Oxidative stress and male reproductive health

Abstract

One of the major causes of defective sperm function is oxidative stress, which not only disrupts the integrity of sperm DNA but also limits the fertilizing potential of these cells as a result of collateral damage to proteins and lipids in the sperm plasma membrane. The origins of such oxidative stress appear to involve the sperm mitochondria, which have a tendency to generate high levels of superoxide anion as a prelude to entering the intrinsic apoptotic cascade. Unfortunately, these cells have very little capacity to respond to such an attack because they only possess the first enzyme in the base excision repair (BER) pathway, 8-oxoguanine glycosylase 1 (OGG1). The latter successfully creates an abasic site, but the spermatozoa cannot process the oxidative lesion further because they lack the downstream proteins (APE1, XRCC1) needed to complete the repair process. It is the responsibility of the oocyte to continue the BER pathway prior to initiation of S-phase of the first mitotic division. If a mistake is made by the oocyte at this stage of development, a mutation will be created that will be represented in every cell in the body. Such mechanisms may explain the increase in childhood cancers and other diseases observed in the offspring of males who have suffered oxidative stress in their germ line as a consequence of age, environmental or lifestyle factors. The high prevalence of oxidative DNA damage in the spermatozoa of male infertility patients may have implications for the health of children conceivedin vitro and serves as a driver for current research into the origins of free radical generation in the germ line.

Full text

Asian Journal of Andrology (2014) 16, (31–38)
© 2014 AJA, SIMM & SJTU. All rights reserved 1008-682X
www.asiaandro.com; www.ajandrology.com
and Curson5 con rmed the presence of such an oxidase in bovine
spermatozoa and demonstrated that it was the dead cells in any given
ejaculate that were particularly active in generating hydrogen peroxide
in response to phenylalanine and that the oxidative stress generated
in this manner could have an impact on the live cells present in the
immediate vicinity.  e cytotoxic e ect of ROS generated on exposure
to the phenylalanine in cryostorage medium could be rescued by the
concomitant presence of catalase, con rming hydrogen peroxide as
the cytotoxic principle.
 e notion that oxidative stress might also be a factor in the etiology
of defective sperm function in our species was advanced independently
by Aitken and Clarkson6 and Alvarez etal.7 in 1987. An important but
o en overlooked catalyst for this discovery was the development of
a technique for objectively measuring sperm function, in the form of
the zona-free hamster oocyte penetration assay introduced by another
pioneer of modern andrology, Ryuzo Yanagimachi.8 Up until this point,
the  eld had lacked objective methods for the measurement of human
sperm function aside from motility.  e hamster oocyte penetration
assay provided an objective means of determining the competence
of human spermatozoa to capacitate, undergo the acrosome reaction
and generate a fusogenic equatorial segment capable of initiating
fusion with the vitelline membrane of the oocyte. In the age of
intracytoplasmic sperm injection(ICSI), the hamster oocyte model
can also provide critical information on the ability of spermatozoa
to form a pronucleus.9–11 When combined with objective methods
for assessing sperm motility, this assay has been shown to give a very
accurate assessment of the fertilizing potential of human ejaculates.12,13
One of the interesting results secured with this assay was to demonstrate
INTRODUCTION
Male infertility is a relatively common condition a ecting approximately
1 in 20 of the male population. In a vast majority of infertile subjects
su cient numbers of spermatozoa are generated to initiate a pregnancy;
however, the functionality of the spermatozoa has been compromised.
