org/10.5534/wjmh.200196 0 1 0 evidence evidence Quality of published 3 5 3 JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 16 20 13 CONSORT CONSORT (out of 25) (out Guidelines - - allocation allocation - conceal selec ment, reporttive other ing, no sources; blindness of participants and person - nel identified of bias for of bias for allocation - conceal other ment, high sources; risk of bias blinding for (participants and person - outcome nel, assessment) Unclear risk of No risk of bias Unclear risk Cochrane Risk Cochrane of Bias for RCTof Bias for 3 7 7 Checklist (out of 7) (out for causal for risk factors 3 3 3 of 3) for risk for Checklist factors (out 0 2 2 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - treatment antioxidant antioxidant Reproductive Reproductive tile sperm count tile sperm count after treatment A in group concentration No change in SDF pregnancy and birthlive rates parameters outcomes after outcomes Increased total mo total Increased Improved sperm Improved No change in Improved semen Improved - - nozoospermic nozoospermic patients mic patients Study population Study 31 oligoasthe 174 oligozoosper 51 OAT patients 51 OAT g), μ Antioxidant Antioxidant formulation formulation of treatment (750.1 mg), zinc (30 mg), astaxan - thin (16.05 mg), (90.26 CoQ10 mg), vitamin C (1 g), vitamin B12 (60.1 vitamin E (150 mg) not (dosage to reported) mg), vitamin E (400 mg), selenium (0.20 mg), L-carnitine (1 g), zinc (20 acid (1 mg), folic (10 g), lycopene mg), and vitamin D (2,000 IU)/daily a maximum for of 6 months (250 mg sa - chets) sachets) A) L-carnitine L-carnitine A) B) Hochu-ekki- 3 months For Vitamin C (500 Vitamin A) Glutathione Glutathione A) (200 mg B) CoQ10 6 months For dosage and length design Clinical trial RCT unblinded RCT blinded RCT unblinded Reference (2020) [98] (2020) [99] (2020) [100] Terai et al Terai Steiner et al Steiner Alkumait et al Alkumait 73 74 75 SN Clinical condition Continued 22 2. Continued Table

26 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 0 0 0 1 0 evidence evidence Quality of published 4 5 3 N/A N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 18 25 16 N/A N/A CONSORT CONSORT (out of 25) (out Guidelines - N/A N/A of bias for of bias for allocation - conceal other ment, sources identified of bias for allocation - conceal other ment, sources; high risk of bias for selective re porting and incomplete outcome data Unclear risk No risk of bias Unclear risk Cochrane Risk Cochrane of Bias for RCTof Bias for 3 3 7 7 4 Checklist (out of 7) (out for causal for risk factors 3 3 3 3 3 of 3) for risk for Checklist factors (out 2 2 2 0 0 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - treatment antioxidant antioxidant Reproductive Reproductive parameters lycopene. No lycopene. increase in TAC levels MDA and TAC Catalase, IL-1b, decreased TNF-a IL-8, IL-6, increased increased motilityforward and vitality progressive and progressive normal morphol - ogy SDF% cyte in all count groups reduced showed ROS nancy rate=8.2% C, 1 in (6 in group B and D) groups outcomes after outcomes Improved semen Improved Increased seminal Increased Decreased ROS, Decreased SOD, Increased Group C showed C showed Group leuko Reduced Improved % of fast Improved in No difference B and C Groups - preg Spontaneous - idiopathic OAT idiopathic abacterial prostatovesiculo epididymitis and high seminal leukocytes (>1 million cells/ml) Study population Study 6 healthy men 6 healthy 59 patients with 59 patients 39 healthy men 39 healthy 60 healthy men 60 healthy 98 patients with 98 patients - -

- Antioxidant Antioxidant formulation formulation of treatment 22.8 mg/daily 2 weeks for ment (Daru Dar ment man Parmida, Iran) twice daily 3 months for in water (70 g) in water 14 mg/daily for 14 mg/daily for 3 months sulide 200 mg/ daily+ serratio peptidase 10 mg/daily) tines (2 months each) NSAID (2 months each) 1 g/daily) - anti-inflam matory drugs (nime (NSAID) (Carnitene 2 g/ (Carnitene daily+Nicetile Lycopene Androferti supple Honey dissolved Honey dissolved Lactolycopene C) NSAID+ carni - Carnitines+ D) 4 months For B) Nonsteroidal B) Nonsteroidal A) Carnitines Carnitines A) dosage and length design Clinical trial (open label) (open Prospective study Prospective Uncontrolled Uncontrolled RCT blinded RCT blinded RCT unblinded Reference (2020) [101] (2007) [102] and Maleki (2012) [103] (2020) [104] (2002) [105] Nazari et al Goyal et al Goyal Tartibian Tartibian Williams et al Williams Vicari et al Vicari 76 77 78 79 80 SN Clinical condition inflamma - tion Healthy men Healthy Urogenital Urogenital Continued 23 2. Continued Table

www.wjmh.org 27 https://doi.org/10.5534/wjmh.200196 0 1 evidence evidence Quality of published 5 N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 22 N/A CONSORT CONSORT (out of 25) (out Guidelines N/A identified No risk of bias Cochrane Risk Cochrane of Bias for RCTof Bias for 3 3 Checklist (out of 7) (out for causal for risk factors 3 3 of 3) for risk for Checklist factors (out 3 0 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - treatment antioxidant antioxidant Reproductive Reproductive parameters and parameters activityacrosin - sperm concentra nor tion, motility, mal morphology outcomes after outcomes Improved semen Improved No difference in No difference (n=36) (n=84) Study population Study Chronic prostatitis prostatitis Chronic Leukocytospermia Leukocytospermia - - Antioxidant Antioxidant formulation formulation of treatment gelica Sinensis), Chuan Xiong (Ligusticum Chuanxiong Hort), Chi Shao Veitchii (Paeonia Wu Ling Lynch), Zhi (Trogopterus MiXanthipes - line- Edwards), Huang Pu - Angus (Typha Linne), tata - (Com My Yao Molmol miphora Engler), Yuan Hu (Corydalis Gan Yanhusuo), Jiang (Zingiber Officinale Rosec oe), Guan Gui (Cinnamomum Presl), Cassia and Hui Xiang (Foenicunum Miller) Vulgare Sun-Ten Yu-Tang, Pharmaceutical Taic Company, Taiwan hung, 200 mg/day for for 200 mg/day 1 month - Dang Gui (An A) B) Shao-Fu-Zhu- 2 months For CoQ10 dosage and length design Clinical trial (open label) (open Uncontrolled Uncontrolled RCT blinded Reference (2003) [106] et al (2020) [107] Yang et al Yang Chayachinda 81 82 SN Clinical condition Continued 24 2. Continued Table

28 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 0 0 1 0 evidence evidence Quality of published 5 N/A N/A N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 18 N/A N/A N/A CONSORT CONSORT (out of 25) (out Guidelines N/A N/A N/A identified No risk of bias Cochrane Risk Cochrane of Bias for RCTof Bias for 3 3 3 7 Checklist (out of 7) (out for causal for risk factors 3 3 3 3 of 3) for risk for Checklist factors (out 3 0 2 2 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - - treatment antioxidant antioxidant Reproductive Reproductive concentration concentration and motility rate to morphology, - tal and progres motility sive concentration concentration and acrosome reaction and 8-OH- levels dG levels motility, total total motility, scav oxyradical capacityenging of the semen outcomes after outcomes Improved sperm Improved Higher pregnancy normal Improved Improved sperm Improved ROS Reduced Improved sperm Improved - patients patients tients 11 varicocele patients History of cryptor chidism (n=2), pa - with male tients accessory gland infection (n=7), immunological infertility (n=4), endocrine cause (n=1) patients Study population Study 30 idiopathic 30 idiopathic 52 idiopathic 7 idiopathic pa - 7 idiopathic 60 idiopathic 60 idiopathic - - -carotene β -tocopherol -tocopherol Antioxidant Antioxidant formulation formulation of treatment 4 mg/daily for 3 4 mg/daily for months 3 times 50 mg, 3 weekly for months (600 mg) or cap sules providing a daily amount of (30 mg) and α (180 mg)/daily. In addition, - capsules con taining essential a fatty acids for of daily intake docosahexae noic acid (1 g), gammalinolenic acid (0.25 g) and acid arachidonic 6 (0.10 g) for months (Sigma Tau, Tau, (Sigma Italy) Pomezia, (Sigma B) Zibren Tau) and of carnitene zibren Lycopene Saffron Acetylcysteine Acetylcysteine A) Carnitene Carnitene A) C) A combination 6 months For dosage and length design Clinical trial (open label) (open label) (open (open label) (open Uncontrolled Uncontrolled RCT blinded Uncontrolled Uncontrolled Uncontrolled Reference Kumar Kumar (2002) [108] (2005) [109] (2008) [110] (2000) [47] Gupta and et al Balercia Heidary et al Comhaire et al Comhaire 84 85 86 83 SN Clinical condition IMI Continued 25 2. Continued Table

www.wjmh.org 29 https://doi.org/10.5534/wjmh.200196 0 1 0 0 evidence evidence Quality of published 3 5 N/A N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 12 20 N/A N/A CONSORT CONSORT (out of 25) (out Guidelines N/A N/A of bias for of bias for blinding (partici - and pants personnel); high risk of bias other for sources identified Unclear risk No risk of bias Cochrane Risk Cochrane of Bias for RCTof Bias for 3 3 6 7 Checklist (out of 7) (out for causal for risk factors 1 2 3 3 of 3) for risk for Checklist factors (out 2 0 2 2 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - treatment antioxidant antioxidant Reproductive Reproductive reduced MDA reduced men parameters and SDF motility, semen motility, semen viscosity, - and serum oxida (TAC, stress tive peroxide, total stress oxidative index) concentration and TAC; motility, MDA reduced levels outcomes after outcomes Increased catalase, catalase, Increased No changes in se Improved volume, volume, Improved sperm Improved patients patients patients patients Study population Study 29 idiopathic 29 idiopathic 55 idiopathic 120 idiopathic 120 idiopathic 44 idiopathic - -lipoic acid, -lipoic acid, Antioxidant Antioxidant formulation formulation α of treatment extract for mg/daily 600 3 months ( glutathione, zinc, acid, folic B2, vitamins and B3, B6, B12) Dosage not specified for single compo length of nent, not treatment reported 600 mg/daily for for mg/daily 600 3 months for mg/daily 600 3 months -lipoic acid Grape seed Grape SOD FertiPlus N-acetylcysteine α dosage and length design Clinical trial (open label) (open label) (open RCT unblinded RCT blinded Uncontrolled Uncontrolled Reference (2009) [111] et al (2015) [112] Masoumi (2017) [113] (2017) [114] Ciftci et al Ciftci Haghighian Soleimani and et al Negri 87 88 89 90 SN Clinical condition Continued 26 2. Continued Table

