Accepted: 10 October 2016

DOI: 10.1111/and.12762

ORIGINAL ARTICLE

Oxidative stress status and sperm DNA fragmentation in fertile

and infertile men

M. Dorostghoal1 | S. R. Kazeminejad2 | N. Shahbazian3 | M. Pourmehdi4 | A. Jabbari5

1
Toxicology Research Center, Ahvaz
Jundishapur University of Medical Sciences, Summary
Ahvaz, Iran Evidence suggests that disturbing the balance between reactive oxygen species levels
2
Department of Genetics, Faculty of
and antioxidant contents in seminal plasma leads to oxidative stress resulting in male
Science, Shahid Chamran University of Ahvaz,
Ahvaz, Iran infertility. This study was carried out to identifying clinical significance of seminal oxi-
3
Department of Obstetrics and dative stress and sperm DNA fragmentation in treatment strategies of male infertility
Gynecology, Imam Khomeini Hospital, Ahvaz
in southwest Iran. Sperm parameters, lipid peroxidation and activity of antioxidant
Jundishapur University of Medical Sciences,
Ahvaz, Iran enzymes were assessed in fertile (n = 105) and infertile (n = 112) men. Malondialdehyde
4
Department of Food Hygiene and Public (MDA) levels in seminal plasma were found to be higher significantly (p < .001) in pa-
Health, Faculty of Veterinary Medicine, Shahid
Chamran University of Ahvaz, Ahvaz, Iran tients. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in
5
Department of Biology, Faculty of seminal plasma were significantly (p < .001) lower in infertile men. Significant negative
Science, Shahid Chamran University of Ahvaz, correlations were observed between MDA levels and sperm motility and normal mor-
Ahvaz, Iran
phology. Spermatozoa with fragmented DNA were higher (p < .001) in infertile men
Correspondence and significantly correlated with MDA levels and SOD and GPx activities. MDA of
Mehran Dorostghoal, Toxicology Research
Center, Ahvaz Jundishapur University of 4.2 nmol/ml, SOD of 4.89 U/ml and GPx of 329.6 mU/ml were optimum cut-­off limits
Medical Sciences, Ahvaz, Iran. to discriminate infertile patients from fertile men. The results show the leading role of
Email: dorostghoal@gmail.com
oxidative stress in aetiology of male infertility in southwest Iran and indicate that eval-
Funding information
Research grant was supported by Toxicology uation of seminal antioxidant status and DNA integrity can be helpful in men attending
Research Center of Ahvaz Jundishapur infertility clinics during fertility assessment.
University of Medical Sciences (no. TRC9102).

KEYWORDS
DNA fragmentation, male infertility, oxidative stress, sperm parameters

1 | INTRODUCTION of ROS production in ejaculated human spermatozoa (Musset et al.,

Oxidative stress reflects an imbalance between reactive oxygen spe-
cies (ROS) production and antioxidant defence ability and is thought
to have clinical significance in the pathophysiology of male infertil-
ity (Tremellen, 2008). It has been reported that 25%–40% of infertile
men have higher levels of seminal ROS (Padron et al., 1997). ROS is
produced by spermatozoa in physiological amounts and plays a role
in functional processes such as capacitation, acrosomal reaction and
spermatozoon–oocyte fusion (Agarwal & Allamaneni, 2004). However,
excessive production of ROS impairs the antioxidant defence of sper-
matozoa and seminal plasma resulting in oxidative stress (Henkel,
2011). Leucocytes and morphologically abnormal spermatozoa are the
main sources of ROS in semen (Garrido, Meseguer, Simon, Pellicer, &
Remohi, 2004). NADPH oxidase 5 (NOX5) was detected as the source
2012), and correlations were observed between abnormal sperm
morphology and both the percentage of NOX5-­positive spermatozoa
and the magnitude of NOX5 expression (Ghani, Keshtgar, Habibagahi,
Ghannadi, & Kazeroni, 2013). Higher levels of seminal ROS and lower
antioxidant potential were reported in men with idiopathic infertility
than healthy fertile controls (Pasqualotto et al., 2001). Lack of the
necessary cytoplasmic-­enzyme repair systems and high amounts of
polyunsaturated fatty acids in plasma membrane increase susceptibil-
ity of spermatozoa to excessive ROS (Gharagozloo & Aitken, 2011).
Excessive generation of ROS not only affects the plasma membrane
but also the integrity of DNA and can thus result in abnormal sperm
function. ROS are known to attack DNA bases and phosphodiester
backbones; destabilising this molecule leads to DNA fragmentation
(Villegas et al., 2005). Sperm DNA fragmentation as a potential risk