As a result, defective sperm function is held to be the largest, single
and de ned cause of human infertility.1  e primary causes of defective
sperm function are undoubtedly multifactorial, involving a range of
primary genetic, lifestyle and environmental factors, acting alone or,
more frequently, in combination. However at the level of the gamete,
the integration of these various forces frequently culminates in a state
of oxidative stress that impairs the functional and structural integrity
of these highly di erentiated cells.  e  rst suggestion that oxidative
stress might play a role in the etiology of defective sperm function came
from one of the pioneers of modern andrology, DrJohn MacLeod.2
He published an important paper in 1943 which demonstrated that
in oxygenated medium human spermatozoa rapidly lost motility via
mechanisms that could be rescued by the concomitant presence of
catalase, a speci c scavenger of hydrogen peroxide.  e fundamental
notion that spermatozoa could generate reactive oxygen species(ROS),
speci cally hydrogen peroxide, was con rmed by Tosic and Walton in
a paper published in Nature in 1946.3 In this, and a follow-up paper
published in 1950,4 these authors presented impressive biochemical
evidence that bovine spermatozoa could not only generate hydrogen
peroxide but also that this reactive oxygen metabolite was damaging
to sperm function. In this speci c case, the authors demonstrated the
involvement of an L-amino acid oxidase with a particular a nity for
aromatic amino acids such as phenylalanine. Many years later Shannon
INVITED REVIEW
Oxidative stress and male reproductive health
Robert J Aitken, Tegan B Smith, Matthew S Jobling, Mark A Baker , Geo ry N DeIuliis
One of the major causes of defective sperm function is oxidative stress, which not only disrupts the integrity of sperm DNA but also
limits the fertilizing potential of these cells as a result of collateral damage to proteins and lipids in the sperm plasma membrane.
The origins of such oxidative stress appear to involve the sperm mitochondria, which have a tendency to generate high levels of
superoxide anion as a prelude to entering the intrinsic apoptotic cascade. Unfortunately, these cells have very little capacity to
respond to such an attack because they only possess the fi rst enzyme in the base excision repair (BER) pathway, 8-oxoguanine
glycosylase 1 (OGG1). The latter successfully creates an abasic site, but the spermatozoa cannot process the oxidative lesion further
because they lack the downstream proteins (APE1, XRCC1) needed to complete the repair process. It is the responsibility of the
oocyte to continue the BER pathway prior to initiation of S-phase of the fi rst mitotic division. If a mistake is made by the oocyte
at this stage of development, a mutation will be created that will be represented in every cell in the body. Such mechanisms may
explain the increase in childhood cancers and other diseases observed in the offspring of males who have suffered oxidative stress
in their germ line as a consequence of age, environmental or lifestyle factors. The high prevalence of oxidative DNA damage in the
spermatozoa of male infertility patients may have implications for the health of children conceived
in vitro
and serves as a driver
for current research into the origins of free radical generation in the germ line.
Asian Journal of Andrology (2014) 16, (31–38); doi: 10.4103/1008-682X.122203; published online: 16 December 2013
Keywords: DNA damage; oxidative stress; oxoguanine glycosylase 1; oocyte; spermatozoa
Priority Research Centre in Reproductive Science, Discipline of Biological Sciences, Faculty of Science and IT, University of Newcastle, Callaghan, NSW 2308, Australia.
Correspondence: Prof. RJ Aitken (john.aitken@newcastle.edu.au)
Received: 09-08-2013; Revised: 12-09-2013; Accepted: 22-09-2013
Open Access
Reproductive Health

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Asian Journal of Andrology
that defective sperm function was evident in infertile men, even
when their spermatozoa had been treated with the divalent cation
ionophore, A23187 in order to induce an acrosome reaction.14  is
result indicated that whatever the lesions are in defective spermatozoa,
they lay downstream of the calcium in ux normally triggered when
the spermatozoa make contact with the cumulus-oocyte complex.
Such results suggested that there must be some defect in the plasma
membrane of functionally compromised human spermatozoa that
prevents them from fusing with the vitelline membrane of the oocyte.
It was this quest for an explanation of failed membrane fusion in the
hamster oocyte assay that led us to the concept that lipid peroxidation
was a key factor in the etiology of defective sperm function.
Spermatozoa are particularly vulnerable to lipid peroxidation because
they contain high concentrations of unsaturated fatty acids, particularly
docosahexaenoic acid with six double bonds per molecule.15  e latter
are vulnerable to free radical attack because the carbon hydrogen
dissociation energies are lowest at the bisallylic methylene position.
