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

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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.
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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: agarwaa@ccf.org, Website: www.Clevelandclinic.org/ReproductiveResearchCenter
Copyright © 2021 Korean Society for Sexual Medicine and Andrology
Review Article
pISSN: 2287-4208 / eISSN: 2287-4690
World J Mens Health Published online Jan 15, 2021
https://doi.org/10.5534/wjmh.200196
Male reproductive health and infertility
Utility of Antioxidants in the Treatment of Male
Infertility: Clinical Guidelines Based on a Systematic
Review and Analysis of Evidence
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 addi-
tional double-blind, randomized, placebo-controlled trials, and 2) proposed updated evidence-based clinical guidelines for anti-
oxidant 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: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.
https://doi.org/10.5534/wjmh.200196
2www.wjmh.org
INTRODUCTION
Infertility affects approximately 15% of couples glo-
bally, with 2.5%–12% believed to be solely due to male
factors. The incidence of male factor infertility varies
according to the geographical location, ranging from
20% to 70% [1]. The causes of male infertility are diverse,
including genetic causes, varicocele, reproductive tract
infections, obstructive or non-obstructive azoospermia,
male hypogonadism, and anti-sperm antibodies. How-
ever, a large proportion of cases remain unexplained
(unexplained male infertility, UMI) (±15%) or idiopathic
(idiopathic male infertility, IMI) (30%–50%) in the ab-
sence of identifiable female factors [2,3]. In addition,
numerous environmental and lifestyle factors have been
associated with poor reproductive outcomes in males [4].
Importantly, oxidative stress has been established as a
signif icant mediator in many known causes and risk
factors of male infertility, and has further been associ-
ated with 30% to 80% of IMI cases in a condition termed
male oxidative stress infertility (MOSI) [2,5]. Therefore,
the use of antioxidants to reduce oxidative stress across
a range of etiologies and risk f actors of male inf ertility
has gained increasing attention. This is supported by the
wide availability of oral antioxidants, excellent safety
and bioavailability profiles, and that antioxidants are
considered relatively cost ef fective [5,6]. Therefore, there
is a growing trend of prescribing antioxidants to all
males with infertility, even without complete evaluation
or relevant guidelines [7].
Exogenous administration of antioxidants has been
explored for decades, and the ef f ects of several antioxi-
dants on male fertility have been extensively reported.
Numerous trials of different qualities using various
antioxidants as mono- or poly-formulations, which may
include pharmacologically-active herbal extracts, have
been reported [8-11]. The topic was f irst summarized
by a Cochrane meta-analysis in 2011, with updated
reviews in 2014 and 2019 [8,9,11]. These reviews inves-
tigated the therapeutic benefit of male antioxidant
treatment f or couples undergoing assisted reproduc-
tive technology (A RT). Based on limited randomized
controlled trials (RCTs), the reviews concluded that low
level evidence supports antioxidant therapy in infer-
tile males to increase pregnancy and live birth rates,
with no evidence for increased risk of miscarriage
[8,9,11]. Majzoub and Agarwal (2018) [10] conducted a
systematic review on antioxidant treatment in infer-
tile men, concluding that antioxidants have a positive
eff ect on male f ertility, including semen parameters
and advanced sperm f unction, ART outcomes and live
birth rates. Antioxidants that are commonly used clini-
cally and investigated scientifically as either an indi-
vidual application or in combination include vitamin
A, vitamin C, vitamin E, carnitine, N-acetyl cysteine,
coenzyme Q10 and lycopene, along with important
antioxidant co-factors zinc, selenium, and f olic acid, as
these compounds are significantly involved in essential
sperm f unctions (Fig. 1) [12-15]. However, the outcomes
of clinical trials included in the systematic reviews are
not consistent, ranging f rom clear benefit to no clinical
effect of the treatment, or even having significant de-
trimental effects [16-20]. The reasons for this inconsis-
tency are multifactorial and include: small numbers of
participants in the studies, variable treatment regimens,
dosages, treatment duration, and the lack of placebo-
controlled studies. In addition, many of the trials did not
evaluate final reproductive outcomes, such as live birth
rate, but only certain specif ic aspects such as seminal
volume, sperm concentration and motility, morpho-
logy, seminal levels of reactive oxygen species (ROS) or
oxidative stress. Furthermore, these antioxidants were
Vitamin C
Vitamin E
Thiamin
L
ycopene
N-Acetyl-L-cysteine
L-Arginine
L-carnitine
CoQ10
Molybdenum
Sperm motility
+morphology
Zinc
selenium
Folic acid
Acetyl-L-carnitine
Motility
Motility
Chromatin
DNA damage
DNA damage
DNA damage
Concentration
Sperm
Vitality
Lipid peroxidation
Protamination &
Fig. 1. Individual antioxidant com-
pounds that have significant effects on
sperm functions. CoQ: coenzyme Q.
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
3
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often given in non-proven dosages, thus neglecting the
fine bodily redox balance that is necessary for normal
physiology, including reproductive functions [21]. If the
dosage is too low, the treatment might be inef f ective; if
it is too high, it can result in an excess of antioxidants
causing ‘reductive stress’, which is as detrimental as
oxidative stress [22,23]. Reductive stress due to inappro-
priate antioxidant dosage may lead to infertility [23,24].
In this regard, high dosages of vitamin E have been
shown to have adverse ef fects [20]. Recently more ba-
lanced antioxidant f ormulations have shown promising
results whereby seminal oxidative stress was reduced,
sperm f unction improved and pregnancies achieved
[ 25 ,2 6 ].
With the current rationale and increased use of anti-
oxidants to counteract male infertility, and the hetero-
geneous and inconsistent data currently available, this
study aims to: 1) systematically review the current evi-
dence for antioxidant use to ameliorate male infertility;
and 2) propose updated evidence-based clinical guide-
lines for the use of antioxidants in male infertility.
MATERIALS AND METHODS
1. Literature search strategy
In order to support the development of clinical
guidelines for antioxidant use in male infertility, a sys-
tematic review of the available clinical evidence was
performed to systematically identify relevant clinical
trials investigating the impact of antioxidant therapy
on semen quality. A literature search was performed
according to the Preferred Reporting Items for System-
atic Reviews and Meta-Analyses (PRISMA) guidelines
[27]. The Scopus database was chosen as it currently in-
cludes over 1.4 billion cited references, with over 70,000
indexed articles (https://www.elsevier.com/solutions/
scopus/how-scopus-works/content). The search was con-
ducted on July 15th, 2020 to identify studies investi-
gating the use of antioxidants in the treatment of male
infertility, with no restriction on publication date.
The f ollowing keyword strings and Boolean Opera-
tors were used: TITLE-ABS-KEY (“antioxidant*”)
AND TITLE-ABS-KEY (“male infertil*” OR “infertile
male*” OR “inf ertile men” OR “male subfertil*” OR
“male steril*” OR “sperm*” OR “seminal” OR “semen”).
Further specifications of the search are presented in
Supplementary Table 1. Automatic f ilters were used in
the database to specifically include only English origi-
nal articles and exclude other types of publication such
as book chapters, conf erence papers, editorials, notes,
letters, short surveys, erratum, and books.
The articles identified through the keyword search
were subsequently screened manually by title, key-
words and abstract for eligibility. This screening was
independently done by three researchers (RF; KL;
MKPS), and the number of articles excluded through
screening was recorded. Full text articles were then
reviewed for eligibility using the inclusion and exclu-
sion criteria provided in Table 1 , and the number of
articles excluded based on these criteria was recorded.
Data was subsequently extracted from the eligible
articles, including the clinical trial design, the type of
antioxidant or antioxidant formulation used, the clini-
cal condition under investigation, the evaluation of se-
men parameters and/or sperm f unction tests (i.e ., sperm
DNA f ragmentation [SDF], oxidative stress markers,
capacitation/acrosome reaction, and zona binding test)
as well as reproductive outcomes (i.e., fertilization,
implantation, pregnancy, miscarriage, and live birth
ra t es).
2. Evaluation of study quality
The quality of all studies included was evaluated by
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-
phology) and/or sperm function parameters (sperm DNA fragmen-
tation, seminal oxidative stress markers, mitochondrial membrane
integrity) reported after antioxidant treatment
Non-english studies
https://doi.org/10.5534/wjmh.200196
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over, the quality of RCT was further evaluated by
using the Cochrane Risk of Bias [29] and the JADAD
score [30], as well as by evaluating the adherence to
CONSORT guidelines [31]. Based on a combination of
these quality evaluation tools, the studies were catego-
rized into “low” (0) and “high” (1) quality. All uncon-
trolled studies were considered “low-quality”, in com-
parison with controlled studies, which were evaluated
according to the criteria reported in Supplementary
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
the effect of antioxidant treatment on male infertility,
published f rom Januar y 2019 to July 2020, were f urther
ranked based on study design, the sample size analysed,
the inclusion/exclusion criteria used for selecting the
population, the antioxidant regimen used, the length
of treatment, the assessment of oxidative stress mark-
ers, pregnancy and live birth rates. This range of time
was specifically selected to gain an understanding of
the most recent evidence on the antioxidant therapy
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
this study. The system provides a total score of a maxi-
mum of 12 points and a classification of the articles
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
(Oxford Centre f or 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-
loads/2014/06/CEBM-Levels-of-Evidence-2.1.pdf).
3. Statistical analysis
Statistical analysis was performed using MedCalc
statistical software version 19.5.3. (MedCalc Software
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
to antioxidant treatment on semen parameters and
sperm function such as oxidative stress and SDF are
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
predict the required sample size to attain a statistical
Table 2. Articles investigating the impact of antioxidant treatment on reproductive outcomes
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors
(out of 3)
Checklist
for causal
risk factors
(out of 7)
Infertile men 1 Kessopoulou et
al (1995) [19]
RCT blinded α-tocopheryl ac-
etate (Ephynal,
F. Hoffman-La
Roche ltd)
300 mg/daily for
3 months
30 infertile men No difference in se-
men parameters
before and after
treatment
No difference in
ROS levels
Improved zona
binding
2 3 7 Unclear risk
of bias for
random
sequence
generation,
allocation
conceal-
ment, selec-
tive report-
ing, other
sources and
blinding
(outcome
assessment)
20 4 1
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
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Table 2. Continued 1
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
2 Roseff (2002)
[32]
Uncontrolled
(open label)
Pycnogenol
(Horphag Re-
search, Geneve,
Switzerland)
200 mg daily for
3 months
19 infertile men No difference in se-
men parameters
Improved sperm
binding capacity
2 3 3 N/A N/A N/A 0
3 Keskes-Ammar
et al (2003)
[18]
RCT unblinded Vitamin E (400
mg) (Ephynal
100 mg, 2 tab-
lets) or selenium
(225 µg) for 3
months
54 infertile men Improved sperm
motility
Reduced MDA
levels
1 3 4 No risk of bias
identified
12 3 0
4 Tremellen et al
(2007) [33]
RCT blinded Menevit (Bayer,
Sydney, Austra-
lia)
1 capsule/day
for 3 months
Men with evi-
dence of seminal
oxidative stress
and SDF>25%
by TUNEL. The
total number of
patients is not
clearly reported.
