Advances in Bioscience and Biotechnology, 2013, 4, 30-40 ABB
http://dx.doi.org/10.4236/abb.2013.411A2005 Published Online November 2013 (http://www.scirp.org/journal/abb/)
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.
ABSTRACT
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
1. INTRODUCTION
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
9,10.
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. MATERIAL AND METHODS
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
16.
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

A. Sellami et al. / Advances in Bioscience and Biotechnology 4 (2013) 30-40 32
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
measurement.
2.6. Measurement of Reactive Oxygen Species
(ROS) in Semen by Chemiluminescence
Technique
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.
3. STATISTICAL ANALYSIS
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.
4. RESULTS
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

A. Sellami et al. / Advances in Bioscience and Biotechnology 4 (2013) 30-40 33
(a) (b)
(c) (d)
(e)
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 34
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)
Correlation
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

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.
5. DISCUSSION
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-
matozoa).
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

A. Sellami et al. / Advances in Bioscience and Biotechnology 4 (2013) 30-40 36
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
76.
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.
6. CONCLUSIONS
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.

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.
7. ACKNOWLEDGEMENTS
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.
REFERENCES
[1] Rahman, K. (2007) Studies on free radicals, antioxidants,
and co-factors. Clinical Interventions in Aging, 2, 219-
236.
[2] Brieger, K., Schiavone, S., Miller Jr., F.J. and Krause,
K.H. (2102) Reactive oxygen species: From health to dis-
ease. Swiss Medical Weekly, 142, w13659.
http://dx.doi.org/10.4414/smw.2012.13659
[3] Agarwal, A. and Saleh, R.A. (2002) Role of oxidants in
male infertility: Rationale, significance, and treatment.
Urologic Clinics of North America, 29, 817-827.
http://dx.doi.org/10.1016/S0094-0143(02)00081-2
[4] Cocuzza, M., Sikka, S.C., Athayde, K.S. and Agarwal, A.
(2007) Clinical relevance of oxidative stress and sperm
chromatin damage in male infertility: An evidence based
analysis. International braz j urol, 33, 603-621.
http://dx.doi.org/10.1590/S1677-55382007000500002
[5] Agarwal, A., Makker, K. and Sharma, R. (2008) Clinical
relevance of oxidative stress in male factor infertility: An
update. American Journal of Reproductive Immunology,
59, 2-11.
http :/ /d x. do i.o rg/10 .1111 /j .1600-0897.2007.00559.x
[6] Gharagozloo, P. and Aitken, R.J. (2011) The role of
sperm oxidative stress in male infertility and the signifi-
cance of oral antioxidant therapy. Human Reproduction,
26, 1628-1640. http://dx.doi.org/10.1093/humrep/der132
[7] Agarwal, A. and Said, T.M. (2005) Oxidative stress, DNA
damage and apoptosis in male infertility: A clinical ap-
proach. BJU International, 95, 503-507.
http :/ /d x. do i.o rg/10 .1111 /j .1464-410X.2005.05328.x
[8] Saalu, L.C. (2010) The incriminating role of reactive
oxygen species in idiopathic male infertility: An evidence
based evaluation. Pakistan Journal of Biological Sciences,
13, 413-422. http://dx.doi.org/10.3923/pjbs.2010.413.422
[9] Tremellen, K. (2008) Oxidative stress and male infertile-
ity—A clinical perspective. Human Reproduction Update,
14, 243-258. http://dx.doi.org/10.1093/humupd/dmn004
[10] Aitken, R.J. and Koppers, A.J. (2011) Apoptosis and
DNA damage in human spermatozoa. Asian Journal of
Andrology, 13, 36-42.
http://dx.doi.org/10.1038/aja.2010.68
[11] Kessopoulou, E., Tomlinson, M.J., Barratt, C.L., Bolton,
A.E. and Cooke, I.D. (1992) Origin of reactive oxygen
species in human semen: Spermatozoa or leucocytes? The
Journal of the Society for Reproduction and Fertility, 94,
463-470. http://dx.doi.org/10.1530/jrf.0.0940463
[12] Sharma, R.K. and Agarwal, A. (1996) Role of reactive
oxygen species in male infertility. Urology, 48, 835-850.
http://dx.doi.org/10.1016/S0090-4295(96)00313-5
[13] Anderson, D.J. and Politch, J.A. (1996) Evaluation and
treatment of the infertile male. In: Centola, G.M. and
Ginsburg, K.A., Eds., Cambridge University Press, New
York, 263-277.
