Seminal reactive oxygen species-antioxidant relationship in fertile males with and without varicocele.

Abstract

The aim of this study was to assess seminal reactive oxygen species (ROS)-antioxidants relationship in fertile and infertile men with and without varicocele. One hundred and seventy six males were studied; fertile healthy volunteers (n = 45), fertile men with varicocele (n = 45), infertile oligoasthenozoospermia (OA, n = 44) without varicocele and infertile OA with varicocele (n = 42). In their seminal plasma, two ROS parameters (malondialdehyde, hydrogen peroxide) and five antioxidants (superoxide dismutase, catalase, glutathione peroxidase, vitaminE, vitaminC) were estimated. Compared with fertile healthy men, in all other studied groups, estimated seminal ROS were significantly higher and estimated antioxidants were significantly lower. Infertile men with varicocele showed the same relationship as infertile men without varicocele. Sperm concentration, total sperm motility as well as sperm normal forms were negatively correlated with seminal malondialdehyde and were positively correlated with vitaminC. It is concluded that varicocele has an oxidative stress (OS) in fertile normozoospermic bearing conditions. This may allow understanding that, within men with varicocele, there is a threshold value of OS over which male fertility may be impaired.

Full text

Page 1

ORIGINAL ARTICLE
Seminal reactive oxygen species-antioxidant relationship in
fertile males with and without varicocele
T. Mostafa1, T. Anis1, H. Imam2, A. R. El-Nashar1& I. A. Osman1
1 Andrology & Sexology Department, Faculty of Medicine, Cairo University, Cairo, Egypt;
2 Medical Biochemistry Department, Ophthalmology Institute, Giza, Egypt
Introduction
Oxidative stress (OS) as a result of the imbalance between
reactive oxygen species (ROS) and antioxidants in the
semen can lead to sperm damage, deformity and eventu-
ally male infertility. This involves peroxidative damage to
sperm membrane and DNA fragmentation at both
nuclear and mitochondrial levels. OS has been implicated
as the major aetiological factor leading to sperm DNA
damage (Agarwal et al., 2008).
Varicocele, a varicosity of the pampiniform plexus, is
an important physical finding in infertile men and is
thought to be the most treatable factor of male infertility.
Its overall prevalence is approximately 15% in post-
pubertal populations, with prevalence in infertility clinics
approaching 40% (Levinger et al., 2007). Varicocele may
be associated with a variety of spermatogenetic condi-
tions, ranging from normal semen parameters to moder-
ate oligoasthenoteratozoospermia or azoospermia. The
effect of varicocele on spermatogenesis in subfertile men
is often reflected by low sperm count, decreased sperm
motilityorincreasedsperm
(Pasqualotto et al., 2005; Zucchi et al., 2006). Ko ¨ksal
et al. (2007) added that varicocele-induced testicular
dysfunction may be associated with disruption of the
blood-testis barrier.
Different studies demonstrated high levels of seminal
oxidative stress in men with varicocele, suggesting that
sperm dysfunction may be related to oxidative stress
(Hendin et al., 1999; Pasqualotto et al., 2000; Zini et al.,
2000; Meucci et al., 2003). In addition, oxidative stress
has been shown to affect the integrity of the sperm
genome by causing high frequencies of single- and
double-strand DNA breaks (Mostafa et al., 2001; Saleh &
Agarwal, 2002; Wang et al., 2003; Smith et al., 2006).
Varicocele-associated OS was evidenced also at spermatic
vein blood (Mitropoulos et al., 1996; Ozbek et al., 2000;
Mostafa et al., 2006a) as well as testicular tissues (Ko ¨ksal
abnormal morphology
Keywords
Antioxidants—male infertility—ROS—semen—
varicocele
Correspondence
Dr Taymour Mostafa, MD, Andrology &
Sexology Department, Faculty of Medicine,
Cairo University, Cairo 11562, Egypt.
Tel.: +20 10 515 0297;
E-mail: taymour1155@link.net
Accepted: September 29, 2008
Summary
The aim of this study was to assess seminal reactive oxygen species (ROS)-
antioxidants relationship in fertile and infertile men with and without varico-
cele. One hundred and seventy six males were studied; fertile healthy volunteers
(n = 45), fertile men with varicocele (n = 45), infertile oligoasthenozoospermia
(OA, n = 44) without varicocele and infertile OA with varicocele (n = 42). In
their seminal plasma, two ROS parameters (malondialdehyde, hydrogen perox-
ide) and five antioxidants (superoxide dismutase, catalase, glutathione peroxi-
dase, vitaminE, vitaminC) were estimated. Compared with fertile healthy men,
in all other studied groups, estimated seminal ROS were significantly higher
and estimated antioxidants were significantly lower. Infertile men with varico-
cele showed the same relationship as infertile men without varicocele. Sperm
concentration, total sperm motility as well as sperm normal forms were nega-
tively correlated with seminal malondialdehyde and were positively correlated
with vitaminC. It is concluded that varicocele has an oxidative stress (OS) in
fertile normozoospermic bearing conditions. This may allow understanding
that, within men with varicocele, there is a threshold value of OS over which
male fertility may be impaired.
ª 2009 The Authors
Journal Compilation ª 2009 Blackwell Publishing Ltd Æ Andrologia 41, 125–129
125

