The Incriminating Role of Reactive Oxygen Species in Idiopathic Male Infertility: An Evidence Based Evaluation
The male factor is considered a major contributory factor to infertility. Apart from the conventional causes for male infertility such as varicocoele, cryptorchidism, infections, obstructive lesions, cystic fibrosis, trauma and tumours, a new and important cause has been identified as being responsible for the so-called idiopathic male infertility: oxidative stress. Oxidative Stress (OS) is a condition that occurs when the production of Reactive Oxygen Species (ROS) overwhelms the antioxidant defense produced against them. In male reproductive pathological conditions, the OS significantly impairs spermatogenesis and sperm function, which may lead to male infertility. Reactive Oxygen Species (ROS) known as free radicals are oxidizing agents generated as a result of metabolism of oxygen and have at least one unpaired electron that make them very reactive species. Spermatozoa generate Reactive Oxygen Species (ROS) in physiological amounts, which play a role in sperm functions during sperm capacitation, Acrosome Reaction (AR) and oocyte fusion, but they need to be controlled and their concentrations maintained at a level that is not deleterious to the cells. Administration of antioxidants in patients with male factor infertility has begun to attract considerable interest. The main difficulty of such an approach is our incomplete understanding of the role of free radicals in normal and abnormal sperm function leading to male infertility. The purpose of the present review is to address the relationship between ROS and idiopathic male factor infertility.
Received: January 28, 2010;
Accepted: March 07, 2010;
Published: June 14, 2010
Since the first appearance of humans on earth, infertility has been one of
the most controversial medical and social issues. Infertility is the inability
of a sexually active, non-contracepting couple to achieve pregnancy in one year
(World Health Organization, 2000). Infertility is a major
clinical problem, affecting people medically and psychosocially. Infertility
affects approximately 15% of all couples trying to conceive. Male causes for
infertility are found in 50% of involuntarily childless couples (Rowe,
2006). Reduced male fertility can be the result of congenital and acquired
urogenital abnormalities, infections of the genital tract, increased scrotal
temperature (varicocele), endocrine disturbances, genetic abnormalities and
immunological factors (World Health Organization, 2000).
No causal factor is found in 60-75% of cases (idiopathic male infertility).
Infertility is the main complication arising in male patients with high levels
of OS, apoptosis or sperm DNA damage. Although techniques like Intracytoplasmic
Sperm Injection (ICSI) offer considerable promise to such male factor patients,
the indiscriminate use of such assisted fertility treatments, especially when
the etiology of sperm dysfunction is poorly understood is not warranted. Idiopathic
male factor infertility has been linked with oxidative stress by several research
groups (Said et al., 2004; Kao
et al., 2007; Joffe, 2010). Oxidative stress
originates from the excessive generation of Reactive Oxygen Species (ROS) by
the spermatozoa and results in the peroxidation of unsaturated fatty acids in
the sperm plasma membrane. Oxidative stress is thought to contribute to the
development of a wide range of diseases including Alzheimers disease (Christen,
2000; Nunomura et al., 2006), Parkinsons
disease (Wood-Kaczmar et al., 2006), the pathologies
caused by diabetes (Davi et al., 2005), rheumatoid
arthritis (Hitchon, 2004), neurodegeneration in motor
neurone disease (Cookson and Shaw, 1999; Rao
and Balachandran, 2002), cancer (Valko et al.,
2004, 2007; Nakabeppu et
al., 2006), obesity (Vincent, 2007; Gomez-Cabera,
2008) and cataract (Berthood and Beyer, 2009).
Several forms of sperm DNA damage are caused by ROS, e.g., chromatin cross-linking,
chromosome deletion, DNA strand breaks and base oxidation. Moreover, ROS are
important in mediating apoptosis by inducing cytochrome c and caspases 9 and
3, which in turn result in a high frequency of single- and doublestranded DNA
strand breaks (Said et al., 2004). Human spermatozoa
exhibit a capacity to generate ROS and initiate peroxidation of the unsaturated
fatty acids in the sperm plasma membrane, which plays a key role in the etiology
of male infertility. The short half-life and limited diffusion of these molecules
is consistent with their physiologic role in key biological events such as acrosome
reaction and hyperactivation. The intrinsic reactivity of these metabolites
in peroxidative damage induced by ROS, particularly H2O2
and the superoxide anion, has been proposed as a major cause of defective sperm
function in cases of male infertility. Understanding of how such conditions
affect sperm function will help in designing new and effective treatment strategies.