[14] Depuydt, C.E., Bosmans, E., Zalata, A., Schoonjans, F.
and Comhaire, F.H. (1996) The relation between reactive
oxygen species and cytokines in andrological patients
with or without male accessory gland infection. Journal
of Andrology, 17, 699-707.
[15] Keck, C., Gerber-Schäfer, C., Clad, A., Wilhelm, C. and
Breckwoldt, M. (1998) Seminal tract infections: Impact
on male fertility and treatment options. Human Repro-
duction Update, 4, 891-903.
http://dx.doi.org/10.1093/humupd/4.6.891
[16] World Health Organization (1999) Laboratory manual for
the examination of human semen and sperm-cervical mu-
cus interaction. Cambridge University Press, Cambridge.
[17] World Health Organization (2010) Laboratory manual for
the examination and processing of human semen. WHO
Press, Geneva.
[18] Kashou, A.H., Sharma, R. and Agarwal, A. (2013) As-
sessment of oxidative stress in sperm and semen. In:
Carrell, D.T. and Aston, K.I., Eds., Spermatogenesis: Me-
thods and protocols, Methods in Molecular Biology, Hu-
mana Press, New York, 351-361.
[19] Krausz, C., Mills, C., Rogers, S., Tan, S.L. and Aitken,
R.J. (1994) Stimulation of oxidant generation by human
sperm suspensions using phorbol esters and formyl pep-
tides: Relationships with motility and fertilization in vitro.
Fertility and Sterility, 62, 599-605.
[20] Storey, B.T. (1997) Biochemistry of the induction and
prevention of lipoperoxidative damage in human sper-
matozoa. Molecular Human Reproduction, 3, 203-213.
http://dx.doi.org/10.1093/molehr/3.3.203
[21] Tomlinson, M.J., Barratt, C.L. and Cooke, I.D. (1993)
Prospective study of leukocytes and leukocyte subpopu-
lations in semen suggests they are not a cause of male in-
fertility. Fertility and Sterility, 60, 1069-1075.
[22] Aitken, R.J., Irvine, D.S. and Wu, F.C. (1991) Prospec-
tive analysis of sperm-oocyte fusion and reactive oxygen
species generation as criteria for the diagnosis of infertil-
ity. American Journal of Obstetrics & Gynecology, 164,
542-551. http://dx.doi.org/10.1016/S0002-9378(11)80017-7
[23] du Plessis, S.S., McAllister, D.A., Luu, A., Savia, J.,
Agarwal, A. and Lampiao, F. (2010) Effects of H2O2 ex-
posure on human sperm motility parameters, reactive oxy-
gen species levels and nitric oxide levels. Andrologia, 42,
206-210.
http://dx.doi.org/10.1111/j.1439-0272.2009.00980.x
[24] Sharma, R.K., Pasqualotto, A.E., Nelson, D.R., Thomas
Jr., A.J. and Agarwal A. (2001) Relationship between
seminal white blood cell counts and oxidative stress in

A. Sellami et al. / Advances in Bioscience and Biotechnology 4 (2013) 30-40 38
men treated at an infertility clinic. Journal of Andrology,
22, 575-583.
[25] Barraud-Lange, V., Pont, J.C., Ziyyat, A., Pocate, K., Si-
fer, C., Cedrin-Durnerin, I., Fechtali, B., Ducot, B. and
Wolf, J.P. (2011) Seminal leukocytes are good samaritans
for spermatozoa. Fertility and Sterility, 96, 1315-1319.
http://dx.doi.org/10.1016/j.fertnstert.2011.09.035
[26] Zini, A. and Sigman, M. (2009) Evaluation of sperm
function. In: Lipshultz, L.I., Howards, S.S. and Nieder-
berger, C.S., Eds., Infertility in the Male, Cambridge Uni-
versity Press, New York, 177-198.
http://dx.doi.org/10.1017/CBO9780511635656.012
[27] Agarwal, A. and Prabakaran, S.A. (2005) Oxidative stress
and antioxidants in male infertility: A difficult balance.
Iranian Journal of Reproductive Medicine, 3, 1-8.