Page 2

et al., 2000, 2007). Allamaneni et al. (2004) correlated
increased ROS with varicocele grade, but not with the
testis size. Further, Shiraishi & Naito (2006) speculated
that surgical varicocele repair reduces OS in the testis.
The aim of this study was to assess seminal ROS-
antioxidants relationship in fertile and infertile males with
and without varicocele.
Materials and methods
We enrolled a total of 176 males with matched age who
were recruited prospectively from the Andrology Depart-
ment of the University Hospital after institutional review
board approval. Their mean age was 31.4 ± 6.2 years
(range 23–47 years) and all of them were married for
more than 2 years. They were divided into groups such as
(Gp1, n = 45) fertile healthy volunteers without varicocele
as controls, fertile men with varicocele (Gp2, n = 45),
infertile oligoasthenozoospermia without varicocele (Gp3,
n = 44) and infertile oligoasthenozoospermia with unilat-
eral varicocele (Gp4, n = 42). Inclusion criteria of fertile
men were having a child within the last year in addition
to two successive normozoospermic semen analyses.
Mean duration of infertility was 3.2 ± 0.62 years. Exclu-
sion criteria included secondary infertility, leucocytosper-
mia and smoking that could elevate ROS levels (Mostafa
et al., 2006b; Athayde et al., 2007). A detailed medical
history was gathered and physical examination was car-
ried out. Varicocele was diagnosed by physical examina-
tion confirmedbyusing
ultrasound (Acuson 128XB, Golden, Colorado, USA) in
both supine and upright position. Grades I, II, III were
included.
Ejaculates were obtained (08:00–10:00) after 4 days of
sexual abstinence into sterile containers for immediate
analysis. More than one sample provided by each subject
was examined according to the guidelines of the World
Health Organization (1999) by computer-assisted (Auto-
sperm) method (Kamtchouing et al., 1993). Sperm tracks
in freshly liquefied semen were followed manually with
the help of a cursor on the digitising tablet superimposed
at the centre of the microscope field (Olympus micro-
scope CH30/CH40, Tokyo, Japan) magnified X500. This
tablet generated data that can be analysed by a micro-
computer programme to calculate sperm concentration
and percentage of total sperm motility. Sperm morphol-
ogy was evaluated by phase contrast microscopy of the
native sample. Also, air-dried smears were prepared and
fixed in equal parts of ethanol-ether, then stained using
simplified Papanicolaou stain counting minimal 200 sper-
matozoa using oil-immersion lenses. Samples with white
blood cell >1 million ml)1or positive bacterial culture
were excluded.
scrotal colour Doppler
Semen samples were centrifuged at 1800 g for 15 min
and the supernatant seminal plasma was subjected to esti-
mationof two ROSparameters
[MDA], hydrogen peroxide [H2O2]) and five antioxidants
(superoxide dismutase [SOD], catalase [Cat], glutathione
peroxidase [GPx], vitaminE and vitaminC). MDA was
assayed by thiobarbituric acid reaction (Placer et al.,
1966). H2O2was estimated by the modified spectrophoto-
metric method (Feigl, 1958). SOD was measured accord-
ing to Winterbourn et al. (1975). Cat was measured
calorimetrically (Sinha, 1972). Total PGx was measured
using the method of Paglia & Valentine (1967). Vitamin E
was measured spectrofluorometrically by the method of
Taylor et al. (1976). Saponification and extraction of
vitaminE from seminal plasma were performed according
to Desai (1984). VitaminC was measured calorimetrically
as described by Jagota & Dani (1982).
(malondialdehyde
Statistical analysis
Numerical data were expressed as mean ± SD. Compari-
sons were performed by one-way anova to test the null
hypothesis that there were no differences and then Bon-
ferroni post-hoc test was conducted. Correlation between
variables was calculated using Spearman’s nonparametric
methods. Statistical significance was set at P < 0.05.
Results
Data of the investigated groups are presented in Table 1.
Seminal ROS parameters; MDA, H2O2were significantly
higher and estimated antioxidants were significantly lower
in fertile men with varicocele, OA men with and without
varicocele compared with healthy controls. Also, there
was a significant decrease in seminal ROS parameters and
a significant increase in seminal antioxidants in infertile
men without varicocele compared with infertile men with
varicocele.
Sperm concentration showed a significant negative cor-
relation with seminal MDA (r = )0.857, P = 0.001),
H2O2 (r = )0.680, P = 0.001) and significant positive
correlation with seminal vitaminC (r = 0.629, P = 0.005),
vitaminE (r = 0.619, P = 0.006). Total sperm motility
percentage showed a significant negative correlation with
seminal MDA (r = )0.859, P = 0.001), H2O2(r = )0.718,
P = 0.001) and a significant positive correlation with
seminal vitaminC (r = 0.705, P = 0.001), SOD (r = 0.662,
P = 0.014),catalase(r = 0.538,
abnormal forms percentage showed a significant positive
correlation with seminal MDA (r = 0.733, P = 0.001),
H2O2(r = )0.766, P = 0.001) and a significant negative
correlation with seminal vitaminC (r = )0.569, P =
0.009), SOD (r = )0.509, P = 0466), GPx (r = )0.518,
P = 0.0225).Sperm
Seminal ROS in varicocele
T. Mostafa et al.
ª 2009 The Authors
126
Journal Compilation ª 2009 Blackwell Publishing Ltd Æ Andrologia 41, 125–129

