Review Article

Indian J Med Res 129, February 2009, pp 127-137

Role of reactive oxygen species in the pathogenesis of mitochondrial

DNA (mtDNA) mutations in male infertility
S. Venkatesh, M. Deecaraman*, R. Kumar, M.B. Shamsi & R. Dada

Laboratory for Molecular Reproduction & Genetics, Department of Anatomy, All India Institute of Medical
Sciences, New Delhi & *Dr M.G.R. Educational & Research Institute, Dr M.G.R. University, Chennai, India

Received November 29, 2007

Infertility affects about 15 per cent married couples half of which may be attributed to men with
low sperm motility (asthenozoospermia), low sperm count (oligozoospermia) or abnormal sperm
morphology (teratozoospermia). As mitochondria are the energy source for initiation, differentiation
and function of the germ cells, mutation in mitochondrial genome can impair the formation of mature
spermatozoa. Mutations in mitochondrial genome are identified in patients with fertility problems.
However, mitochondria are also both the source and target of reactive oxygen species (ROS). ROS are
normally generated at low levels by human spermatozoa in order to perform its physiological function.
However, if the generation of these reactive free radicals overwhelm the antioxidant defense system,
this can lead to oxidative stress, which is characterized by mitochondrial and nuclear genome damage.
So both ROS and mtDNA mutations are considered to be the major aetiological factors in a variety of
human diseases including male infertility. Identification of novel mutations in mtDNA of infertile patients
with supraphysiological levels of ROS are considered to be important to gain better understanding of
the aetiology of idiopathic infertility. 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.
Key words Male infertility - mitochondrial mutation - mtDNA damage - oxidative stress - reactive oxygen species - spermatozoa

Infertility is the major concern among married

couples when they fail to achieve conception after
1 year of regular unprotected intercourse1. It affects
approximately 15 per cent of couples, attempting
pregnancy and in half of these cases infertility can
be attributed to be due to male factor2,3. Common
causes of male infertility, include gene mutations,
aneuploides, varicocele, radiation, chemotherapy,
genital tract infections, and erectile dysfunction4,5.
Azoospermia factor (AZF) deletions6 and high

frequency of Y chromosome microdeletion7 in semen

than in blood are reported to be one of the major
aetiology leading to male infertility. In about half of the
male infertile patients, the cause is not clear and hence
such cases are diagnosed with idiopathic infertility.
Though semen analysis is the first diagnostic step
in the male infertility, it fails to detect abnormalities
in up to 20 per cent of subfertile males8. The recent
research in the field of male infertility is focused on the
reactive oxygen species (ROS), which is suspected to
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be one of the major causes of infertility at molecular

level9. ROS are generated both at exogenous and
endogenous levels and can damage different parts
of the spermatozoa including mitochondrial DNA
(mtDNA) and thus impair sperm function. It is well
known that mitochondrial dysfunction is responsible
for a variety of disorders. Mitochondrial mutations are
important aetiopathogenic factors in neurodegenerative
and musculoskeletal disorders10. But still, little
attention has been paid to mitochondrial disorders of
germinal tissues especially mitochondria of sperm. As
mitochondria are the major source of ATP, they play
a critical role in spermatogenesis, differentiation and
optimal functioning of germ cells. However, genetic
alteration of mtDNA may have serious consequences
on normal spermatogenesis and fertilization. Several
reports have shown that mitochondrial dysfunction
could lead to partial or complete spermatogenesis arrest
and be an important causative factor in male infertility11-13,
and recent research points to the mitochondria as the
crucial targets of ROS and as regulator of cell death.
So the present review deals with the role of ROS in the
pathogenesis of mtDNA mutation and the importance
of detection of ROS levels and antioxidant therapy in
male infertility.
Reactive oxygen species
reproductive system