[28] Agarwal, A., Sharma, R.K., Nallella, K.P., Thomas Jr.,
A.J., Alvarez, J.G. and Sikka, S.C. (2006) Reactive oxy-
gen species as an independent marker of male factor in-
fertility. Fertility and Sterility, 86, 878-885.
http://dx.doi.org/10.1016/j.fertnstert.2006.02.111
[29] Doshi, S.B., Khullar, K., Sharma, R.K. and Agarwal, A.
(2012) Role of reactive nitrogen species in male infertile-
ity. Reproductive Biology and Endocrinology, 10, 109.
http://dx.doi.org/10.1186/1477-7827-10-109
[30] Sikka, S.C. (2004) Role of oxidative stress and antioxi-
dants in andrology and assisted reproductive technology.
Journal of Andrology, 25, 5-18.
[31] Ochsendorf, F.R., Thiele, J., Fuchs, J., Schuttau, H., Freis-
leben, H.J., Buslau, M. and Milbradt, R. (1994) Chemilu-
minescence in semen of infertile men. Andrologia, 26,
289-293.
http :/ /d x. do i.o rg/10 .1111 /j .1439-0272.1994.tb00804.x
[32] Williams, A.C. and Ford, W.C. (2005) Relationship be-
tween reactive oxygen species production and lipid per-
oxidation in human sperm suspensions and their associa-
tion with sperm function. Fertility and Sterility, 83, 929-
936. http://dx.doi.org/10.1016/j.fertnstert.2004.11.031
[33] Athayde, K.S., Cocuzza, M., Agarwal, A., Krajcir, N.,
Lucon, A.M., Srougi, M. and Hallak, J. (2007) Develop-
ment of normal reference values for seminal reactive
oxygen species and their correlation with leukocytes and
semen parameters in a fertile population. Journal of An-
drology, 28, 613-620.
http://dx.doi.org/10.2164/jandrol.106.001966
[34] Plante, M., de Lamirande, E. and Gagnon, C. (1994) Re-
active oxygen species released by activated neutrophils,
but not by deficient spermatozoa, are sufficient to affect
normal sperm motility. Fertility and Sterility, 62, 387-
393.
[35] Griveau, J.F. and Le Lannou, D. (1997) Reactive oxygen
species and human spermatozoa: Physiology and pathol-
ogy. International Journal of Andrology, 20, 61-69.
http://dx.doi.org/10.1046/j.1365-2605.1997.00044.x
[36] Armstrong, J.S., Rajasekaran, M., Chamulitrat, W., Gatti,
P., Hellstrom, W.J. and Sikka, S.C. (1999) Characteriza-
tion of reactive oxygen species induced effects on human
spermatozoa movement and energy metabolism. Free
Radical Biology and Medicine, 26, 869-880.
http://dx.doi.org/10.1016/S0891-5849(98)00275-5
[37] Baumber, J., Ball, B.A., Gravance, C.G., Medina, V. and
Davies-Morel, M.C. (2000) The effect of reactive oxygen
species on equine sperm motility, viability, acrosomal in-
tegrity, mitochondrial membrane potential, and mem-
brane lipid peroxidation. Journal of Andrology, 21, 895-
902.
[38] Wang X., Sharma, R.K., Gupta, A., George, V., Thomas,
A.J., Falcone, T. and Agarwal, A. (2003) Alterations in
mitochondria membrane potential and oxidative stress in
infertile men: A prospective observational study. Fertility
and Sterility, 80, 844-850.
http://dx.doi.org/10.1016/S0015-0282(03)00983-X
[39] Agarwal, A., Saleh, R.A. and Bedaiwy, M.A. (2003) Role
of reactive oxygen species in the pathophysiology of hu-
man reproduction. Fertility and Sterility, 79, 829-843.
http://dx.doi.org/10.1016/S0015-0282(02)04948-8
[40] Aitken, R.J., Wingate, J.K., De Iuliis, G.N., Koppers, A.J.
and McLaughlin, E.A. (2006) Cis-unsaturated fatty acids
stimulate reactive oxygen species generation and lipid
peroxidation in human spermatozoa. The Journal of Cli-
nical Endocrinology & Metabolism, 91, 4154-4163.
http://dx.doi.org/10.1210/jc.2006-1309
[41] de Lamirande, E. and Gagnon, C. (1992) Reactive oxy-
gen species and human spermatozoa. I. Effects on the
motility of intact spermatozoa and on sperm axonemes.