Men with idiopathic infertility generally present with significantly higher
seminal ROS levels and lower antioxidant properties than healthy controls (Pasqualotto
et al., 2001). Therefore, it appears that the presence of OS in infertile
normozoospermic men may be the cause behind previously unexplained cases of
infertility. Similarly, sperm DNA damage analysis may reveal hidden sperm DNA
abnormalities in infertile men with normal standard sperm values who were diagnosed
with idiopathic infertility. The increase in sperm DNA damage in these patients
may be partly related to high levels of seminal OS. Finally and importantly,
some conditions may pass unnoticed but still affect the sperm genomic integrity.
Free radicals: origin and oxidative stress:
By definition, a free radical is any chemical compound with one or more
unpaired electrons. The assumption that free radicals can influence male fertility
has received substantial scientific support (Saalu et
al., 2009a, b, 2010).
The free radicals that have been associated with infertility are oxygen and
oxygen-derived oxidants, namely, the superoxide anion (O2¯),
hydrogen peroxide (H2O2), peroxyl radicals (ROO-) and
hydroxyl radicals (OH¯) (Agarwal et al., 2005).
These oxidants are widely known as Reactive Oxygen Species (ROS) and, due to
unpaired electron(s) tend to strongly react with other chemical compounds (Attaran
et al., 2000). The nitrogen derived free radical Nitric Oxide (NO.)
and peroxynitrite anion (ONOO-) also appear to play a significant role in the
reproduction and fertilization. The ultimate effects of (NO.) depend upon its
concentration and interactions with hydrogen peroxide. Peroxynitrite (oxoperoxonitrate)
anion may be formed in vivo from superoxide and nitric oxide and actively
reacts with glutathione, cysteine, deoxyribose and other thiols/thioethers (Koppenol
et al., 1992). This can form a strongly nitrating species in the
presence of metal ions or complexes.
Free radicals seek to participate in chemical reactions that relieve them of
their unpaired electron, resulting in the oxidation of lipids in membranes,
amino acids in proteins and carbohydrates within nucleic acids (Ochsendorf,
1999). The terms free radical and ROS are commonly used in an interchangeable
manner, despite the fact that not all ROS are free radicals (Cheeseman
and Slater, 1993). An expanding body of evidence now supports a role for
oxidative stress as a significant cause of male infertility. However, despite
being a common pathology in infertile men, oxidative stress is ignored by many
infertility practitioners. The currently popular response of resorting to mechanical
techniques such as IVF-ICSI in all cases of male factor infertility is unlikely
to be best practice since ROS damaged paternal DNA will result in
poor quality blastocysts, less than optimal pregnancy rates and an increase
in miscarriage. At present there are over 30 assays of oxidative stress (Ochsendorf,
1999), broadly divided into three different types (Direct methods, Indirect
methods and routine semen analysis). In many complex biological systems including
semen, the true ROS status leading to oxidative stress reflects a relative balance
between the ROS-generated and ROS-scavenged. The measurement of the rate of
ROS generation by luminal induced chemiluminescence has been the most common
method for quantitating ROS. Although this rate measurement is dynamic, it may
not accurately reflect the status of potential sperm damaging ROS. For such
evaluations, the amount of ROS-detected, rather than the ROS-generated will
represent a more physiological assessment of oxidative stress (Iwasaki
and Gagnon, 1992).
Sources of ROS:
There are several sources of reactive oxygen species in the human body.
Production of superoxide in mitochondria is a by-product of the function of
the respiratory chain (Balaban et al., 2005).
The first known example of regulated generation of Reactive Oxygen Species (ROS)
in mammalian cells was through the respiratory burst of phagocytic cells by
Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase. This enzyme complex
uses electrons derived from intracellular NADPH to generate superoxide anion,
which is further processed to form hydrogen peroxide and other ROS-providing
host defense against bacterial and fungal pathogens (Quinn
and Gauss, 2004). The essential role of the phagocytic oxidase in host defense
is well illustrated by the serious phenotype of Chronic Granulomatous Disease
(CGD), in which susceptibility to infections develops in the absence of a functional
phagocytic oxidase (Geiszt et al., 2001). Virtually
every human ejaculate is considered to be contaminated with potential sources
of ROS as human semen is known to contain different types of cells, such as
mature and immature spermatozoa, round cells from different stages of spermatogenesis,
leukocytes and epithelial cells. Of these different cell types, leukocytes and
spermatozoa have been shown to be the two main sources of ROS (Garrido
et al., 2004).