Andrologia 2017; e12762; wileyonlinelibrary.com/journal/and © 2017 Blackwell Verlag GmbH | 1 of 9

DOI: 10.1111/and.12762
2 of 9 | DOROSTGHOAL et al.
factor for normal embryo development is associated with poor fertili-
sation, increased miscarriage rates, birth anomalies and cancer in chil-
dren (Aitken, De Iuliis, & McLachlan, 2009; Hansen, Kurinczuk, Bower,
& Webb, 2002; Lewis & Aitken, 2005). Furthermore, higher fraction
of sperm DNA damage has been reported in infertile men (Sheikh,
Amiri, Farimani, Rezvan Najafi, & Hadeie, 2008; Zhang et al., 2010).
Recent evidence that shows the association between degree of DNA
fragmentation and sperm parameters (Cassuto et al., 2012; Oleszczuk,
Augustinsson, Bayat, Giwercman, & Bungum, 2013; Omran, Bakhiet,
& Dashti, 2013) suggests that assessment of DNA fragmentation in
fertile and infertile men has significant clinical implications, improved
predictive values and may lead to the development of new therapeu-
tic approaches for male infertility. Thus, in this study, oxidative stress
status, sperm DNA fragmentation and their possible associations with
sperm parameters were analysed in fertile and infertile men in south-
west Iran.
2 | MATERIALS AND METHODS
2.1 | Subjects
The study was approved by the institutional review board of the
Toxicology Research Center, Ahvaz Jundishapur University of Medical
Sciences (no. TRC9102). All participants gave written informed con-
sent and completed a questionnaire which included information
regarding age, body weight, height, general health, lifestyle, con-
sumption of alcohol and drugs and smoking. Semen specimens were
obtained from fertile volunteer donors (n = 105), with normal semen
characteristics whose wives achieved a full-­term pregnancy within the
last 2 years, and from infertile men (n = 112) attending the infertility
clinic in the Arya Hospital of Ahvaz between 2013 and 2015. Inclusion
criteria were infertile men with abnormal semen parameters from
couples with a history of inability to conceive a child after 1 year of
regular unprotected sexual intercourse and a normal female reproduc-
tive function. Exclusion criteria were smoking, alcohol consumption
and use of drugs such as habitual drugs, consumption of certain foods,
antioxidant supplementation and occupational and environmental ex-
posures to potential reproductive toxins. Patients were also excluded
from analysis if they had azoospermia, varicocele, cryptorchidism,
prostatitis, urinary tract infection, genital trauma, testicular torsion,
inguinal or genital surgery, sexually transmitted disease, chronic illness
and serious systemic diseases.
2.2 | Semen analysis
Semen samples were collected by masturbation in wide-­mouth ster-
ile plastic containers after 3 days of sexual abstinence. Semen speci-
mens were allowed to liquefy at room temperature for 30 min, and
then, their macroscopic and microscopic analyses were performed
according to the World Health Organization’s guidelines (WHO,
2010). Sperm parameters were considered normal when sperm con-
centration was ≥15 × 106/ml, motility was ≥40% and normal sperm
forms were ≥4%. Semen samples containing >1 × 106 WBC/ml were
T A B L E 1 Median and interquartile range of age and semen
parameters in fertile and infertile men
Fertile men Infertile men p
Parameters (n = 105) (n = 112) Value
Age (years) 36.0 (32.0–39.5) 38.0 (33.0–40.0) .105
Volume (ml) 4.0 (3.5–5.0) 4.0 (3.0–4.5) .014
Sperm count 60.0 (55.0–64.0) 12.0 (8.0–45.0) <.001
(million/ml)
Motility (%) 48.0 (42.0–59.0) 15.0 (10.0–29.25) <.001
Normal 10.0 (6.0–12.0) 4.0 (3.0–8.0) <.001
morphology (%)
excluded. The characteristics of patients and their semen quality are
presented in Table 1.
2.3 | Single cell gel electrophoresis (comet) assay
The sperm DNA integrity was assessed using the modified neutral
comet assay as described by Singh, Muller, and Berger (2003). Briefly,
5 μl of sperm cell suspension in PBS was mixed with 50 μl of 0.7% low-­
melting point agarose (Sigma, USA), layered onto a pre-­coated slide
with 1.0% low-­melting point agarose. Then, the slide was covered
with a coverslip and the agarose was allowed to solidify at 4°C for at
least 5 min. The cover glass was then removed, and the low-­melting
point agarose (200 μl) was layered on slide as a third layer. After com-
plete solidification, the cover glass was removed and the slides were
dipped in lysis buffer (50 mm tetrasodium ethylenediaminetetraacetic
acid, 0.01% sodium N-­lauroyl sarcosinate, 100 mm Tris, 1.25 m NaCl,
2 mg/ml reduced glutathione, 0.5 mg/ml DNAse free proteinase K,
pH 10) at room temperature for 2 hr. The slides were then immersed
in an electrophoresis unit (Bio-­Rad, USA) filled with freshly prepared
neutral electrophoresis solution (100 mm Tris HCl, 500 mm NaCl and
500 mm EDTA, pH 9), allowed to equilibrate for 20 min and elec-