No differences
between treated
and placebo
for fertilization,
implantation,
pregnancy, and
miscarriage rates
Live pregnancy
rate higher in
treated patients
0 3 7 No risk of bias
identified
19 3 0
5 Ménézo et al
(2007) [20]
Uncontrolled
(open label)
Vitamins C and E
(400 mg each),
ß-carotene (18
mg), zinc (500
µmoL), selenium
(1 µmoL) for 3
months
58 patients experi-
encing 2 previous
failures of IVF or
ICSI, and DFI and
chromatin decon-
densation>15%
Reduced SDF but
higher sperm
decondensation
0 3 3 N/A N/A N/A 0
6 Tunc et al
(2009) [34]
Uncontrolled
(open label)
Menevit (Bayer
Australia Ltd,
Sydney, Austra-
lia)
1 capsule/daily
for 3 months for
a maximum of 3
months
50 infertile men
with high OS
No difference in se-
men parameters
Reduced SDF, ROS
and apoptotic
markers
Improved DNA
protamination
3 3 3 N/A N/A N/A 0
https://doi.org/10.5534/wjmh.200196
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Table 2. Continued 2
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
7 Shukla et al
(2010) [35]
Uncontrolled
(open label)
Mucuna pruriens
seed powder
5 g/daily for 3
months
120 infertile men Improved sperm
count and
motility, seminal
plasma lipid
peroxide levels,
SOD, catalase,
GSH and ascorbic
acid
1 3 6 N/A N/A N/A 0
8 da Silva et al
(2013) [17]
RCT blinded Folic acid
5 mg/daily for 3
months
70 infertile men No difference in se-
men parameters
3 3 7 No risk of bias
identified
15 5 1
9 Bejarano et al
(2014) [36]
Uncontrolled
(open label)
Melatonin
6 mg/daily for
45 days
30 infertile men Improved semen
parameters, uri-
nary and semen
TAC
Reduced SDF
Improved embryo
quality
3 3 3 N/A N/A N/A 0
10 Martínez-Soto
et al (2016)
[37]
Uncontrolled
(open label)
1.5 g capsules of
docosahexae-
noic acid oil/
daily for 10
weeks
57 infertile men No changes in se-
men parameters
Increased seminal
TAC, reduced
DNA damage
0 3 3 N/A N/A N/A 0
11 Chattopadhyay
et al (2016)
[16]
Uncontrolled
(open label)
L-Carnitine,
Acetyl-L-Car-
nitine, CoQ10,
Lycopene,
Zinc, Folic acid,
Vitamin B12,
Selenium, Fruc-
tose, and citric
acid (dosage not
reported) for 6
months
115 infertile men Increased sperm
count, motility,
TAC
Reduced ROS
levels
0 3 3 N/A N/A N/A 0
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
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Table 2. Continued 3
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
12 Hosseini et al
(2016) [38]
RCT blinded Ginger powder
250 mg/daily for
3 months
100 patients with
SDF≥15%
No difference in se-
men parameters
Decreased SDF
2 3 7 Unclear risk
of bias
for other
sources
23 5 1
13 Stenqvist et al
(2018) [39]
RCT blinded Vitamin C (30
mg), vitamin E
(5 mg), vitamin
B12 (0.5 ug), l-
carnitine (750
mg), coenzyme
Q10 (10 mg),
folic acid (100
ug), zinc (5 mg),
selenium (25
ug) with malto-
dextrin, calcium
carbonate, citric
acid, ste-
viol glycoside,
flavours, beta-
carotene, silicon
dioxide/daily for
6 months
77 infertile men
with DFI ≥25%
Improved sperm
concentration,
no change in
DNA damage
4 3 7 No risk of bias
identified
19 5 1
14 Ahmad et al
(2008) [40]
Uncontrolled
(open label)
Mucuna pruriens
seed powder
5 g/daily for 3
months
60 infertile men Improved volume,
sperm concen-
tration, count,
motility
Reduced MDA
levels
0 3 5 N/A N/A N/A 0
15 Alizadeh et al
(2018) [41]
RCT blinded Curcumin
80 mg/daily for
10 weeks
60 infertile men Increased sperm
count, concen-
tration, total mo-
tility and vitality,
TAC
Reduced MDA and
inflammatory
biomarkers
0 3 7 Unclear risk
of bias
for other
sources
19 4 0
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Table 2. Continued 4
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
16 Salehi et al
(2019) [42]
Uncontrolled
(open label)
Vitamin E (50 mg),
vitamin C (500
mg) and CoQ10
(100 mg) for 3
months
485 infertile men
with DFI>27% by
SCSA
Improved semen
parameters
Reduced DNA
damage
Pregnancy
rate=16.8%
5 3 3 N/A N/A N/A 0
17 Hasoon
(2019)[43]
Uncontrolled
(open label)
L-arginine (1 g)
and CoQ10
(200 mg) for 8
months
24 infertile men Improved volume,
sperm count,
motility, and
normal morphol-
ogy
2 3 3 N/A N/A N/A 0
18 Nurmawati et
al (2020) [44]
RCT blinded Astaxanthin
8 mg/daily for 1
month
25 infertile men Improved sperm
concentration,
motility, and
morphology
Reduced MDA and
8-OHdG levels
4 3 7 Unclear risk
of bias for
selective
reporting
and other
sources;
high risk
of bias for
random
sequence
generation,
allocation
conceal-
ment and
blinding
(outcome
assessment)
15 3 0
19 Hadi et al
(2020) [45]
Uncontrolled
(open label)
L-carnitine
2 g/daily for 3
months
58 infertile men Improved sperm
count, total mo-
tility, and normal
morphology
In serum: reduced
FSH and LH
levels, increased
testosterone, and
inhibin levels
2 3 3 N/A N/A N/A 0
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
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Table 2. Continued 5
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
20 Schisterman et
al (2020) [46]
RCT blinded Folic acid 5 mg/
daily and 30
mg zinc for 6
months
1,185 male partners
of couples plan-
ning IVF for infer-
tility treatment
No changes in se-
men parameters;
improved SDF
by Comet assay;
no significant
differences in
β-HCG–detected
pregnancy, clini-
cal intrauterine
pregnancy, ec-
topic pregnancy,
pregnancy with
multiple fetuses,
live birth rate
2 3 7 Unclear risk
of bias for
random
sequence
generation,
allocation
conceal-
ment, other
sources,
selective re-
porting and
blinding
14 3 0
Varicocele 21 Comhaire et al
(2000) [47]
Uncontrolled
(open label)
Acetylcysteine
(600 mg) or cap-
sules providing
a daily amount
of β-carotene
(30 mg) and
α-tocopherol
(180 mg)/daily.
In addition,
capsules con-
taining essential
fatty acids for a
daily intake of
docosahexae-
noic acid (1 g),
gammalinolenic
acid (0.25 g) and
arachidonic acid
(0.10 g) for 6
months
7 idiopathic pa-
tients
11 varicocele
patients
History of cryptor-
chidism (n=2), pa-
tients with male
accessory gland
infection (n=7),
immunological
infertility (n=4),
endocrine cause
(n=1)
Improved sperm
concentration
and acrosome
reaction
Reduced ROS
levels and 8-OH-
dG levels
2 3 3 N/A N/A N/A 0
https://doi.org/10.5534/wjmh.200196
10 www.wjmh.org
Table 2. Continued 6
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors
(out of 3)
Checklist
for causal
risk factors
(out of 7)
22 Paradiso
Galatioto et
al (2008)[48]
RCT unblinded Ccommercial
preparation of:
NAC (10 mg/kg/
die), vitamin C
(3 mg/kg/die), vi-
tamin E (0.2 mg/
kg/die), vitamin
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
(8 IU/kg/die), cal-
cium (1 mg/kg/
die), magnesium
(0.35 mg/kg/die),
phosphate (0.45
mg/kg/die), iron
(0.2 mg/kg/ die),
manganese (0.01
mg/kg/die), cop-
per (0.02 mg/kg/
die), zinc (0.01
mg/kg/die) for a
minimum of 90
days
42 varicocele
patients with
persistent oligo-
spermia 6 months
after retrograde
embolization
Improved semen
parameters
No change in
pregnancy rate
3 3 7 No risk of bias
identified
16 5 1
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
11
www.wjmh.org
Table 2. Continued 7
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors
(out of 3)
Checklist
for causal
risk factors
(out of 7)
23 Oliva et al
(2009) [49]
Uncontrolled
(open label)
Pentoxifylline (1.2
g), folic acid (5
mg) and zinc
sulfate (66 mg)
for 3 months
36 varicocele
patients
Improved semen
parameters
2 3 3 N/A N/A N/A 0
24 Festa et al
(2014)[50]
Uncontrolled
(open label)
CoQ10
100 mg/daily for
3 months
38 varicocele
patients
Sperm concentra-
tion, progressive
motility, and TAC
0 3 4 N/A N/A N/A 0
25 Pourmand et al
(2014)[51]
RCT unblinded L-carnitine
750 mg/daily for
6 months
100 varicocele
patients
No changes in se-
men parameters,
SDF and prot-
amine damage
assay
0 3 7 Unclear risk
of bias for
random
sequence
generation,
allocation
conceal-
ment, and
incomplete
outcome
data
14 2 0
26 Nematollahi-
Mahani et al
(2014)[52]
RCT unblinded A) Zinc sulphate/
folic acid
B) Folic acid (5
mg/daily)
C) Zinc sulphate
(66 mg/daily)
D) Placebo
For 6 months
160 varicocele
patients
No difference in
TAC between the
groups
Increased SOD
activity in group
A and C
0 3 7 Unclear risk
of bias for
random
sequence
generation,
allocation
conceal-
ment, selec-
tive report-
ing, other
sources, and
incomplete
outcome
data
12 2 0
https://doi.org/10.5534/wjmh.200196
12 www.wjmh.org
Table 2. Continued 8
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
27 Cyrus et al
(2015)[53]
RCT blinded Vitamin C
250 mg/daily for
3 months
115 varicocele
patients
Improved semen
parameters
2 3 6 Unclear risk
of bias for
random
sequence
generation,
allocation
conceal-
ment, se-
lective
reporting,
other sourc-
es, blinding
(partici-
pants and
personnel,
outcome
assess-
ment), and
incomplete
outcome
data
18 5 0
28 Gual-Frau et al
(2015) [54]
Uncontrolled
(open label)
L-Carnitine (1,500
mg), vitamin C
(60 mg), CoQ10
(20 mg), vitamin
E (10 mg),
vitamin B9 (200
μg), vitamin B12
(1 μg), zinc (10
mg), selenium
(50 μg) for 3
months
20 varicocele
patients
Improved total
sperm count and
reduced SDF
2 3 3 N/A N/A N/A 0
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
13
www.wjmh.org
Table 2. Continued 9
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
29 Barekat et al
(2016)[55]
RCT blinded N-acetyl-L-cyste-
ine (NAC)
200 mg/daily for
3 months
35 varicocele
patients
No changes for
sperm concen-
tration, motility,
morphology, %
of ROS negative
sperm and inten-
sity of sperm ROS
Improved normal
protamine
content and DNA
integrity
Pregnancy
rate: NAC
group=33.4%,
control
group=10%. No
p-value reported
1 3 4 Unclear risk
of bias for
random
sequence
generation,
allocation
conceal-
ment, selec-
tive report-
ing, other
sources and
blinding
(partici-
pants and
personnel
as well as
outcome
assessment)
12 2 0
30 Kızılay and
Altay
(2019)[56]
RCT unblinded L-carnitine
fumarate (2 g),
Acetyl-L- carni-
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
mg)/daily for 6
months
90 varicocele
patients
Improved semen
parameters
Higher pregnancy
rate
3 3 7 High risk of
bias for
random
sequence
generation,
allocation
conceal-
ment, other
sources,
blinding
(partici-
pants and
personnel,
outcome as-
sessment),
incomplete
outcome
data
19 2 0
https://doi.org/10.5534/wjmh.200196
14 www.wjmh.org
Table 2. Continued 10
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
31 Ardestani
Zadeh et al
(2019)[57]
RCT unblinded Folic acid (5 mg),
Selenium (200
µg) and vitamin
E (400 IU)/daily
for 6 months
60 varicocele
patients
Improved sperm
count and motil-
ity
2 3 7 Unclear risk
of bias for
allocation
conceal-
ment, other
sources;
high risk
of bias for
blinding
(partici-
pants and
personnel,
outcome
assessment)
24 4 1
Abnormal se-
men quality
32 Suleiman et al
(1996)[58]
RCT blinded Vitamin E
300 mg/daily for
6 months
Oligoastheno-
(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)
Improved sperm
motility
Reduced MDA
levels
Higher pregnancy
and live birth
rates
4 3 7 Unclear risk
of bias for
random
sequence
generation,
allocation
conceal-
ment, other
sources,
blinding
(partici-
pants and
personnel,
outcome as-
sessment),
incomplete
outcome
data
12 3 0
33 Rolf et al
(1999)[59]
RCT blinded Vitamin C (1,000
mg) and Vitamin
E (800 mg)/daily
for 56 days
31 asthenozoosper-
mic patients
No changes in se-
men parameters
0 3 7 No risk of bias
identified
21 5 1
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
15
www.wjmh.org
Table 2. Continued 11
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
34 Vicari and
Calogero
(2001)[60]
Uncontrolled
(open label)
Carnitene®
(Sigma-Tau,
Pomezia-Rome,
Italy)
Twice/day
for3 months,
followed by
a treatment-
free period of 3
months
54 OAT patients
with prostato-
vesiculo-epididy-
mitis
Improved sperm
progressive mo-
tility and viability
Reduced ROS
production
Higher pregnancy
rate
3 3 3 N/A N/A N/A 0
35 Suzuki et al
(2003)[61]
Uncontrolled
(open label)
Sairei-to
9.0 g/daily for 3
months
16 healthy men
47 non-nor-
mozoospermic
patients
Improved sperm
concentration,
and total motility
No change in SOD
activity
3 3 3 N/A N/A N/A 0
36 Balercia et al
(2004)[62]
Uncontrolled
(open label)
CoQ10 (Phar-
maNord, Veyle,
Denmark)
400 mg/daily for
6 months
22 asthenozoosper-
mic patients
Improved progres-
sive motility after
treatment, which
reduced after 6
months of wash-
out
Pregnancy
rate=2.4%, with
3 out of 22 pa-
tients achieving
a spontaneous
pregnancy
4 3 4 N/A N/A N/A 0
