Advances in Bioscience and Biotechnology, 2013, 4, 30-40 ABB Published Online November 2013 (
Evaluation of the impact of leucocytospermia on semen
oxidative status by chemiluminescence technique in
infertile men*
Afifa Sellami1,2#†, Nozha Chakroun1,2#, Yemna Rtaib1,2, Hajer Hdhili1,2, Riadh Ben Mansour3,
Louati Dolira4, Kais Chaabene4, Leila Keskes1, Saloua Lassoued3, Tarek Rebai1,2
1Laboratory of Histology and Biology of Reproduction, Medical School, Sfax, Tunisia
2Histology Embryology Research Unit, Medical School, Sfax, Tunisia
3Laboratory of Cell Culture, Biotechnology Institute, Sfax, Tunisia
4Gynaecology and Obstetrics Department, Hedi Chaker Academic Hospital, Sfax, Tunisia
Received 9 September 2013; revised 10 October 2013; accepted 26 October 2013
Copyright © 2013 Afifa Sellami et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The presence of high reactive oxygen species (ROS)
levels in semen is a major factor involved in the de-
cline of male fertility. In seminal plasma, ROS are
mainly produced by activated leucocytes. Spermato-
zoa were the first cell type reported to show a poten-
tial susceptibility to oxidative damage. The aim of
our study was to evaluate the impact of leucocyto-
spermia on basal and FMLP (Formyl-Methionyl-
Leucyl-Phenylalanine) induced oxidative status in
semen of infertile men. We also analyzed the correla-
tions of the spermatic parameters with amounts of
ROS in semen. Our study included 50 semen samples
of infertile men. Sperm analysis was performed using
WHO standardized method. Seminal leucocytes were
quantified using peroxidase technique. The meas-
urement of ROS levels in semen was made by che-
miluminescence assay. We measure respectively ROS
amounts in neat semen and in washed sperm cells
suspension from the same ejaculate. We also applied
the test of provocation of leucocytes by FMLP on neat
and washed samples to assess the spermatic oxidative
status after leucocyte stimulation. Our results showed
significant correlations between ROS levels in neat
semen and many sperm parameters: motility, sperm
concentration, leucocytes concentration and the rate
of sperm cytoplasmic droplets. The studied samples
were divided into 2 groups: (G1) composed of 36
samples without leucocytospermia and (G2) com-
posed of 14 leucospermic samples. ROS levels were
significantly lower in G1 than in G2 (p = 0.002). ROS
production was significantly increased after applica-
tion of FMLP in washed leucospermic samples (p =
0.001). The measurement of ROS in neat semen is a
considerable contribution to explore the impairment
of semen quality in infertile men. ROS levels in
washed semen reflect the oxidative status generated
by sperm preparation techniques used in assisted re-
productive procedures. Levels of ROS are highly in-
fluenced by the presence of leucocytes and associated
with decreased seminal parameters.
Keywords: Semen; Oxidative Stress; Leucocytospermia;
Chemiluminescence; ROS; FMLP
Oxidative stress induced by reactive oxygen species
(ROS) plays crucial roles in a wide range of physiologi-
cal processes and is also implicated in various diseases,
such as cancer, cardiovascular pathology, neurodegen-
erative disorders, and other chronic conditions 1,2. In
semen, the controlled generation of very low amounts of
ROS appears to regulate sperm normal functions 3,4.
Oxidative stress appears in semen once an imbalance
between the production of ROS and their destruction by
different enzymatic and non-enzymatic seminal antioxi-
dant systems is created 5. Interestingly, spermatozoa
were the first cell type reported to show a potential sus-
ceptibility to oxidative damage 6. While the presence
of high levels of ROS in the ejaculate is among the risk
factors involved in reducing male fertility 7,8. It has
evolved that three inter-related mechanisms account for
*Disclosure: The authors declare that there are no conflicts of interest.
#The first two authors contribute equally to the article.
Corresponding author.