trophoresed under 12 V and 100 mA at room temperature for 1 hr.
Slides were immersed in freshly prepared neutralising buffer (20 mm
Tris [pH 7.4], 50% ethanol and 1 mg/ml of spermine) twice and each
time for 15 min. Air-­dried slides were stained with DNA safe Stain
(CinnaGene, Iran) for 5 min and covered with a 24 × 50 mm coverslip.
Two slides were prepared for each subject, 200 randomly chosen nu-
clei analysed using a fluorescence microscope (Olympus BX51, Japan)
and presented as the per cent of spermatozoa with fragmented DNA.
2.4 | Measurement of lipid peroxidation
Lipid peroxidation in seminal plasma was assayed using the thiobarbi-
turic acid (TBA) test as described by Ohkawa, Ohishi, and Yagi (1979).
This method is based on the reaction of malondialdehyde (MDA), as
an index of lipid peroxidation, with TBA. After liquefaction, semen
samples were centrifuged at 1000 g for 10 min and 500 μl of seminal
plasma added to 3 ml of phosphoric acid 1%. Then, 1 ml of TBA 0.67%
was added to the mixture and heated at 95°C in a warm water bath
for 1 hr. After the mixture was cooled, 2 ml of n-­butanol added to
DOROSTGHOAL et al.
tube, centrifuged at 600 g for 10 min and absorbance of the superna-
tant measured at 532 nm by spectrophotometer. The seminal levels of
lipid peroxidation were expressed as nmol/ml.
2.5 | Antioxidant enzyme activity
Activity of superoxide dismutase (SOD) and glutathione peroxidase
(GPx) enzymes were analysed using colorimetric assay kits according
to the recommendations of the manufacturer. Ransod kit (Randox,
UK) was used to analyse SOD activity in seminal plasma. Superoxide
dismutase activity was measured by the degree of inhibition of red
formazan dye formation following the reaction of superoxide radi-
cals with 2-­(4-­iodophenyl)-­3-­(4-­nitrophenol)-­5-­phenyltetrazolium
chloride that was detected at 505 nm and expressed as U/ml seminal
plasma (Woolliams, Wiener, Anderson, & Mc Murray, 1983). Ransel
kit (Randox) was utilised to analyse GPx activity in seminal plasma.
The oxidation of gluthatione by cumene hydroperoxide catalyses by
GPx enzyme. In the presence of NADPH and glutathione reductase,
NADPH was oxidised to NADP+ in concomitant with oxidised glu-
tathione reduction. This reaction was detected at 340 nm and GPx ac-
tivity expressed as mU/ml seminal plasma (Paglia & Valentine, 1967).
2.6 | Statistical analysis
Data analysis was performed using SPSS 16.0 (SPSS Inc., Chicago, IL,
USA) for Windows. For normal distribution, variables were analysed
by the Kolmogorov–Smirnov test. The statistical significance was as-
sessed using Mann–Whitney U-­test and presented as median values
and interquartile ranges. The correlations were assessed using the
Spearman’s correlation analysis. Values were considered to be sig-
nificant when p < .05. Receiver operating characteristic (ROC) curve
F I G U R E 1 Median and interquartile
range of malondialdehyde (MDA) levels
in seminal plasma, superoxide dismutase
(SOD) and glutathione peroxidase (GPx)
activities in seminal plasma and sperm
DNA fragmentation in fertile (n = 105) and
infertile (n = 112) men. *p < .001
| 3 of 9
analysis by assessment area under curve (AUC) was used to assess
the discriminative ability of MDA, SOD and GPx with respect to male
infertility.
3 | RESULTS
Seminal parameters in fertile and infertile men are presented as me-
dian and interquartile ranges. Table 1 shows the semen profiles of
fertile and infertile subjects. There was no significant difference in
the age of the subjects of the two studied groups. The median val-
ues of semen volume and sperm parameters (concentration, motility
and morphology) were significantly different between control and
infertile men. The median levels of MDA, GPx and SOD in seminal
plasma and sperm DNA fragmentation of the two groups are shown
in Figure 1. Significantly higher levels of MDA in seminal plasma
were seen in infertile men compared to the fertile controls. Levels
of GPx and SOD activity in seminal plasma were significantly lower
in infertile men. Infertile men showed significantly higher sperma-
tozoa with fragmented DNA in comparison with fertile subjects
(Figure 2).
Significant negative correlations were seen between levels of
MDA in seminal plasma and sperm concentration, motility and nor-
mal morphology in healthy control and infertile men (Table 2). MDA
levels in seminal plasma showed significant negative correlations with
SOD and GPx activities in seminal plasma in fertile (r = −.229, p = .019
and r = −.262, p = .007) and infertile (r = −.338, p < .001 and r = −.234,
p = .013) men respectively. In fertile and infertile men, there were
significant negative correlations between sperm DNA fragmentation
and sperm concentration, motility and normal morphology (Table 3).
SOD and GPx activities in seminal plasma showed significant positive
4 of 9 | DOROSTGHOAL et al.