As a consequence, the hydrogen abstraction event that initiates lipid
peroxidation is promoted, generating a carbon-centered lipid radical
that then combines with oxygen to generate peroxyl(ROO•) and
alkoxyl(RO•) radicals that, in order to stabilize, abstract hydrogen
atoms from adjacent carbons. These chemical reactions create
additional lipid radicals that then perpetuate the lipid peroxidation
chain reaction, culminating in the generation of small molecular
mass electrophilic lipid aldehydes such as 4-hydroxynonenal(4HNE),
acrolein and malondialdehyde. Added to this vulnerability, we have
shown that sperm mitochondria respond to the presence of free
unsaturated fatty acids with a dramatic increase in ROS generation; the
greater the level of unsaturation, the greater the level of the stimulatory
e ect. Esteri cation of the fatty acid counters this pro-oxidant e ect
suggesting that it is the amphiphilic properties of these molecules
that are central to their ROS-inducing activity, possibly by de ning
the orientation of the fatty acids in relation to the mitochondrial
electron transport chain. In this context, it is signi cant that defective
human spermatozoa possess abnormally high cellular contents of free
polyunsaturated fatty acids, the levels of which are positively correlated
with mitochondrial superoxide generation.17
 us, defective human spermatozoa are particularly vulnerable
to oxidative stress because they contain a superabundance of free
unsaturated fatty acids that trigger ROS generation by the sperm
mitochondria and induce high levels of lipid peroxidation. To make
matters worse, the products of lipid peroxidation in the form of small
molecular mass electrophilic aldehydes such as 4HNE or acrolein, are
also capable of triggering ROS generation by the sperm mitochondria.18
 is ability of lipid aldehydes generated as a consequence of lipid
peroxidation to trigger mitochondrial ROS generation appears to be a
function of their capacity to adduct onto proteins in the mitochondrial
electron transport chain, such as succinic acid dehydrogenase.18 As a
consequence of these interactions, it is evident that oxidative stress in
human spermatozoa is a self-propagating cycle that, once initiated,
will inevitably lead to oxidative damage, a loss of functionality and
ultimately, cell death(Figure1).
OXIDATIVE STRESS SPERM FUNCTION, DNA INTEGRITY AND
CELL DEATH
One of the first functions affected by oxidative stress and lipid
peroxidation is sperm motility. Correlations between lipid peroxide
formation and sperm movement have been repeatedly observed in a
variety of di erent species.15,19,20 Experiments involving exposure of
mammalian spermatozoa to a variety of ROS using the xanthine oxidase
ROS-generating system have also clearly demonstrated the susceptibility
of sperm motility to oxidative attack and identi ed hydrogen peroxide
as the most cytotoxic oxygen metabolite in this context; catalase, but
not superoxide dismutase, preventing sperm motility loss under such
circumstances.21–24  e mechanisms by which motility is lost when
spermatozoa are under oxidative stress is not known with certainty, but
both oxidative damage to the axoneme and depletion of intracellular
adenosine triphosphate(ATP) appear to be involved.25–27
Notwithstanding the dramatic e ects that high levels of exposure to
ROS have on sperm motility, it is also evident that oxidative stress can
compromise the fertilizing capacity of spermatozoa under conditions
where motility is normal.28,29 Under these circumstances, it is the
capacity of the spermatozoa to fuse with the vitelline membrane of
the oocyte which is impaired. Acareful dose-dependent analysis of
the impact of oxidative stress on sperm-oocyte fusion demonstrated
a biphasic response which beautifully encapsulates the complex
relationship between ROS and sperm function.30  us, at low levels of
oxidative stress, sperm-oocyte fusion rates were enhanced, presumably
as a consequence of:(i) the positive role that ROS are known to play
in driving the tyrosine phosphorylation events associated with sperm
