Middle East Fertility Society Journal 23 (2018) 19–22

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Middle East Fertility Society Journal

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Original Article

Effect of serum antioxidant levels on sperm function in infertile male

Ayad F. Palani
Department of Chemistry, College of Science, University of Garmian, Kalar (46021), Sulaymaniyah, Iraq

a r t i c l e i n f o a b s t r a c t

Article history: Antioxidant and reactive oxygen species (ROS) has been implicated in male infertility. Antioxidants are
Received 4 September 2016 compounds which scavenge formation or oppose of the actions ROS. Spermatozoa are particularly vulner-
Revised 16 June 2017 able to ROS because of inherent deficiencies in intracellular antioxidant enzyme protection, thus total
Accepted 19 July 2017
body antioxidant capacity become more substantial to protect sperm. In this study to try to evaluate
Available online 8 August 2017
the role of serum total antioxidant capacity, uric acid, glutathione, total thiols, ferritin, albumin and malo-
dialdehyde in male infertility. 66 men included in this study 40 were infertile and 26 were healthy fertile.
Keywords:
Results showed a significant decrease in sperm progressive motility and a significant increase in sperm
Serum antioxidants
Progressive motility
tail abnormality, also there were a significant decrease in serum total antioxidant capacity, uric acid
Sperm morphology and albumin in infertile group. Because of lack of the intracellular antioxidant enzymes sperm cannot
protect plasma membrane that surrounds the tail, the protection depends on the antioxidant defense
afforded by the seminal plasma and on the entire body antioxidant capacity. Decrease of serum antiox-
idant capacity, uric acid and albumin may cause destroy of sperm tail membrane and this may lowering
of sperm progressive motility.
Ó 2017 Middle East Fertility Society. Production and hosting by Elsevier B.V. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction peroxidse, and glutathione reductase), which catalyze free radical

Spermatozoal dysfunction is the single most common cause of
infertility [1]. New researches focused on the investigation of the
causes of spermatozoal dysfunction. In recent years, Oxidative
stress (OS) has become the major interest in the pathophysiology
of human sperm function and male infertility [2,3]. OS develops
as a result of an imbalance between reactive oxygen species ROS
(e.g. H2O2, O
2 , OH
, etc.) generating and antioxidants defense
[4]. The excessive generation of ROS has been identified as one of
the most important defined etiologies for male infertility [5]. ROS
have a potential to damage the sperm membrane particularly on
membrane polyunsaturated fatty acid (PUFA) lipids, when ROS
damage the unsaturated bonds of PUFA, a lipid peroxidation chain
reaction begins which in turn reduces the sperm motility [2–6]. To
prevent the damaging effect of excessive ROS, enzymatic and non-
enzymatic antioxidant pathways scavenge excess ROS and allow a
balance to be achieved between beneficial oxidant generation and
damaging harmful ROS [7]. Antioxidants are the most important
defense substances against free radical induced infertility [8].
Human body provides a complete antioxidant defense system
composed of enzymatic and non-enzymatic antioxidants [9].
Enzymatic antioxidant, (e.g., superoxide dismutase, glutathione
Peer review under responsibility of Middle East Fertility Society.
E-mail address: Ayad.palani@garmian.edu.krd
quenching reactions [10]. A number of compounds can be consid-
ered as non-enzymatic antioxidants such as; ascorbate, urate, a-
tocopherol, pyruvate, glutathione, albumins, and b-carotene and
ubiquinol [11,12]. These molecules have the capability to take free
electrons from the radicals without being as reactive as free oxy-
gen radicals [13].
2. Materials and methods
A total of 66 serum samples were included in this study, 40
samples were obtained from patients who attended to clinical lab-
oratories for infertility treatment and 26 samples were obtained
from normal fertile men.
Semen samples have been collected in the laboratory or home
by masturbation after 3–5 days of sexual abstinence and ejaculated
in a wide mouth plastic container, delivered samples incubated at
(37 °C). Blood samples were collected using a sterile syringe and
serum were separated by centrifugation and frozen in ( 45 °C)
deep freeze for further analysis.
2.1. Semen analysis
Semen analysis was performed according to (WHO, 2010).
Semen examinations include; sperm count, viability activity and
morphology [14].

http://dx.doi.org/10.1016/j.mefs.2017.07.006
1110-5690/Ó 2017 Middle East Fertility Society. Production and hosting by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
20 A.F. Palani / Middle East Fertility Society Journal 23 (2018) 19–22

