|
International Journal of Reproductive BioMedicine
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences of Yazd
ISSN: 1680-6433 EISSN: 2008-2177
Vol. 7, Num. 1, 2009, pp. 29-34
|
Iranian Journal of Reproductive Medicine Vol. 7, No. 1, Winter, 2009, pp. 29-34
Correlation of sperm morphology and oxidative stress in
infertile men
Sundararajan Venkatesh1,4
M.Pharm, Gurdeep Singh2 M.D., Narmada Prasad Gupta3 M.D.,
Rajeev Kumar3 M.D., Munusamy Deecaraman4 Ph.D., Rima Dada1
M.D., Ph.D.
1Laboratory for Molecular Reproduction and Genetics,
Department of Anatomy, AIIMS, New Delhi, India.
2Department of Pathology, Air Force Central Medical
Establishment, New Delhi, India.
3Department of Urology, AIIMS, New Delhi, India.
4Dr. MGR University, Maduravoyal, Chennai, India.
Corresponding Author: Rima Dada, Department of Anatomy, All India
Institute of Medical Sciences, New Delhi-110019, India. Tel: +91 11 16541416,
Fax: +91 11 16588663. E-mail: rima_dada@rediffmail.com
Received: 22 October 2008; accepted: 10 March
2009
Code Number: rm09006
Abstract
Background: Excess reactive oxygen species (ROS) in the semen is
believed to affect fertility in men. Morphologically abnormal sperms and their
relation to seminal oxidative stress in infertile and subfertile men are not
clear.
Objective: To correlate various sperm morphological defects with
seminal oxidative stress in infertile and subfertile men.
Materials and Methods: The study included 25 primary, 21 secondary
infertile men of idiopathic infertility and 15 fertile controls. Standard semen
analysis was performed according to WHO (1999) guidelines. Sperm inter-morphological
defects were evaluated in 100 sperms per sample by Giemsa staining. ROS in spermatozoa
was measured by the chemiluminescence assay.
Results: Significant difference in percent sperm amorphous head was
found between secondary infertile group and control men. The study showed a
significantly higher percent spermatozoa with residual cytoplasm between
primary [11.61 (6.6, 3.9)], secondary [7.49 (0.8, 13)] and fertile controls
[2.44 (0.8, 3.7)] similar to sperm count, percent sperm progressive motility,
and ROS levels. A non significant but strong positive correlation (r=0.3479, p=0.0884)
between percent cytoplasmic retained spermatozoa and ROS levels was observed in
the primary infertile group. However, no correlation between other sperm
morphological defects and oxidative stress was observed.
Conclusion: Sperm morphology was not found to be associated with
oxidative stress in the present study. However, retained cytoplasmic residues
in the sperm may be an important source of ROS in both primary and secondary
infertile men. These immature spermatozoa are believed to be associated with impaired
fertility.
Key words: Infertility, Oxidative stress, Spermatozoa, Reactive
Oxygen Species.
Introduction
Declining male reproductive health is a
major concern among the population of reproductive age (1).
Sperm counts are falling at an
alarming rate of 1% per annum for the last 10 years and concomitant with this is a decline in
percent of morphologically normal sperm. Though semen analysis is the first
diagnostic step routinely employed in the evaluation of the male infertility,
it fails to predict the exact cause behind impaired fertility.
However, sperm count and sperm motility
are the first and most important predictors of fertility potential rather than
sperm morphology. There are number of common causes of male infertility, which
includes gene mutations, aneuploides, varicocele, radiation, chemotherapy,
genital tract infections, and erectile dysfunction (2, 3) and azoospermia
factor (AZF) deletions (4).
In half of the male infertile patients, the
cause is not clear and hence such cases are diagnosed with idiopathic
infertility.
In male factors, sperm
morphological defects are rarely evaluated for in vivo and in vitro evaluation
of male infertility because of unavailability of universal methods, reliability
and predictability. Abnormal sperm morphology has been associated with
cytogenetic anomalies, reproductive toxicants, smoking etc (5, 6), but its role
in idiopathic infertility is not clear.
Though overall sperm
morphological defects could give a basic idea about the fertility status, sperm
inter-morphological defects may provide some additional information about the
idiopathic cases.
Moreover, idiopathic
infertile cases are blindly treated and selected for assisted reproductive
techniques without understanding the basic mechanism behind the fertility impairment.