Physiological role of ROS:
Low levels of ROS have been shown to be essential for fertilization, acrosome
reaction, hyperactivation, motility and capacitation (Griveau
and Le Lannou, 1997; Agarwal et al., 2004).
Capacitation has been shown to occur in the female genital tract, a process
carried out to prepare the spermatozoa for interaction with the oocyte. During
this process, the levels of intracellular calcium, ROS and tyrosine kinase all
increase, leading to an increase in cyclic adenosine monophosphate (cAMP). This
increase in cAMP facilitates hyperactivation of spermatozoa, a condition in
which they are highly motile (Aitken, 1995). However,
only capacitated spermatozoa exhibit hyperactivated motility and undergo a physiological
acrosome reaction, thereby acquiring the ability to fertilize (De
Lamirande et al., 1997).
Consequences of excessive generation of ROS:
Excessive ROS production by immature, morphologically abnormal spermatozoa
with cytoplasmicresidues such as those confronted in teratozoospermic semen
specimens may induce oxidative damage of mature spermatozoa during sperm migration
from the seminiferous tubules to the epididymis and may be an important cause
of male infertility (Sikka, 2004). ROS is involved in
many physiological functions of human spermatozoa, their excess production in
semen especially during leukocytospermia can overwhelm the antioxidant defense
mechanisms of spermatozoa and seminal plasma resulting in oxidative stress.
Spermatozoa are also particularly susceptible to the damage induced by excessive
ROS because their plasma membranes contain large quantities of polyunsaturated
fatty acids (PUFA), which readily experience lipid peroxidation by ROS, resulting
in a loss of membrane integrity (Halliwell, 1990; Buettner,
Oxidative stress and apoptosis:
Apoptosis is a non-inflammatory response to tissue damage characterized
by a series of morphological and biochemical changes (Wyllie
et al., 1980; Sinha-Hikim and Swerdloff, 1999;
Sakkas et al., 1999a, b,
2002; Shen et al., 2002;
Said et al., 2004; Grunewald
et al., 2005). Apoptosis during spermatogenesis has been assessed,
discussed and supported in several studies (Hikim et
al., 1998; Print and Loveland, 2000) and has
been associated with male infertility (Lin et al.,
1997; Hikim et al., 1998; Jurisicova
et al., 1999).
Apoptosis appears to be strictly regulated by extrinsic and intrinsic factors
and can be triggered by a wide variety of stimuli. Apoptosis in sperm may also
be initiated by ROS-independent pathways involving the cell surface receptor
called as Fas or CD 95. Fas is a type I membrane protein that mediates apoptosis.
When Fas ligand or anti-Fas antibody binds to Fas, apoptosis occurs (Lee
et al., 1999). Moustafa et al. (2004)
determined that infertile patients had high ROS levels in their seminal plasma
and higher percentage of apoptosis than normal healthy donors. When the molecular
framework of apoptosis is identified, specific apoptotic inhibitors may have
a role in promoting germ-cell survival.
Lipid peroxidation (LPO) is the most extensively studied manifestation of
oxygen activation in biology. LPO is broadly defined as oxidative deterioration
of PUFA which are fatty acids that contain more than two carbon double bonds
(Halliwell, 1990). Most membrane PUFA contain unconjugated
double bonds that are separated by methylene groups. The presence of a double
bond adjacent to a methylene group makes the methylene carbon-hydrogen bond
weaker and as a result, the hydrogen is more susceptible to abstraction. Once
this abstraction has occurred, the radical produced is stabilized by the rearrangement
of double bonds. The PUFA rearranges to form a conjugated diene radical that
subsequently can be oxidized (Aitken et al., 1989,
1993; Aitken and Fisher, 1994;
Aitken, 1995; Alvarez and Storey,
1995; Griveau et al., 1995; Kodama
et al., 1996; Ochsendorf, 1999). The most
common types of LPO are: (1) nonenzymatic membrane LPO and (2) enzymatic (NADPH
and ADP dependent) LPO. The enzymatic reaction involves NADPH cytochrome P-450
reductase and proceeds via an ADP-Fe3+O2¯ (perferryl)
complex (Ernster, 1993; Oborna et
al., 2010). Mammalian spermatozoa membranes are very sensitive to free
radical induced damage mediated by lipid peroxidation, as they are rich in polyunsaturated
fatty acids. In spermatozoa, production of malondialdehyde (MDA), an end product
of LPO induced by ferrous ion promoters, has been reported by Darley-Usmar
et al. (1995). Formation of MDA can be assayed by the thiobarbituric
acid (TBA) reaction which is a simple and useful diagnostic tool for the measurement
of LPO for in vitro and in vivo systems (Taourel
et al., 1992). Lipid peroxidation caused by low levels of ROS leads
to modification of the plasma membrane, thus facilitating sperm-oocyte adhesion
(Kodama et al., 1996).