A. Sellami et al. / Advances in Bioscience and Biotechnology 4 (2013) 30-40 31
oxidative stress-mediated male infertility: impaired mo-
tility, impaired fertilization and oxidative DNA damage
The principle sources of excessive generation of ROS
in semen are activated leucocytes and abnormal sper-
matozoa 11,12. Indeed, the prevalence of leucocyto-
spermia in infertile men varies from 2% to 40% de-
pending on the patient population, the detection method
and threshold values used 13. Moreover, it has been
shown that depending on their activation status, leuco-
cytes are capable of producing ROS and cytokines and
there seems to be a relation between ROS formation and
cytokine production 14,15.
The importance of seminal ROS production has been
already stressed in the World Health Organization manu-
als (WHO 1999 and WHO 2010) 16,17. The chemilu-
minescence method is the most commonly employed
technique as a direct measurement of ROS generation in
semen according to the standardized method recom-
mended by the WHO 1999 16. This assay is capable of
quantifying both intracellular and extracellular ROS 18.
Furthermore, the use of leukocyte-specific stimulant
(FMLP: formyl-methionyl-leucyl-phenylalanine) can en-
hance the chemiluminescent signal to measure low amounts
of light generated by leucocytes 16,19.
The degree of sperm damage by leucocytes products
depends on the location of the inflammatory reaction, the
duration of exposure of sperm to these products and the
ability of spermatozoa to activate its intrinsic anti-lipop-
eroxidative defense systems 20. The clinical signify-
cance of both ROS and leucocytes levels continues to be
debated. Some authors have found ROS levels or leuko-
cyte counts to be of little prognostic help in either in vivo
or in vitro reproduction 21. These findings are in dis-
agreement with other in vivo 22 and in vitro studies
19,23 that reported the significant prognostic value of
semen ROS levels in reproduction. Much of the contro-
versy centers on the best definition of pathological leu-
cocytospermia and the correlation of leucocytes with
seminal oxidative stress are unclear 15,24,25.
The aims of our study were to evaluate the impact of
leucocytospermia on basal and FMLP induced semen
oxidative status using the chemiluminescence technique
in infertile men. We also analyzed the correlations of the
routine spermatic parameters with amounts of ROS gen-
eration in semen.
2.1. Patients
Our study was carried out in 50 semen samples from
male partner of infertile couple attending the Histology-
Embryology laboratory of Sfax medical school (Tunisia)
for semen investigations. The patients were aged be-
tween 27 and 51 years old with a mean age (± Standard
Deviation (SD)) of 36.16 ± 0.57 years.
2.2. Collection of Semen Samples
Semen samples were collected by masturbation after 3 -
5 days of sexual abstinence and allowed to liquefy for 30
minutes at 37˚C.
2.3. Semen Analysis
Basic semen analysis consisted in the measurement of
the following semen parameters: volume, sperm concen-
tration, percentage of motile spermatozoa, sperm vitality
and percentage of normal spermatozoa. For sperm con-
centration, diluted semen samples were mixed before
transferring a drop to the chamber of the hemocytometer.
The spermatozoa were counted under a light microscope
at 400× magnification. To determine the percentage of
motile spermatozoa, a 10 µl drop of mixed semen was
placed on a heated glass slide (37˚C) under a square
cover glass (22 mm) and observed at 400× magnifica-
tion. The percentage of motile spermatozoa was evalu-
ated immediately and four hours after semen liquefaction,
we evaluated total motility, rapid progressive motility
(type a), slow progressive motility (type b), and no pro-
gressive motility (type c) according to WHO guidelines
16. Sperm vitality was assessed using eosin-nigrosin
staining technique. A 20 µl of liquefied semen was mixed
with 20 µl of eosin (1%) and 20 µl of nigrosin (10%).