Since spermatogenesis is a complex process involving various stages in the
formation of mature spermatozoa, disruption at any stage would result in morphologically
abnormal spermatozoa, which have been associated with fertilization failure,
poor embryo cleavage and increased rate of abortions (7, 8). Recently oxidative
stress (OS) has been considered as one of the major factors believed to be
involved in idiopathic male infertility. Low levels of ROS are necessary for
normal functions of spermatozoa like capacitation, hyperactivation, motility,
acrosome reaction, oocyte fusion and fertilization (9, 10). OS is a condition
where the production of ROS overwhelms antioxidant levels (11).
Though various sperm
morphological defects are routinely evaluated as a part of semen analysis, its
correlation with seminal oxidative stress in different infertile population is
not clear. For the past two decades the pathological role of ROS in the semen
has been studied but not well established because of various possible sources
associated with excess production of ROS including abnormal spermatozoa. Several
studies (12, 13)reported sperm morphology as the best predictor,
whereas another study reported it as a poor predictor (14) of male infertility.
Though studies have
been reported an association between increased ROS production and overall
abnormal sperm morphology (15, 16),
the role of specific inter-morphological defects in association with oxidative
stress is unclear. Moreover, it is also
important to understand if there are any morphological defects involved in
differentiating infertile and subfertile men. So, the present study was aimed
to correlate various sperm morphological defects and seminal oxidative stress in
idiopathic infertile and subfertile men.
Materials
and methods
Study population
The study included
25 primary infertile (PI) and 21 secondary infertile (SI) patients. Primary
infertile patients were those unable to conceive their partner with normal
female factor. Secondary infertile cases were those experienced at least one
spontaneous abortion with normal female partner.
15 fertile men who have
fathered in the past 2 years have been included as controls. Patients with
varicocele, hypogonadism, obstructive and non-obstructive azoospermia,
cytogenetic abnormalities, history of smoking, alcohol, recent drug intake,
prolonged illness and exposure to reproductive toxicants were excluded from the
study. The study was approved by the ethical committee of All India Institute
of Medical Sciences (AIIMS). Subjects were enrolled in the study after
obtaining informed consent.
Semen analysis
All the participants
were asked to observe sexual abstinence for 3-5 days before collection of semen.
Samples were collected in a sterile plastic container and delivered to the
laboratory before 30 minutes had elapsed. Semen was incubated at room
temperature and standard semen analysis was performed according to WHO (1999)
guidelines (17).
For sperm morphology study, 10µl of liquefied ejaculate was placed on the slide and a
smear was made using a cover slip. The smear was dried in air and fixed by 90%
ethanol. The slide was dipped in the Giemsa stain for 3 5 minutes and washed
under running tap water and then dried in air.
Classification of
spermatozoa morphological defects
Sperm inter-morphological
defects were evaluated in at least 100 spermatozoa per sample and defects expressed
as a percentage. Six abnormalities of the head (large head, small head,
pyriform head, pointed head, double head and amorphous head), two abnormalities
of the mid piece (cytoplasmic droplets and bent neck) and four abnormalities of
the tail (coiled tail, bent tail, broken tail and double tail) were evaluated
in each group of men.
Measurement of ROS by
chemiluminescence assay (18)
Fresh
liquefied semen was centrifuged at 300 x g for 7 minutes and the pellet was
washed with phosphate- buffered saline (PBS- pH 7.4). The washed pellet was
resuspended in the same washing media at a concentration of 20 x 106
sperm/ml.
Ten
microliters of 5M luminol (5-amino-2,3,-dihydro-1,4-phthalazinedione; Sigma),
prepared in dimethyl sulfoxide (DMSO), was added to the mixture and served as a
probe.
A
negative control was prepared by adding 10 µL of 5 mM luminol to 400 µL of PBS.
Levels of ROS were assessed by measuring the luminol-dependant
chemiluminescence with the luminometer (Sirius, Berthold) in the integrated
mode for 15 minutes. The results were expressed as 106 counted photons
per minute (cpm) / 20 x 106 sperm.
Statistical
analysis
Sperm parameters, percent
inter-morphological defects and ROS values between the groups were expressed as
the median (minimum range, maximum range).
The significant
difference of sperm parameters between the groups was calculated using a Newmann
Kuels test.
