Association of Seminal Plasma Total Antioxidant Capacity and
Malondialdehyde Levels With Sperm Parameters in Infertile Men With Varicocele
author: Faramarz Fazeli, Department of Urology, School of Medicine,
Zahedan University of Medical Sciences, Zahedan, IR Iran. Tel:
+98-5433425728, Fax: +98-5433425728, Email: email@example.com
is one of the most common reasons for male infertility and could impair
spermatogenesis through mechanisms that are not well known. Recently,
oxidative stress has been introduced as a major reason for male
infertility caused by varicocele.
the current study, we aimed to assess the TAC (total antioxidant
capacity) and MDA (malondialdehyde) as stress oxidative markers in
infertile men with varicocele and fertile men, and moreover, their
correlation with sperm parameters.
Patients and Methods: This
case control study was performed on 43 infertile men with varicocele
and 46 men with proven fertility. The ferric reducing ability of plasma
(FRAP) and thiobarbituric acid (TBA) reaction methods were used for
seminal plasma TAC and MDA assay, respectively.
TAC levels (1.7 ± 0.2 vs. 1.3 ± 0.4 mmol/L, P = 0.0004) and higher MDA
levels (2.5 ± 1.1 vs. 5.8 ± 1.9 mmol/L, P < 0.0001) were observed in
infertile men with varicocele compared to fertile men. There was no
correlation between TAC and MDA in fertile men (r = 0.02, P = 0.9),
however, a negative correlation was found between TAC and MDA levels in
varicocele infertile men (r = −0.44, P = 0.003). Moreover, a positive
correlation was found between sperm count and sperm motility with TAC
levels in varicocele infertile men (r = 0.4, P = 0.02 and r = 0.6, P
< 0.0001, respectively). There was a correlation between sperm
motility and TAC levels in fertile men (r = 0.5, P = 0.001), but other
parameters did not correlate with TAC in this group. A negative
correlation was shown between semen volume, sperm count, total sperm,
sperm motility, and sperm morphology with MDA levels in varicocele
infertile men (r = 0.3, P = 0.045; r = −0.4, P = 0.009; r = −0.5, P =
0.002; r = −0.5, P = 0.001 and r = −0.4, P = 0.008, respectively). There
was no correlation between these parameters and MDA in fertile men.
findings indicated that oxidative stress could play an essential role
in male infertility caused by varicocele and may impair spermatogenesis
leading to infertility.
Keywords: Malondialdehyde; Semen; Total Antioxidant Capacity; Varicocele
According to the world
health organization (WHO), infertility is defined as the inability to
achieve pregnancy within 12 months of regular sexual intercourse for
couples trying to conceive. Infertility affects 10% - 20% of couples
endeavoring to conceive, regardless of race or ethnicity (1). It is estimated that the male factor of couple infertility is nearly 50% (2).
Male fertility disorder is attributed to environmental factors such as
exposure to certain chemicals, heavy metals, pesticides, and heat, or
electromagnetic radiation. Smoking, alcohol abuse, chronic stress,
obesity, urogenital trauma, and inflammation in the male reproductive
system are also associated with decreased male fertility. Anatomical
abnormalities, such as varicocele, semen outflow tract obstruction, or
neurological disorders of ejaculation may cause abnormal spermatogenesis
and failure in sperm function. Varicocele is the most prevalent
abnormal physical finding and most common surgically correctable risk
factor for male infertility (2, 3).
Varicocele is a pathologic enlargement of the pampiniform venous plexus
within the spermatic cord. Fifteen percent of the normal male
population has been shown to have varicocele and approximately 40% of
these present with infertility (2).
Although the exact pathophysiology of varicocele-induced infertility is
not completely understood, there are several mechanisms that are
thought to contribute to the pathogenesis of this complication such as
hypoperfusion leading to hypoxia, oxidative stress, heat stress hormonal
imbalances, and exogenous toxicants (2).
Oxidative stress is created from the imbalance between the
production of so-called reactive oxygen species (ROS) and the protective
action of the antioxidant system that is responsible for their
neutralization and removal. A pathological response to ROS leads to
damage of cells and tissues. Spermatozoa are particularly susceptible to
the damaging effects of ROS (4, 5). ROS are essential byproducts of aerobic life, which are detoxified by natural processes in the body (4). Oxidative stress occurs when ROS overcome our natural ability to detoxify ROS-induced damage (4, 5).
It is understood that controlled levels (physiological levels) of ROS
are required for sperm physiology, maturation, capacitation, acrosomal
reactions, and normal fertilization. However uncontrolled production of
ROS (pathological levels) leads to sperm dysfunction including, lipid
peroxidation, sperm DNA damage and loss of motility (6).
The excessive amount of ROS could be produced from endogenous
sources including immature or abnormal spermatozoa and leukocytes or
from environmental factors such as cigarette smoking and alcohol (7).