37 Piomboni et al
(2008)[63]
Uncontrolled
(open label)
Fattore M
(Progine, Flor-
ence, Italy)
2 tables/day for
3 months
51 asthenotera-
tozoospermic
patients
Improved semen
parameters and
leukocytosper-
mia
Reduced SDF
1 3 6 N/A N/A N/A 0
https://doi.org/10.5534/wjmh.200196
16 www.wjmh.org
Table 2. Continued 12
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
38 Ghanem et al
(2010)[64]
RCT blinded Clomiphene
citrate (25 mg/
day) and vitamin
E (400 mg/day)
for 6 months
60 oligoasthe-
nozoospermic
patients
Increased sperm
concentration
and motility
Higher pregnancy
rate
0 3 7 Unclear risk
of bias
for other
sources and
blinding
(partici-
pants and
personnel,
outcome
assessment)
17 4 0
39 Ahmad et al
(2010)[65]
Uncontrolled
(open label)
Withania som-
nifera
5 g/daily for 3
months
Oligo- (n=25),
astheno- (n=25)
and normozoo-
spermic (n=25)
patients
Improved sperm
count and motil-
ity, SOD, catalase,
and glutathione
levels
Decreased MDA
and Protein
Carbonyl levels
2 3 4 N/A N/A N/A 0
40 Nadjarzadeh et
al (2011)[66]
RCT blinded CoQ10 capsules
(Nutraceutical
Science Insti-
tute, NC, USA)
200 mg/daily for
3 months
60 OAT patients No changes in se -
men parameters
Reduced MDA and
improved TAC
4 3 7 Unclear risk
of bias for
random
sequence
generation,
allocation
conceal-
ment, other
sources and
blinding
(partici-
pants and
personnel);
high risk
of bias for
incomplete
outcome
data
20 4 1
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
17
www.wjmh.org
Table 2. Continued 13
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
41 Shukla et al
(2011)[67]
Uncontrolled
(open label)
Withania som-
nifera
5 g/daily for 3
months
Oligo- (n=25),
astheno- (n=25)
and normozoo-
spermic (n=25)
patients
Decreased
intracellular ROS
and apoptosis;
increased levels
of Cu2+, Zn2+,
Fe2+ and Au2+
2 3 4 N/A N/A N/A 0
42 Safarinejad
(2011)[68]
RCT blinded Eicosapentanoic
(1.12 g) and
docosahexae-
noic (0.72 g)
acid/daily for
8 months
211 OAT patients Improved total
sperm count,
concentration,
motility, normal
morphology,
seminal SOD, and
catalase
3 3 7 Unclear risk
of bias
for other
sources and
incomplete
outcome
data
21 5 1
43 Safarinejad
(2011)[69]
RCT blinded Pentoxifylline
800 mg/daily for
6 months
278 OAT patients No changes in
semen param-
eters, seminal
SOD, catalase,
and reproductive
hormones
3 3 7 Unclear risk
of bias
for other
sources and
incomplete
outcome
data
19 5 0
44 Moslemi and
Tavanbakhsh
(2011) [70]
Uncontrolled
(open label)
Selenium (200
µg), vitamin E
(400 units)/daily
for 100 days
690 asthenoterato-
spermic patients
Improved semen
parameters
Higher spontane-
ous pregnancy
1 3 3 N/A N/A N/A 0
45 Safarinejad et
al (2011)[71]
RCT blinded Crocus sativus
60 mg/daily for
26 weeks
260 OAT patients No changes in
semen param-
eters, SOD and
catalase-like
activity, LH, FSH,
PRL, TSH, testicu-
lar volume
2 3 7 Unclear risk
of bias for
random
sequence
generation,
selective
reporting
20 5 1
46 Safarinejad
(2012) [72]
Uncontrolled
(open label)
CoQ10
300 mg/daily for
12 months
287 OAT patients Improved semen
parameters
No change in
pregnancy and
miscarriage rates
2 3 4 N/A N/A N/A 0
https://doi.org/10.5534/wjmh.200196
18 www.wjmh.org
Table 2. Continued 14
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
47 Abad et al
(2013)[73]
Uncontrolled
(open label)
Androferti (Q
Pharma Labora-
tories, Alicante,
Spain)
1 capsule/daily
for 3 months
20 asthenotera-
tozoospermic
patients
Improved sperm
concentration,
motility, vital-
ity, morphology,
DNA integrity
Pregnancy
rate=5%
2 3 3 N/A N/A N/A 0
48 Ajayi et al
(2013)[74]
Uncontrolled
(open label)
Vitamin C (200
mg), vitamin E
(200 mg), folic
acid (1 mg), zinc
(50 mg), sele-
nium (200 μg), n-
acetyl-L-cysteine
(100 mg), L-
carnitine (600
mg), citrulline
(600 mg), gluta-
thione red. (100
mg), lycopene (8
mg), CoQ10 (30
mg)/daily for at
least 2 months
Oligo- (n=20),
astheno- (n=33),
OAT (n=42)
patients
65 healthy men
Improved semen
parameters
3 3 3 N/A N/A N/A 0
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
19
www.wjmh.org
Table 2. Continued 15
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
49 Nadjarzadeh et
al (2014)[75]
RCT blinded CoQ10 (Nutra-
ceutical Science
Institute, NC,
USA)
200 mg/daily for
3 months
60 OAT patients No changes in se -
men parameters
Increased seminal
level of CoQ10,
catalase and SOD
activity; reduced
level of seminal
plasma 8-iso-
prostane
4 3 7 Unclear risk
of bias for
random
sequence
generation,
allocation
conceal-
ment, se-
lective
reporting,
other sourc-
es, blinding
(participants
and person-
nel, outcome
assess-
ment), and
incomplete
outcome
data
18 3 0
50 Raigani et al
(2014)[76]
RCT blinded Folic acid (5
mg) and zinc
sulphate (220
mg)/daily for 4
months
83 OAT patients No difference in se-
men parameters
Increased sperm
chromatin integ-
rity
2 3 7 Unclear risk
of bias for
allocation
conceal-
ment, other
sources
20 4 1
51 Kobori et al
(2014)[77]
Uncontrolled
(open label)
CoQ10 (120 mg),
vitamin C (80
mg), vitamin E
(40 mg)/daily
for 6 months
169 OAT patients Improved sperm
concentration
and motility
48 (28.4%)
pregnancies
achieved; of
those, 16 were
spontaneous and
32 by using ART
0 3 3 N/A N/A N/A 0
https://doi.org/10.5534/wjmh.200196
20 www.wjmh.org
Table 2. Continued 16
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
52 Thakur et al
(2015)[78]
Uncontrolled
(open label)
Ubiquinol
150 mg/daily for
6 months
60 OAT patients Improved sperm
concentration,
total and pro-
gressive motility
Testosterone
unchanged
0 3 3 N/A N/A N/A 0
53 Kobori et al
(2015)[79]
Uncontrolled
(open label)
Edicare (KOBAYAS-
HI Pharmaceu-
tical Co., Ltd,
Japan)
6 pills/daily for 3
months
47 OAT patients Improved sperm
concentration
3 3 3 N/A N/A N/A 0
54 Hadwan et al
(2015)[80]
Uncontrolled
(open label)
Zinc sulphate
440 mg/daily for
3 months
60 asthenozoosper-
mic patients
60 healthy men
Improved volume,
progressive
motility, total
sperm count, and
catalase activity
3 3 3 N/A N/A N/A 0
55 Al-Hilli et al
(2009)[81]
Uncontrolled
(open label)
Simvastation
tablet
40 mg/daily for
3 months
Astheno- (n=1),
oligoastheno-
(n=2), OAT (n=7),
terato- (n=7),
asthenonecro-
(n=2), asthe-
noterato- (n=20),
asthenoteratone-
cro- (n=4), 0ligo-
asthenoteratone-
crozoospermic
(n=2) patients
Improved sperm
motility and
normal sperm
morphology
Decreased MDA
level
2 3 4 N/A N/A N/A 0
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
21
www.wjmh.org
Table 2. Continued 17
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
56 Martinez et al
(2015)[82]
RCT blinded Resveratrol 25 mg/
daily
SG1002 (Nuevas
Alternativas
Naturales Ther-
mafat, S.A. de
C.V., Monterrey,
Mexico) 750 mg/
daily for 75 days
54 oligoasthe-
nozoospermic
patients
Improved sperm
concentration
and motility
0 3 7 Unclear risk
of bias for
allocation
conceal-
ment, other
sources;
high risk
of bias for
incomplete
outcome
data
18 5 0
57 Gvozdjáková et
al (2015)[83]
Uncontrolled
(open label)
Carni-Q-Nol
(Tishcon Corp.,
Westbury, NY,
USA)
2 softsules
for the first
3 months, 3
softsules for the
last 3 months
40 oligoasthe-
nozoospermic
patients
Improved sperm
concentration
Reduced con-
centrations of
α-tocopherol
and γ-tocopherol
in seminal fluid,
as well as TBARS,
a marker of lipid
peroxidation
Pregnancy in 45%
of couples
0 3 3 N/A N/A N/A 0
58 ElSheikh et al
(2015)[84]
RCT unblinded A) Vitamin E (400
mg/daily)
B) Clomiphene
citrate (25 mg/
daily)
C) Vitamin
E+clomiphene
citrate for 6
months
90 oligoasthe-
nozoospermic
patients
Improved sperm
concentration
in group B and
C, while total
sperm motility
improved in all
groups
0 3 7 Unclear risk
of bias
for other
sources
15 3 0
59 Montanino
Oliva et al
(2016)[85]
Uncontrolled
(open label)
(Andrositol, Lo.Li.
Pharma s.r.l.,
Rome, Italy)
2 capsules/daily
for 3 months
45 asthenozoosper-
mic patients
Improved concen-
tration, motility,
normal morphol-
ogy
0 3 3 N/A N/A N/A 0
https://doi.org/10.5534/wjmh.200196
22 www.wjmh.org
Table 2. Continued 18
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
60 Singh et al
(2016)[86]
Uncontrolled
(open label)
Tablet Fertisure M
(Sun Pharma)
Twice/day for 3
months
7 oligozoospermic
patients
31 oligoasthe-
nozoospermic
patients
2 OAT patients
Improved sperm
count and motil-
ity, glutathione
level
Reduced MDA level
2 3 3 N/A N/A N/A 0
61 Alahmar
(2017)[87]
Uncontrolled
(open label)
Hansal A–Z Vital
(Hansal Pharm
GmbH, Germa-
ny) for 3 months
32 oligoasthe-
nozoospermic
patients
Improved sperm
concentration,
total and pro-
gressive motility
3 3 3 N/A N/A N/A 0
62 Yamamoto et al
(2017)[88]
RCT unblinded A) Natsushibori
(Kagome Co.,
Ltd., Japan)
B) CINAL Combi-
nation Tablet
(600 mg/
day, Shionogi
Pharmaceuti-
cal Co., Japan),
Juvela N Soft
Capsule (200
mg/day, Tanabe
Seiyaku Hanbai
Co., Japan), and
Tathion Tablet
(300 mg/day,
Eisai Co., Japan)
For 3 months
54 oligoasthe-
nozoospermic
patients
Improved sperm
motility
2 3 7 Unclear risk
of bias for
allocation
conceal-
ment, other
sources;
high risk
of bias for
blinding
(partici-
pants and
personnel,
outcome
assessment)
16 2 0
63 Magdi et al
(2017)[89]
Uncontrolled
(open label)
Vitamin C (1 g),
vitamin E (400
mg) and L-
carnitine (2
g)/daily for 6
months
210 OAT patients Improved sperm
count, total and
progressive
motility, normal
morphology
after treatment
0 3 3 N/A N/A N/A 0
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
23
www.wjmh.org
Table 2. Continued 19
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
64 Alsalman et al
(2018)[90]
Uncontrolled
(open label)
Zinc
220 mg/daily for
3 months
60 asthenozoosper-
mic patients
Improved volume,
progressive
motility, normal
morphology,
total thiol con-
centration, total
disulfide linkage
concentration,
GPx levels
3 3 6 N/A N/A N/A 0
65 Busetto et al
2018[26]
RCT blinded Proxeed Plus
(Sigma-Tau
HealthScience,
Utrecht, the
Netherlands)
2 sachets/daily
for 6 months
104 patients with
semen abnormali-
ties (of those, 52
with varicocele)
Increased semen
parameters,
except sperm
morphology
10 spontaneous
pregnancy in
treated couples
vs. 2 in the
placebo
0 3 7 Unclear risk
of bias
for other
sources
20 4 1
66 Lu et al
(2018)[91]
RCT blinded Melatonin
400 mg/daily for
3 months
54 oligozoospermic
patients
Improved semen
parameters
Improved TAC
1 3 6 Unclear risk
of bias for
random
sequence
generation,
allocation
conceal-
ment, se-
lective
reporting,
other sourc-
es, blinding
(participants
and person-
nel, outcome
assess-
ment), and
incomplete
outcome
data
15 5 0
https://doi.org/10.5534/wjmh.200196
24 www.wjmh.org
Table 2. Continued 20
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
67 Jannatifar et al
(2019)[92]
Uncontrolled
(open label)
N-acetylcysteine
600 mg/daily for
3 months
50 asthenozoosper-
mic patients
Improved volume,
sperm concen-
tration, total
and progressive
motility, normal
morphology
Reduced MDA, SDF
and protamine
deficiency;
improved TAC
3 3 3 N/A N/A N/A 0
68 Gambera et al
(2019)[93]
Uncontrolled
(open label)
Arginine (3 g),
CoQ10 (200 mg),
vitamin C (240
mg), vitamin B3
(27 mg), Tribulus
terrestris (60
mg), ginseng
(12 mg), inositol
(100 mg), vita-
min E (36 mg)
for 2 months
32 OAT patients Improved sperm
concentration,
sperm count, pro-
gressive motility,
normal morphol-
ogy and vitality
after therapy
Oxisperm; reduced
seminal oxida-
tive stress after
therapy
Unclear capacita-
tion check
0 3 2 N/A N/A N/A 0
69 Micic et al
(2019)[94]
RCT blinded Proxeed Plus, con-
sisting of 1 g LC,
0.5 g ALC, 0.725
g fumarate, 1 g
fructose, 50 mg
citric acid, 10
mg zinc, 20 mg
coenzyme Q10,
50 μg selenium,
90 mg vitamin
C, 200 μg folic
acid and 1.5 μg
vitamin B12 for
6 months
175 oligoasthe-
nozoospermic
patients
Improved semen
parameters; in-
creased seminal
carnitine and
α‐glucosidase
activity; reduced
SDF
3 3 7 Unclear risk
of bias for
selection,
other sourc-
es of bias,
blinding of
participants
and person-
nel, and
outcome
assessment
16 3 0
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
25
www.wjmh.org
Table 2. Continued 21
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
70 Nouri et al
(2019)[95]
RCT blinded Lycopene
25 mg/daily for 3
months
44 oligozoospermic
patients
Improved volume,
total sperm count,
concentration,
total motility, TAC
2 3 7 Unclear risk
of bias for
allocation
concealment,
other sources
18 4 0
71 Busetto et al
(2020)[96]
RCT blinded L-carnitine (1 g),
fumarate (725