The overall
significance between all the groups in terms of percentage of
inter-morphological defects was calculated by using a Kruskal-wallis test,
where the significance between any of the two groups was calculated by a Newmann
Kuels test. Correlation between the parameters was found using Spearman's
correlation co-efficient method.
The statistical
analysis was performed using Stata 9.0 version software. In all the cases
p<0.01 was considered as significant unless otherwise stated.
Results
Sperm
count, percent progressive sperm motility and ROS levels of all the three
groups are shown in the table I. Median sperm count was found to be
significantly (p<0.001) lower in PI group [15.9 (3.8, 61.8)] compared to SI
[57.4 (18, 89)] and control groups [60.2 (45.3, 91.6)]. Similarly, percent
progressive sperm motility was also found to have significantly (p<0.001)
lower in PI group when compared to SI and control groups as shown in the table I.
Table I. Seminal parameters of
infertile and control groups.
Group |
Sperm count
( x 106/ ml) |
Progressive motility (%) |
ROS
(106 cpm)
|
Primary
infertile
|
15.9
(3.8, 61.8) *
|
13.5
(3.5, 42) *
|
20.3
(6.89, 35.9) *
|
Secondary
infertile
|
57.4
(18, 89) *
|
55
(20, 90) *
|
3.4
(0.74. 11.2) *
|
Control |
60.2
(45.3, 91.6)
|
73.6
(44.6, 78.5)
|
0.164 (0.15, 1.166) |
cpm- counted photons per
minute, *p < 0.001 is considered as significant compared to controls.
And
also significant difference was found in all the above parameters between PI
and SI group.
However, median (minimum, maximum) range ROS levels in the
semen expressed as 106 counted photons per minute/20 million
spermatozoa was found to be significantly higher in PI group [20.3 (6.89,
35.9)] compared to SI [3.4 (0.74. 11.2)] and controls [0.164 (0.15,1.166)].
Median ROS levels in
the semen of PI and SI group was found to be approximately 124 and 20 times
respectively higher than the control groups.
Among the sperm inter
morphological defects, percent sperm with cytoplasmic droplets (CyD) was found
to be significantly higher in PI group followed by SI and controls which may be
responsible for corresponding seminal ROS levels in their respective groups.
However, no other defects
were found to differ significantly except percent amorphous head in SI group
which was found to be significantly higher compared to control group (Table II).
Progressive
sperm motility showed a strong non significant (p=0.0235) negative correlation
with ROS levels in PI group. Among the other parameters percent sperm CyD
showed strong positive correlation (r=0.3479, p=0.0884) with ROS levels similar
to percent sperm pyriform head (PyH) (r=0.4282, p=0.0327) and sperm broken tail
(BrT) (r=0.3699, p=0.0687).
However
none of the parameters in SI group showed any correlation with ROS levels.
Table
II. Comparison of sperm
inter-morphological defects between PI, SI, and control groups.
|
PI |
SI |
Control |
Head (%)
|
|
|
|
Large head (LH)
|
2.78 (0, 9.5)
|
2.17 (0, 5.8)
|
3.5 (0.6, 4.9)
|
Small head (SH)
|
4.26 (0, 11.8)
|
4.07 (0,11.8)
|
1.86 (0, 4.9)
|
Pyriform head (PyH)
|
2.37 (0, 5.7)
|
2.25 (0, 4.6)
|
2.72 (1.1, 4.3)
|
Pointed head (PnH)
|
6.59 (0.9,15.7)
|
6.67(0.9,12.5)
|
3.64 (1.1,4.6)
|
Amorphous head (AH)
|
2.9 (0, 4.5)
|
3.56 (0, 4.5)b
|
1.86 (0.3, 3.4)
|
Double head (DH)
|
0.74 (0, 4.5)
|
0.28 (0, 1)
|
0.37 (0, 1.4)
|
Round head (RH) |
0.92 (0, 4.8)
|
1.73 (0, 6)
|
1.46 (0, 6)
|
Mid piece (%)
|
|
|
|
Cytoplasmic droplets (CyD)
|
11.61(6.6,3.9)a,c
|
7.49(0.8,13)d
|
2.44 (0.8, 3.7)
|
Bent neck (BN)
|
3.96 (0, 12.5)
|
3.39 (0, 6.5)
|
4.82 (0, 9.2)
|
Tail (%)
|
|
|
|
Broken tail (BrT)
|
3.6 (0, 7.4)
|
3.57 (0, 6)
|
1.86 (0, 6)
|
Bent tail
(BnT)
|
5.52 (0.9, 10.8)
|
5.24 (2.7, 8)
|
3.8 (0, 8.2)
|
Coiled tail
(CT)
|
3.83 (0, 8.8)
|
3.19 (0, 6.1)
|
2.29 (0.8, 6)
|
Double tail (DT)
|
0.47 (0,1.9)
|
0.50 (0, 1.8)
|
0.21 (0, 0.8)
|
PI- Primary infertile and SI- Secondary
infertile group, Values are expressed as median (minimum, maximum) range,
a&d p<0.0001 vs. control, b p<0.001 vs.