The suspension was incubated for 30 s at room tempera-
ture. Then, a 20 µl of the solution was smeared on a mi-
croscope slide. The smear was air dried and examined at
1000× magnification under oil immersion. Unstained
sperm (white) were classified as viable and those that
showed any pink or red coloration were classified as
dead. Sperm morphology was assessed in Shorr-stained
semen smears. All parameters were carried out according
to the standardized methods recommended by the WHO
2.4. Assessment of Leucocytes in Semen by
Peroxidase Method
A leukocyte count was carried out by using the cyto-
chemical peroxidase method, as described in the WHO
laboratory manual 16. This assay identifies polymor-
phonuclear granulocytes, the most prevalent leucocyte
type in semen, as peroxidase-positive cells. A working
solution was prepared by combining 250 µl of Benzidine
with 50 µl of Hydrogen peroxide (H2O2). The procedure
consisted of mixing 50 µl of neat semen with 50 µl of the
working solution. This mixture was allowed for 30 min-
utes at room temperature. We transferred 20 µl of the
mixture onto a hemocytometer chamber and the number
of peroxidase-positive leucocytes which stained brown
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A. Sellami et al. / Advances in Bioscience and Biotechnology 4 (2013) 30-40
was counted at 400× magnification. Leucocytospermia
was defined as the presence of more than 1 × 106 leuco-
cytes per milliliter of semen 16.
2.5. Semen Preparation
From each sample, one aliquot of 0.5 ml of liquefied neat
semen was used for immediate ROS measurement and
another 0.5 ml aliquot of semen was centrifuged at 300 g
for 7 minutes. Seminal plasma was removed and the pel-
let of cells was washed twice in 3 ml of PBS (Phosphate
Buffer Saline, isotonic solution, pH = 7.4). The super-
natant was then removed and the cells pellet was sus-
pended in a volume of 0.5 ml of PBS. Sperm concentra-
tion in the final solution was evaluated before ROS
2.6. Measurement of Reactive Oxygen Species
(ROS) in Semen by Chemiluminescence
The measurement of ROS in semen was made by che-
miluminescence using a microplate luminometer (Lumi-
noskan Ascent, Thermo Electron Corporation). Chemilu-
minescence probe used is luminol (5-amino-2, 3-dihydro
1, 4-phthalazinedione; Sigma chemical Co) which is a
redox-sensitive, light emitting probe 26. A 10 mM
stock solution of Luminol was prepared in DMSO (Di-
methyl Sulfoxide; Sigma-Aldrich). For each semen sam-
ple, we measured the basal levels of ROS in neat semen
and in a suspension of washed semen cells obtained from
the same ejaculate.
The basal ROS production was measured respectively
in 200 µl of liquefied neat semen and 200 µl of washed
cells suspension after addition of 5 µl of the working
solution of Luminol (0.1 mM) obtained by dilution of the
stock solution with the HBSS (Hank’s Balanced Salt
Solution). Negative controls for estimation of back-
ground signals were prepared for each assay by adding 5
µl of 0.1 mM Luminol to 200 µl of PBS. Also, 200 µl of
PBS served as a blank.
We also applied the test of specific provocation of
leucocytes by FMLP (Formyl-Methionyl-Leucyl-Pheny-
lalanine; Sigma Aldrich) on all our samples (neat and
washed). The FMLP is used to stimulate a chemilu-
minescent signal from any polymorphonuclear granulo-
cytes that are present in the sperm suspension 16. Since
FMLP receptors are not present on the surface of human
spermatozoa, this signal is specific for the leucocyte
population 16. A 0.1 mM stock solution of FMLP in
DMSO was prepared.
The ROS production was measured respectively in
200 µl of liquefied neat semen and 200 µl of washed
cells suspension after addition of 5 µl of the working
solution of Luminol (0.1 mM) and stimulation with 2 µl
of FMLP working solution (0.2 µM) obtained by dilution
of the FMLP stock solution with the HBSS. The che-
miluminescence signal was monitored for 15 minutes.
Results were expressed as relative light units (RLU) per
minute and per 20 × 106 spermatozoa.