mg), acetyl-L-
carnitine (500
mg), fructose
(1 g), citric acid
(50 mg), sele-
nium (50 μg),
coenzyme Q10
(20 mg), vitamin
C (90 mg), zinc
(10 mg), folic
acid (200 μg),
vitamin B12 (1.5
μg)/daily for 6
months
104 patients with
altered semen
quality. Of those,
52 showed grade
I-III varicoceles
Improved total
sperm count, to-
tal and progres-
sive motility
Higher pregnancy
rate
4 3 7 No risk of bias
identified
22 5 1
72 Alahmar et al
(2020)[97]
Uncontrolled
(open label)
CoQ10
200 mg/daily for
3 months
65 oligoasthe-
nozoospermic
patients
Improved sperm
concentration,
progressive and
total motility,
CoQ 10 level, TAC
and GPx
Reduced ROS
levels and SDF
4 2 4 N/A N/A N/A 0
https://doi.org/10.5534/wjmh.200196
26 www.wjmh.org
Table 2. Continued 22
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
73 Terai et al
(2020)[98]
RCT unblinded A) L-carnitine
(750.1 mg), zinc
(30 mg), astaxan-
thin (16.05 mg),
CoQ10 (90.26
mg), vitamin C
(1 g), vitamin
B12 (60.1 μg),
vitamin E (150
mg)
B) Hochu-ekki-
to (dosage not
reported)
For 3 months
31 oligoasthe-
nozoospermic
patients
Increased total mo-
tile sperm count
after treatment
in group A
0 3 3 Unclear risk of
allocation
conceal-
ment, selec-
tive report-
ing, other
sources; no
blindness of
participants
and person-
nel
16 3 0
74 Steiner et al
(2020)[99]
RCT blinded Vitamin C (500
mg), vitamin
E (400 mg),
selenium (0.20
mg), L-carnitine
(1 g), zinc (20
mg), folic acid (1
g), lycopene (10
mg), and vitamin
D (2,000 IU)/daily
for a maximum
of 6 months
174 oligozoosper-
mic patients
Improved sperm
concentration
No change in
SDF
No change in
pregnancy and
live birth rates
2 3 7 No risk of bias
identified
20 5 1
75 Alkumait et al
(2020)[100]
RCT unblinded A) Glutathione
(250 mg sa-
chets)
B) CoQ10 (200 mg
sachets)
For 6 months
51 OAT patients Improved semen
parameters
2 3 7 Unclear risk
of bias for
allocation
conceal-
ment, other
sources; high
risk of bias
for blinding
(participants
and person-
nel, outcome
assessment)
13 3 0
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
27
www.wjmh.org
Table 2. Continued 23
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
76 Nazari et al
(2020)[101]
Prospective study Androferti supple-
ment (Daru Dar-
man Parmida,
Iran) twice daily
for 3 months
59 patients with
idiopathic OAT
Improved semen
parameters
2 3 3 N/A N/A N/A 0
Healthy men 77 Goyal et al
(2007)[102]
Uncontrolled
(open label)
Lycopene
22.8 mg/daily
for 2 weeks
6 healthy men Increased seminal
lycopene. No
increase in TAC
levels
2 3 3 N/A N/A N/A 0
78 Tartibian
and Maleki
(2012)[103]
RCT blinded Honey dissolved
in water (70 g)
39 healthy men Decreased ROS,
MDA
Increased SOD,
Catalase, TAC and
decreased IL-1b,
IL-6, IL-8, TNF-a
2 3 7 Unclear risk
of bias for
allocation
conceal-
ment, other
sources
18 4 0
79 Williams et al
(2020)[104]
RCT blinded Lactolycopene
14 mg/daily for
3 months
60 healthy men Improved % of fast
progressive and
normal morphol-
ogy
No difference in
SDF%
0 3 7 No risk of bias
identified
25 5 1
Urogenital
inflamma-
tion
80 Vicari et al
(2002)[105]
RCT unblinded A) Carnitines
(Carnitene 2 g/
daily+Nicetile
1 g/daily)
B) Nonsteroidal
anti-inflam-
matory drugs
(NSAID) (nime-
sulide 200 mg/
daily+ serratio-
peptidase 10
mg/daily)
C) NSAID+ carni-
tines (2 months
each)
D) Carnitines+
NSAID (2 months
each)
For 4 months
98 patients with
abacterial
prostatovesiculo-
epididymitis and
high seminal
leukocytes (>1
million cells/ml)
Group C showed
increased
forward motility
and vitality
Reduced leuko-
cyte count in all
groups
Groups B and C
showed reduced
ROS
Spontaneous preg-
nancy rate=8.2%
(6 in group C, 1 in
groups B and D)
0 3 4 Unclear risk
of bias for
allocation
conceal-
ment, other
sources;
high risk
of bias for
selective re-
porting and
incomplete
outcome
data
16 3 0
https://doi.org/10.5534/wjmh.200196
28 www.wjmh.org
Table 2. Continued 24
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
81 Yang et al
(2003)[106]
Uncontrolled
(open label)
A) Dang Gui (An-
gelica Sinensis),
Chuan Xiong
(Ligusticum
Chuanxiong
Hort), Chi Shao
(Paeonia Veitchii
Lynch), Wu Ling
Zhi (Trogopterus
Xanthipes Mi-
line- Edwards),
Pu Huang
(Typha Angus-
tata Linne),
My Yao (Com-
miphora Molmol
Engler), Yuan
Hu (Corydalis
Yanhusuo), Gan
Jiang (Zingiber
Officinale Rosec-
oe), Guan Gui
(Cinnamomum
Cassia Presl),
and Hui Xiang
(Foenicunum
Vulgare Miller)
B) Shao-Fu-Zhu-
Yu-Tang, Sun-Ten
Pharmaceutical
Company, Taic-
hung, Taiwan
For 2 months
Chronic prostatitis
(n=36)
Improved semen
parameters and
acrosin activity
3 3 3 N/A N/A N/A 0
82 Chayachinda
et al
(2020)[107]
RCT blinded CoQ10
200 mg/day for
1 month
Leukocytospermia
(n=84)
No difference in
sperm concentra-
tion, motility, nor-
mal morphology
0 3 3 No risk of bias
identified
22 5 1
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
29
www.wjmh.org
Table 2. Continued 25
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
IMI 83 Comhaire et al
(2000)[47]
Uncontrolled
(open label)
Acetylcysteine
(600 mg) or cap-
sules providing
a daily amount
of β-carotene
(30 mg) and
α-tocopherol
(180 mg)/daily.
In addition,
capsules con-
taining essential
fatty acids for a
daily intake of
docosahexae-
noic acid (1 g),
gammalinolenic
acid (0.25 g) and
arachidonic acid
(0.10 g) for 6
months
7 idiopathic pa-
tients
11 varicocele
patients
History of cryptor-
chidism (n=2), pa-
tients with male
accessory gland
infection (n=7),
immunological
infertility (n=4),
endocrine cause
(n=1)
Improved sperm
concentration
and acrosome
reaction
Reduced ROS
levels and 8-OH-
dG levels
2 3 3 N/A N/A N/A 0
84 Gupta and
Kumar
(2002)[108]
Uncontrolled
(open label)
Lycopene
4 mg/daily for 3
months
30 idiopathic
patients
Improved sperm
concentration
and motility
Higher pregnancy
rate
3 3 3 N/A N/A N/A 0
85 Balercia et al
(2005)[109]
RCT blinded A) Carnitene
(Sigma Tau,
Pomezia, Italy)
B) Zibren (Sigma
Tau)
C) A combination
of carnitene and
zibren
For 6 months
60 idiopathic
patients
Improved sperm
motility, total
oxyradical scav-
enging capacity
of the semen
2 3 7 No risk of bias
identified
18 5 1
86 Heidary et al
(2008)[110]
Uncontrolled
(open label)
Saffron
50 mg, 3 times
weekly for 3
months
52 idiopathic
patients
Improved normal
morphology, to-
tal and progres-
sive motility
0 3 3 N/A N/A N/A 0
https://doi.org/10.5534/wjmh.200196
30 www.wjmh.org
Table 2. Continued 26
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
87 Ciftci et al
(2009)[111]
RCT unblinded N-acetylcysteine
600 mg/daily for
3 months
120 idiopathic
patients
Improved volume,
motility, semen
viscosity, semen
and serum oxida-
tive stress (TAC,
total peroxide,
oxidative stress
index)
2 3 6 Unclear risk
of bias for
blinding
(partici-
pants and
personnel);
high risk
of bias
for other
sources
12 3 0
88 Haghighian
et al
(2015)[112]
RCT blinded α-lipoic acid
600 mg/daily for
3 months
44 idiopathic
patients
Improved sperm
concentration
and motility, TAC;
reduced MDA
levels
2 3 7 No risk of bias
identified
20 5 1
89 Soleimani and
Masoumi
(2017)[113]
Uncontrolled
(open label)
Grape seed
extract
600 mg/daily for
3 months
29 idiopathic
patients
Increased catalase,
reduced MDA
2 1 3 N/A N/A N/A 0
90 Negri et al
(2017)[114]
Uncontrolled
(open label)
FertiPlus SOD
(α-lipoic acid,
glutathione,
folic acid, zinc,
and vitamins B2,
B3, B6, B12)
Dosage not
specified for
single compo-
nent, length of
treatment not
reported
55 idiopathic
patients
No changes in se-
men parameters
and SDF
0 2 3 N/A N/A N/A 0
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
31
www.wjmh.org
Table 2. Continued 27
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
91 Kopets et al
(2020)[115]
RCT blinded L-carnitine/l-
acetyl-carnitine
(1,990 mg), l-
arginine (250
mg), glutathi-
one (100 mg),
co-enzyme
Q10 (40 mg),
zinc (7.5 mg),
vitamin B9 (234
mcg), vitamin
B12 (2 mcg),
selenium (50
mcg)/daily for 6
months
83 idiopathic
patients
Increased % of
normozoosper-
mia in treated
patients after 2
and 4 months in
comparison with
placebo
Higher pregnancy
rate
0 3 7 No risk of bias
identified
24 5 1
92 Arafa et al
(2020)[25]
Uncontrolled
(open label)
FH PRO for Men
(Fairhaven
Health LLC,
Bellingham, WA,
USA)
Twice/day for 3
months
119 idiopathic
patients
29 unexplained
infertile men
Improved progres-
sive motility and
seminal oxida-
tion reduction
potential
Reduced SDF
3 3 3 N/A N/A N/A 0
UMI 93 Greco et al
(2005)[116]
Uncontrolled
(open label)
Vitamin C (1 g)
and vitamin E
(1 g)/daily for 2
months
Oligoterato- (n=6),
OAT (n=26)
patients, 6 unex-
plained infertile
men
Improved semen
parameters and
SDF
No change in
fertilization and
cleavage rates
after treatment
Higher implanta-
tion and preg-
nancy rates
2 3 3 N/A N/A N/A 0
94 Greco et al
(2005)[117]
Uncontrolled
(open label)
Vitamin C and E
1 g/daily for 2
months
64 unexplained
infertile men
No difference in se-
men parameters
Reduced SDF
1 3 7 N/A N/A N/A 0
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32 www.wjmh.org
Table 2. Continued 28
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
95 Safarinejad et al
(2012)[118]
RCT blinded CoQ10
200 mg/daily
for 26 week,
followed by a
treatment-free
period of 12-
week
228 unexplained
infertile men
Improved semen
parameters,
seminal catalase,
and SOD
4 3 7 No risk of bias
identified
18 5 1
96 Khani et al
(2013)[119]
Uncontrolled
(open label)
Sesame
0.5 mg/kg body
weight for 3
months
25 unexplained
infertile men
Improved sperm
concentration,
motility
Pregnancy: 3 out
of 25 patients
Live birth rate:
3 out of 25
patients
0 3 3 N/A N/A N/A 0
97 Arafa et al
(2020) [25]
Uncontrolled
(open label)
FH PRO for Men
(Fairhaven
Health LLC,
Bellingham, WA,
USA)
Twice/day for 3
months
119 idiopathic
patients
29 unexplained
infertile men
Improved progres-
sive motility and
seminal oxida-
tion reduction
potential
Reduced SDF
3 3 3 N/A N/A N/A 0
Hyperinsu-
linaemic
male
patients
98 Bosman et al
(2015)[120]
Uncontrolled
(open label)
A) metformin
(500–2,000 mg
daily
B) Metformin+ Sta-
minoGro (Georen
Pharmacuti-
cals PTY LTD,
Fontainebleau,
South Africa)
For 3 months
34 hyperinsulinae-
mic male patients
Improved sperm
morphology in
both groups
Decreased CMA3
assay results in
both groups after
treatment
0 3 6 N/A N/A N/A 0
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
33
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Table 2. Continued 29
Clinical
condition SN Reference Clinical trial
design
Antioxidant
formulation
dosage and length
of treatment
Study population
Reproductive
outcomes after
antioxidant
treatment
Cambridge Quality Checklist
Cochrane Risk
of Bias for RCT
CONSORT
Guidelines
(out of 25)
JADAD
(Oxford
Quality)
(out of 5)
Quality of
evidence
published
Checklist
for
correlate
(out of 5)
Checklist
for risk
factors (out
of 3)
Checklist
for causal
risk factors
(out of 7)
RPL 99 Hamidian et al
(2020)[121]
Uncontrolled
(open label)
Vitamin C
250 mg/daily for
3 months
20 patients with
recurrent preg-
nancy loss
Improved sperm
morphology
Reduced SDF
Changes in mRNA
levels of PRM1,
PRM2, and the
PRM1/PRM2 ratio
after treatment
2 3 4 N/A N/A N/A 0
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 and the risk of bias
have been determined for each study by applying the Cambridge Quality Checklist, the Cochrane Risk of Bias for RCTs, CONSORT guidelines, and JADAD score.
SN: serial number, RCT: randomized controlled trial, 8-OHdG: 8-hydroxy-2' -deoxyguanosine, IMI: iopathic male infertility, UMI: unexplained male infertility, RPL: recurrent pregnancy loss, ROS,
reactive oxygen species, N/A: not available, MDA: malondialdehyde, TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labelling, SDF: sperm DNA fragmentation, ICSI: intracytoplasmic
sperm injection, DFI: DNA fragmentation index, OS: oxidative stress, SOD: superoxide dismutase, GSH: gluthatione, TAC: total antioxidant capacity, SCSA: sperm chromatin structure assay, CoQ: co-
enzyme Q, 8-OHdG: 8-hydroxy-2' -deoxyguanosine, FSH: follicle-stimulating hormone, LH: luteinizing hormone, IVF: in vitro fertilization, β-HCG: beta-uman chorionic gonadotropin, OAT: oliasthe-
noteratozoospermia, PRL: prolactin, TSH: thyroid-stimulating hormone, ART: assisted reproductive techniques, TBARS: thiobarbituric acid reactive substances, GPx: glutathione peroxidase, PRM:
protamine, CMA3: chromomycin A3.
https://doi.org/10.5534/wjmh.200196
34 www.wjmh.org
signif icance of p<0.05.