(ROS)

and

male

A free radical is defined as any atom or molecule

that possesses one or more unpaired electrons.
Reactive oxygen species are highly reactive oxidizing
agents belonging to the class of free radicals. They
are highly unstable oxidants that react with many
biochemical substances like lipids, amino acids,
carbohydrates, protein, and DNA. Therefore ROS
are considered as a causative factor for a variety of
diseases. Though the presence of free radicals in
the spermatozoa was reported in 194314, their role
in male reproductive physiology was reported later
in 198915. Low levels of ROS are necessary for
normal functions of spermatozoa like capacitation,
hyperactivation, motility, acrosome reaction, oocyte
fusion and fertilization16,17. It has been reported that
normal functions of spermatozoa are stimulated when
they are incubated with low concentration of H2O218.
Other species such as nitric oxide (NO) and superoxide
anion (O2) have also been shown to promote sperm
capacitation and the acrosome reaction19. Though
it is well demonstrated that human spermatozoa
can generate ROS for its normal function, excess
production in some pathological conditions adversely

affect sperm function20. The mechanism behind this

effect is ROS induced lipid peroxidation of sperm
plasma membrane21, 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 proteins22. Approximately
40 to 80 per cent of non selected infertile patients
have high levels of seminal reactive oxygen species23.
Hence, uncontrolled and excessive production of ROS
may be one of the major factors leading to infertility.
The cause for the excess production of ROS in the
semen was proposed to be seminal leukocytes as
well as abnormal spermatozoa24. A study25 has also
reported the positive correlation of seminal ROS levels
with age in the healthy men. Among the suspected
radicals, hydrogen peroxide (H2O2) is found to be the
major ROS producer in human spermatozoa. During
infection and inflammation, activated leukocytes in the
seminal plasma can produce significantly high amounts
of ROS than non-activated leukocytes26. Spermatozoa
may also generate ROS by the nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase system
at the level of the sperm plasma membrane and
nicotinamide adenine dinucleotide (NADH) dependent
oxidoreductase at the level of mitochondria27, which is
both the target and source of ROS.
Role of antioxidants in male fertility
Antioxidants are group of defense system that
comprises both enzymatic and non-enzymatic molecules.
The enzymatic antioxidants include superoxide dismutase
(SOD), glutathione peroxidase/glutathione reductase
(SPx/SRD) system and catalase9. The non-enzymatic
antioxidants include ascorbate, urate, -tocopherol,
pyruvate, glutathione, taurine and hypotaurine9.
Normally, antioxidants are produced against oxidizing
molecules to keep them in normal level by the
mechanisms of prevention, interception and repair28 to
perform its physiological functions. Since ROS have
both physiological and pathological roles, these groups
of antioxidants in the seminal plasma and spermatozoa
maintain a steady state of ROS level in the semen by
converting free radicals into non reacting substances. It
is a well known fact that unlike somatic cells, mature
spermatozoa lack cytoplasm. Since cytoplasm is the
major source of antioxidants, lack of cytoplasm in
the mature spermatozoa causes deficiency in both
antioxidant defense and endogenous repair mechanism.
However, naturally the deficiency of antioxidant system
is compensated by the enzymatic and non enzymatic
components of seminal fluid29. It has been reported that

VENKATESH et al: ROS & mtDNA MUTATION IN MALE INFERTILITY

seminal plasma and spermatozoa from an infertile men

has lower antioxidant capacity than seminal plasma
from fertile men30. In many pathological conditions,
these combined antioxidant capacity of enzymatic
and non enzymatic substances i.e., total antioxidant
capacity (TAC) was found to be decreased. Yadav
et al31 reported that the seminal total antioxidant power
was decreased in leukocytospermic patients. Another
study showed that the levels of TAC were also found
to be lowered in varicocele, vasectomy reversal,
varicocele with prostatitis and idiopathic infertile
group32. Some of the reports revealed that semen of
infertile men had increased level of ROS rather than
reduced antioxidant capacity33. Though activities and
levels of antioxidant systems decline with age34 and
other male factors, the mechanism behind its elevation
in male infertility is not well established. Apart from
the above mentioned antioxidants, recently the role
of L-carnitine and L-acetyl carnitine in scavenging
the free radicals and protecting the cell membrane
has gained much importance in the treatment of male
infertility35,36. Highest level of L-carnitine in the man
is found in epididymal fluid. Since spermatozoa are
stored and gain final maturity in epididymis hence
sperms are thus protected from ROS induced damage
in epididymis. Carnitine also provides energy readily
to the spermatozoa for motility and maturation through
-oxidation of long chain fatty acids in mitochondria.
So its deficiency may cause defective sperm function
and immotility. Apart from having antioxidant
property37, it is also believed to have an antiapoptotic
effect38. Thus L-carnitine and L-acetyl carnitine have
been used in the treatment of certain OAT patients35.
A double blind random study showed increased sperm
motility after treatment with combined L-carnitine and
L-acetyl carnitine in patient with lower baseline sperm
parameters36.
Oxidative stress (OS): its pathogenic role in male
infertility
Oxidative stress (OS) is a condition that occurs when
the production of 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 infertility5,39. It has been reported that
infertile men are more likely under seminal oxidative
stress than the fertile men as a measure of seminal
total antioxidant capacity40. Several studies have used
ROS-TAC (Reactive oxygen species- Total antioxidant
capacity) score as a measure of seminal oxidative stress