capacitation,31 and(ii) the importance of sterol oxidation in facilitating
the e ux of cholesterol from the sperm plasma membrane.32 However,
at higher levels of oxidative stress the induction of lipid peroxidation in
the plasma membrane is associated with a decline in the competence for
sperm-oocyte fusion, possibly due to the direct induction of oxidative
damage to proteins involved in the fusion process, rather than any
change in the  uidity of the sperm plasma membrane.33
Figure 1: Proposed cycle of cause and effect by which oxidative stress
in the male germ line impacts upon the health and well-being of future
generations. (1) A variety of primary factors can initiate oxidative stress
in the male germ line including infection, age, obesity and exposure to a
variety of adverse environmental infl uences. (2) This initial oxidative stress
induces lipid peroxidation culminating in the production of lipid aldehydes
such as 4HNE, which bind to proteins in the mitochondrial electron
transport chain, stimulating the generation of reactive oxygen species
(ROS). The latter stimulate yet more lipid peroxidation in a self-propagating
cycle that culminates in apoptosis. (3) One of the most sensitive targets
of oxidative stress is the DNA in the sperm nucleus, generating 8-hydroxy,
2’deoxyguanosine (8OHdG) base adducts. (4) The fi rst enzyme in the base
excision repair pathway, 8-oxoguanine glycosylase 1 (OGG1), is present in
spermatozoa and its activity creates abasic sites. The remainder of the DNA
repair pathway is present in the oocyte. The oocyte has to repair the DNA
damage brought into the zygote by the fertilizing spermatozoon before the
initiation of S-phase for the fi rst mitotic division. (5) If the oocyte makes a
mistake at this stage of DNA repair, it has the potential to create a mutation
that will be represented in every cell in the body and could account for the
range of pathologies seen in the offspring of fathers exhibiting high levels
of oxidative DNA damage in their spermatozoa. Abbreviations: IVF, in vitro
fertilization; ROS, reactive oxygen species.

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OXIDATIVE STRESS AND DNA DAMAGE
When human spermatozoa were exposed to increasing levels of
hydrogen peroxide it was not just the fertilizing potential of the cells
that followed a biphasic pattern of change, the DNA in the sperm
nucleus behaved similarly. At low levels of oxidative stress DNA
damage was diminished, possibly because of the powerful role played
by glutathione peroxidase in e ecting the cross linking of sperm
chromatin. However, at higher levels of oxidative stress, the sperm
chromatin started to fragment.30 Importantly, the losses of fertilizing
potential and DNA integrity occurred at di erent rates, with the latter
being the more sensitive. As a result, spermatozoa that had been driven
to a high state of readiness for fertilization by low levels of oxidative
stress were also found to exhibit signi cantly elevated levels of DNA
damage.30  is is an extremely signi cant observation, since it suggests
a mechanism by which environmental in uences on the paternal germ
line could have a major impact on the health trajectory of any progeny.
 e oxidized base adduct, 8-hydroxy, 2′deoxyguanosine(8OHdG),
has been used in studies to demonstrate that oxidative DNA damage is
signi cantly elevated in the spermatozoa of patients attending infertility
clinics.34,35 Furthermore, the levels of 8OHdG expression have been
shown to correlate highly with the measurement of DNA damage
in spermatozoa, as measured by the TUNEL or sperm chromatin
dispersion assays.34,36 Indeed, the correlation between 8OHdG
formation and DNA damage is so high that we have been forced
to conclude that most DNA damage in spermatozoa is oxidatively
induced. In order to understand why this would be the case we need
to appreciate the particular architecture of human spermatozoa and
the major points of di erence with somatic cells in terms of the
mechanisms regulating apoptosis.