RESULTS
A total number of 1,978 articles were identified
through the application of the keyword search strat-
egy. Through manual screening of the title, keywords
and abstract, non-relevant articles (n=1,864) were ex-
cluded (Fig. 2).
Following full-text review f or eligibility using the in-
clusion and exclusion criteria, 17 articles were f urther
excluded, resulting in 97 articles that were eligible for
inclusion (Fig. 2). Two of the studies are each repeated
as they included both IMI and UMI participants respec-
tively, resulting in a total of 99 studies included in Table
2. Relevant data were extracted from the articles and
summarized in Table 2, including the studied popula-
tion, reported impact of treatment on reproductive out-
comes, evaluation of quality, and risk of bias.
Of the 97 articles collected, 52 (53.6%) were uncon-
trolled (open label) clinical trials, 12 (12.4%) were un-
blinded RCTs and 33 (34.0%) were blinded RCTs. Based
on the type of antioxidants investigated, 44 (45.4%) of
the articles tested individual antioxidants, 31 (32.0%)
tested a combination of several products in variable
dosages, and 22 (22.7%) used registered antioxidants
products. Semen parameters were evaluated after an-
tioxidant treatment in 92.8% (n=90 out of 97) of the
included publications, while the remaining 7 studies
evaluated markers of sperm function.
Based on the statistical analysis, it is reported that
85.7% and 89.6% of the low-quality studies showed sig-
nif icant improvement (p<0.0001) in semen and sperm
function parameters, respectively, in inf ertile men
after antioxidant supplementation, whereas 65.0% and
58.3% of the high-quality studies, respectively, reported
positive effect of antioxidant treatment on semen and
sperm function parameters (Table 3). However, these
results were not significant due to the availability of
a small number of studies in the literature report-
ing semen parameters (n=20) and those reporting
sperm functions (n=12) and this has led to the under-
powering of statistical analysis. Sample size calculation
predicted that a total number of 95 and 292 studies
reporting the outcome of semen parameters and sperm
functions, respectively, will allow it to gain a statistical
signif icance of p<0.05. Furthermore, statistical analysis
revealed that 78.6% (p=0.0733) and 60% (p=0.6949) of
low and high-quality studies, respectively, reported a
positive eff ect of antioxidant treatment on reproduc-
tive outcomes. However, these values were not signif i-
cant (p≥0.05) due to the availability of very f ew studies
(n=14 for low-quality and n=5 for high-quality) in the
literature. Sample size calculation predicted that a to-
tal number of 33 low and 202 high-quality studies are
required to attain a statistical significance of p<0.05
for reproductive outcomes.
1. Varicocele
A total of 11 studies investigated a male population
affected by varicocele (Table 2). Of those, semen pa-
rameters after antioxidant treatment were reported
Articles identified through
Scopus database
(n=1,978)
Articles screened
(n=1,978)
Full-text articles assessed
for eligibility
(n=114)
Studies included in
qualitative synthesis
(n=97)
Non-relevant articles excluded by screening the title,
keywords and abstract (n=1,864)
Non-relevant full-text articles excluded (n=17)
Studies not reporting the included outcomes (n=7)
Non-clinical trials (n=9)
study (n=1)
In vitro
Fig. 2. Preferred Reporting Items for
Systematic Reviews and Meta-Analyses
(PRISMA) workflow reporting the litera-
ture search strategy.
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
35
www.wjmh.org
in 90.9% (n=10 out of 11) of the included publications.
Based on these studies, antioxidant supplementation
seems to be beneficial in varicocele patients as 75.0%
and 83.0% of low-quality studies, respectively, available
in the literature, reported positive ef f ect of antioxidant
treatment on semen and sperm f unction parameters
(Table 3). However, these values were not signif icant.
Sample size calculation predicted that a total number
of 41 and 24 studies reporting the outcome of semen
parameters and sperm f unction, respectively are need-
ed to reach a statistical significance of p<0.05.
2. Abnormal semen quality
A total of 45 studies investigated a male population
with abnormal semen quality (Table 2). Of those, semen
parameters after antioxidant treatment were reported
in 97.8% (n=44 out of 45) of the included publications,
whereas sperm function biomarkers were reported in
25 out of 45 studies (55.6%) (Table 3). The majority of
the studies showed significant improvement in semen
and sperm function parameters of men with abnormal
semen quality after antioxidant supplementation, al-
though these results were not statistically significant
in case of the high-quality studies (Table 3). Sample
size calculation predicted that a total number of 204
studies reporting the outcome of sperm function are
required to reach a statistical significance of p<0.05.
3. Idiopathic male infertility
A total of 10 studies investigated idiopathic infertile
men (Table 2). Of those, semen parameters af ter anti-
oxidant treatment were reported in 90.0% (n=9 out of
10) of the included publications, whereas sperm function
biomarkers were reported in 7 out of 10 studies (70.0%)
(Table 3). Our statistical analysis showed that all the
high-quality studies reported improvement in the semen
and sperm function parameters (p<0.0001) after anti-
oxidant treatment in men with IMI. Although a high
percentage of low-quality studies showed improvement
in semen and sperm function parameters in men with
IMI after antioxidant supplementation, these values
were not significant. Sample size calculation revealed
that a total number of 24 and 30 studies, respectively,
reporting the outcome of semen parameters and sperm
function, may allow to reach statistical significance.
4. Unexplained male infertility
A total of 5 studies investigated the eff ect of antioxi-
dant therapy in unexplained infertile men (Table 2).
All of those studies reported semen parameters af ter
antioxidant treatment (100%), whereas sperm function
biomarkers were reported in 4 out of 5 studies (80.0%)
(Table 3). A ll the low-quality studies showed improve-
ment in sperm function, while sample size calculation
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-
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
Group Category
Report of semen parameters Report of sperm function
Number of articles on
the total of studies
% of studies reporting
an improvement after
AOX treatment
Number of articles on
the total of studies
% of studies reporting
an improvement after
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.
https://doi.org/10.5534/wjmh.200196
36 www.wjmh.org
Table 4. Articles published between January 2019 and July 2020 investigating the impact of antioxidant treatment on reproductive outcomes
SN Reference Study design
Study
population/
sample size
Inclusion criteria Exclusion criteria
Strict male
inclusion/
exclusion
Female
factor
Main outcomes
reported
Power of statistical
analysis
Study
quality score
(out of 4)
Study
outcome
(out of 3)
1Terai et al
(2020)[98]
RCT unblinded 31 oligoasthe-
nozoospermic
patients
Age: 20–60 years
old; presence
of oligozoo-
spermia and/
or asthenozoo-
spermia
Azoospermia
Sperm concentration
<5×106/mL
Sperm motility<5%
TMSC>30×106
Clinical conditions result-
ing in infertility
History of cancer, chemo-
therapy, drug abuse
Administration of andro-
gens, anti-androgens,
and immunosuppres-
sants
0N/A Improved TMSC
(p=0.04)
N/A 0 1
2Schisterman et al
(2020)[46]
Double-blind RCT Treatment
(n=1,185)
vs. placebo
(n=1,185)
Male partners
of couples
planning IVF
for infertility
treatment
Planning of donor sperm
use or a gestational
surrogate
Pregnancy at enrollment
Obstructive azoospermia
Chronic diseases
0N/A No difference in
semen parameters
between both
groups.
Increase in SDF by
Comet assay in
treatment group
vs. placebo group
(Adjusted MD 2.4,
95% CI 0.5–4.4)
No significant differ-
ences in β-HCG–de-
tected pregnancy,
clinical intrauterine
pregnancy, ectopic
pregnancy, preg-
nancy with multiple
fetuses
LBR: Treatment group
404 (34%) vs.
placebo group 416
(35%) (ns)
90% power at a
2-sided α level of
0.05 to detect a risk
difference of 7% in
LBR (implying a risk
ratio of 1.10), with
continuity correc-
tion and allowing
for a dropout rate of
15%
Esteem of risk differ-
ences and risk ratios
Sequential ap-
proach of Lan
and DeMets with
Bonferroni adjust-
ment to distribute
the 1-sided type I
error rate among 3
continuous semen
quality parameters
Post hoc sensitivity
analyses
2 0
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
37
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Table 4. Continued 1
SN Reference Study design
Study
population/
sample size
Inclusion criteria Exclusion criteria
Strict male
inclusion/
exclusion
Female
factor
Main outcomes
reported
Power of statistical
analysis
Study
quality score
(out of 4)
Study
outcome
(out of 3)
3Steiner et al
(2020)[99]
Double-blind RCT Treated (n=85) vs.
placebo (n=86)
Infertile men
with abnormal
semen analysis
in the last 6
months or
DFI≥25%
Sperm concentration
<5×106/mL
Consumption of fertility
medication or testos-
terone
0Yes No difference in
semen parameters,
DFI by SCSA and PR
LBR: 15% AOX vs.
24% placebo (ns)
LBR=35% in the
treated group and
25% in the placebo
group with a 17%
dropout
Sample size calcula-
tion, assuming a
20% dropout rate,
≥80% power at
α=0.05
3 0
4Kopets et al
(2020)[115]
Double-blind RCT Treated (n=42) vs.
placebo (n=41)
Age: 21–50 years,
with IMI
Allergy to any compo-
nent
Any clinical cause of male
or female infertility
Alcohol or drug addic-
tion
Use of any investiga-
tional product within
the previous 3 months
1Yes Significant difference
between both
groups as regards
normalization of
semen parameters
at 2 months (26/42
[61.9%]) males in
treatment group
vs. 8/41 [19.5%]
males in pla-
cebo group) and
at 4 months (29/42
[69.0%] vs. 9/41
[22.0%]).
Significant change
from baseline in
mean values for all
main semen pa-
rameters at 2 and 4
months, except for
sperm morphology
At 6 months higher
PR in treatment
than placebo
group (10/42
[23.8%] vs. 2/41
[4.9%])
Sample size calcula-
tion assuming
1-beta error 0.80
and type I error
alpha 5%
Control for con-
founders by
ANCOVA analysis
2 1
https://doi.org/10.5534/wjmh.200196
38 www.wjmh.org
Table 4. Continued 2
SN Reference Study design
Study
population/
sample size
Inclusion criteria Exclusion criteria
Strict male
inclusion/
exclusion
Female
factor
Main outcomes
reported
Power of statistical
analysis
Study
quality score
(out of 4)
Study
outcome
(out of 3)
5Arafa et al
(2020)[25]
Prospective study Idiopathic
(n=119) and
unexplained
male infertility
(n=29)
Infertile men
(20–50 years)
with unknown
etiology and
female infertil-
ity factor
Azoospermia
Sperm concentration
<1×106/mL
Leucocytospermia
Any cause for infertility
Chemotherapy
Clinical endocrinopathy
Abnormal hormonal
profile
AOXs in the past 6
months
Dietary, social habits or
medical conditions
which may impact on
oxidative stress
Use of drugs
1Yes IMI: significant
improvement in
sperm concentra-
tion (p<0.001),
total motility
(p=0.001), normal
morphology
(p<0.001), ORP
(p<0.001), SDF
(p=0.001) by Halo-
sperm
UMI: significant
improvement
in progressive
motility (p=0.002),
ORP (p=0.03), SDF
(p=0.02)
N/A 3 3
6Nazari et al
(2020)[101]
Prospective study 59 patients with
idiopathic OAT
Infertile patients
with at least
1 abnormal
semen param-
eter; age<45
years, BMI<30
Azoospermia
Prostatitis
Any clinical condition
causing infertility
History of hormonal
therapy, drug addi-
tion, alcohol abuse,
smoking, exposure to
potential reproductive
toxins
1No Significant improve-
ments in sperm
concentration
(p=0.004) and
normal morphol-
ogy (p=0.01)
N/A 1 1
7Nurmawati et al
(2020)[44]
Single-blinded
RCT
25 infertile men Inclusion criteria
not clearly
stated
Exclusion criteria not
clearly stated
0No Improved sperm
concentration, mo-
tility, and morphol-
ogy (p<0.05)
Reduced levels of
8-OHdG levels
(p<0.01) and MDA,
with the val-
ue<1.98 being able
to predict 100% of
the normal sperm
motility level (>40)
Sample size calcula-
tion assuming that
the prevalence
of male infertile
couples with
idiopathic causes
in the world is 15%
and in Indonesia
1.11%
2 2
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
39
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Table 4. Continued 3
SN Reference Study design
Study
population/
sample size
Inclusion criteria Exclusion criteria
Strict male
inclusion/
exclusion
Female
factor
Main outcomes
reported
Power of statistical
analysis
Study
quality score
(out of 4)
Study
outcome
(out of 3)
8Hadi et al
(2020)[45]
Uncontrolled
(open label)
58 infertile men Inclusion criteria
not clearly
stated
Presence of varicocele,
orchitis, cryptorchidism
Consumption of herbals
or medications that
might affect seminal
parameters in the last
3 months prior to the
study
0No Improved sperm
volume, count,
total motility, and
normal morphol-
ogy (p<0.05)
N/A 1 1
9Busetto et al
(2020)[96]
Double-blinded
RCT
104 patients
with altered
semen quality.