Page 3

P = 0.0047). Other relations demonstrated nonsignificant
correlations.
Discussion
Oxidative stress results from the production of oxygen
radicals in excess of the antioxidant capacity of the
stressed tissue. Varicocele has been demonstrated to
increase testicular oxidative stress, which leads to an
increase in germ cell apoptosis and subsequent hyposper-
matogenesis. Such stress conditions can cause changes in
the dynamics of testicular microvascular blood blow,
endocrine signaling and germ cell apoptosis (Turner &
Lysiak, 2008).
Most of the articles that discussed the influence of vari-
cocele in infertile males demonstrated increased seminal
ROS in varicocele-associated conditions being negatively
correlated with sperm concentration, sperm motility and
sperm normal forms percentage (Hendin et al., 1999;
Mostafa et al., 2001; Mancini et al., 2004). In this study,
this issue was investigated as well in fertile normozoo-
spermic males with and without varicocele. Estimated
seminal ROS parameters were significantly higher and
seminal antioxidants were significantly lower in fertile
normozoospermic men with varicocele compared with
those without varicocele.
Several studies suggested that an individual with vari-
cocele even with a normal semen analysis or documenta-
tion of previous fertility is at risk of subsequent loss of
testicular function and fertility status (Cozzolino & Lipsh-
ultz, 2001; Marmar, 2001). Pasqualotto et al. (2001)
showed increased ROS-total antioxidant capacity (TAC)
scores in normozoospermic infertile patients compared
with their levels in the controls concluding that presence
of OS in infertile normozoospermia may explain the
previously unexplained cases of infertility attributed
to female factors. Nakamura et al. (2002) added that
elevated OS in subfertile groups was shown even in the
sperm fraction considered being mature and normal. In
normal rats, Cam et al. (2004) found that increased rate
of apoptosis with experimental varicocele suggested a
molecular alteration that involved ROS overproduction as
a triggering mechanism. Smith et al. (2006) showed that
varicocele is associated with high levels of oxidative dam-
age and DNA-damaged spermatozoa even in the presence
of normal semen profile. Compared with healthy subjects,
Pasqualotto et al. (2008) observed that infertile men with
varicocele had significantly lower ROS-TAC scores, but