Journal of Andrology, 13, 368-378.
[42] Tavilani, H., Doosti, M. and Saeidi, H. (2005) Malondial-
dehyde levels in sperm and seminal plasma of astheno-
zoospermic and its relationship with semen parameters.
Clinica Chimica Acta, 356, 199-203.
http://dx.doi.org/10.1016/j.cccn.2005.01.017
[43] Kothari, S., Thompson, A., Agarwal, A. and du Plessis,
S.S. (2010) Free radicals: Their beneficial and detrimen-
tal effects on sperm function. Indian Journal of Experi-
mental Biology, 48, 425-435.
[44] Aitken, R.J., Buckingham, D., West, K.M., Wu, F.C., Zi-
kopoulos, K. and Richardson, D.W. (1992) Differential
contribution of leucocytes and spermatozoa to the gene-
ration of reactive oxygen species in the ejaculates of oli-
gozoospermic patients and fertile donors. The Journal of
the Society for Reproduction and Fertility, 94, 451-462.
http://dx.doi.org/10.1530/jrf.0.0940451
[45] Zalata, A., Hafez, T. and Comhaire, F. (1995) Evaluation
of the role of reactive oxygen species in male infertility.
Human Reproduction, 10, 1444-1451.
http://dx.doi.org/10.1093/HUMREP/10.6.1444
[46] Pasqualotto, F.F., Sharma, R.K., Nelson, D.R., Thomas,
A.J. and Agarwal, A. (2000) Relationship between oxida-
tive stress, semen characteristics, and clinical diagnosis in
men undergoing infertility investigation. Fertility and
Sterility, 73, 459-464.
http://dx.doi.org/10.1016/S0015-0282(99)00567-1
[47] Boonstra, J. and Post, J.A. (2004) Molecular events asso-
ciated with reactive oxygen species and cell cycle pro-
gression in mammalian cells. Gene, 337, 1-13.
http://dx.doi.org/10.1016/j.gene.2004.04.032
[48] Tvrdá, E., Kňažická, Z., Bárdos, L., Massányi, P. and
Lukáč, N. (2011) Impact of oxidative stress on male ferti-
lity—A review. Acta Veterinaria Hungarica, 59, 465-484.

A. Sellami et al. / Advances in Bioscience and Biotechnology 4 (2013) 30-40 39
http://dx.doi.org/10.1556/AVet.2011.034
[49] Ochsendorf, F.R. (1999) Infections in the male genital
tract and reactive oxygen species. Human Reproduction
Update, 5, 399-420.
http://dx.doi.org/10.1093/humupd/5.5.399
[50] Fraczek, M. and Kurpisz, M. (2007) Inflammatory medi-
ators exert toxic effects of oxidative stress on human
spermatozoa. Journal of Andrology, 28, 325-333.
http://dx.doi.org/10.2164/jandrol.106.001149
[51] Villegas, J., Schulz, M., Soto, L., Iglesias, T., Miska, W.
and Sánchez, R. (2005) Influence of reactive oxygen spe-
cies produced by activated leukocytes at the level of apo-
ptosis in mature human spermatozoa. Fertility and Steril-
ity, 83, 808-810.
http://dx.doi.org/10.1016/j.fertnstert.2004.09.022
[52] Wang, A., Fanning, L., Anderson, D.J. and Loughlin, K.R.
(1997) Generation of reactive oxygen species by leukocy-
tes and sperm following exposure to urogenital tract in-
fection. Archives of Andrology, 39, 11-17.
http://dx.doi.org/10.3109/01485019708987896
[53] Saleh, R.A., Agarwal, A., Kandirali, E., Sharma, R.K.,
Thomas, A.J., Nada, E.A., Evenson, D.P. and Alvarez,
J.G. (2002) Leukocytospermia is associated with in-
creased reactive oxygen species production by human
spermatozoa. Fertility and Sterility, 78, 1215-1224.
http://dx.doi.org/10.1016/S0015-0282(02)04237-1
[54] Krausz, C., West, K., Buckingham, D. and Aitken, R.J.
(1992) Development of a technique for monitoring the
contamination of human semen samples with leukocytes.