30 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 1 0 0 0 evidence evidence Quality of published 5 N/A N/A N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 24 N/A N/A N/A CONSORT CONSORT (out of 25) (out Guidelines N/A N/A N/A identified No risk of bias Cochrane Risk Cochrane of Bias for RCTof Bias for 7 3 3 7 Checklist (out of 7) (out for causal for risk factors 3 3 3 3 of 3) for risk for Checklist factors (out 0 3 2 1 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out - - treatment antioxidant antioxidant Reproductive Reproductive sive motility and sive - seminal oxida tion reduction potential normozoosper mia in treated mia in treated after 2 patients in and 4 months with comparison placebo rate parameters and parameters SDF fertilization and rates cleavage after treatment - tion and preg nancy rates men parameters outcomes after outcomes - progres Improved SDF Reduced Increased % of Increased Higher pregnancy Improved semen Improved No change in Higher- implanta in se No difference Reduced SDF Reduced - patients 29 unexplained infertile men patients OAT (n=26) OAT 6 unex patients, plained infertile men infertile men Study population Study 119 idiopathic 119 idiopathic 83 idiopathic 83 idiopathic Oligoterato- (n=6), Oligoterato- 64 unexplained Antioxidant Antioxidant formulation formulation of treatment (Fairhaven (Fairhaven Health LLC, Bellingham, WA, USA) 3 for Twice/day months acetyl-carnitine (1,990 mg), l- (250 arginine - mg), glutathi one (100 mg), co-enzyme Q10 (40 mg), zinc (7.5 mg), vitamin B9 (234 mcg), vitamin B12 (2 mcg), selenium (50 6 mcg)/daily for months and vitamin E 2 (1 g)/daily for months 2 1 g/daily for months FH PRO for Men FH PRO for L-carnitine/l- Vitamin C (1 g) Vitamin C and E Vitamin dosage and length design Clinical trial (open label) (open (open label) (open label) (open RCT blinded Uncontrolled Uncontrolled Uncontrolled Uncontrolled Uncontrolled Reference (2020) [115] (2020) [25] (2005) [116] (2005) [117] Kopets et al Kopets Arafa et al Arafa Greco et al Greco et al Greco 91 92 94 93 SN Clinical condition UMI Continued 27 2. Continued Table

www.wjmh.org 31 https://doi.org/10.5534/wjmh.200196 1 0 0 0 evidence evidence Quality of published 5 N/A N/A N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out 18 N/A N/A N/A CONSORT CONSORT (out of 25) (out Guidelines N/A N/A N/A identified No risk of bias Cochrane Risk Cochrane of Bias for RCTof Bias for 3 7 3 6 Checklist (out of 7) (out for causal for risk factors 3 3 3 3 of 3) for risk for Checklist factors (out 0 4 3 0 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out treatment antioxidant antioxidant Reproductive Reproductive concentration, concentration, motility of 25 patients 3 out of 25 patients motility and sive - seminal oxida tion reduction potential parameters, parameters, seminal catalase, and SOD morphology in both groups in results assay after both groups treatment outcomes after outcomes Improved sperm Improved Pregnancy: 3 out birth Live rate: - progres Improved SDF Reduced Improved semen Improved Improved sperm Improved CMA3 Decreased - infertile men patients 29 unexplained infertile men infertile men mic male patients Study population Study 25 unexplained 25 unexplained 119 idiopathic 228 unexplained 228 unexplained 34 hyperinsulinae Antioxidant Antioxidant formulation formulation of treatment 0.5 mg/kg body 0.5 mg/kg 3 for weight months (Fairhaven Health LLC, Bellingham, WA, USA) 3 for Twice/day months 200 mg/daily 26 week, for a by followed treatment-free period of 12- week (500–2,000 mg daily (Georen minoGro - Pharmacuti cals PTY LTD, Fontainebleau, South Africa) Sesame Men FH PRO for CoQ10 A) metformin metformin A) - Sta B) Metformin+ 3 months For dosage and length design Clinical trial (open label) (open label) (open (open label) (open RCT blinded Uncontrolled Uncontrolled Uncontrolled Uncontrolled Reference (2012) [118] (2013) [119] (2020) [25] (2015) [120] Safarinejad et al Khani et al et al Arafa Bosman et al 95 96 97 98 SN Clinical condition linaemic male patients Hyperinsu - Continued 28 2. Continued Table

32 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility - - 0 evidence evidence Quality of published N/A JADAD JADAD (Oxford (Oxford Quality) (out of 5) (out N/A CONSORT CONSORT (out of 25) (out Guidelines N/A Cochrane Risk Cochrane of Bias for RCTof Bias for 4 -HCG: beta-uman chorionic gonadotropin, OAT: oliasthe OAT: beta-uman chorionic gonadotropin, -HCG: β Checklist (out of 7) (out for causal for risk factors 3 of 3) for risk for Checklist factors (out 2 for for Cambridge QualityCambridge Checklist correlate correlate Checklist (out of 5) (out treatment antioxidant antioxidant Reproductive Reproductive morphology of PRM1, levels PRM2, and the PRM1/PRM2 ratio after treatment outcomes after outcomes Improved sperm Improved SDF Reduced Changes in mRNA - preg recurrent nancy loss Study population Study 20 patients with 20 patients Antioxidant Antioxidant formulation formulation of treatment 250 mg/daily for for mg/daily 250 3 months Vitamin C Vitamin dosage and length design Clinical trial (open label) (open Uncontrolled Uncontrolled Reference (2020) [121] Hamidian et al 99 SN Clinical condition RPL Continued 29 2. Continued Table fertilization, in vitro IVF: hormone, LH: luteinizing FSH: follicle-stimulating -deoxyguanosine, hormone, 8-OHdG: 8-hydroxy-2' enzyme Q, Data is summarized based on the clinical trial design, the antioxidant formulation and the study population tested as well as the impact on reproductive outcomes. The quality The and the risk of bias outcomes. as the impact as well on reproductive tested and the study population formulation the antioxidant based on the clinical trial design, is summarized Data score. and JADAD guidelines, CONSORT Quality RiskRCTs, applying the Cambridge of Bias for Checklist, each study by the Cochrane for been determined have ROS, pregnancy loss, RPL: recurrent male infertility, unexplained UMI: male infertility, IMI: iopathic -deoxyguanosine, 8-OHdG: trial, 8-hydroxy-2' controlled randomized RCT: SN: serial number, ICSI: intracytoplasmic sperm DNA fragmentation, SDF: dUTP nick end labelling, deoxynucleotidyl transferase TUNEL: terminal malondialdehyde, MDA: N/A: not available, species, oxygen reactive co CoQ: structure assay, SCSA: sperm chromatin capacity, antioxidant total TAC: GSH: gluthatione, dismutase, SOD: superoxide stress, OS: oxidative index, sperm injection, DFI: DNA fragmentation noteratozoospermia, PRL: prolactin, TSH: thyroid-stimulating hormone, ART: assisted reproductive techniques, TBARS: thiobarbituric acid reactive substances, GPx: glutathione peroxidase, PRM: peroxidase, GPx: glutathione substances, TBARS: thiobarbituric acid reactive techniques, reproductive assisted ART: hormone, thyroid-stimulating TSH: PRL: prolactin, noteratozoospermia, A3. chromomycin CMA3: protamine,

www.wjmh.org 33 https://doi.org/10.5534/wjmh.200196 significance of p<0.05. 85.7% and 89.6% of the low-quality studies showed sig nificant improvement (p<0.0001) in semen and sperm RESULTS function parameters, respectively, in infertile men after antioxidant supplementation, whereas 65.0% and A total number of 1,978 articles were identified 58.3% of the high-quality studies, respectively, reported through the application of the keyword search strat positive effect of antioxidant treatment on semen and egy. Through manual screening of the title, keywords sperm function parameters (Table 3). However, these and abstract, non-relevant articles (n=1,864) were ex results were not significant due to the availability of cluded (Fig. 2). a small number of studies in the literature report Following full-text review for eligibility using the in ing semen parameters (n=20) and those reporting clusion and exclusion criteria, 17 articles were further sperm functions (n=12) and this has led to the under- excluded, resulting in 97 articles that were eligible for powering of statistical analysis. Sample size calculation inclusion (Fig. 2). Two of the studies are each repeated predicted that a total number of 95 and 292 studies as they included both IMI and UMI participants respec reporting the outcome of semen parameters and sperm tively, resulting in a total of 99 studies included in Table functions, respectively, will allow it to gain a statistical 2. Relevant data were extracted from the articles and significance of p<0.05. Furthermore, statistical analysis summarized in Table 2, including the studied popula revealed that 78.6% (p=0.0733) and 60% (p=0.6949) of tion, reported impact of treatment on reproductive out low and high-quality studies, respectively, reported a comes, evaluation of quality, and risk of bias. positive effect of antioxidant treatment on reproduc Of the 97 articles collected, 52 (53.6%) were uncon tive outcomes. However, these values were not signifi trolled (open label) clinical trials, 12 (12.4%) were un cant (p≥0.05) due to the availability of very few studies blinded RCTs and 33 (34.0%) were blinded RCTs. Based (n=14 for low-quality and n=5 for high-quality) in the on the type of antioxidants investigated, 44 (45.4%) of literature. Sample size calculation predicted that a to the articles tested individual antioxidants, 31 (32.0%) tal number of 33 low and 202 high-quality studies are tested a combination of several products in variable required to attain a statistical significance of p<0.05 dosages, and 22 (22.7%) used registered antioxidants for reproductive outcomes. products. Semen parameters were evaluated after an tioxidant treatment in 92.8% (n=90 out of 97) of the 1. Varicocele included publications, while the remaining 7 studies A total of 11 studies investigated a male population evaluated markers of sperm function. affected by varicocele (Table 2). Of those, semen pa Based on the statistical analysis, it is reported that rameters after antioxidant treatment were reported