A statistical analysis was performed using SPSS 13.0
software. Statistical tests including Student’s t test, Pear-
son’s and Spearman’s correlations, linear regression were
used. The statistical significance was considered for p
values < 0.05.
The mean values (± SD) and ranges of semen parameters,
basal and after FMLP stimulation ROS levels in neat and
washed semen are summarized in Table 1.
The levels of basal ROS in neat semen was negatively
correlated with the immediate and late total motility, the
immediate and late rapid progressive motility and the
sperm concentration (Table 2; Figures 1(a)-(c)). We also
found significant and positive correlations of levels of
basal ROS in neat semen with the leucocytes concen-
tration in semen firstly and with the rates of cytoplasmic
droplets in the mid piece area of spermatozoa secondly
(Table 2; Figures 1(d) and (e)).
There was a marked increase of ROS in semen after
washing (Table 1) with significant correlations of the
Table 1. Means and ranges of semen parameters, basal ROS
levels in neat and washed semen and ROS levels in neat and
washed semen after FMLP stimulation (n = 50).
Means ± SD Ranges
Volume (ml) 3.9 ± 1.5 1.9 - 8.5
Total motility (%) 38.7 ± 14.6 0 - 60
Rapid progressive mobility “a”
(%) 10.6 ± 7.1 0 - 25
Sperm concentration
(Millions/ml) 45.9 ± 39.8 0.02 - 189.6
Vitality (%) 68.9 ± 14.9 26 - 89
Leucocytes (millions/ml) 0.9 ± 2.1 0.01 - 9.5
Normal morphology (%) 7.2 ± 5.3 0 - 22
Cytoplasmic droplets (%) 5.1 ± 4.9 0 - 20
Basal ROS (RLU/mn/ 20.106 spz)
In neat semen 3.9 ± 21.4 0.01 - 150.5
In washed semen 164.6 ± 1136.2 0.03 - 8037
ROS after FMLP stimulation
(RLU/mn/20 × 106 spz)
In neat semen 3.9 ± 21.7 0.01 - 153.0
In washed semen 222.7 ± 1542.7 0.02 - 10912.5
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A. Sellami et al. / Advances in Bioscience and Biotechnology 4 (2013) 30-40
Copyright © 2013 SciRes.
(a) (b)
(c) (d)
Figure 1. Correlations of basal ROS levels (log transformed) in neat semen with the following semen parameters: imme-
diate total motility (a), immediate progressive motility (b), sperm concentration (c), leucocyte concentration (d), and cyto-
plasmic droplets (e). Empirical distributions of the ROS levels were highly skewed on the original scale, and so
log-transformed data were used for all statistical analyzes. Basal ROS in neat semen was positively correlated with the
leucocytes concentration in semen and with the rates of cytoplasmic droplets in the mid piece area of spermatozoa.
A. Sellami et al. / Advances in Bioscience and Biotechnology 4 (2013) 30-40
ROS levels in neat semen with those in sperm suspension
after washing (Figure 2).
The studied samples were divided into 2 groups: (G1)
composed of 36 samples without leucospermia (leuco-
cytes ˂ 106/ml and (G2) composed of 14 leucospermic
samples (leucocytes 106/ml). ROS levels were signify-
cantly higher in the group of leucospermic samples
compared with the group of leucocyte free samples (Ta-
ble 3) and a significant and positive correlation of basal
Table 2. Correlations between ROS levels in neat semen and
semen parameters (n = 50). The levels of basal ROS in neat
semen were negatively correlated with the immediate and late
total motility, the immediate and late rapid progressive motility
and the sperm concentration.
ROS in neat semen (n = 50)
coefficient P value
Sperm concentration 0.77 <0.001
Immediate total Motility 0.45 0.001
Immediate rapid progressive
motility “a” 0.27 0.04
Late total Motility 0.46 0.001
Late rapid progressive motility “a”0.29 0.04
Sperm vitality +0.02 NS
Leucocytes +0.32 0.02
Sperm morphology 0.02 NS
Cytoplasmic droplets +0.51 <0.001
NS = No significant. Pearson’s and Spearman’s correlations tests was used
and statistical significance was assessed at p < 0.05.