Total antioxidant capacity (TAC) and ROS production are in
balance in fertile men, however, in pathological conditions, such as
chronic disease, autoimmune disorders, alcohol consumption, smoking,
infections, advanced age, and stress, the oxidative stress has been
In the seminal fluid, antioxidants play a crucial role in antioxidant
defense mechanisms. Therefore, relatively low amounts 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 (8).
As a result, the most essential form of antioxidant defense available
to spermatozoa is antioxidants in seminal fluid. The most important
antioxidants in semen are vitamins, glutathione, thioredoxin, and
superoxide dismutase (8).
Several studies have indicated that high levels of ROS in seminal fluid
increase the risk of male infertility including varicocele (9, 10).
Elevated levels of ROS in semen samples of varicocele infertile men
were observed, which were associated with an increased DNA fragmentation
index (9, 11, 12).
These findings suggest that the imbalance between oxidant capacity and
the natural antioxidant defense system could play a role in
Among various sperm parameters, sperm count and sperm motility
are essential parameters that show the functional ability of
spermatozoa. Asthenozoospermia (low sperm motility) is considered to be
associated with male infertility. Although the factors that affect sperm
motility are not clear, the oxidative stress that is produced by ROS is
a key factor in this complication (13).
The antioxidant power of biological fluids, including seminal
fluid, can be analyzed either with measurement of each antioxidant or
with total antioxidant capacity. In addition, a malondialdehyde (MAD)
assay is a useful method to display the amount of peroxidation damage of
Since the findings
regarding the correlation of seminal plasma TAC and MAD with varicocele
infertility and sperm parameters are controversial, the purpose of this
study was to explore the total antioxidant capacity and malondialdehyde
status in seminal fluid of varicocele infertile and fertile men and
their correlation with sperm parameters.
3. Patients and Methods
This study was performed
on 43 consecutive infertile men who presented with varicocele 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.
The diagnosis of a clinically significant varicocele was made on
physical examination of the scrotum and its contents. The patients were
examined in the supine and standing position in a warm room that
promotes relaxation of the scrotal dartos muscle and facilitates
accurate evaluation for varicocele.
Forty-six men with proven fertility were considered as
controls. All patients and fertile controls with specific genital
infection, genital diseases, undescended testis, testicular atrophy, and
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 the world health organization guidelines of
A written informed consent was obtained from the patients and
controls. This study was approved by the ethics committee of research of
Zahedan University of Medical Sciences.
The hematoxylin-eosin (H and E) staining method was used to
determine the percentage of normal morphology of spermatozoa. Morphology
of the spermatozoa was assessed using Kruger’s criteria, and morphology
< 14% was considered abnormal (16).
3.1. TAC Assay
TAC was assessed using the ferric reducing ability of plasma (FRAP) method, as discussed by Benzie et al. (17). In the FRAP method the ability of seminal plasma antioxidants to reduce ferric-tripyridyltriazine (Fe3+-TPTZ) to a ferrous form (Fe2+)
was measured. The working FRAP reagent was prepared using 10 volumes 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 1 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 1.5 mL FRAP reagent and heated to 37°C for 10 minutes. Absorbance was
measured at 593 nm using a spectrophotometer (UV-visible). The final
results were shown as mmol/L.
3.2. MDA Assay
Seminal MDA levels were measured according to the method that was defined by Rao et al. (18). This method was based on a thiobarbituric acid (TBA) reaction and extraction with normal
butanol. As the standard, 1, 1, 3, 3-tetramethoxypropane was
used. Spectrophotometric detection of absorbance was done at 532 nm
wavelength and compared with a standard curve. The TBA was purchased
3.3. Statistical Analysis
Data was analyzed using the statistical software SPSS-18 (SPSS,
Chicago, IL). Data are shown as mean ± SD. The normal distribution of
data was evaluated using the Kolmogorov-Smirnov (K-S) statistical test.
The independent sample t-test or Mann-Whitney U test were used for
comparison between two groups, whenever appropriate. In addition,
Pearson’s correlation coefficient test was performed to determine the
correlation among different factors. Values of P < 0.05 were
considered statistically significant.
The semen parameters, TAC, and MDA levels are presented in Table 1.
The semen volume, sperm motility, sperm morphology, and sperm count
between varicocele infertile men and fertile men were significantly
different. A higher TAC level was observed in fertile men compared to
varicocele infertile men (1.7 ± 0.2 vs. 1.3 ± 0.4 mmol/L, P = 0.0004).
Moreover the MDA level was significantly lower in fertile men compared
to varicocele infertile men (2.5 ± 1.1 vs. 5.8 ± 1.9 mmol/L, P <
Semen Parameters, Total Antioxidant Capacity (TAC) and Malondialdehyde (MAD) Levels in Varicocele Infertile Men and Fertile Men
Although there was no correlation between TAC and MDA in fertile men (Figure 1A; r = 0.02, P = 0.9), a negative correlation was found between TAC and MDA levels in varicocele infertile men (Figure 1B; r = −0.4, P = 0.002).