Articles identified through Scopus database (n=1,978)

Articles screened Non-relevant articles excluded by screening the title, (n=1,978) keywords and abstract (n=1,864)

Non-relevant full-text articles excluded (n=17) Full-text articles assessed Studies not reporting the included outcomes (n=7) for eligibility Non-clinical trials (n=9) (n=114) In vitro study (n=1)

Fig. 2. Preferred Reporting Items for Studies included in Systematic Reviews and Meta-Analyses qualitative synthesis (PRISMA) workflow reporting the litera- (n=97) ture search strategy.

34 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility

Table 3. Number of low and high-quality studies analysing semen parameters and/or sperm function after antioxidant treatment, overall as well as in each clinical condition Report of semen parameters Report of sperm function % of studies reporting % of studies reporting Group Category Number of articles on Number of articles on an improvement after an improvement after the total of studies the total of studies AOX treatment AOX treatment Overall (n=97) Low quality 70/90 (77.8) 85.7*** 50/60 (83.3) 89.6*** High quality 20/90 (22.2) 65.0 12/60 (20.0) 58.3 Varicocele (n=11) Low quality 9/11 (81.8) 75.0 6/11 (54.5) 83.0 High quality 2/11 (18.2) - 0/11 (0) - Abnormal semen quality (n=45) Low quality 36/44 (81.8) 94.4*** 20/25 (80.0) 90.0** High quality 8/44 (18.2) 50.0 5/25 (20.0) 60.0 Idiopathic male infertility (n=10) Low quality 6/9 (66.7) 83.0 5/7 (71.4) 80.0 High quality 3/9 (33.3) 100*** 2/7 (28.6) 100*** Unexplained male infertility (n=5) Low quality 4/5 (80.0) 83.3 3/4 (75.0) 100*** High quality 1/5 (20.0) 100*** 1/4 (25.0) 100*** Values are presented as number (%) or percentage only. AOX: antioxidant, -: not available. Chi-square test: **p<0.01, ***p<0.0001. in 90.9% (n=10 out of 11) of the included publications. 3. Idiopathic male infertility Based on these studies, antioxidant supplementation A total of 10 studies investigated idiopathic infertile seems to be beneficial in varicocele patients as 75.0% men (Table 2). Of those, semen parameters after anti and 83.0% of low-quality studies, respectively, available oxidant treatment were reported in 90.0% (n=9 out of in the literature, reported positive effect of antioxidant 10) of the included publications, whereas sperm function treatment on semen and sperm function parameters biomarkers were reported in 7 out of 10 studies (70.0%) (Table 3). However, these values were not significant. (Table 3). Our statistical analysis showed that all the Sample size calculation predicted that a total number high-quality studies reported improvement in the semen of 41 and 24 studies reporting the outcome of semen and sperm function parameters (p<0.0001) after anti parameters and sperm function, respectively are need oxidant treatment in men with IMI. Although a high ed to reach a statistical significance of p<0.05. percentage of low-quality studies showed improvement in semen and sperm function parameters in men with 2. Abnormal semen quality IMI after antioxidant supplementation, these values A total of 45 studies investigated a male population were not significant. Sample size calculation revealed with abnormal semen quality (Table 2). Of those, semen that a total number of 24 and 30 studies, respectively, parameters after antioxidant treatment were reported reporting the outcome of semen parameters and sperm in 97.8% (n=44 out of 45) of the included publications, function, may allow to reach statistical significance. whereas sperm function biomarkers were reported in 25 out of 45 studies (55.6%) (Table 3). The majority of 4. Unexplained male infertility the studies showed significant improvement in semen A total of 5 studies investigated the effect of antioxi and sperm function parameters of men with abnormal dant therapy in unexplained infertile men (Table 2). semen quality after antioxidant supplementation, al All of those studies reported semen parameters after though these results were not statistically significant antioxidant treatment (100%), whereas sperm function in case of the high-quality studies (Table 3). Sample biomarkers were reported in 4 out of 5 studies (80.0%) size calculation predicted that a total number of 204 (Table 3). All the low-quality studies showed improve studies reporting the outcome of sperm function are ment in sperm function, while sample size calculation required to reach a statistical significance of p<0.05. predicted that a total number of 41 low-quality studies reporting the outcome of semen parameters would allow to attain a statistical significance of p<0.05. Further

www.wjmh.org 35 https://doi.org/10.5534/wjmh.200196 1 0 Study Study outcome outcome (out of 3) (out

0 2 Study Study (out of 4) (out quality score quality score - - - - level of level α N/A analysis 2-sided detect0.05 to a risk of 7% in difference LBR (implying a risk of 1.10), with ratio correc continuity tion and allowing of rate a dropout for 15% and risk ratios ences Sequential ap of Lan proach and DeMets with adjust Bonferroni distribute to ment the 1-sided type I among 3 rate error semen continuous quality parameters hoc sensitivity Post analyses Power of statistical of statistical Power 90% power at a at 90% power of risk differ Esteem - - -HCG–de β reported Main outcomes Main outcomes (p=0.04) semen parameters semen parameters both between groups. in assay Comet group treatment group vs . placebo MD 2.4, (Adjusted 95% CI 0.5–4.4) in ences tected pregnancy, tected pregnancy, clinical intrauterine ectopic pregnancy, - preg pregnancy, nancy multiple with fetuses 404 (34%) vs . 416 group placebo (35%) (ns) Improved TMSC TMSC Improved No difference in No difference in SDF by Increase differ No significant LBR: Treatment group LBR: Treatment N/A N/A factor Female Female 0 0 exclusion inclusion/ Strict male - - - 6 /mL 6 Exclusion criteria Exclusion ing in infertility therapy, drug abuse therapy, use or a gestational surrogate gens, anti-androgens, anti-androgens, gens, - and immunosuppres sants <5×10 Clinical conditions result Clinical conditions chemo History of cancer, Administration of andro Administration of donor sperm Planning enrollment at Pregnancy Obstructive azoospermia diseases Chronic Sperm motility<5% TMSC>30×10 Azoospermia Sperm concentration - - of couples of couples planning IVF infertilityfor treatment old; presence old; presence of oligozoo spermia and/ or asthenozoo spermia Male partners Age: 20–60 years 20–60 years Age: Inclusion criteria - Study Study sample size population/ population/ (n=1,185) vs . placebo (n=1,185) nozoospermic nozoospermic patients Treatment Treatment 31 oligoasthe Study design Study Double-blind RCT RCT unblinded Reference (2020) [46] (2020) [98] Schisterman et al Schisterman Terai et al Terai 2 1 SN Articles published between January 2019 and July 2020 investigating the impact of antioxidant treatment on reproductive outcomes on reproductive 4. Articles treatment the impact January published between of antioxidant 2019 and July 2020 investigating Table

36 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 0 1 Study Study outcome outcome (out of 3) (out

3 2 Study Study (out of 4) (out quality score quality score analysis =0.05 tion, assuming a rate, 20% dropout at ≥80% power α tion assuming 0.80 1-beta error and type I error alpha 5% by founders analysis ANCOVA Power of statistical of statistical Power Sample size calcula - Sample size calcula - Sample size - con for Control vs . 9/41 vs . 2/41 reported Main outcomes Main outcomes between both between as regards groups of normalization semen parameters (26/42 2 months at [61.9%]) males in group treatment vs . 8/41 [19.5%] males in pla - and group) cebo (29/42 4 months at [69.0%] [22.0%]). baseline in from all for mean values main semen pa - 2 and 4 at rameters for except months, sperm morphology PR in treatment than placebo (10/42 group [23.8%] [4.9%]) semen parameters, semen parameters, SCSA and PR DFI by (ns) 24% placebo and group treated 25% in the placebo with a 17% group dropout Significant difference difference Significant change Significant higher 6 months At No difference in No difference vs . LBR: 15% AOX LBR=35% in the Yes Yes factor Female Female 1 0 exclusion inclusion/ Strict male - - /mL 6 Exclusion criteria Exclusion nent infertilityor female tion tional product within 3 months the previous - or testos medication terone <5×10 Allergy to any compo any to Allergy Any clinical cause of male Any or drug addic Alcohol - investiga of any Use Consumption of fertilityConsumption Sperm concentration Sperm concentration with IMI with abnormal semen analysis in the last 6 or months DFI≥25% Age: 21–50 years, 21–50 years, Age: Infertile men Inclusion criteria . . vs vs Study Study sample size population/ population/ placebo (n=41) placebo placebo (n=86) placebo Treated (n=42) (n=42) Treated Treated (n=85) (n=85) Treated Study design Study Double-blind RCT Double-blind RCT Reference (2020) [115] (2020) [99] Kopets et al Kopets Steiner et al Steiner 4 3 SN Continued 1 4. Continued Table

www.wjmh.org 37 https://doi.org/10.5534/wjmh.200196 2 3 1 Study Study outcome outcome (out of 3) (out