Fertility and Sterility, 57, 1317-1325.
[55] Sukcharoen, N., Keith, J., Irvine, D.S. and Aitken, R.J.
(1996) Prediction of the in-vitro fertilization (IVF) poten-
tial of human spermatozoa using sperm function tests:
The effect of the delay between testing and IVF. Human
Reproduction, 11, 1030-1034.
http://dx.doi.org/10.1093/oxfordjournals.humrep.a019291
[56] Gomez, E., Buckingham, D.W., Brindle, J., Lanzafame,
F., Irvine, D.S. and Aitken, R.J. (1996) Development of
an image analysis system to monitor the retention of re-
sidual cytoplasm by human spermatozoa: Correlation
with biochemical markers of the cytoplasmic space, oxi-
dative stress, and sperm function. Journal of Andrology,
17, 276-287.
[57] Said, T.M., Agarwal, A., Sharma, R.K., Thomas Jr., A.J.
and Sikka, S.C. (2005) Impact of sperm morphology on
DNA damage caused by oxidative stress induced by beta-
nicotinamide adenine dinucleotide phosphate. Fertility
and Sterility, 83, 95-103.
http://dx.doi.org/10.1016/j.fertnstert.2004.06.056
[58] Fisher, H.M. and Aitken, R.J. (1997) Comparative analy-
sis of the ability of precursor germ cells and epididymal
spermatozoa to generate reactive oxygen metabolites.
Journal of Experimental Zoology, 277, 390-400.
http://dx.doi.org/10.1002/(SICI)1097-010X(19970401)27
7:5<390::AID-JEZ5>3.0.CO;2-K
[59] Said, T.M., Agarwal, A., Sharma, R.K., Mascha, E., Sik-
ka, S.C. and Thomas Jr., A.J. (2004) Human sperm su-
peroxide anion generation and correlation with semen
quality in patients with male infertility. Fertility and Ste-
rility, 82, 871-877.
http://dx.doi.org/10.1016/j.fertnstert.2004.02.132
[60] Pasqualotto, F.F., Sharma, R.K., Kobayashi, H., Nelson,
D.R., Thomas Jr., A.J. and Agarwal, A. (2001) Oxidative
stress in normospermic men undergoing infertility eva-
luation. Journal of Andrology, 22, 316-322.
[61] Agarwal, A., Ikemoto, I. and Loughlin, K.R. (1994) Ef-
fect of sperm washing on levels of reactive oxygen spe-
cies in semen. Archives of Andrology, 33, 157-162.
http://dx.doi.org/10.3109/01485019408987819
[62] Fingerova, H., Oborna, I., Novotny, J., Svobodova, M.,
Brezinova, J. and Radova, L. (2009) The measurement of
reactive oxygen species in human neat semen and in sus-
pended spermatozoa: A comparison. Reproductive Biol-
ogy and Endocrinology, 7, 118.
http://dx.doi.org/10.1186/1477-7827-7-118
[63] Venkatesh, S., Shamsi, M.B., Dudeja, S., Kumar, R. and
Dada, R. (2011) Reactive oxygen species measurement in
neat and washed semen: Comparative analysis and its sig-
nificance in male infertility assessment. Archives of Gy-
necology and Obstetrics, 283, 121-126.
http://dx.doi.org/10.1007/s00404-010-1645-4
[64] Hughes, C.M., Lewis, S.E., McKelvey-Martin V.J. and
Thompson, W. (1998) The effects of antioxidant supple-
mentation during percoll preparation on human sperm
DNA integrity. Human Reproduction, 13, 1240-1247.
http://dx.doi.org/10.1093/humrep/13.5.1240
[65] Donnelly, E.T., McClure, N. and Lewis, S.E. (1999) The
effect of ascorbate and alpha-tocopherol supplementation
in vitro on DNA integrity and hydrogen peroxide-induced
DNA damage in human spermatozoa. Mutagenesis, 14,
505-512. http://dx.doi.org/10.1093/mutage/14.5.505
[66] Chi, H.J., Kim, J.H., Ryu, C.S., Lee, J.Y., Park, J.S.,
Chung, D.Y., Choi, S.Y., Kim, M.H., Chun, E.K. and
Roh, S.I. (2008) Protective effect of antioxidant supple-
mentation in sperm-preparation medium against oxidative
stress in human spermatozoa. Human Reproduction, 23,
1023-1028. http://dx.doi.org/10.1093/humrep/den060
[67] Donnelly, E.T., McClure, N. and Lewis, S.E. (2000) Glu-
tathione and hypotaurine in vitro: Effects on human
sperm motility, DNA integrity and production of reactive
oxygen species. Mutagenesis, 15, 61-68.
http://dx.doi.org/10.1093/mutage/15.1.61
[68] Baumber, J., Ball, B.A. and Linfor, J.J. (2005) Assess-
ment of the cryopreservation of equine spermatozoa in
the presence of enzyme scavengers and antioxidants.