129

to predict male infertility34,41. Oxidative stress has been

reported to cause abnormal denaturation of DNA
into single stranded DNA and double-stranded DNA
breaks, DNA base-pair oxidation, chromatin crosslinking, and chromosome microdeletion42. Also many
factors like drug intake, smoking, pollution, radiation
etc., were reported to increase seminal oxidative stress
which causes spermatozoa dysfunction leading to
male infertility41. Metals like cadmium and lead have
also been reported to increase OS and damage vital
parts like protein and antioxidants in the semen43.
Shen et al44 reported DNA damage as a cause of
oxidative stress which resulted in increased levels of
specific forms of oxidative damage such as 8-hydroxy
deoxy guanosine in sperm DNA. Moreover ROS can
cause various types of gene mutations including
deletion, point mutation or polymorphism of both
mitochondrial and nuclear DNA of spermatozoa45,46.
One of the common oxidative byproducts of DNA,
8-hydroxy-2-deoxy guanosine (8-OHdG) has been
reported as a biomarker of oxidative DNA damage
in the spermatozoa44 and also ROS produced by
spermatozoa were negatively correlated with the
quality of sperm in the original semen47.
Presence of xenobiotics, defect in the cellular
mechanism that regulates free radical generation,
spermatozoa with defective cytoplasmic extrusion,
varicocele, defect in Sertoli cell that removes residual
cytoplasm of spermatozoa48 are proposed to be the
main causes for origin of oxidative stress. But the exact
mechanism behind this origin is yet to be established.
Oxidative stress has also been reported to decrease
mitochondrial respiratory function49. Endogenous OS
by electron leakage of the mitochondrial respiratory
chain adversely affects mitochondrial function49.
Thus oxidative stress not only affects the fertilization
capacity of spermatozoa but also alters the mtDNA of
the sperm. Increased numbers of mtDNA mutations
have been reported in infertile men50-52 with increased
lipid peroxidation, as increased malondialdehyde
(MDA) levels were found in the same population of
men51,52.
Mitochondrial genome and oxidative phosphorylation
The human mtDNA is a closed circular
extranulcear genome and contains 16,569 base pairs53.
It contains two strands, a guanine-rich heavy (H) and
a cystosine-rich light (L) strand. mtDNA comprises
37 genes of which 13 encode essential components
of oxidative phosphorylation (OXPHOS), 22 encode

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tRNA genes, 1 for 12s and 1 for 16s tRNA genes