ROS and motility:
Sperm count and sperm motility are the first and most important predictors
of fertility potential.
The underlying pathology behind free radicals ability to reduce sperm motility
was first reported by Jones et al.(1979).
They reported that ROS-induced peroxidation of the sperm membrane decreasing
its flexibility and therefore tail motion. Sperm membranes are vulnerable to
this type of damage as they contain large amounts of unsaturated fatty acids.
Direct ROS damage to mitochondria, decreasing energy availability, may also
impede sperm motility (Lamirande and Gagnon, 1992; De
Lamirande et al., 1997, 1998). The link between
ROS and reduced motility may be due to a cascade of events that results in a
decrease in axonemal protein phosphorylation and sperm immobilization, both
of which are associated with a reduction in membrane fluidity that is necessary
for sperm oocyte fusion (De Lamirande and Gagnon, 1995).
The mechanism behind this effect is ROS induced lipid peroxidation of sperm
plasma membrane (Aitken and Baker, 2006), which affects
membrane fluidity and mobility. In addition ROS may also affect the sperm axoneme,
inhibit mitochondrial function and affect the synthesis of DNA, RNA and proteins
(Lamirande and Gagnon, 1992). Sperm cell dysfunction,
a result of ROS damage, is dependent on the nature, amount and duration of exposure
to ROS. The extent of ROS damage is also dependent upon surrounding environmental
factors such as oxygen tension and temperature as well as the concentrations
of molecular components such as ions, proteins and ROS scavengers (Agarwal
and Saleh, 2002).
OS and DNA damage:
Sperm genetic material is structured in a special manner that keeps the
nuclear chromatin highly stable and compact. The normal DNA structure is capable
of decondensation at appropriate time transferring the packaged genetic information
to the egg without defects in the fertilization process. The cause of DNA damage
in sperm can be attributed to various pathological conditions including cancer
(ODonovan, 2005), varicocele (Saleh
et al., 2003), high prolonged fever (Evenson
et al., 2000), advanced age (Singh et al.,
2003) or leukocytospermia (Erenpreiss et al.,
2002). Also a variety of environmental conditions can be involved as radiation
(Aitken et al., 2005), air pollution, smoking
(Said et al., 2005), pesticides, chemicals, heat
and ART prep protocols (Potts et al., 1999; Aitken
et al., 2005; Bennetts and Aitken, 2005;
Aitken and De Iuliis, 2010). Abnormal sperm morphology
has been reported to be associated with high sperm DNA fragmentation in infertile
men (Nicopoullos et al., 2008). The oxidative
damage to mitochondrial DNA is well known to occur in all aerobic cells, which
are rich in mitochondria and this, may include spermatozoa. Two factors protect
the sperm deoxyribonucleic acid (DNA) from oxidative insult: the characteristic
tight packing of the DNA and the antioxidants present in the seminal plasma
(Twigg et al., 1998). ROS attacks the fluidity
of the sperm plasma membrane and the integrity of DNA in the sperm nucleus.
ROS induced DNA damage accelerate the germ cell apoptosis. Unfortunately spermatozoa
are unable to repair the damage induced by excessive ROS as they lack the cytoplasmic
enzymes required to accomplish the repair. Free radicals have the ability to
directly damage sperm DNA by attacking the purine and pyrimidine bases and the
deoxyribose backbone. Normally, sperm DNA is tightly packaged by protamines
protecting it from free radical attack. Oxidative stress also is associated
with high frequencies of single- and double-strand DNA breaks (Duru
et al., 2000; Aitken and Krause, 2001). ROS
also can cause various types of gene mutations such as point mutations and polymorphism,
resulting in decreased semen quality (Spiropoulos et
al., 2002; Sharma et al., 2004; Thomson
et al., 2009). However, infertile men often exhibit deficient protamination,
leaving the sperm DNA particularly vulnerable to ROS attack (Oliva,
2006). Early detection and prompt antioxidant therapy can prevent ROS induced
DNA damage. This has far reaching impact if such men opt for assisted reproductive
technology (ART)/in vitro fertilization. The assessment of sperm DNA
damage appears to be a potential tool for evaluating semen samples prior to
their use in ART as besides impairment of fertility DNA damage is likely to
increase the transmission of genetic diseases during the assisted reproductive
procedures. In addition, the redox status of human spermatozoa is likely to
affect phosphorylation and ATP generation with a profound influence on its fertilizing
potential (Aitken and Baker, 2003).
