Correlation of Seminal Plasma Total Antioxidant Capacity and
Malondialdehyde Levels With Sperm Parameters in Men With
author: Saeedeh Salimi, Department of Clinical Biochemistry, School of
Medicine, Zahedan University of Medical Sciences, Zahedan, IR Iran. Tel:
+98-5433425728, Fax: +98-5433425728, Email: email@example.com
stress is the result of an imbalance between the production and
scavenging of reactive oxygen species (ROS). Recently, oxidative stress
has been introduced as a major cause of male infertility.
aim of the present study was to determine the correlation between total
antioxidant capacity (TAC) and malondialdehyde (MDA) as markers of
oxidative stress in relation to idiopathic male infertility and sperm
Patients and Methods: This
case control study was conducted using 35 men with idiopathic
infertility and 34 men with proven fertility. Seminal plasma TAC and MDA
were measured by ferric reducing ability of plasma (FRAP) and
thiobarbituric acid (TBA) reaction methods, respectively.
TAC levels were significantly lower and seminal MDA levels were
significantly higher in men with idiopathic infertility than in fertile
men (P < 0.0001 and P = 0.004, respectively). A positive correlation
was shown between sperm motility, sperm morphology, and TAC levels in
men with idiopathic infertility (P = 0.002 and P = 0.002, respectively).
In addition, there was a correlation between sperm motility and TAC
levels in fertile men (P = 0.005). There was no correlation between
sperm count and TAC levels in either men with idiopathic infertility or
in fertile men. Negative correlations were observed between MDA levels
and sperm motility, morphology, and sperm count only in men with
idiopathic infertility (P = 0.003, P = 0.001, and P = 0.006,
results show that oxidative stress could play an important role in male
infertility as well as in sperm motility and sperm morphology.
Keywords: Infertility; Male; Malondialdehyde; Semen; Total Antioxidant Capacity
Infertility is a major
social and medical problem worldwide. Studies in normal couples reveal
that 90% of them will conceive within one year of unprotected
intercourse. Thus, the classic definition of infertility became the
absence of conception after one year of regular and unprotected
However, because a small number of normal couples will conceive between
12 and 24 months, the world health organization (WHO) has suggested
that absence of conception after 24 months of unprotected intercourse is
the preferred definition of infertility (2). The prevalence of infertility is increasing, and at present 10 - 15% of all couples in worldwide suffer from infertility (3). The male factor seems to be responsible for approximately 50% of the cases (4).
There are several known etiologies of infertility, such as male
accessory gland infection, hypogonadotropic hypogonadism, retrograde
ejaculation, and positive anti-sperm antibody, but despite technical
advances, the exact etiology of male infertility is not clear in 25% of
all cases. Idiopathic male infertility is also known as idiopathic
oligoasthenospermia, which indicates that the men have an unexplained
reduction in semen quality (5).
Recently, oxidative stress has been shown to be an important cause of idiopathic male infertility (6).
Oxidative stress is a result of an imbalance between the production of
reactive oxygen species (ROS) and their neutralization or scavenging by
the antioxidant system. ROS are inherent byproducts of aerobic life, and
oxidative stress occurs when they overcome our natural ability to
detoxify ROS-induced damage (7, 8).
It is believed that controlled levels (physiological levels) of these
ROS are necessary for sperm physiology, maturation, capacitation,
acrosomal reactions, and normal fertilization. However, uncontrolled
production of ROS (pathological levels) causes sperm dysfunction,
including lipid peroxidation, sperm DNA damage, and loss of motility (9).
The excessive production of ROS could originate from endogenous
sources, including immature/abnormal spermatozoa and leukocytes, or
from environmental sources such as cigarette smoking and alcohol (10)
In fertile men, total antioxidant capacity (TAC) and ROS
production remain in balance. However, pathological conditions such as
autoimmune disorders, chronic disease, alcohol consumption, advanced
age, smoking, infections, and stress result in oxidative stress (7).