2 3 1 Study Study (out of 4) (out quality score quality score N/A N/A analysis tion assuming that tion assuming that the prevalence of male infertile with couples causes idiopathic is 15% in the world and in Indonesia 1.11% Power of statistical of statistical Power Sample size calcula - Sample size - - - reported Main outcomes Main outcomes tility, and morphol - tility, ogy (p<0.05) 8-OHdG levels (p<0.01) and MDA, - with the val ue<1.98 being able predict 100% of to the normal sperm (>40) motility level concentration, mo concentration, improvement in improvement - sperm concentra tion (p<0.001), motilitytotal (p=0.001), normal morphology (p<0.001), ORP (p<0.001), SDF Halo (p=0.001) by sperm improvement in progressive motility (p=0.002), ORP (p=0.03), SDF (p=0.02) in sperm ments concentration (p=0.004) and normal morphol - ogy (p=0.01) Reduced levels of levels Reduced Significant improve Significant sperm Improved IMI: significant IMI: significant significant UMI: No No Yes factor Female Female 1 0 1 exclusion inclusion/ Strict male /mL 6 Exclusion criteria Exclusion profile months medical conditions impactwhich may on stress oxidative <1×10 causing infertility History of hormonal drug addi - therapy, abuse, tion, alcohol to exposure smoking, reproductive potential toxins clearly stated Leucocytospermia infertility cause for Any Chemotherapy Clinical endocrinopathy hormonal Abnormal in the past 6 AOXs social habits or Dietary, of drugs Use Azoospermia Sperm concentration Azoospermia Prostatitis clinical condition Any not criteria Exclusion (20–50 years) (20–50 years) with unknown etiology and infertilfemale - ity factor with at least with at 1 abnormal - semen param age<45 eter; BMI<30 years, not clearly stated Infertile men Infertile patients Inclusion criteria Inclusion criteria Study Study sample size population/ population/ (n=119) and unexplained male infertility (n=29) idiopathic OAT idiopathic Idiopathic Idiopathic 59 patients with 59 patients 25 infertile men Study design Study RCT Prospective study Prospective Prospective study Prospective Single-blinded Reference (2020) [25] (2020) [101] (2020) [44] Arafa et al Arafa Nazari et al et al Nurmawati 5 6 7 SN Continued 2 4. Continued Table

38 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 1 1 Study Study outcome outcome (out of 3) (out

3 1 Study Study (out of 4) (out quality score quality score =0.20 β N/A analysis =0.05 (sig - tion assuming α nificance), of 80%), (power 15% of and up to dropping patients out of the study esteemed Power of statistical of statistical Power Sample size calcula - Sample size . placebo vs . placebo reported - vs . 2 pregnan Main outcomes Main outcomes sperm count sperm count (p<0.0001), total (p<0.0001) and motil - progressive ity (p=0.0012) group (10 respectively; cies, p=0.0141) volume, count, count, volume, and motility, total normal morphol - ogy (p<0.05) Improved total total Improved Higher PR in treated Improved sperm Improved No Yes factor Female Female 0 0 exclusion inclusion/ Strict male - - Exclusion criteria Exclusion any of the compound any or cancer, testes endocrine disorders, post-pubertal mumps, genitourinary surgery, obstructive azoosper mia or obstructive of the pathology system, urogenital disease, autoimmune cystic fibrosis affecting fertility, apy or drug abuse alcohol special diet or taking AOXs other clinical in any trials orchitis, cryptorchidism orchitis, that or medications affect might seminal in the last parameters the prior to 3 months study Known hypersensitivity to to hypersensitivity Known History of undescended History ther of taking any any Subjects following Involvement Presence of varicocele, of varicocele, Presence of herbals Consumption - - astheno- and/ or teratozoo spermia, with or without varicocele (not surgically and treated) infer men from tile couples not clearly stated Oligo- and/or Inclusion criteria Inclusion criteria Study Study sample size population/ population/ with altered with altered semen quality. Of 52 those, grade showed I–III varicoceles 104 patients 104 patients 58 infertile men Study design Study RCT (open label) (open Double-blinded Uncontrolled Uncontrolled Reference (2020) [96] (2020) [45] Busetto et al Busetto Hadi et al 9 8 SN Continued 3 4. Continued Table

www.wjmh.org 39 https://doi.org/10.5534/wjmh.200196 2 1 1 Study Study outcome outcome (out of 3) (out

2 1 3 Study Study (out of 4) (out quality score quality score N/A N/A N/A analysis Power of statistical of statistical Power reported Main outcomes Main outcomes concentration, concentration, and progressive motilitytotal of (p<0.05), levels 10 (p<0.001), CoQ (p<0.01) and TAC GPx (p<0.001) (p<0.05) and SDF SCD assay by (p<0.01) concentration, concentration, motility (p=0.01) and morphology (p=0.03) fast progressive fast progressive (p=0.006) and normal morphol - ogy (p<0.001) TUNEL by Improved sperm Improved ROS levels Reduced Improved sperm Improved Improved % of Improved in SDF No difference No No No factor Female Female 1 1 0 exclusion inclusion/ Strict male - - - Exclusion criteria Exclusion of genital tract, varico genital infection, cele, systemic surgery, scrotal diseases and selective ntioxidant reuptake serotonin in the intake inhibitors last 6 months diseases, neoplasm, diseases, hypospadias, trauma, obstruc deference vas and tion, varicocele, genital tract infection recently gery cancer - deriva or soy protein tives Azoospermia abnormalities Anatomical Smoking factor Female of Consumption Presence of chronic of chronic Presence treatment Receiving Previous testicular sur testicular Previous Existing or previous Existing or previous whey tomato, to Allergy - - showing showing oligoastheno zoospermia factor with idio OAT pathic unteers, aged unteers, 18–30 years, within 1 h lived of the clinic or live planning to in the region the duration for of the study Infertile patients Normal female Normal female - male vol Healthy Inclusion criteria - Study Study sample size population/ population/ nozoospermic nozoospermic patients 65 oligoasthe 51 OAT patients 51 OAT 60 healthy men 60 healthy Study design Study (open label) (open RCT Uncontrolled Uncontrolled RCT unblinded Double-blinded Reference (2020) [97] (2020) [100] (2020) [104] Alahmar et al Alahmar Alkumait et al Alkumait Williams et al Williams 10 11 12 SN Continued 4 4. Continued Table

40 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility 3 2 1 1 Study Study outcome outcome (out of 3) (out

2 2 0 2 Study Study (out of 4) (out quality score quality score N/A N/A N/A N/A analysis Power of statistical of statistical Power - reported Main outcomes Main outcomes morphology (p=0.000) TUNEL (p=0.00) defi - protamine ciency assessed by assay CMA3-based (p=0.00) concentration concentration (p=0.003), total motility (p=0.001). DFI by Reduced SCSA (p=0.001) patients treated sperm count, sperm count, and nor motility, mal morphology (p<0.005) count (p=0.021) count and motility (p=0.003) Improved sperm Improved SDF by Reduced sperm Reduced Improved sperm Improved AOX PR=16.8% for Improved volume, volume, Improved Improved sperm Improved No No No Yes factor Female Female 1 1 1 0 exclusion inclusion/ Strict male - - - - Exclusion criteria Exclusion varicocele or other with AOXs medications of the presence ners, hormonal imbalance, - altera chromosomal tubal obstruc tions, tion, and bacterial or infections viral surgery, and inflamma - surgery, tion obstructive infertility tions - complica and delayed tions of varicocelec tomy diction, smoking hormonal disorders, or active infec chronic Obesity, diabetes, and diabetes, Obesity, treatments Previous part the female For History of varicocele, History of varicocele, Presence of organic or of organic Presence Presenting side effects, side effects, Presenting Usage of supplements Usage and/or drug ad - Alcohol mellitus, Diabetes - - pregnancy loss, pregnancy loss, age<40 years, no history of alcohol/drug abuse or smok altered ing, semen quality fertility patients who patients underwent sub-inguinal varicocelec tomy Recurrency of Aged 20–40 years Aged Unexplained sub-Unexplained Varicocele Varicocele Inclusion criteria Study Study sample size population/ population/ with recurrent with recurrent pregnancy loss with DFI>27% SCSA by patients 20 patients 20 patients 485 infertile men 24 infertile men 60 varicocele 60 varicocele Study design Study (open label) (open (open label) (open (open label) (open Uncontrolled Uncontrolled Uncontrolled Uncontrolled Uncontrolled Uncontrolled Single blind RCT Reference (2020) [121] (2019) [42] (2019) [43] et al (2019) [57] Hamidian et al Salehi et al Hasoon Ardestani Zadeh Ardestani 14 15 16 13 SN Continued 5 4. Continued Table

www.wjmh.org 41 https://doi.org/10.5534/wjmh.200196 1 2 3 Study Study outcome outcome (out of 3) (out