these scores were nonsignificantly different from those in
fertile men with varicocele. They warned that the fertility
potential in fertile varicocele patients can decline due to
OS.
There is consensus that there are two main sources of
increased ROS in semen; seminal leucocytes and sperma-
tozoa with abnormal morphology, particularly mid-piece
defects. As these two factors were excluded in our fertile
men with varicocele cases, the origin of excess ROS in
this group is suggested to be due to qualitative defect in
the voided spermatozoa. Another explanation could be
related to the association between the disturbances in the
concentration of metabolites by metabolomic profiling in
semen reported in varicocele associated conditions (Saleh
et al., 2003; Deepinder et al., 2007).
With the background of evidence-based medicine,
several controlled studies were performed on the effective-
ness of varicocele repair with controversial results (Mad-
gar et al., 1995; Hargreave, 1997; Nieschlag et al., 1998;
Heaton, 2006). Proving that OS in varicocele-associated
cases is present even in normozoospermic men may shed
a light on the impact of such a condition. Marmar (2001)
Table 1 Data of the studied groups (mean ± SD)
Gp1 (n = 45)
Controls
Gp2 (n = 45)
Fertile with varicocele
Gp3 (n = 44)
OA without varicocele
Gp4 (n = 42)
OA with varicocele
Age (years)
Sperm concentration (106per ml)
Total sperm motility (%)
Sperm abnormal forms (%)
MDA (nmol ml)1)
H2O2(lmol ml)1)
SOD (U ml)1)
Cat (lmol ml)1)
GPx (U ml)1)
Vit. E (lg dl)1)
Vit. C (mg dl)1)
30.3 ± 4.5
89.5 ± 41.3
69.5 ± 2.1
25.0 ± 8.8
5.2 ± 0.6
18.0 ± 3.7
22.1 ± 3.2
15.2 ± 2.1
5.4 ± 1.8
45.5 ± 5.0
0.50 ± 0.09
27.7 ± 6.7
59.8 ± 24.5a
66.0 ± 5 .0
35.7 ± 6.6a
12.5 ± 1.2a
37.0 ± 4.1a
14.9 ± 3.5a
8.6 ± 2.0a
4.1 ± 1.3a
32.3 ± 4.2a
0.24 ± 0.09a
34.8 ± 5.6
12.2 ± 11.1a,b
33.0 ± 11.7a,b
52.2 ± 11.1a,b
15.1 ± 2.1a
34.2 ± 4.2a
14.3 ± 2.9a
6.9 ± 2.1a
3.6 ± 1.4a,b
33.5 ± 33.1a
0.25 ± 0.04a
32.5 ± 6.7
8.0 ± 6.0a,b,c
23.3 ± 10.4a,b,c
68.6 ± 14.6a,b
23.5 ± 2.2a,b,c
40.7 ± 4.6a,c
6.1 ± 1.4a,b,c
5.7 ± 0.7a,b
1.1 ± 0.2a,b,c
31.5 ± 4.7a
0.3 ± 0.07a,b
aSignificant comparison Gr1 versus all groups (P < 0.05).
bSignificant comparison Gr2 versus all groups (P < 0.05).
cSignificant comparison Gr3 versus all groups (P < 0.05).
T. Mostafa et al.
Seminal ROS in varicocele
ª 2009 The Authors
Journal Compilation ª 2009 Blackwell Publishing Ltd Æ Andrologia 41, 125–129
127