Antioxidants in the seminal fluid play a key role in antioxidant
defense mechanisms. Therefore, relatively low concentrations of
scavenging enzymes or non-enzymatic antioxidants within the cytoplasm
and large amounts of polyunsaturated fatty acids in membranes make
spermatozoa susceptible to ROS from lipid peroxidation (11).
As a result, the most important form of antioxidant defense available
to spermatozoa is the antioxidants in seminal fluid. Antioxidants
naturally found in semen include vitamins, glutathione, thioredoxin, and
superoxide dismutase (11). Several studies have indicated that high levels of ROS in seminal fluid increase the risk of male infertility (12, 13).
Sperm count and sperm motility are important parameters that
determine the functional ability of spermatozoa. Low sperm motility,
which is called asthenozoospermia, is considered to be associated with
male infertility. Although the factors that could affect sperm motility
are not well known, oxidative stress, which is induced by ROS, may be an
important factor in this condition (14).
The antioxidant power of biological fluids such as seminal
fluid can be assessed either with measurement of each antioxidant or
with total antioxidant capacity (TAC). Malondialdehyde (MAD) is used to
measure the degree of peroxidation damage in spermatozoa (15).
Because the findings
about the correlation of seminal plasma TAC and MAD with sperm
parameters are controversial, the purpose of this study was to explore
the total antioxidant capacity (TAC) and malondialdehyde (MAD) levels in
the seminal fluid of infertile and fertile men and their relationship
with sperm parameters.
3. Patients and Methods
This study was performed
using 35 consecutive infertile men with the complaint of infertility
(absence of conception after 12 months of regular and unprotected
intercourse) within the last year that were referred to the urology
clinic in Ali-ebne-Abitaleb and Khatamolanbia hospitals in Zahedan from
August, 2013 to March, 2014. Thirty-four men with proven fertility were
considered as controls. All patients and healthy controls with specific
genital diseases, genital infections, undescended testes, testicular
atrophy, or systemic disease were excluded from the study.
All semen samples were collected by masturbation in sterile
polypropylene containers after three to five days of abstinence. Semen
specimens were liquefied at 37°C for 30 minutes. Routine semen analysis
was performed according to world health organization guidelines 2010 (2).
Written informed consent was obtained from the patients and
controls. The project has been approved by the ethics committee of
research of the Zahedan university of medical sciences.
For determination of the percent normal morphology of
spermatozoa, the hematoxylin-eosin (H and E) staining method was used.
Morphology of the spermatozoa was assessed using Kruger’s criteria that
morphology < 14% is considered abnormal (16).
3.1. TAC assay
TAC was assessed using ferric reducing ability of plasma (FRAP) according to the method of Benzie et al. (17). We measured the ability of seminal plasma antioxidants in reduction of ferric-tripyridyltriazine (Fe3+-TPTZ) to a ferrous form (Fe2+).
The working FRAP reagent was prepared by 10 vol. of 300 mmol/L acetate
buffer; pH 3.6 with 1 volume of 10 mmol/L 2,4,6-tripyridyl-s-triazine in
40 mmol/L HCl with one volume of 20 mmol/L FeCl3.6H2O.
Then, 1.5 mL of the working FRAP reagent was aliquoted into a glass
tube and warmed to 37°C for five minutes. Subsequently, 50 μL of seminal
plasma and 50 μL of distilled water (reagent-free) as well as 50 μL of
each of the standard solutions (FeSO4.7H2O; 1000,
500, 250, 125 μM) were added to the 1.5 mL of FRAP reagent and heated to
37°C for 10 minutes. Absorbance was measured at 593 nm using a
spectrophotometer (UV-visible). The final results are shown as mmol/L.
3.2. MDA Assay
Seminal MDA levels were measured according to the method described by Rao et al. (18).
This method is based on thiobarbituric acid (TBA) reaction and
extraction with normal butanol. 1,1,3,3-tetramethoxypropane was used as
the standard. Spectrophotometric detection of absorbance was performed
at 532 wave length and compared with the standard curve. TBA was
purchased from Merck.