Table 1 Sperm activity has been examined by taking 10 µL of semen

Semen analysis variables of infertile and fertile groups. sample directly after liquefaction on a pre-warmed slide, the result
Semen analysis variables Infertile men Fertile men P value described as progressive motility (PR), Non progressive motility
(n = 40) (n = 26) (NP) and immotility (IM). Morphology examined by staining with
Mean ±SEM Mean ±SEM India ink, the slides has been seen by oil emersion lens. The abnor-
Count (million/ml) 60.66 ±8.01 55.72 6.53 n.s
mality recorded as abnormal head, midpiece and tail.
T. count (million/ejaculate) 215.23 32.32 257.89 38.51 n.s
Viability 2.2. Serum biochemical parameters
Live % 70.94 2.78 73.00 2.12 n.s
Dead % 29.06 2.78 27.00 2.12 n.s
Activity Frozen serum samples thawed and allowed to reach room tem-
Active % 47.56 2.75 54.73 2.71 n.s perature for biochemical analysis. TAC estimated using a kit of
Progressive % 27.79 2.35 39.84 3.28 0.003 (CUPRAC) method [15]. The uric acid/uricase assay based kit is
Non-progressive % 19.78 2.27 14.90 2.64 n.s used for measuring uric acid concentrations [16]. Glutathione mea-
Sluggish 23.38 2.07 18.27 1.65 n.s
Morphology
sured using colorimetric method [17], based on the reaction of 5,5-
Normal % 53.24 2.34 58.29 2.65 n.s dithiobis(2-nitro-benzoic acid) [DTNB] with the aliphatic thiols at
Abnormal % 46.76 2.34 40.88 2.25 n.s pH 8.0. The estimation of total thiols has carried out as the same
Head % 31.12 2.13 27.02 1.72 n.s as glutathione method except in the step of the addition of TCA
M.P % 4.60 0.52 5.53 1.05 n.s
which have not been added in the total thiols assay. Total protein
Tail % 8.36 0.78 5.66 0.64 0.016
C.D % 1.47 0.30 1.03 0.36 n.s is determined using sensitive lowery method [18]. Albumin esti-
mated using commercially available kit demanding on bromocre-
sol green (BCG) reagent. Ferritin determined by enzyme-linked
immunosorbent assay (ELISA) [19], and MDA measured using a col-
Sperm count calculated using a Hematocytometer after 10 µl of orimetric method depends on the reaction of thiobarbituric acid
semen diluted with 190 µl of sample diluent and sperm concentra- (TBA) with MDA [20].
tion described as millions per ml.
Sperm viability examined by (Hypo-osmotic swelling test) test,
2.3. Statistical analysis
semen sample mixed with hypo-osmotic solution in a ratio (1:9)
and incubated at 37 °C for 30 min slides prepared and seen under
Results presented as Mean ± SE, and the differences between
phase contrast microscopy, the live and dead spermatozoa were
groups determined by ANOVA table using SPSS computer software
calculated and expressed as percent.
program, P  0.05 has been considered statistically significant.

3. Results
80
Inferle Results of semen analysis are showed in Table 1 and Fig. 1. Data
70
showed both groups have no difference in sperm count and viabil-
Ferle
60 ity. The groups have high significant difference in progressive
motility, Mean ± SEM (27.79 ± 2.35% and 39.84 ± 3.28%, P  0.003)
50
Percent %

for infertile and fertile groups respectively.

40 The following evidences suggest that the cause of infertility in
this study is the lack of sufficient motility as shown in Table 1.
30 Total motility was lower but not significant in infertile group
20 (P  0.08). This results also reported by Roya Rozati et al. [21]. Also
results showed high significant difference in the morphology of
10 sperm tail Mean ± SEM (8.36 ± 0.78 and 5.66 ± 0.64, P  0.01).
0 Table 2 and Fig. 2 show serum biochemical variables. The
groups have high significant difference in TAC levels, Mean ± SEM
(665.55 ± 32.25 and 813.70 ± 36.72 µM, p  0.015) and significant
difference in uric acid (261.80 ± 20.99 and 451.20 ± 24.97 µmol/l,
p  0.03) for infertile and fertile groups respectively. Also albumin
was significantly decreased in infertile men as compared to fertile
Fig. 1. Mean ± SEM of semen analysis variables of infertile and fertile groups.
men (41.5 ± 2.2 and 49.2 ± 1.4 g/l, p  0.05) respectively.

Table 2
Serum biochemical variables of infertile and fertile groups.