Page 4

supported a ‘co-factor’ hypothesis that for varicocele to
be associated with infertility, they exist as ‘co-factors’
along with other molecular/genetic problems. Gallardo
(2007) showed that the measurement of the antioxidative
and oxidative agents could serve to evaluate human infer-
tility in those men in whom the result of the spemato-
bioscopy appears normal.
It is concluded that varicocele has an OS impact even
in fertile normozoospermic bearing conditions. It could
be understood that within men with varicocele, there is a
threshold value of OS over which male fertility may be
influenced.
References
Agarwal A, Makker K, Sharma R (2008) Clinical relevance
of oxidative stress in male factor infertility: an update.
Am J Reprod Immunol 59:2–11.
Allamaneni SS, Naughton CK, Sharma RK, Thomas AJ,
Agarwal A (2004) Increased seminal reactive oxygen species
levels in patients with varicoceles correlate with varicocele
grade but not with testis size. Fertil Steril 82:1684–1686.
Athayde KS, Cocuzza M, Agarwal A, Krajcir N, Lucon AM,
Srougi M, Hallak J (2007) Development of normal reference
values for seminal reactive oxygen species and their correla-
tion with leucocytes and semen parameters in a fertile popu-
lation. J Androl 28:613–620.
Cam K, Simsek F, Yuksel M, Turkeri L, Haklar G, Yalcin S,
Akdas A (2004) The role of reactive oxygen species and
apoptosis in the pathogenesis of varicocele in a rat model
and efficiency of vitamin E treatment. Int J Androl 27:
228–233.
Cozzolino DJ, Lipshultz LI (2001) Varicocele as a progressive
lesion: positive effect of varicocele repair. Hum Reprod
Update 7:55–58.
Deepinder F, Chowdary HT, Agarwal A (2007) Role of
metabolomic analysis of biomarkers in the management of
male infertility. Expert Rev Mol Diagn 7:351–358.
Desai ID (1984) Vitamin E analysis methods for animal
tissues. Method Enzymol 105:138–142.
Feigl F (1958) Spots in Inorganic Analysis, 5th edn. Elsevier,
Amsterdam, Netherlands.
Gallardo JM (2007) Evaluation of antioxidant system in
normal semen. Rev Invest Clin 59:42–47.
Hargreave TB (1997) Varicocele: overview and commentary
on the results of the WHO varicocele trial. In: Current
Advances in Andrology. Waites GMH, Frick J, Baker GUH
(eds). Monduzzi, Bologna, Italy, pp 31–44.
Heaton JP (2006) Varicocelectomy, evidence-based medicine
and fallibility. Eur Urology 49:217–219.
Hendin BN, Kolettis PN, Sharma RK, Thomas AJ Jr, Agarwal
A (1999) Varicocele is associated with elevated spermatozoal
reactive oxygen species production and diminished seminal
plasma antioxidant capacity. J Urol 16:1831–1834.
Jagota SK, Dani HM (1982) A new colorimetric technique for
the estimation of vitamin C using Folin phenol reagent.
Anal Biochem 127:178–182.
Kamtchouing P, Vermeulen L, Schoonjans F, Comhaire F