Figure 2. The correlation of basal ROS levels (log transformed)
in neat semen with basal ROS levels (log transformed) in
washed semen was positively significant.
ROS levels in washed semen with seminal leucocytes
concentrations was noted (Figure 3). ROS production
was significantly increased after FMLP stimulation in
leucospermic washed samples (Figure 4).
Figure 3. Correlation of basal ROS levels in washed semen
with seminal leucocytes concentrations in neat semen. Positive
correlation was noted between basal ROS levels (log trans-
formed) in washed semen with seminal leucocytes concentra-
tions (log transformed) in neat semen.
Figure 4. Correlation of FMLP stimulated ROS levels in
washed semen with leucocytes concentrations in neat semen.
Positive correlation was noted between FMLP stimulated ROS
levels (log transformed) in washed semen with leucocytes con-
entrations (log transformed) in neat semen. c
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A. Sellami et al. / Advances in Bioscience and Biotechnology 4 (2013) 30-40 35
Table 3. Comparison of semen parameters, basal and FMLP induced semen oxidative status between leucocyte free samples (G1)
and leucospermic samples (G2); the ROS levels were significantly enhanced in washed semen and after FMLP application in
leucospermic group.
Means ± SD
G1 (n = 36)
Means ± SD
G2 (n = 14) P value
Volume (ml) 4.2 ± 1.6 3.6 ± 1.2 NS
Sperm concentration (Millions/ml) 42.3 ± 37.4 54.9 ± 45.6 NS
Immediate total Motility (%) 39.7 ± 14.3 36.1 ± 15.6 NS
Immediate rapid progressive motility “a” (%) 10.4 ± 6.8 11.1 ± 7.9 NS
Late total Motility (%) 35.1 ± 15.8 28.5 ± 16.5 NS
Late rapid progressive motility “a” (%) 7.7 ± 6.2 7.3 ± 6.9 NS
ROS (RLU/20.106 spz)
Neat semen FMLP () 1.06 10.96 0.002
Neat semen FMLP (+) 0.95 11.22 0.002
Wasched semen FMLP () 3.12 579.72 0.001
Wasched semen FMLP (+) 3.49 786.11 0.001
FMLP (): without FMLP stimulation; FMLP (+): after FMLP stimulation; NS: No significant. Student’s t test was used for comparison and statistical signifi-
cance was assessed at p < 0.05.
It is well established that the production of excessive
quantities of reactive oxygen species in the male genital
tract, essentially by leucocytes, can overwhelm the semi-
nal antioxidant defenses and give rise to a harmful oxi-
dative stress altering the microenvironment in which
spermatozoa develop and mature 5,18,27. Currently,
there are growing evidences that genital oxidative stress
is involved in the pathogenesis of many reproductive
processes through sperm damage, deformity and eventu-
ally male infertility 28,29. We used chemiluminescence
technique in our study because it is the most commonly
described assay to detect ROS production within semen
30. It is very sensitive and has the advantage of rela-
tively well established reported ranges for both the fertile
and infertile population 31-33.
As regards the relationship between semen quality and
its oxidative status evaluated by chemiluminescence as-
say, we found significant correlations between basal
ROS levels produced in neat semen of infertile men and
conventional sperm parameters such as motility, sperm
concentration and one of sperm morphological abnor-
malities (cytoplasmic droplets in mid piece area of sper-
The decrease of sperm motility associated to an oxida-
tive stress in infertile men semen was reported previ-
ously 23,34-36 and was related to the negative effect of
ROS on sperm mitochondrial membrane potential and
the reduction of ATP production 37-39. In addition, the
sperm membranes are characterized by the abundance of
unsaturated fatty acids having a double-bonded nature
and predisposing them to lipid peroxidation 26,40. This
peroxidation reaction affects membrane structure and
fluidity and causes damage to axonemal proteins leading
to a permanent impairment in sperm motility 41-43.