3.3. Statistical Analysis
Data were analyzed using the statistical software SPSS-18 (SPSS,
Chicago, IL). Data have been presented as mean ± SD. The normal
distribution of data was analyzed using the Kolmogorov-Smirnov (KS)
statistical test. Comparison between two groups was performed using the
independent sample t-test or Mann-Whitney U test whenever appropriate.
In addition, Pearson’s correlation coefficient test was used to
determine the correlation among different factors. Values of P < 0.05
were considered statistically significant.
The semen parameters, TAC, and MDA levels are shown in Table 1.
There were significant differences in semen volume, sperm motility,
sperm morphology, and sperm count between men with idiopathic
infertility and fertile men. The TAC level was higher in fertile men
than in men with idiopathic infertility (1.8 ± 0.4 vs. 1.3 ± 0.2 mmol/L,
P < 0.0001). In addition MDA level was significantly lower in
fertile men than in men with idiopathic infertility (2.6 ± 1 vs. 3.4 ±
1.5 mmol/L, P = 0.004).
Semen Parameters, TAC, and MDA Levels in Men With Idiopathic Infertility and Fertile Mena
A positive correlation was observed between sperm motility,
sperm morphology, and TAC levels in men with idiopathic infertility (Figure 1A and B). Although there was a correlation between sperm motility and TAC levels in fertile men (Figure 2),
sperm morphology was not correlated with TAC in this group. There was
no correlation between sperm count and TAC levels in either men with
idiopathic infertility fertile men.
The Correlation of Seminal TAC Level
The Correlation of Seminal TAC Level and Sperm Motility in Fertile Men
In addition, a negative correlation was identified between
sperm motility, normal sperm morphology, and sperm count and MDA level
in men with idiopathic infertility (Figure 3A - C). There was no correlation between these parameters and MDA in fertile men.
The Correlation of Seminal MDA Level
In the current study,
higher levels of MDA and lower levels of TAC were observed in men with
idiopathic infertility than in fertile men. A positive correlation was
shown between sperm motility, normal sperm morphology, and TAC levels
men with idiopathic infertility infertile men and between sperm motility
and TAC levels in fertile men. A negative correlation was observed
between sperm motility, normal sperm morphology, and sperm count and MDA
levels only in men with idiopathic infertility.
Male infertility is a serious health problem and, in spite of
major advances in its diagnosis and treatment, its specific etiology
remains unknown. However, it is believed that seminal oxidative stress
could be one of the main factors in the pathogenesis of this condition (7).
In addition, it is reported that 25% of infertile men have high levels
of seminal ROS, which could cause lipid peroxidation, loss of motility,
and DNA sperm damage (19, 20).
Because spermatozoa possess high amounts of polyunsaturated fatty acids
in their plasma membranes, they are predisposed to oxidative injury.
Therefore, they are susceptible to radical attack and consequently to
lipid peroxidation in the plasma membrane (8, 21). ROS could also induce base alterations, DNA strand breaks, DNA cross-links, and chromosomal rearrangements (22).
Different studies have revealed that seminal antioxidant
capacity is lower in infertile men with high ROS levels. In 2009,
Mahfouz et al. evaluated cutoff value, sensitivity, specificity, and
intra- and inter-observer variability of TAC in the seminal plasma of
fertile and infertile men and reported that seminal plasma TAC was
higher in fertile men. Moreover, they showed that the best cutoff point
to distinguish between the two groups was 1420 microM (specificity 64%
and sensitivity 76%) (23).
Hosseinzadeh Colagar et al. also demonstrated a correlation
between lower levels of TAC and low sperm count, motility, and low rates
of normal morphology in the seminal plasma of asthenoteratozoospermic
and oligoasthenoteratozoospermic men. In addition, they documented
higher MDA levels in the seminal plasma of asthenoteratozoospermic and
oligoasthenoteratozoospermic men and MDA’s negative correlation with
sperm count, motility, and normal morphology (21).
In another study, Patel et al. observed elevated levels of MDA in all
infertile groups, except the azoospermic group, compared to fertile men.
Indeed, they demonstrated a negative correlation between seminal MDA
and normal sperm motility and morphology and suggested that free
radicals could damage sperm membrane integrity (24).