required for mitochondrial protein synthesis49. Unlike
nuclear genes, introns are absent in mtDNA and all of
the coding sequence is contiguous and overlapping.
The only non coding segment of mtDNA is the
displacement loop (D-loop), a region of 1121bp that
contains the origin of replication of the H-strand and
the promoters for L and H strand transcription54. The
remaining mitochondrial OXPHOS proteins, the
metabolic enzymes, the DNA and RNA polymerases,
the ribosomal proteins and the mtDNA regulating
factors are all encoded by nuclear genes. OXPHOS unit
is an essential part of mitochondria that are the main
source of ATP55, and also the main source of ROS56.
OXPHOS or electron transport chain (ETC) associated
with the generation of energy consists of 13 subunits,
which include complex I (NADH-Q oxidoreductase),
seven subunits of complex II (ND1 to ND6 and 4L),
complex III (cytochrome b), 3 subunits of cytochrome
oxidase (COX I, COX II and COX III) complex IV, and
complex V (ATPase 6 and 8 subunits). Most of these
protein subunits are encoded by mtDNA except for the
complex II, which is encoded exclusively by nuclear
DNA57. So nuclear DNA also plays an indirect role
in the function of mitochondria. ATP synthesis starts
with the electron separated by the hydrogen atoms
and then undergoes series of reactions to set up an
electrochemical gradient which supplies energy for the
synthetic process. As mitochondrial DNA is the first
site of ROS induced attack, majority of mitochondrial
genes have migrated to the nuclear genome during
the course of evolution, and nature has selected only
paternal nuclear DNA to be transmitted to offspring58.
As human cells rely on ATP for growth,
differentiation and several physiological functions,
generation of ATP by mitochondria is the major and
only source for cellular homeostasis59. Glycolsis,
citric acid cycle and -oxidation biochemical
pathways produce a high potential energy yielding
reducing substance (NADH) that enters respiratory
chain (RC). The electron transport through the RC
complex from NADH to oxygen molecule results
in release of free energy which is utilized for the
production of ATP in OXPHOS60. The importance of
OXPHOS has been studied by using mitochondrial
inhibitors. Specific OXPHOS complex inhibitors
showed a decreased sperm motility54. It has been
demonstrated that certain chemical ETC inhibitors
produce high ROS in the mitochondria61. Moreover,
increased sperm motility has also been observed

with addition of complex II substrates like pyruvate

and succinate54. These studies have demonstrated the
importance of OXPHOS in the production of ATP for
the sperm motility. Since OXPHOS complexes are
encoded by both mitochondrial and nuclear genome,
pathogenic mutation in any or both of them may lead
to dysfunctional RC complex resulting deficient ATP
production. mtDNA deletion and mutation have been
reported in a variety of diseases10. However, any
mutation in mitochondrial genome would ultimately
affect the production of ATP, consequently may lead
to abnormal spermatogenesis, impaired differentiation
and hypospermatogenesis.
Role of mitochondria in spermatogenesis
Mammalian spermatogenesis occurs continuously
with individual maturation of sperm through meiotic
division of spermatocytes. However, it has not been
well understood whether mitochondrial respiratory
function is essential for the meiotic process. But during
the phase of meiosis the mitochondria are round shaped
and relatively small with the electron-dense matrix
flattened to the outer part of the organelle. Some of the
mitochondria move in the direction of the developing
flagellum, whereas the remaining mitochondria
progressively group together62,63. These mitochondria
leave the developing spermatozoa on the residual
bodies and are probably phagocytosed by the Sertoli
cells. Then the mitochondria, by elongation and fusion
form a twisted structure in the form of chain around
the flagellum in the mid-piece. Hence the mitochondria
lose its somatic morphological appearance to typical
for the condensed form in the spermatozoa. Then the
mitochondria reach their maximum elongation in order
to assume a close association with outer dense fibres
of the middle piece62,63. Finally, the mitochondria are
arranged in a helix of 11-13 gyri, with two mitochondria
per gyrus, and deliver ATP to the axoneme for flagellar
propulsion64. It is clear that morphological change of
mitochondria takes place in the differentiation of germ
cells to the haploid spermatozoa and the process is said
to be influenced by ATP65. So mitochondrial genome
mutation and associated respiratory chain dysfunction
may disrupt normal differentiation of mitochondria
and could lead to defective spermatogenesis.
Mitochondria of germ cells modify their
morphological organization, number and location
during the process leading to sperm production. An
average of 1000 mtDNA copies have been reported
to be present in both progressive and non progressive