Of those, 52
showed grade
I–III varicoceles
Oligo- and/or
astheno- and/
or teratozoo-
spermia, with
or without
varicocele
(not surgically
treated) and
men from infer-
tile couples
Known hypersensitivity to
any of the compound
History of undescended
testes or cancer,
endocrine disorders,
post-pubertal mumps,
genitourinary surgery,
obstructive azoosper-
mia or obstructive
pathology of the
urogenital system,
autoimmune disease,
cystic fibrosis
History of taking any ther-
apy affecting fertility,
alcohol or drug abuse
Subjects following any
special diet or taking
AOXs
Involvement
in any other clinical
trials
0Yes Improved total
sperm count
(p<0.0001), total
(p<0.0001) and
progressive motil-
ity (p=0.0012)
Higher PR in treated
group vs. placebo
(10 vs. 2 pregnan-
cies, respectively;
p=0.0141)
Sample size calcula-
tion assuming
α=0.05 (sig-
nificance), β=0.20
(power of 80%),
and up to 15% of
patients dropping
out of the study
esteemed
3 1
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40 www.wjmh.org
Table 4. Continued 4
SN Reference Study design
Study
population/
sample size
Inclusion criteria Exclusion criteria
Strict male
inclusion/
exclusion
Female
factor
Main outcomes
reported
Power of statistical
analysis
Study
quality score
(out of 4)
Study
outcome
(out of 3)
10 Alahmar et al
(2020)[97]
Uncontrolled
(open label)
65 oligoasthe-
nozoospermic
patients
Infertile patients
showing
oligoastheno-
zoospermia
Azoospermia
Anatomical abnormalities
of genital tract, varico-
cele, genital infection,
scrotal surgery, systemic
diseases
Smoking
Female factor
Consumption of
ntioxidant and selective
serotonin reuptake
inhibitors intake in the
last 6 months
1No Improved sperm
concentration,
progressive and
total motility
(p<0.05), levels of
CoQ 10 (p<0.001),
TAC (p<0.01) and
GPx (p<0.001)
Reduced ROS levels
(p<0.05) and SDF
by SCD assay
(p<0.01)
N/A 2 2
11 Alkumait et al
(2020)[100]
RCT unblinded 51 OAT patients Normal female
factor with idio-
pathic OAT
Presence of chronic
diseases, neoplasm,
trauma, hypospadias,
vas deference obstruc-
tion, varicocele, and
genital tract infection
Receiving treatment
recently
1No Improved sperm
concentration,
motility (p=0.01)
and morphology
(p=0.03)
N/A 1 1
12 Williams et al
(2020)[104]
Double-blinded
RCT
60 healthy men Healthy male vol-
unteers, aged
18–30 years,
lived within 1 h
of the clinic or
planning to live
in the region
for the duration
of the study
Previous testicular sur-
gery
Existing or previous
cancer
Allergy to tomato, whey
protein or soy deriva-
tives
0No Improved % of
fast progressive
(p=0.006) and
normal morphol-
ogy (p<0.001)
No difference in SDF
by TUNEL
N/A 3 1
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
41
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Table 4. Continued 5
SN Reference Study design
Study
population/
sample size
Inclusion criteria Exclusion criteria
Strict male
inclusion/
exclusion
Female
factor
Main outcomes
reported
Power of statistical
analysis
Study
quality score
(out of 4)
Study
outcome
(out of 3)
13 Hamidian et al
(2020)[121]
Uncontrolled
(open label)
20 patients
with recurrent
pregnancy loss
Recurrency of
pregnancy loss,
age<40 years,
no history of
alcohol/drug
abuse or smok-
ing, altered
semen quality
Obesity, diabetes, and
varicocele
Previous treatments
with AOXs or other
medications
For the female part-
ners, the presence of
hormonal imbalance,
chromosomal altera-
tions, tubal obstruc-
tion, and bacterial or
viral infections
1Yes Improved sperm
morphology
(p=0.000)
Reduced SDF by
TUNEL (p=0.00)
Reduced sperm
protamine defi-
ciency assessed by
CMA3-based assay
(p=0.00)
N/A 2 3
14 Salehi et al
(2019)[42]
Uncontrolled
(open label)
485 infertile men
with DFI>27%
by SCSA
Aged 20–40 years History of varicocele,
surgery, and inflamma-
tion
1No Improved sperm
concentration
(p=0.003), total
motility (p=0.001).
Reduced DFI by
SCSA (p=0.001)
PR=16.8% for AOX
treated patients
N/A 2 2
15 Hasoon
(2019)[43]
Uncontrolled
(open label)
24 infertile men Unexplained sub-
fertility
Presence of organic or
obstructive infertility
1No Improved volume,
sperm count,
motility, and nor-
mal morphology
(p<0.005)
N/A 0 1
16 Ardestani Zadeh
et al (2019)[57]
Single blind RCT 60 varicocele
patients
Varicocele
patients who
underwent
sub-inguinal
varicocelec-
tomy
Usage of supplements
Alcohol and/or drug ad-
diction, smoking
Diabetes mellitus,
hormonal disorders,
chronic or active infec-
tions
Presenting side effects,
and delayed complica-
tions of varicocelec-
tomy
0No Improved sperm
count (p=0.021)
and motility
(p=0.003)
N/A 2 1
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Table 4. Continued 6
SN Reference Study design
Study
population/
sample size
Inclusion criteria Exclusion criteria
Strict male
inclusion/
exclusion
Female
factor
Main outcomes
reported
Power of statistical
analysis
Study
quality score
(out of 4)
Study
outcome
(out of 3)
17 Kızılay and Altay
(2019)[56]
RCT unblinded 90 varicocele
patients
Varicocele
patients treated
with varicoce-
lectomy, with
spouses<35
years old, regu-
lar hormone
profiles and
menstrual
cycles and no
identified cause
of infertility
Previous genitourinary
system and/or varico-
cele surgery
IMI
Any clinical condition af-
fecting fertility for the
previous 3 months
Patients following a
fertility specific diet
Alcohol or drug abuse,
smoking
0Yes Improved TSC, sperm
concentration,
sperm count in
normal morphol-
ogy, and total
and progressive
motile sperm count
(p<0.05)
Higher PR in AOX
treated patients
than placebo
group (29% vs.
17.9%, respectively;
p=0.029)
Study powered to
detect an effect
size of d≥0.70 as
statistically signifi-
cant in a two-tailed
test with α=0.05
and power of 0.80
with n=24 per
condition.
3 1
18 Gambera et al
(2019)[93]
Uncontrolled
(open label)
32 OAT patients Infertile patients
with normal
sexual develop-
ment, medical
history, serum
hormone levels
and physical
examination
Azoospermia and infertil-
ity due to the female
factor
0Yes Improved sperm
concentration,
sperm count, pro-
gressive motility,
normal morphol-
ogy, and vitality
Oxisperm test:
reduced seminal
oxidative stress
after therapy (no p-
values reported)
N/A 2 2
19 Jannatifar et al
(2019)[92]
Uncontrolled
(open label)
50 astheno-
zoospermic
patients
Infertile couples
with no previ-
ous report of
pregnancy, nor-
mal female and
male partners
Varicocele, leukospermia,
hormonal abnormali-
ties, and/or obstruc-
tion, cryptorchidism,
vasectomy, abnormal
liver function
Smoking, alcohol con-
sumption
Anatomical disorders,
Klinefelter’s syndrome,
cancer, fever in the 90
days prior to sperm
analysis, seminal sperm
antibodies
1No Improved sperm con-
centration (p=0.02),
total (p=0.01)
and progressive
motility (p=0.001),
normal morphol-
ogy (p=0.001), TAC
(p=0.01)
Reduced levels of
MDA (p=0.01),
SDF by TUNEL
(p=0.001), % of
sperm showing
protamine deficien-
cy by CMA3-based
assay (p=0.009)
N/A 1 3
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
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Table 4. Continued 7
SN Reference Study design
Study
population/
sample size
Inclusion criteria Exclusion criteria
Strict male
inclusion/
exclusion
Female
factor
Main outcomes
reported
Power of statistical
analysis
Study
quality score
(out of 4)
Study
outcome
(out of 3)
20 Nouri et al
(2019)[95]
Double-blind RCT 44 oligozoosper-
mic patients
Infertile men
(25–45
years), sperm
count<20×106/
mL, normal
sperm <65%
and average
motility <60%
History of anatomical
disorders, endocri-
nopathy, previous hor-
monal therapy, use of
androgens, antiandro-
gens, anticoagulants,
cytotoxic drugs, or
immunosuppressants
Alcohol and drug abuse
BMI≥30 kg/m2
1No Improved volume,
TSC, concentration,
total motility, TAC
(p<0.05)
N/A 2 2
21 Micic et al
(2019)[94]
Double-blind RCT Treatment
(n=125) vs.
placebo (n=50)
Total sperm num-
ber ≤15×106/
mL; progres-
sive motility
<32%; normal
viscosity and
normal leuco-
cytes number
(<1×106/mL);
sperm vitality
≤58%; normal
sperm mor-
phology <4%
Motility<5%
Sperm concentration
<1×106/mL
History of therapy for
infertility within the
last 2 months
Alcohol consumption
Undescended testes,
post‐pubertal mumps,
endocrine and autoim-
mune diseases, cystic
fibrosis, or testicular
cancer
Hypersensitivity to ingre-
dients in Proxeed Plus
Presence of endocrine
disorders, anti-sperm
antibodies, leukocyto-
spermia
Use of antioxidant agents
or vitamins
Involvement in other
clinical trials
0Yes Improved ejaculated
volume (p=0.001),
progressive
motility (p<0.001),
vitality (p=0.002)
after treatment
Reduced SDF by
Halosperm test
Increased seminal
carnitine and
α‐glucosidase
activity, positively
correlated with
improved progres-
sive motility
N/A 4 3
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
power of the statistical analysis.
SN: serial number, RCT: randomized controlled trial, TMSC: total motile sperm count, N/A: not available, IVF: in vitro fertilization, SDF: sperm DNA fragmentation, MD: median, CI: confidence inter-
val, β-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
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,
CoQ: coenzyme Q, TAC: total antioxidant capacity, GPx: glutathione peroxidase, ROS: reactive oxygen species, SCD: sperm chromatin dispersion, OAT: oligoasthenoteratozoospermia, TUNEL: termi-
nal deoxynucleotidyl transferase dUTP nick end labeling, CMA3: chromomycin A3, TSC: total sperm count.
https://doi.org/10.5534/wjmh.200196
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more, all the high-quality studies reported a significant
improvement in the semen and sperm function para-
meters after antioxidant treatment in men with UMI.
5. Analysis of the most recent publications
A total of 21 articles published between January 2019
and July 2020 investigated the effects of antioxidant
treatment on semen quality (Table 4) [25,42-46,56,57,92-
101,104,115,121]. Based on our analysis, 13 and 8 studies
were ranked as low and high-quality, respectively. Of
these, 19 out of 21 (90.5%) showed improvement in se-
men parameters, while 4 out of 6 (66.7%) reported a
signif icant improvement in sperm function. The num-
ber of studies investigating reproductive outcomes af-
ter antioxidant treatment was very limited, with only
3 out of 5 (60.0%) reporting an improvement in preg-
nancy rate, while birth rate showed no variation in the
two studies reporting its evaluation.
DISCUSSION
Male infertility is a relatively common concern, con-
tributing significantly to poor reproductive outcomes
in couples. Oxidative stress has been increasingly iden-
tified as a common mechanism that mediates not only
the pathophysiology, but also the many etiologies and
risk factors associated with male infertility [1,2,5,6].
Within this context, there is increased use of antioxi-
dants as a therapeutic option in male infertility, how-
ever, there remains no consensus on the ef f icacy, indi-
cations, dosage or length of treatment [8-11]. Therefore,
the objective of this study was to systematically review
the literature of trials investigating antioxidant use in
male infertility, and to propose some broad guidelines
for the practicing clinicians based on the currently
available evidence. The results (Table 2) were stratified
based on the currently available evidence on the clini-
cal conditions investigated and the data were further
analysed. Most studies reported men with abnormal
semen quality (n=45) and inf ertile men (n=20) as well
as male infertility conditions such as varicocele (n=11),
IMI (n=10), UMI (n=5), and urogenital inflammation
(n=3). Although there is no doubt that assessing sperm
quality is just a first approach to a diagnosis and that
evaluation of it as a predictor of f ecundity or a couple’s
fertility success may lead to imprudent conclusions,
there is no consensus whether the intake of exogenous
antioxidants should be routinely done in clinical prac-
tice.
Majzoub and Agarwal (2018) [10] perf ormed a sys-
tematic review and identified 26 studies showing posi-
tive ef fects of exogenous antioxidant intake on sperm
quality and relevant outcomes of assisted reproduction
such as live birth rates. The authors critically discussed
the studies and highlighted that the treatment was
given only for a short period to a small number of men.
In addition, the lack of a standardized test to estimate
oxidative stress levels in sperm and seminal fluid was
another flaw in these studies, while the heterogeneity
of the study designs made it particularly challenging
to compare the effects and reach a robust conclusion.
This has also been observed in our study, where 60
studies (61.9%) analyzed a variety of seminal oxidative
stress markers, including the levels of seminal ROS
and/or several endogenous antioxidants (i.e., total anti-
oxidant capacity assay, superoxide dismutase, catalase,
glutathione), markers of lipid peroxidation (i.e ., malo-
ndialdehyde), oxidative DNA damage (8-hydroxy-2' -de-
oxyguanosine), and oxidation-reduction potential (ORP)
(Table 2). The lack of standardization in the evaluation
of oxidative stress in seminal fluid before and af ter
therapy hinders a definitive conclusion regarding the
implementation of oral antioxidant supplementation
for infertile men in the clinical practice. Moreover, the
lack of detailed methodological descriptions in most
articles testing oral antioxidants supplementation is a
major shortcoming that makes comparisons between
diff erent studies difficult. The evaluation of the length
of treatment in the analyzed studies also does not help
in this regard, as it is variable, with 15 (15.5%) studies
reporting the treatment for an unclear amount of time
or less than 3 months (Table 2). This might result in
diff iculties to observe any significant influence on hu-
man spermatogenesis.
More recently, a systematic review and meta-
analysis with data from seven RCTs using L-carnitine
(LC) and L-acetyl carnitine (LAC) as treatment (LC
2 g/day+L AC 1 g/day in six studies and LC 150 mg/
day+LAC 50 mg/day in one study during 12 or 24
weeks) enrolling a total of 693 patients, concluded that
a combined therapy of LC and LAC is ef fective in men
with idiopathic oligoasthenoteratozoospermia [122].