(a) (b)

(c) (d) F I G U R E 2 Sperm DNA integrity

analysed by neutral single cell gel
electrophoresis (comet) assay (×400). The
displacement between the genetic content
of the nucleus and the resulting tail is
proportional to the extent of damage on
the cell. Undamaged (white arrows in a–d)
and damaged cells (red arrows in a: low
level of DNA damage, b and c: moderate
level of DNA damage and d: high level of
DNA damage)

T A B L E 2 Spearman’s correlations between malondialdehyde (MDA) levels in seminal plasma, sperm DNA fragmentation and sperm
parameters in fertile and infertile men

Fertile men (n = 105) Infertile men (n = 112)

Parameters Count (million/ml) Motility (%) Morphology (%) Count (million/ml) Motility (%) Morphology (%)

MDA (nmol/ml) r = −.244 r = −.257 r = −.364 r = −.178 r = −.218 r = −.517

p = .012 p = .008 p < .001 p = .060 p = .021 p < .001
DNA fragmentation r = −.205 r = −.236 r = −.207 r = −.524 r = −.480 r = −.487
(%) p = .036 p = .015 p = .034 p < .001 p < .001 p < .001

T A B L E 3 Spearman’s correlations between superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in seminal plasma,
sperm concentration, motility and normal morphology in fertile and infertile men

Fertile men (n = 105) Infertile men (n = 112)

Parameters Count (million/ml) Motility (%) Morphology (%) Count (million/ml) Motility (%) Morphology (%)

SOD (U/ml) r = .211 r = .374 r = .372 r = .422 r = .499 r = .244

p = .031 p < .001 p < .001 p < .001 p < .001 p = .010
GPx (mU/ml) r = .305 r = .224 r = .210 r = .290 r = .508 r = .140
p = .002 p = .022 p = .032 p = .002 p < .001 p = .142

correlations with sperm concentration, motility and normal morphol-
ogy in both groups (Table 3). Sperm DNA fragmentation showed sig-
nificant positive correlation with MDA levels in seminal plasma and
significant negative correlations with SOD and GPx activities in semi-
nal plasma in both groups (Figure 3). ROC analysis revealed a diagnos-
tic value for MDA index with respect to male infertility, with an AUC of
0.762 (95% confidence interval = 0.700–0.817), sensitivity = 44.64%
and specificity = 97.14% with a cut-­off value of 4.2 nmol/ml (Figure 4).
In addition, ROC analysis revealed a diagnostic value for SOD activity
with respect to male infertility, with an AUC of 0.874 (95% confidence
interval = 0.822–0.915), sensitivity = 69.64% and specificity = 93.33%
with a cut-­off value of 4.89 U/ml (Figure 4). Also, GPx activity showed
a diagnostic value regarding male infertility with an AUC of 0.969 (95%
confidence interval = 0.936–0.987), sensitivity = 92.86% and specific-
ity = 98.10% with a cut-­off value of 329.6 mU/ml (Figure 4).
4 | DISCUSSION
It has been suggested that all parameters of male fertilising ability
do not adequately reflect during traditional semen analysis and a
combined assessment of semen quality and a more comprehensive
evaluation of fertility status promote a better management of infer-
tile couples (Samplaski, Agarwal, Sharma, & Sabanegh, 2010). In this
regard, assessment of seminal oxidative stress and sperm DNA frag-
mentation have been suggested as complementary tools in clinical
DOROSTGHOAL et al. | 5 of 9