VENKATESH et al: ROS & mtDNA MUTATION IN MALE INFERTILITY

spermatozoa of fertile men, whereas a 7-fold increase

in mtDNA number per cubic micrometer of cell
takes place during spermatozoa maturation which
demonstrates the higher ATP requirements66 and the
importance of mtDNA for the sperm function. The
orientation and the structure of mitochondrion in the
midpiece of mature spermatozoa is also an important
factor in the sperm motility. Shorter midpiece and less
number of gyres was observed in asthenozoospermic
men when compared with the fertile controls67,68. It
has been suspected that degeneration of mitochondria
may also occur during epididymal passage69. The
position of mitochondria in the upper part of the sperm
flagellum is unique. As mitochondria are the major
source of the ATP that helps in the movement of the
sperm, the mitochondria produce ATP that diffuses
downwards to the axonemal microtubules and their
dynein arms70. Various defects or pathological changes
in the arrangement of mitochondria are believed to
affect the motility of the spermatozoa in idiopathic
asthenozoospermic and oligoasthenozoospermic (OA)
patients71. Since there is an interaction between nuclear
and mitochondrial gene, a study also proposed the DNA
fragmentation and mitochondrial swelling as a more
indicative parameter of impaired motility72. Moreover,
abnormal or complete absence of mitochondria or its
functional impairment, sometimes all of the above
may act as a sole reason for the sperm immotility due
to the inability to produce ATP for the flagellar beat.
Mitochondrial structure and DNA studies are also
recently focused as paternal mtDNA inheritance could
occur at low level in intracytoplasmic sperm injection
(ICSI)54.
Mitochondrial DNA (mtDNA) mutation in male
infertility
The location of mitochondria is unique in case of
spermatozoa, where it is located at the site of maximum
energy requirement i.e., around the midpiece of
the spermatozoa. It has been well established that
mitochondria make ATP by the coupling of respiration
generated proton gradient with the proton-driven
phosphorylation of ADP. However, on the other
hand unlike nuclear DNA, mitochondrial DNA is not
protected by histones and are physically associated
with the inner mitochondrial membrane, where highly
mutagenic oxygen radicals are generated as byproduct
of OXPHOS in the respiratory chain73 and leakage of
these free radicals from the respiratory chain makes
the mitochondria as a major intracellular source of
ROS. These unique features are probably the cause

131

of the about 10 to 17 times faster accumulation of

polymorphisms and mutation in mitochondrial DNA
than in nuclear DNA74. Also lack of an efficient repair
system in mitochondria75 and abnormal mitochondrial
metabolism may accelerate the rate of mitochondrial
DNA mutation76 and therefore, higher amount of 8OHdG have been reported in mitochondrial DNA than
in nuclear DNA of infertile patient77. Several single
nucleotide polymorphisms (SNPs) in mtDNA have
been reported in many of the diseases including male
infertility50-52,78. Studies79-82 have also reported low
levels of mtDNA mutation in the semen of the infertile
men. Cell division in the testis may be the reason for
the accumulation of tissue specific mosaicism at higher
level in spermatozoa83.
In most of the mitochondrial disease, the expression
of abnormal phenotype occurs only when the ratio of
mutated or deleted mtDNA and wild mtDNA exceeds
a critical threshold. For example >60 per cent mutant
mtDNA are expected to express the phenotype in
Lebers hereditary optic neuropathy (LHON) patients84.
Some of the heteroplasmic associated disorders are
mitochondrial myopathy, encephalopathy, lactic
acidosis, and stroke (MELAS)85, rhabdomyolysis86,
dystonia87, etc. The mixture of wild type mtDNA
and altered mtDNA in the same mitochondrion
called heteroplasmy has gained much importance
recently in the expression of the abnormal genotype,
because most of the pathogenic mtDNA mutations are
heteroplasmic but not all. Usually the phenotype is
normal until the proportion of mutant mtDNA and the
threshold for genotype expression exceeds88. Though
energy requiring organs like brain, muscle and heart
are mostly affected by heteroplsamy89,90, such effect
on sperm mtDNA is not well studied. One study has
attempted to reverse the heteroplasmic mutation in
mtDNA91. More studies are needed to understand the
role of heteroplasmy in sperm mtDNA of infertile
men, where homoplasmy mutant mtDNA has also been
reported in OA infertile patients92. As there are a few
mtDNA in sperm as compared to somatic cell, mtDNA
mutations manifest early and have more deleterious
consequences.
In 1988, seven years after the complete sequence
of human mtDNA was published, the first pathogenic
mutations were discovered93. Now, more than 100 point
mutations and many rearrangements have been known
to be associated with various human mitochondrial
diseases. But so far only a few studies have reported
mutation in mtDNA of the spermatozoa50-52,94-96.