control, c p<0.01 vs. control by two-sample Wilcoxon rank-sum
(Mann-Whitney) test.
Discussion
Oxidative stress in the semen has been
found to be elevated in infertile men as reported in many studies (19, 20).
Since OS is greatly associated with idiopathic infertile men, the exact source
of ROS in the semen is still unknown. Though the sperm count and percent sperm motility
are the most accessed sperm parameters during infertility evaluation, sperm morphology
is rarely considered. It is not clearly known whether morphological defects are
linked to ROS production, whereas as other contaminants in the semen like
leukocytes, bacteria and immature germ cells produce high ROS levels (2).
Moreover, single sperm may have variousdeformities
that may be necessary to evaluate instead classifying as once. Though several
studies have reported the association between abnormal sperm morphology and ROS,
it has not been extensively used in the diagnostic evaluation of male
infertility. Sperm deformity index (SDI) has been used to evaluate infertile
group and it has been found to distinguish the semen sample with impaired
fertility (21). Therefore, we evaluated detailed morphological defects to find
out if they have any role in ROS production.
Sperm with retained cytoplasmic residues
were found to be significantly different between the groups similar to their ROS
levels. These results are in support of earlier studies showing that abnormal
morphology is highly associated with the production of ROS in idiopathic
infertile men (22). Moreover, superoxide anion is believed to be the primary
free radical produced by the immature spermatozoa (23). However, activation
of the NADPH system mediated by abundant glucose 6-phosphate dehydrogenase in
the retained cytoplasmic residue of sperm may be involved in the production of
ROS (22). Same study reported that ROS production of NADPH has been greatly
increased in immature spermatozoa (22). But the role of mitochondria in ROS production
cannot be omitted as genomic mutation or alteration in mitochondria may increase
ROS production through a vicious cycle (24).
Moreover, abnormal sperm morphology has
been reported to be associated with high sperm DNA fragmentation in infertile
men (25-27). In our subfertile group, though they have normal sperm parameters,
difference in the percent cytoplasmic residues and ROS levels with the PI and
control group could give the better understanding in the impaired fertility mechanism.
This impaired fertility may be due to production of ROS by the immature cytoplasm
retained spermatozoa. Though the percent amorphous head in SI group was
significantly higher compared to control, the mechanism is not clear. The
capacity of spermatozoa to produce ROS has also been inversely depends on
maturational stage (28, 29).
In the present study, no differences
have seen in the inter-morphological defects between the PI and SI groups
revealed the absence of their role in the ROS production.
Large numbers of studies with large
samples are warranted in future for better understanding in this aspect. Though
30% normal sperm morphology is the criteria for normal fertile men (17), the
effect of ROS production by abnormal sperm on normal sperm parameters/DNA
integrity is not well studied.
Moreover, increased poor sperm morphology
has been reported to reduce successful fertilization rates with increased miscarriages even after successful embryo
transfer (30). Spermatozoa exposed to ROS for a short period may have less DNA
and membrane damage than exposed for a long duration.
Therefore,
subfertile population may have less DNA damage, hence these DNA damage may be
reversed by neutralizing free radicals like ROS with suitable antioxidant
supplements unlike in PI men, who may have high sperm DNA damage due to prolonged
presence of OS in semen. Hence SI cases in our study have been found to contain
significantly less percent of cytoplsamic retained sperm and ROS levels which
could be used to distinguish them from PI men. Treatment with antioxidants has
also been reported significant improvement in sperm count, motility and
morphology (31).
Therefore, infertile
men identified with OS and increased cytoplasmic retained sperm can yet benefit
from AO therapy treatment.