(a) (d)

(b) (e)

F I G U R E 3 Spearman’s correlations
between sperm DNA fragmentation,
malondialdehyde (MDA) levels in seminal
plasma and superoxide dismutase (SOD)
and glutathione peroxidase (GPx) activities
(c) (f)
in seminal plasma in fertile (n = 105) (a–c)
and infertile (n = 112) (d–f) men

F I G U R E 4 Receiver operating characteristic curve analysis of malondialdehyde (MDA) levels in seminal plasma (AUC = 0.762, cut-­off
point = 4.2 nmol/ml) and activity of superoxide dismutase (SOD) (AUC = 0.874, cut-­off point = 4.89 U/ml) and glutathione peroxidase (GPx)
(AUC = 0.969, cut-­off point = 329.6 mU/ml), respectively, in seminal plasma as a predictor power to discriminate infertile subjects from fertile
men

recognition of male infertility (Micinski et al., 2011; Omran et al.,
2013; Venkatesh et al., 2011).
In the present study, the oxidative stress status was evaluated by
assessing the MDA levels in semen of healthy control and infertile
men. Our data showed a significant increase in seminal levels of lipid
peroxidation index in infertile men. ROC analysis showed MDA mea-
surement to be reasonably accurate in differentiation between infertile
subjects versus fertile men at criterion value 4.2 nmol/ml. Benedetti
et al. (2012), Hosen, Islam, Begum, Kabir, and Howlader (2015) and
Shamsi et al. (2009) found that MDA levels in seminal plasma in infertile
6 of 9 | DOROSTGHOAL et al.
men was significantly higher than in fertile group. Ben Abdallah et al.
(2009), Hsieh, Chang, and Lin (2006) and Tavilani et al. (2008) showed
that MDA levels in seminal plasma in normozoosperma were signifi-
cantly higher than asthenozoospermia and oligoasthenozoospermia.
Also, Nabil, Lekaka, Moemen, and Eela (2008) reported higher MDA
levels in seminal plasma in oligozoospermic and azoospermic men.
Moreover, our finding indicated that MDA in seminal plasma was
negatively correlated with sperm parameters in healthy control and
infertile patients. Several studies showed that lipid peroxidation cor-
related with poor quality of spermatozoa and MDA concentration in
seminal plasma correlated negatively with sperm concentration, motil-
ity and morphology (Benedetti et al., 2012; Hosen et al., 2015; Shiva
et al., 2011). In this regard, it has been shown that ROS are negatively
correlated with traditional semen parameters such as sperm concen-
tration, motility and morphology in infertile men (Agarwal et al., 2014)
and with sperm concentration in teratozoospermic patients and con-
trols (Agarwal, Tvrda, & Sharma, 2014). An imbalance between ROS
generation and the antioxidant capacity results in lipid peroxidation
which subsequently leads to the loss of plasma membrane fluidity and
function (Agarwal, Saleh, & Bedaiwy, 2003). Excessive ROS impairs
sperm motility mainly through the inhibition of enzymes such as glyc-
eraldehyde 6-­phosphate dehydrogenase (G-­6-­PDH) due to low levels
of intracellular NADPH availability and also accumulation of oxidised
glutathione and reduced glutathione that in turn increase membrane
phospholipids peroxidation (Griveau, Renard, & Le Lannou, 1995).
Moreover, it has been proposed that increased production of ROS in-
duces a cascade of events that result in a decrease in axoneme protein
phosphorylation and sperm immobilisation (De Lamirande & Gagnon,
1995). However, Suleiman, Ali, Zaki, el-­Malik, and Nasr (1996) have
not found any correlation between MDA levels in seminal plasma with
sperm concentration and motility.
In our study, significantly lower activity of SOD and GPx in seminal
plasma was found in infertile men than in fertile men. ROC analysis
clearly demonstrated that SOD and GPx activities in seminal plasma
are reliable predictors with respect to male infertility at criterion value
4.89 U/ml and 329.6 mU/ml respectively. Similarly, compared to nor-
mozoospermic subjects, significantly lower activity of SOD in seminal
plasma (Hosen et al., 2015; Murawski et al., 2007) and GPx (Alkan et al.,
1997; Giannattasio et al., 2002) was reported in infertile men. Siciliano
et al. (2001) showed that oligoasthenozoospermic patients have lower