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These mutations in spermatozoa could be pathogenic

or common mtDNA variants that only affect male
fertility because mtDNA is maternally inherited. But a
study showed that individual cells must contain a high
percentage of mutant mtDNA before they express a
respiratory chain defect97. As only a few mtDNA per
mitochondrion in spermatozoa are present, an early
expression of phenotype takes place. High levels of
mutant mtDNA were also correlated with low sperm
motility in males who had inherited the A3243G
mtDNA mutation from their mother45.
One study from India95 revealed a 9 misense and
27 silent mutations in the sperm mtDNA, but not in the
blood cells DNA of an oligoasthenoteratozoospermic
(OAT) man, who also had varicocele of the left testis.
Though the patient was not fertile under the normal
criteria, his wife conceived without varicoceloctomy.
But the diminished motility of the sperm has been
suspected due to a 2-nucleotide deletion in the
mitochondrial COII genes. Another recent study92 from
India showed a novel missense mutation (C11994T)
in the ND4 gene of oligoasthenozoospermic samples
and it has been proposed that the wild type allele had
totally replaced by the mutant allele over several
generation in the maternal lineage leading to the
mutantDNA homoplasmy. Kumar et al52 showed
significant nucleotide changes in the mitochondrial
gene (ATPase 6, ATPase 8, ND2, ND3, ND4 and ND
5) in the semen of the infertile men. In contrast, a
study from Portugal98 reported no C11994T mutation
in their OA infertile population suggesting that
such mutation may be population specific. GSTM1
(glutathione S-transferase) in association with
mtDNA 4977 deletion has also been reported in
infertile Indian population99.
During differentiation of germ cells in the
testes, abnormal mitochondria were also reported
in the spermatids and Sertoli cells of the patient
with encephalomyopathy100. This may lead to
depletion in the energy production and thus affects
spermatogenesis and could result in production of
abnormal spermatozoa. However, another study
revealed multiple mtDNA in the biopsied tissue of
an azoospermic or severe oligozoospermic patient101.
Recent ongoing study in our laboratory reported a
high frequency of mitochondrial mutation in men
with severe oligoasthenozoospermia (OA)50,52. These
men were cytogenetically normal and harboured no
Y-chromosome microdeletion50. Thus it is possible
that mitochondrial mutations were involved in the

pathogenesis of OA in this group of infertile men. In

addition, men who harboured multiple mitochondrial
mutations and large deletion had a much more severe
phenotypic defect with the presence of immotile
sperms96,102. But studies failed to explain the exact
causative mechanism behind these mtDNA mutations
in the male germ cells.
A recent study in mito-mice carrying different
proportions of mt DNA showed respiratory
chain defect that leads to meiotic arrest during
spermatogenesis103. Thus respiration deficient
spermatocytes could not complete meiosis, and were
removed by apoptosis. Some of the spermatids showed
abnormalities in middle piece and nucleus suggesting
abnormal sperm formation. The study also confirmed
that mtDNA affected mitochondrial respiration
chain function and resulted in oligozoospermia,
asthenozoospermia and teratozoospermia leading to
infertility in mice103.
ROS associated mtDNA mutation
It is well established that increased production
of ROS adversely affects sperm function leading to
infertility. Production of ROS is significantly increased
in dysfunctional mitochondria. This process may be
due to ROS induced damage to the mitochondrial
genome as ROS are byproduct of OXPHOS system.
Mitochondrial dysfunction in such cases may be
measured as mitochondrial membrane potential
(MMP), which has been reported to decrease in
the spermatozoa of infertile men with raised ROS
level104. Several studies have reported that human cells
harbouring mutated mtDNA have lower respiratory
function and show increased production of superoxide
anions, hydroxyl radicals and H2O2105,106. However,
excess production of ROS further damages mtDNA,
which is more susceptible than the nuclear genome.
Thus during the course of evolution, majority of
mitochondrial genes have been transferred to the
nuclear genome and only those genes essential for
OXPHOS are now located in mtDNA. Ultimately,
impairment of electron transport chain results in
enhanced production of ROS in mitochondria due to
incomplete reduction of oxygen107,108. Selvi Rani et al92
reported for the first time, mutations in the sperms
mtDNA but not in the DNA isolated from blood.
This study clearly suggests that genetic alteration in
sperm mtDNA occurred during the differentiation
or spermatogenesis or due to random segregation of
mtDNA during cell division.