Smoking, oxidative stress and infertility:
Tobacco smoke consists of approximately 4,000 compounds such as alkaloids, nitrosamines
and inorganic molecules and many of these substances are reactive oxygen or
nitrogen species. Significant positive association has been reported between
active smoking and sperm DNA fragmentation (Sun et al.,
1997), as well as axonemal damage (Zoas et al.,
1998) and decreased sperm count (Vine et al.,
In a prospective study, Saleh et al. (2002)
compared infertile men who smoked cigarettes with nonsmoker infertile men. Smoking
was associated with a significant increase (approximately 48%) in seminal leukocyte
concentrations, a 107% ROS level increase and a 10 point decrease in ROS-TAC
score. The authors concluded that infertile men who smoke cigarettes present
higher seminal OS levels than infertile nonsmokers, possibly due to significant
increase in leukocyte concentration in their semen. The link between cigarette
smoking and high seminal ROS can be attributed in part to the associated increase
in seminal leukocytes. Indeed, smoking can increase leukocyte concentrations
by as much as 48% (Saleh et al., 2002). Smokers
have decreased levels of seminal plasma antioxidants such as Vitamin E (Fraga
et al., 1996) and Vitamin C (Mostafa et al.,
2006), placing their sperm at additional risk of oxidative damage. This
has been confirmed by the finding of a significant increase in levels of 8-OHdG
within smoker's seminal plasma (Fraga et al., 1996).
In a study carried out on 655 smokers and 1131 non smokers, cigarette smoking
was associated with a significant decrease in sperm density (-15.3%), total
sperm count (-17.5%) and total number of motile sperm (-16.6%) (Kunzle
et al., 2003). Thus, smoking does, in fact, affect the quality and
quantity of sperm present within a male.
Varicocele and ROS:
Varicocele has long been implicated as a major cause of male infertility
(Schlesinger et al., 1994; Benoff
and Gilbert, 2001; Hauser et al., 2001),
but the pathophysiology remains unclear. Clinical varicocele is found in about
15% of the general population including adolescents and adults: in 35% of men
with primary infertility and in up to 80% of men with secondary infertility
(World Health Organization, 1992; Belloli
et al., 1993; Pfeiffer et al., 2006).
Oxidative stress is now widely believed to be the principal underlying pathology
linking varicocele with male infertility (Barbieri et
al., 1999; Hendin et al., 1999; Saleh
et al., 2003; Nallella et al., 2004;Smith
et al., 2006; Ishikawa et al., 2007;
Smith et al., 2007; Saalu
et al., 2008). It appears that infertile men with varicocele have
significantly greater spermatozoal DNA damage, which can be related to high
levels of OS in semen (Saleh et al., 2003). Levels
of ROS positively correlate with the degree of varicocele and are expected to
decrease after varicocelectomy (Barbieri et al.,
1999). According to one metaanalysis, varicocele patients as compared with
normal sperm donors have significantly increased oxidative stress parameters
such as ROS and lipid peroxidation as well as significantly decreased antioxidant
concentrations. The exact pathways by which a varicocele damages spermatogenesis
and sperm quality remain poorly understood.
Role of ROS in assisted reproductive technique:
By means of in vitro fertilization (IVF), men with very low sperm
counts can be given a reasonable chance of paternity. However, this also increases
the possibility of passing genetic abnormalities on to the next generation because
the sperm of infertile men shows an increase in aneuploidy, other genetic abnormalities
and DNA damage. Although there are prospects for screening of sperm (Griffin
and Finch, 2005), current routine clinical practice is based on the screening
of peripheral blood samples. The use of Assisted Reproductive Technologies (ART)
has the potential to exacerbate sperm oxidative stress. During IVF and intrauterine
insemination (IUI) treatment semen is centrifuged to separate sperm from seminal
plasma. This exacerbates oxidative stress as centrifugation increases sperm
ROS production many fold (Iwasaki and Gagnon, 1992;
Shekarriz et al., 1995a, b)
Spermatozoa selected for ART usually originate from an environment experiencing
oxidative stress and a high percentage of these sperm may have damaged DNA (Kodama
et al., 1997). When IUI or IVF is used; such damage may not be a
cause of concern because the collateral peroxidative damage to the sperm plasma
membrane ensures that fertilization cannot occur with a DNA-damaged sperm.