A study also reported
a significant reduction in abnormal sperm population after glutathione
treatment (32). However at the testicular level the elevated ROS may damage the
sperm membrane that would result in the altered morphology, which has to be
studied in detail.
In
conclusion, various sperm morphological defects were not associated with
oxidative stress in both PI and SI group.
However,
the retained cytoplasmic residues in the sperm may have significant role in the
production of ROS and fertility impairment. However, proper work up of such men
for both OS and detailed sperm morphological evaluation rather either alone
could be useful in treating SI patients with antioxidants to improve their fertilization
rate. Because of highly elevated ROS levels and percent cytoplasmic residues in
PI men compared to the other groups, antioxidant treatment must be evaluated in
detail in the form of clinical trials for the successful pregnancy.
Also
ROS at elevated levels have been reported to cause DNA damage, which is one of
the suspected causes for poor ART outcome including embryonic cleavage,
fragmentation and post implantation loss. Since intra cytoplasmic sperm
injection (ICSI) involves the selection of single sperm for fertilization,
sperm morphology assessment plays a vital role in sperm selection. Since ROS
has also been reported to affect ART, these cytoplasmic residue sperm can be
separated to minimize the OS during gamete preparation for ART.
Since
cytoplasmic residues has been suspected to increase ROS production, higher
sampled studies are required to find their correlation in impaired male fertility.
References
- Carlsen E, Giwercman A, Keiding N, Skakkebaek NE.
Evidence for decreasing quality of semen during past 50 years. BMJ 1992;
305: 609-613.
- Ollero M, Gil-Guzman E, Lopez MC, Sharma RK, Agarwal
A, Larson K, et al. Characterization of subsets of human spermatozoa at
different stages of maturation: implications in the diagnosis and treatment of
male infertility. Hum Reprod 2001; 16: 1912-1921.
- Tremellen K. Oxidative
stress and male infertility--a clinical perspective. Hum Reprod Update 2008; 14:243-258.
- Dada R, Kumar R, Sharma
RK, Gupta NP, Gupta SK. et al. AZF
deletion in varicocele cases with oligospermia. IJMS 2007; 61:505-510.
- Ushijima C, Kumasako
Y, Kihaile PE, Hirotsuru K, Utsunomiya T. Analysis of chromosomal abnormalities
in human spermatozoa using multi-colour fluorescence in-situ hybridization. Hum
Reprod 2000; 15:1107-1111.
- Meeker JD, Rossano MG,
Protas B, Diamond MP, Puscheck E, Daly D, et al. Cadmium, Lead, and Other
Metals in Relation to Semen Quality: Human Evidence for Molybdenum as a Male
Reproductive Toxicant 2008;116:1473-1479.
- Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta
JF, Oehninger S. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 1988; 49:112-117.
- Oehninger S, Acosta AA, Morshedi M, Veeck L, Swanson
RJ, Simmons K,et al. Corrective measures and pregnancy outcome in in
vitro fertilization in patients with severe sperm morphology abnormalities. Fertil
Steril 1988; 50:283-287.
- Griveau JF, Le Lannou D. Reactive oxygen species and
human spermatozoa: Physiology and pathology. Int J Androl 1997; 20:61-69.
- Agarwal A, Nallela KP, Allamaneni SSR, Said TM. Role of
antioxidants in treatment of male infertility: an overview of the literature. Reprod
Biomed online 2004;8:616-627
- Venkatesh S, Deecaraman M, Kumar R, Shamsi MB, Dada R.
Reactive oxygen species and its role in the pathogenesis of mitochondrial DNA
(mtDNA) mutations in male infertility. Ind J Med Res 2009. In Press
- Chia SE, Tay SK, Lim ST. What constitutes a normal
seminal analysis? Semen parameters of 243 fertile men. Hum Reprod 1998; 13:3394-3398.
- Guzick DS, Overstreet JW, Factor-Litvak P, Brazil CK,
Nakajima ST, Coutifaris C,et al. Sperm morphology, motility, and
concentration in fertile and infertile men. N Engl J Med 2001; 345:1388-1393.
- Nallella KP, Sharma RK, Aziz N, Agarwal A. Significance
of sperm characteristics in the evaluation of male infertility. Fertil
Steril 2006; 85:629-634.