(1993) Comparison between cell motion analyser,
autosperm, and conventional semen analysis. Andrologia
25:345–350.
Ko ¨ksal IT, Tefekli A, Usta M, Erol H, Abbasoglu S, Kadioglu
A (2000) The role of reactive oxygen species in testicular
dysfunction associated with varicocele. BJU Int 86:549–552.
Ko ¨ksal IT, Ishak Y, Usta M, Danisman A, Guntekin E,
Bassorgun IC, Ciftcioglu A (2007) Varicocele-induced
testicular dysfunction may be associated with disruption of
blood-testis barrier. Arch Androl 53:43–48.
Levinger U, Gornish M, Gat Y, Bachar GN (2007) Is varicocele
prevalence increasing with age? Andrologia 39:77–80.
Madgar I, Weissenberg R, Lunenfeld B, Karasik A, Goldwasser
B (1995) Controlled trial of high spermatic vein ligation for
varoccocele in infertile men. Fertil Steril 63:120–124.
Mancini A, Meucci E, Milardi D, Giacchi E, Bianchi A,
Pantano AL, Mordente A, Martorana GE, de Marinis L
(2004) Seminal antioxidant capacity in pre- and
postoperative varicocele. J Androl 25:44–49.
Marmar JL (2001) The pathophysiology of varicoceles in the
light of current molecular and genetic information. Hum
Reprod Update 7:461–472.
Meucci E, Milardi D, Mordente A, Martorana GE, Giacchi E,
De Marinis L, Mancini A (2003) Total antioxidant capacity
in patients with varicoceles. Fertil Steril 79(Suppl 3):
1577–1583.
Mitropoulos D, Deliconstantinos G, Zervas A, Villiotou V,
Dimopoulos C, Stavrides J (1996) Nitric oxide synthase
and xanthine oxidase activities in the spermatic vein of
patients with varicocele: a potential role for nitric oxide
and peroxynitrite in sperm dysfunction. J Urol 156:1952–
1958.
Mostafa T, Anis TH, El-Nashar A, Imam H, Othman IA
(2001) Varicocelectomy reduces reactive oxygen species
levels and increases antioxidant activity of seminal plasma
from infertile men with varicocele. Int J Androl 24:261–265.
Mostafa T, Anis TH, Ghazi S, El-Nashar AR, Imam H, Osman
IA (2006a) Reactive oxygen species and antioxidants
relationship in the internal spermatic vein blood of infertile
men with varicocele. Asian J Androl 8:451–454.
Mostafa T, Tawadrous G, Roaia MMF, Amer MK, Kader RA,
Aziz A (2006b) Effect of smoking on seminal plasma ascor-
bic acid in infertile and fertile males. Andrologia 38:221–224.
Nakamura H, Kimura T, Nakajima A, Shimoya K, Takemura
M, Hashimoto K, Isaka S, Azuma C, Koyama M, Murata Y
(2002) Detection of oxidative stress in seminal plasma and
fractionated sperm from subfertile male patients. Eur J
Obstet Gynecol Reprod Biol 105:155–160.
Nieschlag E, Hertle L, Fischedick A, Abshagen K, Behre HM
(1998) Update on treatment of varicocele: counseling as
Seminal ROS in varicocele
T. Mostafa et al.
ª 2009 The Authors
128
Journal Compilation ª 2009 Blackwell Publishing Ltd Æ Andrologia 41, 125–129