This conclusion was supported by a signif icant increase
in forward sperm motility and total motile sperm
count. All the other sperm characteristics analyzed
including semen volume, sperm concentration and per-
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
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centage of abnormal spermatozoa showed no change.
Since most of the selected studies lacked consistent and
detailed information, pregnancy of the female part-
ner as an endpoint was not considered in the analysis.
Nevertheless, the authors found that the combined
therapy of LC+LAC could lead to higher pregnancy
rates. Although this meta-analysis provides evidence
for a positive eff ect of dietary supplementation with
LC+LAC for 3–6 months in men with idiopathic oligo-
asthenoteratozoospermia seeking fertility treatment,
there are several limitations that hamper a robust
conclusion. First, the clinical diagnosis of idiopathic
oligoasthenoteratozoospermia is open to interpretation.
Thus, each study may have considered and recruited
distinct types of patients. In addition, since the studies
had a signif icant variance regarding the number of se-
lected patients (21 patients in two studies and up to 175
patients in one study), this impedes the robustness of
the conclusions. The most striking limitation, common
to most studies, is the fact that the bioavailability of
the compounds is unknown. Moreover, the mechanisms
by which they target testicular function and exert
their action is not well established and these studies do
not provide evidence for a synergistic action ver sus a
single compound action, nor do they clearly show how
the compounds act.
CRITICAL EVALUATION OF THE
NECESSITY OF ADDITIONAL
DOUBLE-BLIND, RANDOMIZED,
PLACEBO-CONTROLLED TRIALS
An important issue that must be pointed out in this
context is that out of 90 studies that reported the ef-
fects of antioxidant treatment on semen parameters, 70
were low-quality studies, whereas only 20 were ranked
as high-quality. The overall statistical analysis of both
low- and high-quality studies indicates that the antioxi-
dant treatment has a significant positive effect on se-
men parameters. A similar result could still be obtained
for the effect of the antioxidant treatment on seminal
oxidative stress and SDF. Only 60 of these studies
evaluated oxidative stress markers. For the reproduc-
tive outcome, however, only a total of 19 studies, 5 of
which were of high-quality, have reported this outcome
parameter. Out of these 5 studies, 3 reported a positive
effect, while in 2 studies either no effect or a nega-
tive effect was observed. These data clearly indicates
that the number of high-quality studies is too low to
obtain significant results. Some of the reasons are that
fertilization is a multifactorial process. In this case the
man is treated for oxidative stress, which ref lects in
improvements of semen parameters and seminal oxida-
tive stress, even in high-quality studies. However, when
looking at fertilization, pregnancy and live birth rates
oocyte quality has to be considered as a confounding
variable. These factors are also the reason why the
number of studies necessary (n=202) to obtain a sig-
nificant result for the high-quality studies is so high
and is therefore unrealistic. Secondly, the general cost
for high-quality double-blind, randomized, and placebo-
controlled studies with a sufficiently high number of
participants is also very high.
Smits et al (2019) [11] performed an extensive meta-
analysis to evaluate if dietary supplementation with
oral antioxidants was ef fective and safe. The authors
analysed 61 studies involving 6,264 subfertile men
seeking fertility treatment. The authors found that 18
diff erent oral antioxidants were used in these studies.
The most relevant conclusion was that oral antioxidant
supplementation can improve the reproductive capac-
ity of subfertile men and even enhance live birth rates.
However, the evidence collected was considered of low
or very low-quality due to serious study limitations,
making the comparison and/or aggregation to perform
robust statistical analysis dif ficult. The studies also
showed a significant variation in the antioxidant sup-
plementation regimens (type, dose, or even combined
intake). Some studies used a placebo group to compare
with, while others chose to compare with no treatment
or treatment with another antioxidant.
The endpoint f or couples attending a fertility treat-
ment has to be clinical pregnancy or live birth, but
most studies fail to present these data. Only 12 of the
44 studies that were included in the previously cited
meta-analysis reported on clinical pregnancy and live
birth, which is considered a major limitation [11]. The
latter evidence has also been highlighted in our sys-
tematic analysis of the literature, where only 22 (22.7%)
and 4 (4.1%) out of 97 studies reported pregnancy and
live birth rates as a clinical outcome, respectively.
Pregnancy and live birth are highly influenced by a
wide variety of embryological and f emale factors. This
highlights one of the major problems in literature as
the studies on the use and eff ectiveness of antioxidants
are mostly focused on the males and not on the couple.
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Studies need standardization not only regarding the
selection of the males, but also the female partners to
evaluate clinical pregnancy, achievement of live birth,
and the influence of the treatment. Many confounding
factors may interfere with such outcomes including
female age, ovarian reserve, anatomic, inf lammatory
and endocrinal disorders, and as such, the impact of
any fertility treatment can only be assessed after ad-
justing for such confounding factors. All these factors
determine the type of ART treatment that is employed,
namely in vitro fertilization (IVF) or intracytoplasmic
sperm injection (ICSI), and eventually also have a sig-
nificant impact on the reproductive outcome. Hence,
the outcomes, positive or negative, can be skewed due
to numerous confounding factors. Consequently, the fo-
cus of the effectiveness of an antioxidant treatment of
men should be whether or not the treatment improves
seminal parameters and sperm function, rather than
reproductive outcomes which are influenced by numer-
ous other variables that are, i) not/insuf f iciently con-
sidered in recent studies, and ii) can have a significant
negative effect on blastulation, embryo development,
onset/continuation of pregnancy, and live birth. Fur-
thermore, in order to be able to judge the effectiveness
of treatment, it is necessary to specify the andrological
condition for which patients are being treated. Without
this, one might include patients with a condition in a
study which are not or only poorly responding to the
treatment. In turn, this would negatively af f ect the
outcome of the study.
We have identified 21 studies investigating the ef-
fects of antioxidant treatment on semen quality that
were published f rom Januar y 2019 to July 2020. The
majority of these studies showed an improvement in
semen parameters and sperm f unction. With a more
detailed assessment of quality reported, 13 of the 21
were ranked as low-quality. Steiner et al [99] and Micic
et al [94] strictly excluded couples with female factors,
while most of the studies inadequately documented
such criteria in their methodologies. From the molecu-
lar point of view, this integrative focus on the couple
is essential. The sperm-oocyte interaction is sensitive
to the redox balance, and excessive ROS in spermato-
zoa can lead to impaired oocyte function [123]. Hence,
the ef fects of sperm oxidative damage go far beyond
fertilization since they can have a negative impact on
embryo development or even pregnancy loss [124,125].
On the other hand, the oocyte has the capability to re-
pair damages in spermatozoa [126], and this is another
unknown variable that needs to be considered when
thinking of reproductive outcomes as a therapeutic
eff ect of treating men with antioxidants. A number
of critical points can further be pointed out from indi-
vidual studies. Schisterman et al [46] lost 31% of their
subjects during follow-up and a substantial number of
couples received non-specif ied treatment of f-site, which
could alter the study results. The Steiner et al’s [99]
study was terminated early as they f ailed to show a
>10% difference in pregnancy/live birth rates. More-
over, the authors f ailed to confirm adherence with
antioxidant treatment, and ovarian stimulation was
used in couples who did not conceive after 3 months of
therapy, which could affect the outcomes. Terai et al [98]
included men between 20 and 60 years of age, which is
atypical of the reproductive age group. Abstinence was
defined as 4 or more days without an upper limit [98].
Advanced male age and delayed abstinence are associ-
ated with increased oxidative stress measures and with
alteration in semen parameters [127,128]. Furthermore,
Terai et al [98] did not include a placebo group, with an
experimental group investigating a Chinese herbal for-
mula. Hadi et al [45], Alahmar et al [97], Hamidian et
al [121], Salehi et al [42], Hasoon [43], Gambera et al [93],
Arafa et al [25], Nazari et al [101], and Jannatifar et al
[92] all reported improved semen parameters, but these
studies are uncontrolled open label trials and are rela-
tively underpowered. Furthermore, Alkumait et al [100]
and Terai et al [98] were unblinded trials with rela-
tively small sample sizes (n=51 and 31, respectively). On
the other hand, the reviewed studies also have several
positive virtues that can be pointed out. For example,
the study by Agarwal et al [129] investigated changes
in protein expression following antioxidant therapy,
thereby enhancing our understanding of the physi-
ologic alterations at the molecular level. Several studies
assessed the eff ect of antioxidant therapy on SDF lev-
els (Table 2). SDF is increasingly being utilized in the
evaluation of male factor infertility and is believed to
be an important determinant of fertility potential [130].
Furthermore, SDF is considered as an indirect measure
of oxidative stress and can validate the benefits of an-
tioxidant therapy in restoring the body’s redox poten-
tial.
Therefore, for the above discussed reasons, having
more double-blind RCT studies with a large enough
sample size are neither feasible nor could they provide
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
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the expected clear result in terms of improved live
birth rates after the antioxidant treatment.
STRENGTH WEAKNESS
OPPORTUNITY THREAT (SWOT)
ANALYSIS
1. Strengths
Antioxidant supplementation for the treatment of
male infertility has been increasingly investigated in
the past decade. Reports suggest that different an-
tioxidant formulations were used to improve sperm
quality and function in inf ertile men with various
clinical circumstances (Table 2). These improvements
were ref lected on reproductive outcomes such as preg-
nancy rate (Table 2). An increasing number of studies
have investigated the ef fect of antioxidant therapy on
measures of oxidative stress, perhaps indicating it is a
feasible treatment approach/option for patients with
alteration in seminal redox potential.
2. Weaknesses
The contradictory results in reproductive outcomes
seen in a number of studies can be considered as the
main factor limiting the routine use of antioxidants
for the treatment of male infertility, while female and
embryological confounding f actors were not taken into
account (Table 2). Yet, in these studies, the male was
treated with the false expectation that this treatment
would automatically increase the success of the repro-
ductive outcome. In addition, the low level of evidence
was extrapolated from the clinical trials reporting
benefit due to non-homogenous study designs, or in-
consistencies in the treatment regimens (individual or
combined) used (Table 2). Furthermore, the majority of
these studies failed to adjust for confounding factors
(e.g., female factors) that are essential for conception or
establishment of pregnancy (Table 2).
3. Opportunities
Selection of suitable candidates for antioxidant sup-
plementation by oxidative stress measurement seems a
logical approach [25]. Indeed, the concept of MOSI has
been recently proposed. The classif ication may guide
the identification of a specific group of idiopathic in-
fertile men who will be more likely to benefit f rom the
treatment [2]. Secondly, evaluation of the sperm pro-
teome of idiopathic infertile men bef ore and af ter oral
oxidant supplementation offers a window to better un-
derstanding the molecular mechanisms associated with
sperm function (Fig. 3) [129]. Oral antioxidants may be
an alternative cost-effective treatment option for infer-
tile couples who desire to avoid assisted reproduction.
4. Threats
Despite the conclusion f rom Cochrane collaborations
Fig. 3. Strength Weakness Opportunity
Threat (SWOT) analysis. SWOT has been
conducted to describe the impact of an-
tioxidant supplementation in the treat-
ment of male infertility. ART: assisted
reproductive technology.
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that oral antioxidant therapy may improve semen pa-
rameters and the likelihood of pregnancy, the lack of
suff icient high-quality evidence still hinders a consen-
sus among clinicians [11]. In addition, the wide varia-
tion in the treatment regimen raises concerns about
overzealous use of antioxidants. The detrimental ef-
fects of reductive stress may be as pathological as that
of oxidative stress [23]. Moreover, the of ten unpredict-
able outcome after antioxidant supplementation in the
context of multiple confounding f actors in reproduction
may delay the definitive treatment, particularly f or
couples of advanced age.
CLINICAL GUIDELINES
While antioxidant supplementation is f requently
utilized for the treatment of male factor infertility, no
clear recommendations exist endorsing their use for
specific clinical indications. Therefore, we aimed to de-
velop clinical practice guidelines based on the available
evidence to help in identif ying the clinical circumstanc-
es in which antioxidant supplementation appears to
be most beneficial. This systematic review included 97
articles, which investigated antioxidant treatment for
various etiologies of male infertility. Very few studies
explored the effect of antioxidants on semen quality of
men with genitourinary inflammation (n=3), a hyper-
insulinemic state (n=1) and recurrent pregnancy loss in
female partners (n=1) (Table 2), and are hence insuf-
ficient for evidence-based recommendations for their
use. On the other hand, there are some perceived high-
quality studies available but, as highlighted above, due
to the diff iculties recruiting a sufficient number of pa-
tients and the small number of these studies, a statisti-
cal analysis will be underpowered and theref ore does
not provide the required answer. As we have shown
in our analysis, the number of such studies conducted
under the given circumstances and with a pre-selected
set of criteria would have to be unreasonably high.
Yet, looking at the diverse groups of conditions where
oxidative stress is significantly involved and an anti-
oxidant treatment would make sense, it is clear that in
absence of prior testing for oxidative stress and with-
out proper identification of suitable patient groups,
the treatment will fail. Hence, appropriate patient
identification is essential for the success of the treat-
ment. We were able to formulate recommendations for
antioxidant treatment for men with abnormal semen
quality, IMI, UMI, and clinical varicocele based on
studies reviewed (Table 2). However, it should be noted
that our recommendations are based on the previously
published studies, which, in our analysis, cannot be
considered as high-quality because a number of vari-
ables (such as female factor, inclusion criteria, sample
size esteem, etc.) were not properly reported. Therefore,
our recommendation is that antioxidant treatment is
possible and can result in improved male seminal pa-
rameters if the condition is caused by oxidative stress.
It is self-evident that the therapy has to be monitored
to not only avoid over-dosage of antioxidants, but also
to see if it is successful or if alternative treatments are
to be considered.