3 2 1 Study Study (out of 4) (out quality score quality score =0.05 α N/A N/A analysis detect an effect of d≥0.70 as size - signifi statistically in a two-tailedcant with test of 0.80 and power with n=24 per condition. Power of statistical of statistical Power Study powered to to powered Study - reported Main outcomes Main outcomes concentration, concentration, in sperm count normal morphol - and total ogy, and progressive motile sperm count (p<0.05) patients treated than placebo (29% vs . group 17.9%, respectively; p=0.029) concentration, concentration, pro sperm count, motility, gressive normal morphol - and vitality ogy, seminal reduced stress oxidative (no p-after therapy reported)values centration (p=0.02), centration (p=0.01) total and progressive motility (p=0.001), normal morphol - ogy (p=0.001), TAC (p=0.01) (p=0.01), MDA SDF TUNEL by (p=0.001), % of sperm showing deficien - protamine cy CMA3-based by (p=0.009) assay Improved TSC, TSC, Improved sperm Higher PR in AOX Improved sperm Improved test: Oxisperm - sperm con Improved of levels Reduced No Yes Yes factor Female Female 0 0 1 exclusion inclusion/ Strict male - - Exclusion criteria Exclusion system and/or varico system surgerycele fecting fertility the for 3 months previous fertility specific diet smoking ity due to the female ity the female due to factor hormonal abnormali - and/or obstruc ties, tion, cryptorchidism, abnormal vasectomy, functionliver sumption syndrome, Klinefelter’s in the 90 fever cancer, sperm prior to days sperm seminal analysis, antibodies Previous genitourinary Previous IMI af - clinical condition Any a following Patients or drug abuse, Alcohol Azoospermia and infertilAzoospermia and - Varicocele, leukospermia, leukospermia, Varicocele, - con alcohol Smoking, disorders, Anatomical - - - patients treated treated patients with varicoce with lectomy, spouses<35 - regu old, years lar hormone and profiles menstrual cycles and no cause identified of infertility with normal develop sexual medical ment, serum history, hormone levels and physical examination - with no previ ous report of nor pregnancy, and mal female male partners Varicocele Varicocele Infertile patients Infertile couples Inclusion criteria - Study Study sample size population/ population/ patients zoospermic zoospermic patients 90 varicocele 90 varicocele 32 OAT patients 32 OAT 50 astheno Study design Study (open label) (open (open label) (open RCT unblinded Uncontrolled Uncontrolled Uncontrolled Uncontrolled Reference (2019) [56] (2019) [93] (2019) [92] Kızılay and Altay and Altay Kızılay Gambera et al Jannatifar et al Jannatifar 17 18 19 SN Continued 6 4. Continued Table

42 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility - 3 2 Study Study outcome outcome (out of 3) (out

4 2 Study Study (out of 4) (out quality score quality score N/A N/A analysis Power of statistical of statistical Power reported ‐glucosidase ‐glucosidase Main outcomes Main outcomes volume (p=0.001), volume progressive motility (p<0.001), vitality (p=0.002) after treatment Halosperm test carnitine and α positively activity, with correlated - progres improved motility sive TSC, concentration, concentration, TSC, total TAC motility, (p<0.05) Improved ejaculated ejaculated Improved SDF by Reduced seminal Increased Improved volume, volume, Improved No Yes factor Female Female 0 1 exclusion inclusion/ Strict male - - - - 2 /mL 6 Exclusion criteria Exclusion infertility within the last 2 months post‐pubertal mumps, - endocrine and autoim cysticmune diseases, or testicular fibrosis, cancer Plus in Proxeed dients anti-sperm disorders, leukocyto antibodies, spermia or vitamins clinical trials <1×10 disorders, endocri - disorders, hor previous nopathy, use of monal therapy, antiandro androgens, anticoagulants, gens, or drugs, cytotoxic immunosuppressants History for of therapy consumption Alcohol testes, Undescended Hypersensitivity ingre to of endocrine Presence agents antioxidant of Use in other Involvement Motility<5% Sperm concentration History of anatomical and drug abuse Alcohol BMI≥30 kg/m / 6 - / 6 - /mL); 6 sperm vitality ≤58%; normal sperm mor phology <4% mL, normal sperm <65% and average motility <60% - mL; progres motility sive <32%; normal viscosity and normal leuco cytes number (<1×10 (25–45 sperm years), count<20×10 ber ≤15×10 Infertile men Total sperm num - Total Inclusion criteria - vs . Study Study sample size population/ population/ mic patients (n=125) (n=50) placebo 44 oligozoosper Treatment Treatment Study design Study Double-blind RCT Double-blind RCT Reference (2019) [95] (2019) [94] Nouri et al Micic et al -HCG: beta-human chorionic gonadotropin, LBR: live birth rate, ns: non-significant, DFI: DNA fragmentation index, SCSA: sperm chromatin structure assay, AOX: antioxidant, IMI: idiopathic idiopathic IMI: antioxidant, AOX: structure assay, sperm chromatin SCSA: index, fragmentation DFI: DNA non-significant, ns: birth live LBR: gonadotropin, rate, beta-human chorionic -HCG: β 20 21 SN male infertility, ORP: oxidation reduction potential, UMI: unexplained male infertility, BMI: body mass index, 8-OHdG: 8-hydroxy-2' -deoxyguanosine, MDA: malondialdehyde, PR: pregnancy rate, PR: pregnancy rate, malondialdehyde, MDA: -deoxyguanosine, 8-OHdG: BMI: body mass index, 8-hydroxy-2' male infertility, unexplained UMI: reduction potential, oxidation ORP: male infertility, - TUNEL: termi oligoasthenoteratozoospermia, dispersion, OAT: SCD: sperm chromatin species, oxygen ROS: reactive peroxidase, GPx: glutathione capacity, antioxidant total TAC: coenzymeCoQ: Q, sperm count. total TSC: A3, chromomycin CMA3: dUTP nick end labeling, nal deoxynucleotidyl transferase Continued 7 4. Continued Table val, val, Data are summarized and ranked based on the study design, the population investigated, the inclusion/exclusion criteria, the analysis of the female partner, the main outcomes reported, and the reported, the main outcomes partner, of the female the analysis criteria, the inclusion/exclusion investigated, the population based on the study design, and ranked summarized are Data analysis. of the statistical power inter MD: median, CI: confidence sperm DNA fragmentation, fertilization, in vitro SDF: IVF: N/A: not available, motile sperm count, total TMSC: trial, controlled randomized RCT: SN: serial number,

www.wjmh.org 43 https://doi.org/10.5534/wjmh.200196 more, all the high-quality studies reported a significant tice. improvement in the semen and sperm function para Majzoub and Agarwal (2018) [10] performed a sys meters after antioxidant treatment in men with UMI. tematic review and identified 26 studies showing posi tive effects of exogenous antioxidant intake on sperm 5. Analysis of the most recent publications quality and relevant outcomes of assisted reproduction A total of 21 articles published between January 2019 such as live birth rates. The authors critically discussed and July 2020 investigated the effects of antioxidant the studies and highlighted that the treatment was treatment on semen quality (Table 4) [25,42-46,56,57,92- given only for a short period to a small number of men. 101,104,115,121]. Based on our analysis, 13 and 8 studies In addition, the lack of a standardized test to estimate were ranked as low and high-quality, respectively. Of oxidative stress levels in sperm and seminal fluid was these, 19 out of 21 (90.5%) showed improvement in se another flaw in these studies, while the heterogeneity men parameters, while 4 out of 6 (66.7%) reported a of the study designs made it particularly challenging significant improvement in sperm function. The num to compare the effects and reach a robust conclusion. ber of studies investigating reproductive outcomes af This has also been observed in our study, where 60 ter antioxidant treatment was very limited, with only studies (61.9%) analyzed a variety of seminal oxidative 3 out of 5 (60.0%) reporting an improvement in preg stress markers, including the levels of seminal ROS nancy rate, while birth rate showed no variation in the and/or several endogenous antioxidants (i.e., total anti two studies reporting its evaluation. oxidant capacity assay, superoxide dismutase, catalase, glutathione), markers of lipid peroxidation (i.e., malo DISCUSSION ndialdehyde), oxidative DNA damage (8-hydroxy-2' -de oxyguanosine), and oxidation-reduction potential (ORP) Male infertility is a relatively common concern, con (Table 2). The lack of standardization in the evaluation tributing significantly to poor reproductive outcomes of oxidative stress in seminal fluid before and after in couples. Oxidative stress has been increasingly iden therapy hinders a definitive conclusion regarding the tified as a common mechanism that mediates not only implementation of oral antioxidant supplementation the pathophysiology, but also the many etiologies and for infertile men in the clinical practice. Moreover, the risk factors associated with male infertility [1,2,5,6]. lack of detailed methodological descriptions in most Within this context, there is increased use of antioxi articles testing oral antioxidants supplementation is a dants as a therapeutic option in male infertility, how major shortcoming that makes comparisons between ever, there remains no consensus on the efficacy, indi different studies difficult. The evaluation of the length cations, dosage or length of treatment [8-11]. Therefore, of treatment in the analyzed studies also does not help the objective of this study was to systematically review in this regard, as it is variable, with 15 (15.5%) studies the literature of trials investigating antioxidant use in reporting the treatment for an unclear amount of time male infertility, and to propose some broad guidelines or less than 3 months (Table 2). This might result in for the practicing clinicians based on the currently difficulties to observe any significant influence on hu available evidence. The results (Table 2) were stratified man spermatogenesis. based on the currently available evidence on the clini More recently, a systematic review and meta- cal conditions investigated and the data were further analysis with data from seven RCTs using L-carnitine analysed. Most studies reported men with abnormal (LC) and L-acetyl carnitine (LAC) as treatment (LC semen quality (n=45) and infertile men (n=20) as well 2 g/day+LAC 1 g/day in six studies and LC 150 mg/ as male infertility conditions such as varicocele (n=11), day+LAC 50 mg/day in one study during 12 or 24 IMI (n=10), UMI (n=5), and urogenital inflammation weeks) enrolling a total of 693 patients, concluded that (n=3). Although there is no doubt that assessing sperm a combined therapy of LC and LAC is effective in men quality is just a first approach to a diagnosis and that with idiopathic oligoasthenoteratozoospermia [122]. evaluation of it as a predictor of fecundity or a couple’s This conclusion was supported by a significant increase fertility success may lead to imprudent conclusions, in forward sperm motility and total motile sperm there is no consensus whether the intake of exogenous count. All the other sperm characteristics analyzed antioxidants should be routinely done in clinical prac including semen volume, sperm concentration and per