In the present study, we founded a significant negative
correlation between sperm concentration and the levels
of ROS in semen. Indeed, it was widely reported that
seminal concentrations of ROS in oligospermic patients
were higher than that of normospermic patients 31,
44-46. ROS and their metabolites attack DNA, lipids
and proteins, alter enzymatic systems and cell signaling
pathways, producing irreparable alterations and may ac-
celerate the process of germ cell apoptosis 47. The lat-
ter process can lead to decline in sperm counts and may
explain the apparent deterioration of semen quality 39,
48. Also, the decrease in the sperm concentration could
be explained by the presence of a genital inflammation
process which often results in the presence of high
number of leucocytes, principle source of ROS in semen
49,50. Indeed, our results showed a significant in-
crease of ROS in leucospermic samples compared with
the samples without leucospermia. These results are con-
sistent with other reports indicating that seminal leuco-
cytes have the potential to cause oxidative stress through
their degranulation and the formation of free radicals 9.
In fact, it was also reported that the presence of leu-
cocytes in semen is associated with a high rate of pro-
duction of ROS with reduction of sperm motility and its
fertilizing ability 34. The activation state of leuco-
cytes must play an important role in determining final
ROS output 51. Pro-inflammatory seminal plasma cy-
tokines such as IL-6, IL-8 and tumour necrosis factor
TNF α seem to be produced locally by activated leuco-
cytes in semen and a marked relationship between these
cytokines, leucocytes and seminal ROS production was
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A. Sellami et al. / Advances in Bioscience and Biotechnology 4 (2013) 30-40
reported 14,50,52. During epididymal transit, the sperm
are not in contact with seminal plasma antioxidants, and
are therefore vulnerable to oxidative damage, especially
when there is an epididymal inflammation 49. It was
also demonstrated that leucocytospermia increased the
production of ROS by human spermatozoa 53.
The application of a leucocyte-specific stimulant
(FMLP) test showed an increase in ROS production
especially in semen samples of leucospermic group.
Leucocytes are the only cells present in the human
ejaculate possessing detectable receptors for FMLP and
capable of generating reactive oxygen species in re-
sponse to this reagent 54. Our results confirm that
leucocytes are the major source of ROS in semen and
present an additional argument that the presence of
leucocytes in semen is associated with a high rate of
production of ROS. Moreover, the results of the FMLP
provocation test have an important bearing on the fertile-
izing capacity of the spermatozoa in vitro 19,55.
The leucocytes are the principle but not the exclusive
source of ROS in semen and in concordance with our
findings, Aitken 44 and Gomez 56 showed a signify-
cant production of free radicals by cytosplasmic droplets
present at the midpiece of the defective morphologically
abnormal spermatozoa. These residues are rich in Glu-
cose-6-phosphate dehydrogenase (G6PD), an enzyme
which controls the rate of glucose flux and intracellular
production of Nicotinamide Adenine Dinucleotide Phos-
phate (NADPH) 57. The latter is used to fuel the gen-
eration of ROS via NADPH oxidase located within the
sperm membrane 56-58. As a result, teratozoospermic
sperm produce increased amounts of ROS with a reduced
antioxidant capacity compared with morphologically
normal sperm 9,59. All these relationships underpin the
well established observation that higher ROS have a
negative impact on sperm quality but even men with
normozoospermic idiopathic infertility exhibit signifi-
cantly higher seminal ROS production and lower anti-
oxidant capacity than fertile men for as yet unknown
reasons 28,60.
Besides the measure of ROS level in neat semen, we
quantified ROS in semen after washing and removal of
seminal plasma. We apply this assay in our study to
evaluate the oxidative status of semen during sperm
preparation techniques for assisted reproductive tech-
nologies. Our results are similar to those reported in
other studies 61-63 that showed a significant increase
of ROS production in semen by repeated cycles of cen-
trifugation and aggravated by the ablation of the natural
antioxidant environment. Some studies 64-66 showed
that the addition of antioxidants such as EDTA, catalase,
ascorbate and tocopherol in sperm preparation medium
may scavenge ROS and decrease their deleterious effects
on spermatozoa but these findings are discussed 67,68.