1. Abnormal semen quality
Antioxidants have long been investigated as a thera-
peutic option to counteract the harmf ul ef f ects of ROS
toxicity on various body systems. The reproductive sys-
tem is one good example, as seminal oxidative stress is
believed to be a common pathophysiology and various
oxidative stress-associated aetiologies can alter sperm
quality and function. Results of our review demon-
strated that the majority of low-quality studies report-
ed a significant improvement in conventional semen
parameters and measures of sperm function. However,
this result was not observed by high-quality studies.
The recent Cochrane review revealed a somewhat simi-
lar result [11]. While an improvement in conventional
semen parameters was noted over time, the f indings
were not reliable as a great deal of heterogeneity was
observed across the included studies. Moreover, antioxi-
dants were found to lower SDF compared to placebo.
Recommendation: antioxidants can improve conven-
tional semen parameters and measures of sperm f unc-
tion (grade C recommendation).
2. Varicocele
Varicocele is the most common correctable cause of
male infertility, prevalent in about 40% of men with
primary infertility and up to 80% of men with second-
ary infertility [131]. Several studies have confirmed the
presence of higher levels of oxidative stress in infertile
men with varicocele in comparison to fertile men with
or without varicocele and infertile men with idiopathic
infertility [132-136]. This finding may justify the util-
ity of antioxidants as a medical treatment strategy f or
varicocele. Nonetheless, in most patients, varicocelec-
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
49
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tomy remains the gold standard modality that results
in sustained improvement in semen parameters and
natural conception [137].
Most of the studies exploring the effect of antioxi-
dant supplementation on semen parameters and sperm
function were of low-quality. While the majority of
these studies reported an improvement in outcome, the
evidence supporting antioxidant use as a sole treat-
ment for varicocele is not suf ficient. Our sample size
calculation confirms the need for further research in
this regard in order to obtain a statistically significant
ef f ect. However, the reported improvement may be
clinically relevant supporting antioxidant use as an ad-
junct therapy to varicocele ligation. A recent systematic
review and meta-analysis explored antioxidant efficacy
on improving semen quality after varicocelectomy [138].
The authors included 6 RCT with 576 patients receiv-
ing various antioxidant regimens or placebo f ollowing
varicocelectomy. Signif icant improvements in sperm
concentration (p<0.001), total motility (p=0.03), progres-
sive motility (p<0.001) and normal morphology (p<0.001)
were reported for the treatment group. However, preg-
nancy rate did not improve (p=0.36). Nonetheless, this
finding confirms the presence of an additive effect of
the antioxidant therapy in patients undergoing varico-
celectomy.
Recommendation: antioxidants in addition to varico-
cele ligation result in further improvement in semen
parameters (grade C recommendation).
3. Unexplained male infertility and idiopathic
male infertility
Antioxidants are also commonly utilized f or the
treatment of patients with UMI or IMI. The former is
defined by failure of conception despite having normal
semen parameters, while the latter is characterized by
the presence of semen abnormalities due to unknown
etiology. The prevalence of UMI and IMI ranges be-
tween 6%–27% and 30%–58%, respectively [3,139]. Oxi-
dative stress is believed to play a significant role in the
pathophysiology of infertility of unknown origin and
has been identified in 30%–40% of patients with UMI
and up to 80% of patients with IMI [140-142].
Our analysis revealed that antioxidant use in men
with IMI and UMI resulted in significant improve-
ment in semen parameters and sperm f unction, as
reported by high-quality studies. While the majority
of low-quality studies echoed similar improvements, a
larger number of studies are required to reach statisti-
cal significance.
A systematic review of 32 studies which assessed the
impact of antioxidant therapy in IMI patients revealed
an improvement of semen parameters, with the biggest
benefit observed in sperm motility [143]. Fewer studies
have assessed antioxidant treatment in patients with
UMI. A recent study included 29 UMI patients who
were treated with a combination of antioxidants for 3
months. The authors reported a significant increase in
progressive motility (p=0.002), and a decrease in SDF
(p=0.03) and ORP levels (p=0.02) following treatment.
Greco et al [117] randomized 64 patients with UMI and
elevated SDF to either treatment with vitamins C and
E f or 2 months or placebo. While there was no signif i-
cant difference in semen parameter results, significant
reduction in SDF was noted in the treatment group
(p<0.001).
Recommendation: antioxidants signif icantly increase
sperm quality in men with IMI and UMI (grade B rec-
ommendation).
CURRENT STATE AND FUTURE
RECOMMENDATIONS FOR
ANTIOXIDANT RESEARCH
The bivalent action of ROS as essential signaling
molecules in physiological functions for sperm to f ertil-
ize an oocyte [144-146], as well as in mediating a detri-
mental ef fect on sperm functionality [147-149], suggests
that appropriate clinical indications f or prescribing
antioxidants and monitoring the treatment are critical
clinical considerations. Therefore, it is crucial to con-
tinue to study the physiological and pathophysiological
mechanisms of ROS in the male reproductive tract and
its relevance for sperm production and conception. In
this regard, the physiological needs of functional sper-
matozoa, e.g., for the induction of capacitation and ac-
rosome reaction, have to be considered. It appears that
three aspects of sperm physiolog y have to be addressed,
namely i) preservation and support of metabolism, ii)
improvement of sperm maturation and function, and
iii) protection against ROS-related trauma. Therefore,
the correct ratio and concentration of antioxidants is
essential f or this therapeutic effect to occur and any
new studies must take this into account.
Standardization of oxidative stress measurement
must also be implemented, not only because a repro-
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50 www.wjmh.org
ducible baseline is needed, but also because many of
the current studies are either lacking or using differ-
ent oxidative stress measurement and are theref ore
difficult to compare. The assessment of oxidative stress
may also facilitate the identification of the best candi-
dates and responders to antioxidant therapy (Fig. 4) [2].
Recently, the MiOXSYS system has be proposed as
a standardized assessment of seminal oxidative stress
due to its ease of use, cost ef fectiveness, and repro-
ducibility, as well as proposed evidence based clinical
guidelines [150,151]. The MiOXSYS system measures
the overall redox balance in seminal fluid to directly
evaluate oxidative or reductive stress. Monitoring the
treatment with antioxidants is another important as-
pect as overtreatment could lead to reductive stress-
related infertility because the little amount of ROS,
which is essential to trigger physiological functions of
spermatozoa for it to f ertilize oocytes, would be scav-
enged if the antioxidants are overdosed.
For a safe implementation of oral antioxidants use
in a clinical setting, it is important not only to discover
the molecular mechanisms of action of the bioactive
compounds, but also the secondary ef f ects that may
arise. Personalized or adjusted prescription of oral an-
tioxidants can enhance efficacy, without promoting
over-dosage and deleterious health ef fects. Methodol-
ogy and couple selection must be well reported. Doses
and duration of the treatment should be adjusted ac-
cording to several factors including the detected levels
of ROS and antioxidant enzymes in seminal f luid and
spermatozoa. Bioavailability of the compounds and
their mechanism of action should also be thoroughly
studied. More time-points to detect the oxidative bal-
ance of spermatozoa and seminal f luid should also be
considered. Finally, one should not only look at the
oral antioxidant therapy as a clinical treatment of the
male and then expect that this treatment will result
in the live birth of a healthy baby, but rather consider
reproduction as a joint responsibility of both partners
where a male and a female equally contribute to the
reproductive outcome. Currently, the female partner is
not examined f or possible oxidative stress, but there is
also a lack of knowledge about the impact of ROS and
the redox level (oxidative and reductive stress) in the
female reproductive system on oocyte development and
maturation as well as on embryo development. A study
by Ufer et al (2010) [152] indicated that proper embryo
development depends on finely tuned redox control. On
the other hand, elevated levels of antioxidants may re-
sult in teratogenic developments [153].
Currently, there is significant heterogeneity in tri-
als reporting antioxidants on male infertility. This
includes the condition investigated, type of antioxidant
used, duration of the study, and the outcomes mea-
sured (Table 2). Furthermore, it is essential to select
previously studied antioxidant candidates as well as
new potential compounds, and also determine if they
act better alone or in synergy with other antioxidants
Basic
semen
analysis
OS evaluation
by
ORP (MiOXSYS)
Oxidative
stress
Inflammation/
leukocytospermia
(prostatitis, MAGI)
Lifestyle
(smoking, obesity,.)etc Varicocele
Testing
ORP>
1.34 mV/10 sperm/mL
6
MOSI
Antioxidant+antibiotics
anti-inflammatory
Retest for MOSI
Counsel on
appropriate changes Varicocelectomy
Treatment
Retest for MOSI
with ORP
Antioxidant
supplementation
Fig. 4. Treatment options for male oxida-
tive stress infertility (MOSI). OS: oxidative
stress, ORP: oxidation-reduction poten-
tial, MAGI: male accessory gland infec-
tion.
Ashok Agarwal, et al: Antioxidant Therapy in Male Infertility
51
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as has been shown in some studies [154]. Due to several
factors, including the high costs and the high number
of participants needed to reach a statistically robust
study, the individuals recruited in any new study must
be well selected based on strict inclusion/exclusion cri-
teria. This means the patients should have the same
infertility diagnosis and similar semen analysis: for
example, mixing patients with azoospermia, mild oli-
gozoospermia or asthenozoospermia should be avoided.
Meeting these criteria will likely be difficult at a
single institution and does not even include recruiting
patients for an adequate control group. Use of placebo
in the control group is also mandatory to avoid bias.
Notably, confounding factors, including dietary habits,
are of ten overlooked in most RCTs, but when patients
are being enrolled in a study with antioxidant supple-
mentation, baseline antioxidant intake and diet should
be evaluated in both partners, male and female. Fur-
thermore, an appropriate primary outcome, such as de-
crease in oxidative stress or SDF, should also be evalu-
ated. Finally, it is critical to set a correct time period
for the study. A short to medium time period of 3 to 6
months is considered ideal to study the antioxidant ef-
fects on semen parameters.
UNRESOLVED QUESTIONS
What makes the situation even more complicated is
that there are no dose-response studies in humans to
pinpoint the optimum antioxidant dosages needed to
produce improvement in semen quality. We also still do
not know the normal physiological range of the redox
levels in both males and females. For spermatozoa, a
recent study by Panner Selvam et al (2020) [24] indicat-
ed normal redox values between -9.76 and 1.48 mV/106
sperm/mL. Although these values already indicate
oxidative or reductive stress conditions, the normal
physiological range will be much narrowed in between
these values. In the female, one might have to consider
variations depending on the menstrual cycle and/or the
onset of pregnancy. Besides, these values mig ht change
with age and/or health status. Furthermore, the effec-
tive bioavailability of antioxidants in the testes, the
epididymis and the semen is still unknown. Since many
antioxidants (e.g., co-enzyme Q10, vitamin E, and carni-
tines) can easily cross the blood-testis barrier, a proper
balance for the redox level has to be achieved because
an over-dosage might lead to reductive stress, which
has been shown to be as harmful as oxidative stress [22].
CONCLUSIONS
This systematic review identified a signif icant num-
ber of well-designed studies that unequivocally show
the beneficial effects of oral antioxidants in improving
semen parameters and pregnancy outcome. However,
despite the safety and efficacy of the antioxidant
therapy, five main factors have hindered its wide ac-
ceptance and implementation in the treatment of male
infertility: i) lack of randomized placebo-controlled
studies that show the safety and efficacy of antioxi-
dants in improving pregnancy rates in infertile couples;
ii) type of antioxidant to be used; iii) dose; iv) duration
of treatment; and v) costs.
Although randomized placebo-controlled studies
are regarded as the gold standard in the validation
of the safety and efficacy of therapies, given the fact
that the occurrence of a pregnancy is a multifactorial
process mainly determined by the genomic quality of
the egg, to show the impact of antioxidant therapy on
pregnancy outcome in randomized placebo-controlled
studies would be unrealistic and extremely difficult to
perform. Given the diff iculty in carrying out these ran-
domized placebo-controlled studies and the existence
of significant clinical evidence supporting the safety
and efficacy of antioxidants in improving pregnancy
outcome in infertile couples, the use of antioxidant
therapy should be recommended.
To answer the question of what type of antioxidant
should be used, we believe that antioxidants that read-
ily cross the blood-testis and blood-epididymis barriers
should be recommended. The formulation should be
well-balanced as lipid-soluble and water-soluble antiox-
idants together with other factors are closely interact-
ing, thereby regenerating lipid-located antioxidants. If
this balance is not given, it may not only result in sub-
optimal antioxidant effects, but also in paradoxical pro-
oxidant eff ects due to interference in redox reactions.
Concerning the dose to be used, the dose should be
high enough to restore the normal physiological cel-
lular functions by reducing oxidative stress without
compromising the physiological role of ROS in sperm
maturation and fertilization reactions. An overdosage
which may lead to reductive stress should be avoided.
The duration of an antioxidant therapy would have
to be adjusted according to the place where the damage
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occurs. If it is in the epididymis, a treatment course of
at least two weeks should be sufficient to counteract
ROS-induced damage. In addition, since oxidative stress
in the epididymis is a constitutive process and antioxi-
dants have no side-effects, antioxidant therapy should
be recommended until pregnancy is achieved. This
would apply to couples undergoing timed intercourse as
well as to couples undergoing in vitro fertilization. On
the other hand, if the oxidative damage is occurring in
the testes such as in the case of clinical varicocele, the
duration of antioxidant treatment should be of at least
three months.
Finally, the cost argument has to be seen from the
perspective of the sponsors of high quality randomized,
double-blind placebo-controlled clinical trials. Natural
antioxidant f ormulations have very low cost, and their
use would be amply justified based on ef f icacy and
safety as well as f or cost-saving aspects for patients
and health systems. The problem for the possible fund-
ing by the pharmaceutical industry of the studies is to
recover the high costs of a trial on a cheap antioxidant,
which can be available over the counter, and for which
not even intellectual property rights are available.
In conclusion, the use of antioxidants that readily
cross the blood-testis and blood-epididymis barriers
should be recommended. Their ef f icacy, lack of side-
effects and low costs should encourage their wider ac-
ceptance and implemen<