- Aziz N, Saleh RA, Sharma RK, Lewis-Jones I,
Esfandiari N, Thomas AJ Jr, et al.Novel association
between sperm reactive oxygen species production, sperm morphological defects,
and the sperm deformity index. Fertil Steril 2004; 81:349-354.
- Moein MR, Dehghani VO, Tabibnejad N, Vahidi
S. Reactive Oxygen Species (ROS) level in seminal plasma of infertile men and
healthy donors. Iranian Journal of Reproductive Medicine 2007; 5:51-55.
- WHO Laboratory Manual for the Examination of Human
Semen and Semen-Cervical Mucus Interaction. 4 ed. Cambridge, UK: Cambridge University Press, 1999.
- Athayde KS, Cocuzza M, Agarwal A, Krajcir N, Lucon AM,
Srougi M,et al. Development of normal reference values for seminal
reactive oxygen species and their correlation with leukocytes and semen parameters
in a fertile population. J Androl 2007; 28:613-620.
- Venkatesh S, Riaz AM, Kumar M, Shamsi MB, Tanwar M, Kumar R, et al. OXIDATIVE STRESS - A Marker of Male infertility. Obs
and Gyn today 2009; 14:34-36.
- Zarghami N, Khosrowbeygi
A. Evaluation of Lipid Peroxidation as an Indirect Measure of Oxidative Stress
in Seminal Plasma. Iranian Journal of Reproductive Medicine 2004; 2:34-39.
- Said TM, Aziz N, Sharma RK, Lewis-Jones I,
Thomas Jr AJ, Agarwal A. Novel association between sperm deformity index and
oxidative stress-induced DNA damage in infertile male patients. Asian J
Androl 2005; 7:121126.
- Said TM, Agarwal A, Sharma RK, Thomas AJ Jr., Sikka SC.
Impact of sperm morphology on DNA damage caused by oxidative stress induced by
beta-nicotinamide adenine dinucleotide phosphate. Fertil Steril 2005; 83:95-103.
- Alvarez J, Touchstone J, Blasco L, Storey B.
Spontaneous lipid peroxidation and production of hydrogen peroxide and
superoxide in human spermatozoa. Superoxide dismutase as major enzyme
protectant against oxygen toxicity. J Androl 1987; 8: 338348.
- Andreyev AYu, Kushnareva YuE, Starkov AA.
Mitochondrial Metabolism of Reactive Oxygen Species. Biochemistry (Moscow) 2005; 70:200-214.
- Nicopoullos JD, Gilling-Smith C, Almeida PA, Homa S,
Norman-Taylor JQ, Ramsay JW. Sperm DNA fragmentation in subfertile men: the
effect on the outcome of intracytoplasmic sperm injection and correlation with
sperm variables. BJU Int 2008; 101:1553-1560.
- Huang CC, Lin DP, Tsao HM, Cheng TC, Liu CH, Lee MS.
Sperm DNA fragmentation negatively correlates with velocity and fertilization
rates but might not affect pregnancy rates. Fertil Steril 2005; 84:130-140.
- Muratori M, Piomboni P, Baldi E, Filimberti
E, Pecchioli P, Moretti E, et al.Functional and Ultrastructural
Features of DNA-Fragmented Human Sperm. Journal of Androl 2000; 21:903-912.
- Fisher H, Aitken R.
Comparative analysis of the ability of precursor germ cells and epididymal
spermatozoa to generate reactive oxygen metabolites. J Exp Zool 1997; 277:390400.
- Gil-Guzman E, Ollero M,
Lopez M, Sharma R, Alvarez J, Thomas AJ, et al. Differential production of reactive oxygen species by
subsets of human spermatozoa at different stages of maturation. Hum Reprod 2001; 16:19221930.
- Kobayashi T, Jinno M, Sugimura K,
Nozawa S, Sugiyama T, Iida E. Sperm morphological assessment based on strict
criteria and in-vitro fertilization outcome. Hum Reprod 1991; 6:983-986.
- Agarwal A,
Nallella KP, Allamaneni SS, Said TM. Role of antioxidants in treatment of male
infertility: an overview of the literature. Reprod Biomed Online 2004; 8:616627.
- Lenzi A, Culasso F,
Gandini L, Lombardo F , Dondero F. Placebocontrolled, double-blind, cross-over
trial of glutathione therapy in male infertility. Human Reprod 1993; 8: 16571662.
© Copyright 2009 - Iranian Journal of Reproductive Medicine
|