Page 5

effective as occlusion of the vena spermatica. Hum Reprod
13:2147–2150.
Ozbek E, Turkoz Y, Gokdeniz R, Davarci M, Ozugurlu F
(2000) Increased nitric oxide production in the spermatic
vein of patients with varicocele. Eur Urol 37:172–175.
Paglia DE, Valentine WN (1967) Studies on the quantitative
and qualitative characterization of erythrocyte glutathione
peroxidase. J Lab Clin Med 70:158–169.
Pasqualotto FF, Sharma RK, Nelson DR, Thomas AJ, Agarwal
A (2000) Relationship between oxidative stress, semen
characteristics, and clinical diagnosis in men undergoing
infertility investigation. Fertil Steril 73:459–464.
Pasqualotto FF, Sharma RK, Kobayashi H, Nelson DR,
Thomas AJ Jr, Agarwal A (2001) Oxidative stress in
normospermic men undergoing infertility evaluation.
J Androl 22:316–322.
Pasqualotto FF, Lucon AM, de Goes PM, Sobreiro BP, Hallak
J, Pasqualotto EB, Arap S (2005) Semen profile, testicular
volume, and hormonal levels in infertile patients with
varicoceles compared with fertile men with and without
varicoceles. Fertil Steril 83:74–77.
Pasqualotto FF, Sundaram A, Sharma RK, Borges E, Pasqualot-
to EB, Agarwal A (2008) Semen quality and oxidative stress
scores in fertile and infertile patients with varicocele. Fertil
Steril 89:602–607.
Placer ZA, Cushman LL, Johnson BC (1966) Estimation
of product of lipid peroxidation (malonyl dialdehyde) in
biochemical systems. Anal Biochem 16:359–364.
Saleh RA, Agarwal A (2002) Oxidative stress and male
infertility: from research bench to clinical practice. J Androl
23:737–752.
Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK,
Meyer A, Nelson DR, Thomas AJ (2003) Negative effects of
increased sperm DNA damage in relation to seminal
oxidative stress in men with idiopathic and male factor
infertility. Fertil Steril 79(Suppl 3):1597–1605.
Shiraishi K, Naito K (2006) Generation of 4-hydroxy-2-nonenal
modified proteins in testes predicts improvement in sper-
matogenesis after varicocelectomy. Fertil Steril 86:233–235.
Sinha AK (1972) Colorimetric assay of catalase. Anal Biochem
47:389–394.
Smith R, Kaune H, Parodi D, Madariaga M, Rios R, Morales
I, Castro A (2006) Increased sperm DNA damage in patients
with varicocele: relationship with seminal oxidative stress.
Hum Reprod 21:986–993.
Taylor SL, Landen MP, Tappel AL (1976) Sensitive fluorimet-
ric method for tissue tocopherol analysis. Lipids 11:530–538.
Turner TT, Lysiak JJ (2008) Oxidative stress: a common factor
in testicular dysfunction. J Androl 29:488–498.
Wang X, Sharma RK, Sikka SC, Thomas AJ Jr, Falcone T,
Agarwal A (2003) Oxidative stress is associated with
increased apoptosis leading to spermatozoa DNA damage in
patients with male factor infertility. Fertil Steril 80:531–535.
Winterbourn CC, Hawkins RE, Brian M, Carrell RW (1975)
The estimation of red cell superoxide dismutase activity. Lab
Clin Med 85:337–341.
World Health Organization (1999) WHO Laboratory Manual
for the Examination of Human Semen and Sperm-Cervical
Mucus Interaction, 4th edn. Cambridge University Press,
Cambridge.
Zini A, Defreitas G, Freeman M, Hechter S, Jarvi K (2000)
Varicocele is associated with abnormal retention of
cytoplasmic droplets by human spermatozoa. Fertil Steril
74:461–464.
Zucchi A, Mearini L, Mearini E, Fioretti F, Bini V, Porena M
(2006) Varicocele and fertility: relationship between
testicular volume and seminal parameters before and after
treatment. J Androl 27:548–551.
T. Mostafa et al.
Seminal ROS in varicocele
ª 2009 The Authors
Journal Compilation ª 2009 Blackwell Publishing Ltd Æ Andrologia 41, 125–129
129