SOD activity in seminal plasma than normozoospermic men. In this
regard, it has been shown that antioxidant capacity is significantly
lower in infertile men compared to healthy controls (Benedetti et al.,
2012; Hosen et al., 2015; Khosravi, Valojerdi, Amanlou, Karimian, &
Abolhassani, 2014). Furthermore, Khosrowbeygi and Zarghami (2007)
found that in comparison with controls, asthenozoospermic, asthe-
noteratozoospermic and oligoasthenoteratozoospermic patients have
significantly lower levels of total antioxidant capacity. Also, Koca,
Ozdal, Celik, Unal, and Balaban (2003) and Lewis, Boyle, McKinney,
Young, and Thompson (1995) reported a reduced seminal antioxidant
capacity in asthenozoospermic men compared with fertile controls.
However, in comparison with normozoospermic controls, no defi-
ciency in SOD (Hsieh et al., 2002; Zini, Garrels, & Phang, 2000) and
GPx activities in seminal plasma (Hsieh et al., 2006; Yeung et al., 1998)
was reported in infertile men. Tavilani et al. (2008) found no significant
difference in SOD and GPx activities in seminal plasma between nor-
mozoospermic and asthenozoospermic men.
The present study showed a positive correlation between SOD
and GPx activities in seminal plasma and sperm concentration, mo-
tility and normal morphology in both fertile and infertile men. Hosen
et al. (2015) and Murawski et al. (2007) showed a positive correlation
between SOD activity in seminal plasma and sperm concentration,
motility and normal morphology. Shamsi et al. (2009) observed a pos-
itive correlation of sperm count and motility and a negative correla-
tion of abnormal morphology with SOD and glutathione. Moreover,
positive correlations were reported between sperm concentration
(Hsieh et al., 2006) and motility (Dandekar, Nadkarni, Kulkarni, &
Punekar, 2002; Hsieh et al., 2006) with GPx activity in seminal
plasma. However, no significant correlation was found between SOD
activity in seminal plasma and sperm concentration and motility
(Hsieh et al., 2002).
Our finding showed significantly higher levels of spermatozoa with
fragmented DNA in infertile men than fertile controls. Sperm DNA
fragmentation as a novel marker of sperm quality has been implicated
to poor sperm function and subfertility (Agarwal et al., 2003) and re-
lated to lower rates of natural conception (Evenson & Wixon, 2008),
higher rates of pregnancy loss after IVF and IVF-­ICSI treatments (Zini
& Libman, 2006; Zini & Sigman, 2009) and offspring morbidity (Aitken
et al., 2009). Sergerie, Laforest, Bujan, Bissonnette, and Bleau (2005),
Sheikh et al. (2008), and Zhang et al. (2010) showed that sperm DNA
damage was significantly higher in infertile patients than fertile men.
Hosen et al. (2015) reported that infertile men have significantly
higher levels of 8-­hydroxy-­2′-­deoxyguanosine (8-­OHdG) in seminal
plasma, as a biomarker of DNA damage, compared with fertile con-
trols. Oligozoospermic, asthenozoospermic and teratozoospermic men
showed significant increase in spermatozoa with fragmented DNA in
comparison with controls (Khosravi et al., 2014; Shamsi et al., 2009).
Also, significantly higher levels of DNA fragmentation were found in
the semen of asthenozoospermic patients as compared to men with
normal sperm motility (Benchaib et al., 2003; Piasecka, Gaczarzewicz,
Laszczyñska, Starczewski, & Brodowska, 2007). Normozoospermic
men exhibited lower levels of DNA fragmentation than their non-­
normozoospermic counterparts (Evgeni, Lymberopoulos, Touloupidis,
& Asimakopoulos, 2015).
An inverse correlation was established between DNA damage and
sperm concentration, motility and normal morphology in both control
fertile and infertile patients. Several studies have shown that a nega-
tive correlation exists between DNA damage and sperm parameters
including sperm count (Oleszczuk et al., 2013; Omran et al., 2013;
Venkatesh et al., 2011), motility (Micinski et al., 2011; Omran et al.,
2013; Sheikh et al., 2008) and normal morphology (Cassuto et al.,
2012; Micinski et al., 2011; Omran et al., 2013; Sharbatoghli, Valojerdi,