44 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility centage of abnormal spermatozoa showed no change. that the number of high-quality studies is too low to Since most of the selected studies lacked consistent and obtain significant results. Some of the reasons are that detailed information, pregnancy of the female part fertilization is a multifactorial process. In this case the ner as an endpoint was not considered in the analysis. man is treated for oxidative stress, which reflects in Nevertheless, the authors found that the combined improvements of semen parameters and seminal oxida therapy of LC+LAC could lead to higher pregnancy tive stress, even in high-quality studies. However, when rates. Although this meta-analysis provides evidence looking at fertilization, pregnancy and live birth rates for a positive effect of dietary supplementation with oocyte quality has to be considered as a confounding LC+LAC for 3–6 months in men with idiopathic oligo variable. These factors are also the reason why the asthenoteratozoospermia seeking fertility treatment, number of studies necessary (n=202) to obtain a sig there are several limitations that hamper a robust nificant result for the high-quality studies is so high conclusion. First, the clinical diagnosis of idiopathic and is therefore unrealistic. Secondly, the general cost oligoasthenoteratozoospermia is open to interpretation. for high-quality double-blind, randomized, and placebo- Thus, each study may have considered and recruited controlled studies with a sufficiently high number of distinct types of patients. In addition, since the studies participants is also very high. had a significant variance regarding the number of se Smits et al (2019) [11] performed an extensive meta- lected patients (21 patients in two studies and up to 175 analysis to evaluate if dietary supplementation with patients in one study), this impedes the robustness of oral antioxidants was effective and safe. The authors the conclusions. The most striking limitation, common analysed 61 studies involving 6,264 subfertile men to most studies, is the fact that the bioavailability of seeking fertility treatment. The authors found that 18 the compounds is unknown. Moreover, the mechanisms different oral antioxidants were used in these studies. by which they target testicular function and exert The most relevant conclusion was that oral antioxidant their action is not well established and these studies do supplementation can improve the reproductive capac not provide evidence for a synergistic action versus a ity of subfertile men and even enhance live birth rates. single compound action, nor do they clearly show how However, the evidence collected was considered of low the compounds act. or very low-quality due to serious study limitations, making the comparison and/or aggregation to perform CRITICAL EVALUATION OF THE robust statistical analysis difficult. The studies also NECESSITY OF ADDITIONAL showed a significant variation in the antioxidant sup DOUBLE-BLIND, RANDOMIZED, plementation regimens (type, dose, or even combined PLACEBO-CONTROLLED TRIALS intake). Some studies used a placebo group to compare with, while others chose to compare with no treatment An important issue that must be pointed out in this or treatment with another antioxidant. context is that out of 90 studies that reported the ef The endpoint for couples attending a fertility treat fects of antioxidant treatment on semen parameters, 70 ment has to be clinical pregnancy or live birth, but were low-quality studies, whereas only 20 were ranked most studies fail to present these data. Only 12 of the as high-quality. The overall statistical analysis of both 44 studies that were included in the previously cited low- and high-quality studies indicates that the antioxi meta-analysis reported on clinical pregnancy and live dant treatment has a significant positive effect on se birth, which is considered a major limitation [11]. The men parameters. A similar result could still be obtained latter evidence has also been highlighted in our sys for the effect of the antioxidant treatment on seminal tematic analysis of the literature, where only 22 (22.7%) oxidative stress and SDF. Only 60 of these studies and 4 (4.1%) out of 97 studies reported pregnancy and evaluated oxidative stress markers. For the reproduc live birth rates as a clinical outcome, respectively. tive outcome, however, only a total of 19 studies, 5 of Pregnancy and live birth are highly influenced by a which were of high-quality, have reported this outcome wide variety of embryological and female factors. This parameter. Out of these 5 studies, 3 reported a positive highlights one of the major problems in literature as effect, while in 2 studies either no effect or a nega the studies on the use and effectiveness of antioxidants tive effect was observed. These data clearly indicates are mostly focused on the males and not on the couple.

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Studies need standardization not only regarding the pair damages in spermatozoa [126], and this is another selection of the males, but also the female partners to unknown variable that needs to be considered when evaluate clinical pregnancy, achievement of live birth, thinking of reproductive outcomes as a therapeutic and the influence of the treatment. Many confounding effect of treating men with antioxidants. A number factors may interfere with such outcomes including of critical points can further be pointed out from indi female age, ovarian reserve, anatomic, inflammatory vidual studies. Schisterman et al [46] lost 31% of their and endocrinal disorders, and as such, the impact of subjects during follow-up and a substantial number of any fertility treatment can only be assessed after ad couples received non-specified treatment off-site, which justing for such confounding factors. All these factors could alter the study results. The Steiner et al’s [99] determine the type of ART treatment that is employed, study was terminated early as they failed to show a namely in vitro fertilization (IVF) or intracytoplasmic >10% difference in pregnancy/live birth rates. More sperm injection (ICSI), and eventually also have a sig over, the authors failed to confirm adherence with nificant impact on the reproductive outcome. Hence, antioxidant treatment, and ovarian stimulation was the outcomes, positive or negative, can be skewed due used in couples who did not conceive after 3 months of to numerous confounding factors. Consequently, the fo therapy, which could affect the outcomes. Terai et al [98] cus of the effectiveness of an antioxidant treatment of included men between 20 and 60 years of age, which is men should be whether or not the treatment improves atypical of the reproductive age group. Abstinence was seminal parameters and sperm function, rather than defined as 4 or more days without an upper limit [98]. reproductive outcomes which are influenced by numer Advanced male age and delayed abstinence are associ ous other variables that are, i) not/insufficiently con ated with increased oxidative stress measures and with sidered in recent studies, and ii) can have a significant alteration in semen parameters [127,128]. Furthermore, negative effect on blastulation, embryo development, Terai et al [98] did not include a placebo group, with an onset/continuation of pregnancy, and live birth. Fur experimental group investigating a Chinese herbal for thermore, in order to be able to judge the effectiveness mula. Hadi et al [45], Alahmar et al [97], Hamidian et of treatment, it is necessary to specify the andrological al [121], Salehi et al [42], Hasoon [43], Gambera et al [93], condition for which patients are being treated. Without Arafa et al [25], Nazari et al [101], and Jannatifar et al this, one might include patients with a condition in a [92] all reported improved semen parameters, but these study which are not or only poorly responding to the studies are uncontrolled open label trials and are rela treatment. In turn, this would negatively affect the tively underpowered. Furthermore, Alkumait et al [100] outcome of the study. and Terai et al [98] were unblinded trials with rela We have identified 21 studies investigating the ef tively small sample sizes (n=51 and 31, respectively). On fects of antioxidant treatment on semen quality that the other hand, the reviewed studies also have several were published from January 2019 to July 2020. The positive virtues that can be pointed out. For example, majority of these studies showed an improvement in the study by Agarwal et al [129] investigated changes semen parameters and sperm function. With a more in protein expression following antioxidant therapy, detailed assessment of quality reported, 13 of the 21 thereby enhancing our understanding of the physi were ranked as low-quality. Steiner et al [99] and Micic ologic alterations at the molecular level. Several studies et al [94] strictly excluded couples with female factors, assessed the effect of antioxidant therapy on SDF lev while most of the studies inadequately documented els (Table 2). SDF is increasingly being utilized in the such criteria in their methodologies. From the molecu evaluation of male factor infertility and is believed to lar point of view, this integrative focus on the couple be an important determinant of fertility potential [130]. is essential. The sperm-oocyte interaction is sensitive Furthermore, SDF is considered as an indirect measure to the redox balance, and excessive ROS in spermato of oxidative stress and can validate the benefits of an zoa can lead to impaired oocyte function [123]. Hence, tioxidant therapy in restoring the body’s redox poten the effects of sperm oxidative damage go far beyond tial. fertilization since they can have a negative impact on Therefore, for the above discussed reasons, having embryo development or even pregnancy loss [124,125]. more double-blind RCT studies with a large enough On the other hand, the oocyte has the capability to re sample size are neither feasible nor could they provide

46 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility the expected clear result in terms of improved live treated with the false expectation that this treatment birth rates after the antioxidant treatment. would automatically increase the success of the repro ductive outcome. In addition, the low level of evidence STRENGTH WEAKNESS was extrapolated from the clinical trials reporting OPPORTUNITY THREAT (SWOT) benefit due to non-homogenous study designs, or in ANALYSIS consistencies in the treatment regimens (individual or combined) used (Table 2). Furthermore, the majority of 1. Strengths these studies failed to adjust for confounding factors Antioxidant supplementation for the treatment of (e.g., female factors) that are essential for conception or male infertility has been increasingly investigated in establishment of pregnancy (Table 2). the past decade. Reports suggest that different an tioxidant formulations were used to improve sperm 3. Opportunities quality and function in infertile men with various Selection of suitable candidates for antioxidant sup clinical circumstances (Table 2). These improvements plementation by oxidative stress measurement seems a were reflected on reproductive outcomes such as preg logical approach [25]. Indeed, the concept of MOSI has nancy rate (Table 2). An increasing number of studies been recently proposed. The classification may guide have investigated the effect of antioxidant therapy on the identification of a specific group of idiopathic in measures of oxidative stress, perhaps indicating it is a fertile men who will be more likely to benefit from the feasible treatment approach/option for patients with treatment [2]. Secondly, evaluation of the sperm pro alteration in seminal redox potential. teome of idiopathic infertile men before and after oral oxidant supplementation offers a window to better un 2. Weaknesses derstanding the molecular mechanisms associated with The contradictory results in reproductive outcomes sperm function (Fig. 3) [129]. Oral antioxidants may be seen in a number of studies can be considered as the an alternative cost-effective treatment option for infer main factor limiting the routine use of antioxidants tile couples who desire to avoid assisted reproduction. for the treatment of male infertility, while female and embryological confounding factors were not taken into 4. Threats account (Table 2). Yet, in these studies, the male was Despite the conclusion from Cochrane collaborations