When Intracytoplasmic Sperm Injection (ICSI) is used, this natural selection
barrier is bypassed and a spermatozoon with damaged DNA is directly injected
into the oocyte (Lamirande and Gagnon, 1992; Aitken,
1999). Sperm DNA damage is critical in the context of success of assisted
reproductive techniques (Sakkas et al., 2003;
Sharma et al., 2004). Advances in ART have helped
in improving treatment of male factor infertility (Alvarez,
2003). Currently, ICSI is the most common ART method, although it is associated
with the highest number of miscarriages. One of the explanations can be the
poor selection of sperm that are possibly damaged by free radicals during ART
procedures. Thus evaluation of seminal ROS levels and extent of sperm DNA damage
especially in an infertile male may help develop new therapeutic strategies
and improve success of ART.
ROS and antioxidants:
Antioxidants, in general, are compounds and reactions which dispose, scavenge
and suppress the formation of ROS, or oppose their actions. A variety of biological
and chemical antioxidants that attack ROS and LPO are presently under investigation.
Among the well known biological antioxidants, superoxide dismutase (SOD) and
its two isozymes and catalase have a significant role. SOD spontaneously dismutates
(O2¯). anion to form O2 and H2O2,
while catalase converts H2O2 to H2O and O2:
H2O2 to O2 and H2O2
(O2¯). + 2H+ SOD H2O2 + O2
H2O2 Catalase H2O + 1/2 O2
The most common antioxidants, that protect spermatozoa from excess concentrations
of ROS and OS-induced damage and altogether represent the Total Antioxidant
Capacity (TAC) of seminal plasma are SOD (Nissen and Kreysel,
1983; Aitken and Krause, 2001), catalase (CAT) (Jeulin
et al., 1989), the glutathione (GSH) peroxidase system selenium and
selenoproteins such as the phospholipids hydroperoxide glutathione peroxidase
(PHGPx) and the glutathione reductase system (Li, 1975;
Alvarez and Storey, 1989), vitamins A, C (Niki,
1991) and E (Chow, 1991), glutathione (Kidd,
1997), spermin, thiols, urate (Ronquist and Niklasson,
1984; Gavella et al., 1997), albumin, taurine
and hypotaurine (Alvarez and Storey, 1983), L-carnitine
and zinc. Antioxidants contained within seminal plasma are obviously helpful
for preventing sperm oxidative attack following ejaculation. However, during
spermatogenesis and epididymal storage, the sperm are not in contact with seminal
plasma antioxidants and must rely on epididymal/testicular antioxidants and
their own intrinsic antioxidant capacity for protection. Sperm are therefore
vulnerable to oxidative damage during epididymal transit, especially when there
is epididymal inflammation such as male genital tract infection. A study of
46 alcoholic men of reproductive age has suggested the presence of oxidative
stress within the testicle by reporting a significant reduction in plasma testosterone,
increase in serum lipid peroxidation byproducts and a drop in antioxidants (Maneesh
et al., 2006). However, no study to date has directly examined the
link between alcohol intake and sperm oxidative damage.
The role of physiological and pathological levels of ROS in male fertility is yet to be clearly established. While ROS have been associated with the pathology of numerous diseases including male infertility, Small controlled amounts of ROS are vital for spermatozoa to develop into normal spermatozoa capable of fertilization structures. Since spermatogenesis is a complex process involving various stages and different type of cells, mutations in mitochondrial genome, as a result of excessive ROS could disturb the formation of morphologically and functionally mature spermatozoa thus leading to infertility. Recently, a phenomenal growth has occurred in our knowledge of male reproduction, sperm function and development of diagnostic tools and treatment modalities for male infertility. In addition, knowledge regarding oxidative stress has given rise to several new treatment modalities that are now being tried to improve male infertility. However a wide gap still remains in our knowledge and future multicentric studies with larger samples are needed to help gain a better insight into this essential problem.
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