Correlation of Total Antioxidant Capacity (TAC) and Malondialdehyde (MAD)
A positive correlation was observed between sperm count and sperm motility with TAC level in varicocele infertile men (Table 2).
Although there was a correlation between sperm motility and TAC level
in fertile men, other parameters were not correlated with TAC in this
group (Table 3).
Correlation of Sperm Parameters With Total Antioxidant Capacity (TAC) and Malondialdehyde (MAD) in Varicocele Infertile Men
Correlation of Sperm Parameters With Total Antioxidant Capacity (TAC) and Malondialdehyde (MAD) in Fertile Men
In addition, a negative correlation was shown between semen
volume, sperm count, total sperm, sperm motility, and sperm morphology
with MDA level in varicocele infertile men (Table 2). There was no correlation between these parameters and MDA in fertile men (Table 3).
In the present study, MDA
levels were significantly higher and TAC levels were significantly
lower in varicocele infertile men compared to fertile men. A positive
correlation was found between sperm count, total sperm, and sperm
motility with TAC level in varicocele infertile men. There was a
correlation between sperm motility and TAC level in fertile men;
however, other parameters were not correlated with TAC in this group.
Furthermore a negative correlation was shown between semen volume, sperm
count, total sperm, sperm motility, and sperm morphology with MDA level
in varicocele infertile men. No correlation between these parameters
and MDA was observed in fertile men.
Varicocele is one of the most frequent reasons for male
infertility, and despite many advances in its diagnosis and treatment
the exact mechanisms by which it leads to changes in spermatogenesis are
unknown (19). It is believed that seminal ROS may be one of the key factors in the pathogenesis of this complication (4).
Higher levels of seminal ROS have been reported in 25% of
infertile men. Excessive amounts of ROS may lead to lipid peroxidation,
loss of motility, and sperm DNA damage (20, 21).
There are high amounts of polyunsaturated fatty acids in the plasma
membranes of spermatozoa, therefore, they are predisposed to oxidative
damage (5, 22). In addition, ROS could induce base alterations, DNA strand breaks, DNA cross-links, and chromosomal rearrangements (23).
Several studies have shown elevated reactive oxygen species and
decreased total antioxidant capacity levels in varicocele infertile
men. In 2010, Abd-Elmoaty et al. observed higher levels of oxidants and
lower levels of antioxidants in the seminal fluid of infertile men with
varicocele compared to controls (24).
Increased ROS and DNA fragmentation, and decreased antioxidant levels
and TAC have been observed both in infertile and varicocele individuals (25, 26).
In addition, several reports have shown a significant association
between ROS levels and TAC with sperm parameters in infertile and
varicocele individuals, and they agreed that these abnormal levels of
antioxidants and ROS are complicated by the occurrence of oligospermia,
sperm motility defects, and/or abnormal sperm morphology (27, 28).
Similar to the results of the current study, Pasqualotto et al.
found that the mean semen quality scores of the infertile men with
varicocele were lower compared to control subjects. In addition the
infertile men with varicocele had higher ROS levels but lower TAC levels
compared to the healthy subjects (29).
Although Giulini et al. observed no significant differences in
peripheral blood TAC concentrations between controls and infertile men
with varicocele, lower TAC concentrations were shown in patients with
varicocele and moderate or severe oligoasthenozoospermia compared to
controls and normozoospermic patients with varicocele. TAC levels were
also positively correlated with sperm concentration and motility (28).
In a meta-analysis, which was performed by Agarwal et al. higher ROS
and lower total antioxidant capacity levels in the varicocele population
compared to fertile men have been shown (30).
In a study by Chen et al. a decrease in ROS level following
varicocele repair has been reported also. Therefore, varicocele repair
may restore spermatogenesis, improve semen parameters, and decrease DNA
damage via decreases in ROS levels (31).
In addition, several studies have shown higher MDA levels as lipid
peroxidation markers in infertile men. Mostafa et al. found higher
levels of MDA in the internal spermatic venous blood compared to those
in the peripheral fluid (32). Similarly, in a study by Altunoluk et al., MDA levels were greater in the varicocele group (33).
In conclusion, lower levels of TAC and higher levels of MDA
were found in infertile men with varicocele. A positive correlation was
revealed between TAC and sperm count, and total sperm and sperm motility
in infertile men with varicocele and sperm motility in fertile men.
In addition a negative correlation was shown between sperm
motility, sperm morphology, and sperm count and MDA level only in
idiopathic infertile men, while volume, sperm count, total sperm, sperm
motility, sperm morphology correlated negatively with MDA level in
varicocele infertile men. No correlation between these parameters and
MDA were observed in fertile men.
Therefore, the TAC and MDA assays, as oxidative stress markers,
could be useful in evaluation of infertility caused by varicocele. In
addition, these markers may serve as a guide in antioxidant therapy.
This article was extracted from the MD thesis
(registered number 1340) at Zahedan University of Medical Sciences. The
authors thank Zahedan deputy of research Affairs for funding this
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