Fig. 3. Strength Weakness Opportunity Threat (SWOT) analysis. SWOT has been conducted to describe the impact of antioxidant supplementation in the treatment of male infertility. ART: assisted reproductive technology.

www.wjmh.org 47 https://doi.org/10.5534/wjmh.200196 that oral antioxidant therapy may improve semen pa quality, IMI, UMI, and clinical varicocele based on rameters and the likelihood of pregnancy, the lack of studies reviewed (Table 2). However, it should be noted sufficient high-quality evidence still hinders a consen that our recommendations are based on the previously sus among clinicians [11]. In addition, the wide varia published studies, which, in our analysis, cannot be tion in the treatment regimen raises concerns about considered as high-quality because a number of vari overzealous use of antioxidants. The detrimental ef ables (such as female factor, inclusion criteria, sample fects of reductive stress may be as pathological as that size esteem, etc.) were not properly reported. Therefore, of oxidative stress [23]. Moreover, the often unpredict our recommendation is that antioxidant treatment is able outcome after antioxidant supplementation in the possible and can result in improved male seminal pa context of multiple confounding factors in reproduction rameters if the condition is caused by oxidative stress. may delay the definitive treatment, particularly for It is self-evident that the therapy has to be monitored couples of advanced age. to not only avoid over-dosage of antioxidants, but also to see if it is successful or if alternative treatments are CLINICAL GUIDELINES to be considered.

While antioxidant supplementation is frequently 1. Abnormal semen quality utilized for the treatment of male factor infertility, no Antioxidants have long been investigated as a thera clear recommendations exist endorsing their use for peutic option to counteract the harmful effects of ROS specific clinical indications. Therefore, we aimed to de toxicity on various body systems. The reproductive sys velop clinical practice guidelines based on the available tem is one good example, as seminal oxidative stress is evidence to help in identifying the clinical circumstanc believed to be a common pathophysiology and various es in which antioxidant supplementation appears to oxidative stress-associated aetiologies can alter sperm be most beneficial. This systematic review included 97 quality and function. Results of our review demon articles, which investigated antioxidant treatment for strated that the majority of low-quality studies report various etiologies of male infertility. Very few studies ed a significant improvement in conventional semen explored the effect of antioxidants on semen quality of parameters and measures of sperm function. However, men with genitourinary inflammation (n=3), a hyper this result was not observed by high-quality studies. insulinemic state (n=1) and recurrent pregnancy loss in The recent Cochrane review revealed a somewhat simi female partners (n=1) (Table 2), and are hence insuf lar result [11]. While an improvement in conventional ficient for evidence-based recommendations for their semen parameters was noted over time, the findings use. On the other hand, there are some perceived high- were not reliable as a great deal of heterogeneity was quality studies available but, as highlighted above, due observed across the included studies. Moreover, antioxi to the difficulties recruiting a sufficient number of pa dants were found to lower SDF compared to placebo. tients and the small number of these studies, a statisti Recommendation: antioxidants can improve conven cal analysis will be underpowered and therefore does tional semen parameters and measures of sperm func not provide the required answer. As we have shown tion (grade C recommendation). in our analysis, the number of such studies conducted under the given circumstances and with a pre-selected 2. Varicocele set of criteria would have to be unreasonably high. Varicocele is the most common correctable cause of Yet, looking at the diverse groups of conditions where male infertility, prevalent in about 40% of men with oxidative stress is significantly involved and an anti primary infertility and up to 80% of men with second oxidant treatment would make sense, it is clear that in ary infertility [131]. Several studies have confirmed the absence of prior testing for oxidative stress and with presence of higher levels of oxidative stress in infertile out proper identification of suitable patient groups, men with varicocele in comparison to fertile men with the treatment will fail. Hence, appropriate patient or without varicocele and infertile men with idiopathic identification is essential for the success of the treat infertility [132-136]. This finding may justify the util ment. We were able to formulate recommendations for ity of antioxidants as a medical treatment strategy for antioxidant treatment for men with abnormal semen varicocele. Nonetheless, in most patients, varicocelec

48 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility tomy remains the gold standard modality that results larger number of studies are required to reach statisti in sustained improvement in semen parameters and cal significance. natural conception [137]. A systematic review of 32 studies which assessed the Most of the studies exploring the effect of antioxi impact of antioxidant therapy in IMI patients revealed dant supplementation on semen parameters and sperm an improvement of semen parameters, with the biggest function were of low-quality. While the majority of benefit observed in sperm motility [143]. Fewer studies these studies reported an improvement in outcome, the have assessed antioxidant treatment in patients with evidence supporting antioxidant use as a sole treat UMI. A recent study included 29 UMI patients who ment for varicocele is not sufficient. Our sample size were treated with a combination of antioxidants for 3 calculation confirms the need for further research in months. The authors reported a significant increase in this regard in order to obtain a statistically significant progressive motility (p=0.002), and a decrease in SDF effect. However, the reported improvement may be (p=0.03) and ORP levels (p=0.02) following treatment. clinically relevant supporting antioxidant use as an ad Greco et al [117] randomized 64 patients with UMI and junct therapy to varicocele ligation. A recent systematic elevated SDF to either treatment with vitamins C and review and meta-analysis explored antioxidant efficacy E for 2 months or placebo. While there was no signifi on improving semen quality after varicocelectomy [138]. cant difference in semen parameter results, significant The authors included 6 RCT with 576 patients receiv reduction in SDF was noted in the treatment group ing various antioxidant regimens or placebo following (p<0.001). varicocelectomy. Significant improvements in sperm Recommendation: antioxidants significantly increase concentration (p<0.001), total motility (p=0.03), progres sperm quality in men with IMI and UMI (grade B rec sive motility (p<0.001) and normal morphology (p<0.001) ommendation). were reported for the treatment group. However, preg nancy rate did not improve (p=0.36). Nonetheless, this CURRENT STATE AND FUTURE finding confirms the presence of an additive effect of RECOMMENDATIONS FOR the antioxidant therapy in patients undergoing varico ANTIOXIDANT RESEARCH celectomy. Recommendation: antioxidants in addition to varico The bivalent action of ROS as essential signaling cele ligation result in further improvement in semen molecules in physiological functions for sperm to fertil parameters (grade C recommendation). ize an oocyte [144-146], as well as in mediating a detri mental effect on sperm functionality [147-149], suggests 3. Unexplained male infertility and idiopathic that appropriate clinical indications for prescribing male infertility antioxidants and monitoring the treatment are critical Antioxidants are also commonly utilized for the clinical considerations. Therefore, it is crucial to con treatment of patients with UMI or IMI. The former is tinue to study the physiological and pathophysiological defined by failure of conception despite having normal mechanisms of ROS in the male reproductive tract and semen parameters, while the latter is characterized by its relevance for sperm production and conception. In the presence of semen abnormalities due to unknown this regard, the physiological needs of functional sper etiology. The prevalence of UMI and IMI ranges be matozoa, e.g., for the induction of capacitation and ac tween 6%–27% and 30%–58%, respectively [3,139]. Oxi rosome reaction, have to be considered. It appears that dative stress is believed to play a significant role in the three aspects of sperm physiology have to be addressed, pathophysiology of infertility of unknown origin and namely i) preservation and support of metabolism, ii) has been identified in 30%–40% of patients with UMI improvement of sperm maturation and function, and and up to 80% of patients with IMI [140-142]. iii) protection against ROS-related trauma. Therefore, Our analysis revealed that antioxidant use in men the correct ratio and concentration of antioxidants is with IMI and UMI resulted in significant improve essential for this therapeutic effect to occur and any ment in semen parameters and sperm function, as new studies must take this into account. reported by high-quality studies. While the majority Standardization of oxidative stress measurement of low-quality studies echoed similar improvements, a must also be implemented, not only because a repro

www.wjmh.org 49 https://doi.org/10.5534/wjmh.200196 ducible baseline is needed, but also because many of of ROS and antioxidant enzymes in seminal fluid and the current studies are either lacking or using differ spermatozoa. Bioavailability of the compounds and ent oxidative stress measurement and are therefore their mechanism of action should also be thoroughly difficult to compare. The assessment of oxidative stress studied. More time-points to detect the oxidative bal may also facilitate the identification of the best candi ance of spermatozoa and seminal fluid should also be dates and responders to antioxidant therapy (Fig. 4) [2]. considered. Finally, one should not only look at the Recently, the MiOXSYS system has be proposed as oral antioxidant therapy as a clinical treatment of the a standardized assessment of seminal oxidative stress male and then expect that this treatment will result due to its ease of use, cost effectiveness, and repro in the live birth of a healthy baby, but rather consider ducibility, as well as proposed evidence based clinical reproduction as a joint responsibility of both partners guidelines [150,151]. The MiOXSYS system measures where a male and a female equally contribute to the the overall redox balance in seminal fluid to directly reproductive outcome. Currently, the female partner is evaluate oxidative or reductive stress. Monitoring the not examined for possible oxidative stress, but there is treatment with antioxidants is another important as also a lack of knowledge about the impact of ROS and pect as overtreatment could lead to reductive stress- the redox level (oxidative and reductive stress) in the related infertility because the little amount of ROS, female reproductive system on oocyte development and which is essential to trigger physiological functions of maturation as well as on embryo development. A study spermatozoa for it to fertilize oocytes, would be scav by Ufer et al (2010) [152] indicated that proper embryo enged if the antioxidants are overdosed. development depends on finely tuned redox control. On For a safe implementation of oral antioxidants use the other hand, elevated levels of antioxidants may re in a clinical setting, it is important not only to discover sult in teratogenic developments [153]. the molecular mechanisms of action of the bioactive Currently, there is significant heterogeneity in tri compounds, but also the secondary effects that may als reporting antioxidants on male infertility. This arise. Personalized or adjusted prescription of oral an includes the condition investigated, type of antioxidant tioxidants can enhance efficacy, without promoting used, duration of the study, and the outcomes mea over-dosage and deleterious health effects. Methodol sured (Table 2). Furthermore, it is essential to select ogy and couple selection must be well reported. Doses previously studied antioxidant candidates as well as and duration of the treatment should be adjusted ac new potential compounds, and also determine if they cording to several factors including the detected levels act better alone or in synergy with other antioxidants