Serum biochemical variables Infertile men (n = 40) Fertile men (n = 26) P value
Mean ±SEM Mean ±SEM
TAC (µM) 665.55 32.25 813.70 36.72 0.015
Uric acid (µmol/l) 361.80 20.99 451.20 24.97 0.030
Glutathione (µmol/l) 197.10 9.32 177.50 13.50 n.s
Thiols (µmol/l) 188.82 9.29 181.67 10.64 n.s
Ferritin (ng/ml) 126.44 13.82 143.82 15.16 n.s
T. Protein (g/l) 64.94 2.77 63.04 10.78 n.s
Albumin (g/l) 41.5 2.2 49.2 1.4 0.050
MDA (nmol/ml) 198.10 15.54 247.06 13.45 n.s
 A.F. Palani / Middle East Fertility Society Journal 23 (2018) 19–22
900
800
700
600
concentraon
500
400
300
200
100
0
Fig. 2. Mean ± SE of semen analysis variables of infertile and fertile groups.
4. Discussion
The fertilization of ovum requires normal healthy progressive
motile sperm. Sperm motility is essential for normal fertilization,
defective sperm motility, is common in infertile men [22]. Sperma-
tozoa acquire motility during passage through the epididymis
[23,24]. Progressive movement requires the presence of a specific
epididymal protein, called the forward motility protein [25].
Spermatozoa protect its self via a complete antioxidant defense
system composed of enzymatic and non-enzymatic antioxidants
[9]. Sperm intracellular antioxidant enzymes cannot protect the
plasma membrane that surrounds the tail, forcing to supplement
their limited intrinsic antioxidant defenses by depending on the
protection afforded by the seminal plasma, this make sperm tail
protection depends on the entire body antioxidant capacity [26].
Several studies have reported the role of OS in various types of
male infertility [27]. The proposed mechanism for loss of sperm
function due to OS has been shown to involve excessive generation
of ROS [28].
Study results showed a decrease in TAC, uric acid and albumin
levels in the serum of infertile men. Uric acid is a main contributor
to the chain-breaking antioxidant capacity of blood and seminal
plasma they scavenge peroxyl (ROO
) and hydroxyl (OH
) radicals,
so we might have expected a decrease in urate concentration in
the presence of ROS [29].
Our results agree with Lewis et al. [30]. Albumin is likely to
serve as an antioxidant by providing thiol groups required for
‘‘chain breaking” antioxidant activity [28]. Low albumin level in
infertile men as compared to fertile men (41.5 ± 2.2 and
49.2 ± 1.4 g/l) may empire antioxidant capacity of serum and con-
sequently on seminal plasma.
Low Antioxidant levels induce OS state; OS in the epididymis
destroys the forward motility protein by oxidation that may cause
the decrease in the progressive motility (27.79 ± 2.35% and
39.84 ± 3.28%, p  0.003) for infertile and fertile groups respec-
tively [25]. Also low antioxidant levels cause destroying of the tail
membrane by ROS since spermatozoa depend on the antioxidant
capacity of seminal plasma to protect itself and low serum antiox-
idant levels decrease seminal plasma antioxidant capacity, this
may be the cause of incensing of abnormal tail in the spermatozoa
of infertile men (8.36 ± 0.78%) as compared to fertile men
(5.66 ± 0.64, p  0.01%). These results improved by some clinical
experiments where exogenously given antioxidants to infertile
men was found to cause significant increase in sperm motility, par-
ticularly forward progression [28–31].
21
Glutathione and protein thiols protects lipids in the sperm cell
membranes from oxidation and prevents the formation of free oxy-
Inferle
gen due reconstruction of thiol groups (–SH), thiol group is a strong
Ferle nucleophile, and provide protection against damage by elec-
trophilic oxidants [32,33]. Glutathione deficiency affects sperm
motility via deterioration of the midpiece of the spermatozoa
[32], no significance difference in glutathione and total thiols, no
significant difference in MDA level, as a result no significant differ-
ence in the morphology of the midpiece (4.60 ± 0.52 and
5.53 ± 1.05%) for infertile men and fertile men respectively.
Ferritin also involved in the mechanism of defense against free
radicals, although there is much information regarding their scav-
enging activity in other biological systems, but no significant role
of ferritin was seen in the seminal plasma [34].
5. Conclusion
Sperm motility has correlated to seminal plasma antioxidant
level; the decrease in the antioxidant capacity of seminal plasma
especially uric acid has a negative effect on sperm motility via
deterioration sperm tail morphology, which consequently
decreases sperm motility.
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