Seminal MDA was significantly higher in infertile men than in
controls in a study by Akbari-Asbagh et al. They observed an association
between MDA and abnormal sperm morphology and seminal TAC and weak
sperm motility. However, they did not find any association between
smoking and sperm parameters in infertile men (25).
Similarly, Pahune et al. observed a positive correlation
between seminal plasma TAC and seminogram parameters, including sperm
concentration, sperm motility, and normal sperm morphology. Therefore,
they suggested that decreased seminal TAC could play a key role in the
etiology of impaired sperm functions (26).
Koca et al. suggested that antioxidant capacity is positively related
to sperm motility and that decreased antioxidant capacity could impair
sperm function due to increased ROS production or insufficient
antioxidant capacity (27).
Appasamy et al. observed a negative correlation between antioxidant
activity and sperm concentration, which suggests that oxidative stress
could influence sperm concentration (28).
In a study by Omran et al. the normozoospermic samples showed
lower DNA fragmentation and higher seminal plasma TAC levels than
abnormal samples. In infertile subjects who had abnormal chromatin
condensation, lower TAC levels were observed (29).
Lower levels of TAC and catalase have been reported by Khosrowbeygi et
al. in patients with abnormal seminal parameters. In addition, catalase
and TAC levels were significantly lower in infertile patients and
positively correlated with sperm motility and normal morphology. In
addition, asthenozoospermic, asthenoteratozoospermic, and
oligoasthenoteratozoospermic groups had significantly lower levels of
catalase activity and TAC than normozoospermic males (30).
Pasqualotto et al. showed that men with idiopathic infertility had
lower sperm concentration, sperm motility, reduced rates of normal
morphology, and lower semen quality scores than controls. The ROS levels
were higher and TAC levels were lower in idiopathically infertile men (31).
Recently, Yousefniapasha et al. reported that sperm parameters
were significantly higher in fertile men than in infertile men; however,
the differences were not significant between the infertile non-smoker
and the fertile non-smoker groups. Although the seminal plasma TAC was
higher in fertile non-smokers than in infertile non-smokers and
infertile smokers, the differences were not significant (32).
Lower total antioxidant capacity and vitamin E levels were
observed in the seminal plasma of infertile men in a study by Benedetti
et al. Like those of the current study, Benedetti et al.’s results
indicated higher MDA levels in infertile patients, which was negatively
correlated with sperm motility and normal morphology. In addition, lower
concentrations of TAC, carotenoids, and vitamin E were documented in
blood samples of infertile men, and TAC and carotenoids were positively
associated with sperm motility, normal morphology, and concentration (33).
Because different studies have demonstrated the effects of
oxidative stress on male infertility, several investigations have
attempted to study the effects of antioxidant supplements on these
conditions. Although the effects of different doses and types of oral
antioxidants in attempts to improve semen parameters have not been
established, it is believed that antioxidant therapy has beneficial
effects on male infertility (34, 35).
However the results of antioxidant therapy alone or in combination are
controversial. For example, some studies have shown that vitamin C,
vitamin E, zinc, folic acid, and selenium alone or in combination could
improve sperm parameters, especially sperm motility and sperm DNA
integrity (36-39). In contrast, several reports refute the effects of these antioxidants on sperm parameters (40, 41).
In conclusion, higher levels of MDA and lower levels of TAC
were observed in men with idiopathic infertility. A positive correlation
was revealed between sperm motility, normal sperm morphology, and TAC
levels in men with idiopathic infertility and between sperm motility and
TAC levels in fertile men. In addition, a negative correlation was
observed between sperm motility, normal sperm morphology, and sperm
count and MDA levels only in idiopathically infertile men. Therefore,
TAC and MDA could be used as oxidative stress markers, and their assays
could be useful in the evaluation of idiopathic infertility. In
addition, these markers may guide us in antioxidant therapy.
This article is an extract from an MD thesis
(registered number 1340) at the Zahedan university of medical sciences.
The authors thank the Zahedan deputy of research affairs for funding
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