Testing ORP> 6 1.34 mV/10 sperm/mL Basic OS evaluation semen by Oxidative analysis ORP (MiOXSYS) stress

Inflammation/ Lifestyle leukocytospermia (smoking, obesity,.etc ) Varicocele (prostatitis, MAGI) MOSI

Treatment Antioxidant+antibiotics Counsel on Varicocelectomy anti-inflammatory appropriate changes

Retest for MOSI Antioxidant supplementation Fig. 4. Treatment options for male oxidative stress infertility (MOSI). OS: oxidative stress, ORP: oxidation-reduction poten- Retest for MOSI tial, MAGI: male accessory gland infec- with ORP tion.

50 www.wjmh.org Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility as has been shown in some studies [154]. Due to several has been shown to be as harmful as oxidative stress [22]. factors, including the high costs and the high number of participants needed to reach a statistically robust CONCLUSIONS study, the individuals recruited in any new study must be well selected based on strict inclusion/exclusion cri This systematic review identified a significant num teria. This means the patients should have the same ber of well-designed studies that unequivocally show infertility diagnosis and similar semen analysis: for the beneficial effects of oral antioxidants in improving example, mixing patients with azoospermia, mild oli semen parameters and pregnancy outcome. However, gozoospermia or asthenozoospermia should be avoided. despite the safety and efficacy of the antioxidant Meeting these criteria will likely be difficult at a therapy, five main factors have hindered its wide ac single institution and does not even include recruiting ceptance and implementation in the treatment of male patients for an adequate control group. Use of placebo infertility: i) lack of randomized placebo-controlled in the control group is also mandatory to avoid bias. studies that show the safety and efficacy of antioxi Notably, confounding factors, including dietary habits, dants in improving pregnancy rates in infertile couples; are often overlooked in most RCTs, but when patients ii) type of antioxidant to be used; iii) dose; iv) duration are being enrolled in a study with antioxidant supple of treatment; and v) costs. mentation, baseline antioxidant intake and diet should Although randomized placebo-controlled studies be evaluated in both partners, male and female. Fur are regarded as the gold standard in the validation thermore, an appropriate primary outcome, such as de of the safety and efficacy of therapies, given the fact crease in oxidative stress or SDF, should also be evalu that the occurrence of a pregnancy is a multifactorial ated. Finally, it is critical to set a correct time period process mainly determined by the genomic quality of for the study. A short to medium time period of 3 to 6 the egg, to show the impact of antioxidant therapy on months is considered ideal to study the antioxidant ef pregnancy outcome in randomized placebo-controlled fects on semen parameters. studies would be unrealistic and extremely difficult to perform. Given the difficulty in carrying out these ran UNRESOLVED QUESTIONS domized placebo-controlled studies and the existence of significant clinical evidence supporting the safety What makes the situation even more complicated is and efficacy of antioxidants in improving pregnancy that there are no dose-response studies in humans to outcome in infertile couples, the use of antioxidant pinpoint the optimum antioxidant dosages needed to therapy should be recommended. produce improvement in semen quality. We also still do To answer the question of what type of antioxidant not know the normal physiological range of the redox should be used, we believe that antioxidants that read levels in both males and females. For spermatozoa, a ily cross the blood-testis and blood-epididymis barriers recent study by Panner Selvam et al (2020) [24] indicat should be recommended. The formulation should be ed normal redox values between -9.76 and 1.48 mV/106 well-balanced as lipid-soluble and water-soluble antiox sperm/mL. Although these values already indicate idants together with other factors are closely interact oxidative or reductive stress conditions, the normal ing, thereby regenerating lipid-located antioxidants. If physiological range will be much narrowed in between this balance is not given, it may not only result in sub these values. In the female, one might have to consider optimal antioxidant effects, but also in paradoxical pro- variations depending on the menstrual cycle and/or the oxidant effects due to interference in redox reactions. onset of pregnancy. Besides, these values might change Concerning the dose to be used, the dose should be with age and/or health status. Furthermore, the effec high enough to restore the normal physiological cel tive bioavailability of antioxidants in the testes, the lular functions by reducing oxidative stress without epididymis and the semen is still unknown. Since many compromising the physiological role of ROS in sperm antioxidants (e.g., co-enzyme Q10, vitamin E, and carni maturation and fertilization reactions. An overdosage tines) can easily cross the blood-testis barrier, a proper which may lead to reductive stress should be avoided. balance for the redox level has to be achieved because The duration of an antioxidant therapy would have an over-dosage might lead to reductive stress, which to be adjusted according to the place where the damage

www.wjmh.org 51 https://doi.org/10.5534/wjmh.200196 occurs. If it is in the epididymis, a treatment course of Author contribution at least two weeks should be sufficient to counteract ROS-induced damage. In addition, since oxidative stress Conceptualization: AA. Writing – original draft: all the au in the epididymis is a constitutive process and antioxi thors. Writing – review & editing: all the authors. dants have no side-effects, antioxidant therapy should be recommended until pregnancy is achieved. This Supplementary Materials would apply to couples undergoing timed intercourse as well as to couples undergoing in vitro fertilization. On Supplementary materials can be found via https://doi. the other hand, if the oxidative damage is occurring in org/10.5534/wjmh.200196 the testes such as in the case of clinical varicocele, the duration of antioxidant treatment should be of at least REFERENCES three months. Finally, the cost argument has to be seen from the 1. Agarwal A, Mulgund A, Hamada A, Chyatte MR. A unique perspective of the sponsors of high quality randomized, view on male infertility around the globe. Reprod Biol Endo- double-blind placebo-controlled clinical trials. Natural crinol 2015;13:37. antioxidant formulations have very low cost, and their 2. Agarwal A, Parekh N, Panner Selvam MK, Henkel R, Shah R, use would be amply justified based on efficacy and Homa ST, et al. Male oxidative stress infertility (MOSI): pro- safety as well as for cost-saving aspects for patients posed terminology and clinical practice guidelines for man- and health systems. The problem for the possible fund agement of idiopathic male infertility. World J Mens Health ing by the pharmaceutical industry of the studies is to 2019;37:296-312. recover the high costs of a trial on a cheap antioxidant, 3. Hamada A, Esteves SC, Nizza M, Agarwal A. Unexplained which can be available over the counter, and for which male infertility: diagnosis and management. Int Braz J Urol not even intellectual property rights are available. 2012;38:576-94. In conclusion, the use of antioxidants that readily 4. Leisegang K, Dutta S. Do lifestyle practices impede male fer- cross the blood-testis and blood-epididymis barriers tility? Andrologia 2020. doi: 10.1111/and.13595 [Epub]. should be recommended. Their efficacy, lack of side- 5. Leisegang K, Henkel R. Oxidative stress: relevance, evalua- effects and low costs should encourage their wider ac tion, and management. In: Rizk B, Agarwal A, Sabanegh ES, ceptance and implementation among infertility special editors. Male infertility in reproductive medicine: diagnosis ists for their use in the treatment of male infertility and management. Boca Raton: CRC Press; 2019;119-28. and infertile couples. Despite these favorable aspects of 6. Cardoso JP, Cocuzza M, Elterman D. Optimizing male fertil- antioxidant treatments for idiopathic and UMI, more ity: oxidative stress and the use of antioxidants. World J Urol high-quality research is needed to understand the ef 2019;37:1029-34. fects of ROS and antioxidants on the human fertilizing 7. Kuchakulla M, Soni Y, Patel P, Parekh N, Ramasamy R. A sys- potential in both male and female. However, the practi tematic review and evidence-based analysis of ingredients in cality of conducting such studies remains questionable. popular male fertility supplements. Urology 2020;136:133-41. 8. Showell MG, Brown J, Yazdani A, Stankiewicz MT, Hart RJ. ACKNOWLEDGEMENTS Antioxidants for male subfertility. Cochrane Database Syst Rev 2011;(1):CD007411. Authors are thankful to the artists from the Cleve 9. Showell MG, Mackenzie-Proctor R, Brown J, Yazdani A, land Clinic’s Center for Medical Art & Photography for Stankiewicz MT, Hart RJ. Antioxidants for male subfertility. their help with the illustrations. The study was sup Cochrane Database Syst Rev 2014;(12):CD007411. ported by the American Center for Reproductive Medi 10. Majzoub A, Agarwal A. Systematic review of antioxidant cine (Andrology Research Fund #500000105879). types and doses in male infertility: benefits on semen parameters, advanced sperm function, assisted reproduction and Conflict of Interest live-birth rate. Arab J Urol 2018;16:113-24. 11. Smits RM, Mackenzie-Proctor R, Yazdani A, Stankiewicz MT, The authors have nothing to disclose. Jordan V, Showell MG. Antioxidants for male subfertility. Co- chrane Database Syst Rev 2019;3:CD007411.

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