Amanlou, Khosravi, & Jafarabadi, 2012). However, Evgeni et al. (2015)
reported that in non-­normozoospermic men, DNA damage has no sig-
nificant correlation with sperm parameters except for the progressive
motility. Moreover, no significant correlations were found between
DOROSTGHOAL et al.
DNA fragmentation and sperm concentration (Sheikh et al., 2008) and
morphology (Muratori et al., 2000).
Our study also showed that sperm DNA fragmentation correlates
positively with lipid peroxidation and negatively with SOD and GPx
levels in seminal plasma. In numerous studies, higher DNA fragmen-
tation have been shown in men with high ROS and low antioxidant
capacity in seminal plasma (Aktan et al., 2013; Khosravi et al., 2014;
Shamsi et al., 2009), which clearly indicate the association between
sperm DNA damage and oxidative stress (Aitken, De Iuliis, Finnie,
Hedges, & McLachlan, 2010; De Iuliis et al., 2009; Shamsi et al., 2009).
DNA strand breaks, DNA cross-­links, and chromosomal rearrange-
ments occur following excessive production of ROS (Duru, Morshedi, &
Oehninger, 2000). ROS can damage DNA directly by generation of oxi-
dised DNA adducts, for example 8-­OHdG and 1, N6-­ethenoadenosine
(Badouard et al., 2008) or indirectly by activation of caspases (Sakkas
& Alvarez, 2010). However, in a study by Verit et al. (2006) in normo-
zoospermic infertile men, no relationship was found between sperm
DNA fragmentation and oxidative stress. In the present study, the
correlations between DNA fragmentation and MDA levels in seminal
plasma and GPx activity in seminal plasma but not SOD activity in sem-
inal plasma in fertile men are almost the same as these associations in
infertile men. However, this conformity could be because that, except
for oxidative stress, there are a variety of molecular mechanisms such
as chromatin packaging abnormalities (Laberge & Boissonneault, 2005)
and apoptosis (Sakkas, Seli, Bizzaro, Tarozzi, & Manicardi, 2003) that
occur during spermatogenesis associated with sperm DNA damage.
Our findings clearly indicate that lowered antioxidant profile and
significant accumulation of MDA in seminal plasma, as a biomarker of
lipid peroxidation, are closely associated with poor sperm parameters
and higher degree of DNA damage in infertile men in southwest Iran.
Air, water and soil pollutions caused by airborne particles, industrial ac-
tivities, insecticides, pesticides and plasticisers, exposure to heat and
lifestyle behaviours such as smoking, alcohol abuse, obesity, psycho-
logical stress and poor nutrition have all been linked to oxidative stress.
Determining the sources and the levels of excessive ROS production in
human semen and precise evaluation of the antioxidant system may be
useful tools in the development of therapeutic strategies such as min-
imising exposure to pollutions and heat, lifestyle modification, taking
antioxidant supplements and addition of anti-­inflammatory drugs to
decrease ROS production in leucocytes for male infertility.
However, our study had some limitations: data were collected from
a single randomised centre, all variables such as smoking and alcohol
consumption were collected through self-­completed questionnaires,
which may have introduced a risk of misclassification of the exposure
variable, and only one semen sample per subject was collected. Further
studies with use of other markers, such as 4-­hydroxy-­2-­nonenal, 8-­
OHdG and protein carbonyl content and also proteomics assays, are
recommended to reveal the role of oxidative stress in the pathogene-
sis of male infertility.
Therefore, the present study highlights that status of seminal ox-
idative stress and extent of sperm DNA damage have clinical signif-
icance and should be considered as the important markers in male
infertility aetiology.
| 7 of 9
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How to cite this article: Dorostghoal M, Kazeminejad SR,
Shahbazian N, Pourmehdi M, Jabbari A. Oxidative stress
status and sperm DNA fragmentation in fertile and infertile
men. Andrologia. 2017;00:e12762. https://doi.org/10.1111/
and.12762