Another point for consideration is the implication of
genital oxidative stress in the sperm DNA fragmentation
commonly observed in spermatozoa of infertile men 69.
In fact, significant correlations of seminal ROS levels
with DNA damage were reported in many studies 43,70,
71. Moreover, other studies reported an increase in DNA
damage in sperm from leucocytospermic samples 53,
72,73. Recently, it was suggested a direct implication of
leucocytes, as an exogenous factor, in generating DNA
base modifications evaluated by the formation of 8-
oxoguanine, a key biomarker of the oxidative DNA
damage 74. Oxidized DNA bases have increased sus-
ceptibility to ROS action which reflects a direct and spe-
cific action of ROS on sperm DNA 9,75. The most re-
cent studies on the origin of sperm DNA damage sug-
gested that there might be a cascade of changes that pro-
gress from the induction of oxidative stress and DNA
base-pair oxidation to DNA fragmentation and cell death
Nevertheless, it seems that the plasma membrane is
less vulnerable to oxidative damage than DNA and
sperm with significant oxidative DNA damage and intact
membranes could preserve its ability to fertilize oocyte
70. Many of these embryos developed from spermato-
zoa with fragmented DNA will unfortunately fail at the
blastocyst or early fetal stage 77,78. Thereby, the cur-
rently use of mechanical techniques such as Intra Cyto-
plasmic Sperm Injection (ICSI) to bypass some male
factor infertility is unlikely to be the ‘best practice’ since
ROS damaged DNA, frequently induced by seminal
leucocytes, will result in poor quality blastocysts and an
increase in miscarriage 9,25. Additionally, the patients
may be urged to consider antioxidant therapy before un-
dergoing reproductive assistance technique that may re-
duce DNA damage levels and improve sperm fertility
potential 9.
Oxidative stress is a major factor in male reproductive
disorders because it may impair the physiological func-
tion of spermatozoa at the molecular level. Understand-
ing the exact mechanisms, by which oxidative stress de-
velops in semen, will improve the management of semen
quality impairment by potential toxic ROS. To establish
a treatment strategy of genital oxidative stress in infertile
men, we must first specify its origin: seminal leucocytes
and/or sperm cells themselves, to guide thereby the op-
timum therapeutic modalities for male infertility particu-
larly in the context of leucocytospermia.
Progress in assisted reproductive technologies contin-
ues to be offered to infertile men, who would otherwise
be unable to conceive chances to have their own off-
spring. However, these “mechanical” procedures are un-
able to compensate for oxidative damage to sperm DNA.
Copyright © 2013 SciRes. OPEN ACCESS
A. Sellami et al. / Advances in Bioscience and Biotechnology 4 (2013) 30-40 37
In addition, direct treatment of oxidative stress and its
source may allow for natural conception.
We would like to thank the technicians of the laboratory of Histol-
ogy-Embryology, Medical school Sfax for their help in semen process-
ing and their technical assistance. We express also our gratitude to the
staff of the laboratory of cell culture; Biotechnology institute, Sfax,
Tunisia for their help. This work was supported by the Histology Em-
bryology research unit, Medical school Sfax and the Tunisian Ministry
of Higher Education, Scientific Research, and Technology.
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ROS: Reactive Oxygen Species
DNA: Deoxyribonucleic Acid
FMLP: Formyl-Methionyl-Leucyl-Phenylalanine
WHO: World Health Organisation
H2O2: Hydrogen Peroxide
SD: Standard Deviation
RLU: Relative Light Units
HBSS: Hank’s Balanced Salt Solution
NADPH: Nicotinamide Adenine Dinucleotide Phosphate
G6PD: Glucose-6-Phosphate Dehydrogenase
ICSI: Intra Cytoplasmic Sperm Injection