American Journal of Veterinary Research, 66, 772-779.
http://dx.doi.org/10.2460/ajvr.2005.66.772
[69] Mahfouz, R., Sharma, R., Thiyagarajan, A., Kale, V., Gu-
pta, S., Sabanegh, E. and Agarwal, A. (2010) Semen cha-
racterristics and sperm DNA fragmentation in infertile
men with low and high levels of seminal reactive oxygen
species. Fertility and Sterility, 94, 2141-2146.
http://dx.doi.org/10.1016/j.fertnstert.2009.12.030
[70] Aitken, R.J. and Krausz, C. (2001) Oxidative stress, DNA
damage and the Y chromosome. Reproduction, 122, 497-
506. http://dx.doi.org/10.1530/rep.0.1220497
[71] Aitken, R.J. and De Iuliis, G.N. (2007) Origins and con-
sequences of DNA damage in male germ cells. Repro-

A. Sellami et al. / Advances in Bioscience and Biotechnology 4 (2013) 30-40 40
ductive Bio Medicine Online, 14, 727-733.
http://dx.doi.org/10.1016/S1472-6483(10)60676-1
[72] Alvarez, J.G., Sharma, R.K., Ollero, M., Saleh, R.A.,
Lopez, M.C., Thomas, A.J. Jr., Evenson, D.P. and Agar-
wal, A. (2002) Increased DNA damage in sperm from
leukocytospermic semen samples as determined by the
sperm chromatin structure assay. Fertility and Sterility,
78, 319-329.
http://dx.doi.org/10.1016/S0015-0282(02)03201-6
[73] Erenpreiss, J., Hlevicka, S., Zalkalns, J. and Erenpreisa J.
(2002) Effect of leukocytospermia on sperm DNA integ-
rity: A negative effect in abnormal semen samples. Jour-
nal of Andrology, 23, 717-723.
[74] Zribi, N., Chakroun, N.F., Elleuch, H., Abdallah, F.B.,
Ben Hamida, A.S., Gargouri, J., Fakhfakh, F. and Keskes,
L.A. (2011) Sperm DNA fragmentation and oxidation are
independent of malondialdheyde. Reproductive Biology
and Endocrinology, 9, 47-54.
http://dx.doi.org/10.1186/1477-7827-9-47
[75] Makker, K., Agarwal, A. and Sharma, R. (2009) Oxida-
tive stress and male infertility. Indian Journal of Medical
Research, 129, 357-367.
[76] Aitken, R.J., De Iuliis, G.N., Finnie, J.M., Hedges, A. and
McLachlan, R.I. (2010) Analysis of the relationships be-
tween oxidative stress, DNA damage and sperm vitality
in a patient population: Development of diagnostic crite-
ria. Human Reproduction, 25, 2415-2426.
http://dx.doi.org/10.1093/humrep/deq214
[77] Muriel, L., Garrido, N., Fernández, J.L., Remohí, J., Pel-
licer, A., de los Santos, M.J. and Meseguer, M. (2006)
Value of the sperm deoxyribonucleic acid fragmentation
level, as measured by the sperm chromatin dispersion test,
in the outcome of in vitro fertilization and intracytoplas-
mic sperm injection. Fertility and Sterility, 85, 371-383.
http://dx.doi.org/10.1016/j.fertnstert.2005.07.1327
[78] Avendaño, C., Franchi, A., Duran, H. and Oehninger S.
(2010) DNA fragmentation of normal spermatozoa nega-
tively impacts embryo quality and intracytoplasmic sperm
injection outcome. Fertility and Sterility, 94, 549-557.
http://dx.doi.org/10.1016/j.fertnstert.2009.02.050
ABBREVIATIONS
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