VENKATESH et al: ROS & mtDNA MUTATION IN MALE INFERTILITY

133

Fig. Many physical, chemical and biological factors disturb mitochondrial respiratory chain and increase the production of reactive oxygen
species beyond the level of antioxidant system. This establishes oxidative stress (OS) in the male reproductive system. mtDNA close to
electron transport (ET) chain gets damaged by ROS and undergoes mutation, which results in the formation of defective ET chain. The altered
ET chain further increases the production of ROS and also impairs the spermatogenesis and sperm function leading to male infertility.

Also mtDNA is more sensitive than nuclear DNA to

H2O2- induced damage and protracted treatment leads
to persistent mtDNA damage and loss of mitochondrial
function. Moreover, ROS induced chain-propagating
reaction is responsible for overall increase in the
steady state level of mtDNA damage and it is also
believed that ROS mediated damage to mitochondria
may inactivate electron transport chain, thus altering
normal mitochondrial function109. Persistence of
mtDNA damage could be due to continuous ROS
production by lipid peroxidation and/or damage at the
electron transport chain (Fig.). Study by Yakes and
Houten109 supports the sensitivity of mtDNA to ROS.
Similar results were also reported in a study from an
Indian population51.

A correlation was found between ROS and

mitochondria in apoptosis, as high levels of ROS
disrupt the inner and outer mitochondrial membrane
and result in release of cytochrome C from the
mitochondria104. Cytochrome C protein activates
the caspases and induces apoptosis, which has been
reported to be significantly higher in oxidative stress
induced infertile men104. Though mitochondria are the
major source of ATP, they are also the major source
of intracellular ROS and mitochondrial disorders
are primarily manifested in high energy demanding
organs or tissues110. Since the number of mtDNA in
sperm is far fewer than in somatic cell, these manifest
as early phenotypic defect such as impaired motility
and abnormal cytoskeleton of axoneme111. Increase

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INDIAN J MED RES, FEBRUARY 2009

in number of mtDNA mutations may also lead to

production of sperm with abnormal morphology and
ultrastructural defects111. However, a recent study from
our laboratory showed raised ROS levels and sperm
DNA (nuclear & mitochondria) damage in idiopathic
infertile men52,112,113.
Conclusions
Several studies suggest that raised ROS levels
and mtDNA mutation play an important role in the
pathogenesis of male infertility. The role of ROS, its
physiological and pathological levels are yet to be
established. Since spermatogenesis is a complex process
involving various stages and different type of cells,
mutations in mitochondrial genome, could disturb the
formation of morphologically and functionally mature
spermatozoa thus leading to infertility. For better
understanding of the aetiology of male infertility, the
correlation of pathological levels of ROS which lead
to mtDNA mutation, needs to be established by larger
studies. Infertile patients identified with elevated level
of ROS may get benefit from the suitable antioxidant
therapy. Since OS imbalance manifests as motility
defects and DNA damage, early diagnosis of OS and
prompt antioxidant treatment may prevent OS induced
DNA damage. This has far reaching and important
implication in men opting for ART. Further studies in
this area are recommended to subgroup the idiopathic
infertile patients with high oxidative stress to achieve
high success rate in infertility treatment and ART.
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Reprint requests: Dr Rima Dada, Associate Professor, Department of Anatomy, All India Institute of Medical Sciences (AIIMS)
New Delhi 110 029, India
e-mail: rima_dada@rediffmail.com