Apoptosis is the default condition for spermatozoa. In the
absence of fertilization, most spermatozoa will become senescent and
default to an apoptotic state. In somatic cells, apoptosis is associated
with extensive nuclear fragmentation as a consequence of nucleases
released from the mitochondria(e.g., endonuclease G) or activated
in the cytosol(e.g.,caspase-activated DNase). However, spermatozoa
are distinguished from every other cell type in biology in having a
nucleus that is physically separated from the mitochondria and most
of the cytoplasm. As a consequence, even when apoptosis is activated
in these cells using inhibitors of PI3 kinase such as wortmannin,37
the nucleases associated with this process remain resolutely locked
within the midpiece of the cell and do not penetrate the nuclear
compartment(Figure 2). Thus, even when apoptosis is induced
in suspensions of human spermatozoa, the DNA does not become
cleaved by nucleases, at least in the short-term.37  e only products
of apoptosis that can damage sperm DNA are the ROS generated
by the mitochondria. Mitochondria are potent generators of ROS
in spermatozoa and this activity becomes enhanced as soon as the
spermatozoa default to an apoptotic state. Indeed mitochondrial ROS
generation is one of the  rst signs that these cells have engaged the
intrinsic apoptotic cascade.37,38 It is for thi s re aso n th at m ost of t he D NA
damage observed in spermatozoa is oxidative in nature.
If nucleases are ever involved, it would be at the very beginning or
the very end of sperm existence. During late spermatogenesis, spermatid
DNA becomes enzymatically cleaved in order to relieve the torsional
stress associated with sperm chromatin compaction. Such endogenous
nicks are thought to be resolved by topoisomerase before spermiation,
however in pathological cases, such repair mechanisms may be de cient
leading to the persistence of nicked DNA into the mature gamete.39,40
 e possibility that DNA damage in spermatozoa has its origins during
spermiation is supported by the profound correlation, which has been
observed between DNA fragmentation and chromatin compaction in
spermatozoa as detected by chromomycin A3  uorescence.34,41 Viewed
in this light, both DNA fragmentation and poor chromatin compaction
may be regarded as independent signs of errors in spermiogenesis. An
alternative explanation is that these two events are causally related.
According to this ‘two-step’ model, errors in spermiogenesis initially lead
to poor chromatin protamination and create a state of vulnerability in the
spermatozoa. In the second step, spermatozoa are exposed to oxidative
stress from a variety of sources including exposure to exogenous ROS
as a consequence of leukocyte in ltration, or endogenous ROS triggered
by entry into the intrinsic apoptotic cascade, ultimately resulting in
enhanced oxidative DNA damage. Of course, this two-step hypothesis42,43
to explain the origins of oxidative DNA damage is not necessarily
exclusive of the concept that nuclease-mediated DNA nicks might
persist in spermatozoa from late spermatogenesis. Nevertheless, the high
correlation that has been observed between oxidative DNA damage and
DNA fragmentation suggests that most of the DNA damage is occurring
following spermiation as a result of enhanced vulnerability to oxidative
stress.42  e only other time that nucleases may contribute to DNA
damage in the male germ line would be at the end of a spermatozoon’s
life when intracellular nucleases released during the perimortem as the
internal structure of these cells starts to break down, or extracellular
nucleases released from the male reproductive tract, may aid in the  nal
disposal of these cells by the phagocytic armies of the immune system.44,45
DNA REPAIR IN SPERMATOZOA
The importance of oxidative stress in the mechanisms by which
sperm DNA becomes damaged is also indicated by a consideration
Figure 2: The unique architecture of spermatozoa infl uences the impact of
apoptosis on DNA integrity. (a) Conventional somatic cells feature a centrally
placed nucleus surrounded by mitochondria embedded in the cytoplasm.
Under these circumstances, endonucleases activated in the cytoplasm or
released from the mitochondria during apoptosis are able to move into
the nucleus (arrows) and cleave the DNA. (b) Spermatozoa are completely
different from such somatic cells because their mitochondria (stained
black) and most of their cytoplasm are located in the midpiece of the
cell, physically separated from the nucleus. (c) As a consequence of this
compartmentalization key effectors of apoptosis such as apoptosis inducing
factor (AIF) or Endonuclease G (Endo G) remain resolutely locked in the
sperm midpiece even when apoptosis is induced by the powerful PI3 kinase
inhibitor, wortmannin and cannot move into the sperm nucleus. Because
of this physical limitation, most DNA damage in mature spermatozoa is
induced by membrane permeant reactive oxygen species emanating from
the mitochondria, rather than nucleases.
b
ac

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of the DNA repair strategies these cells are capable of employing.
Incorporated into the subcellular structure of the sperm nucleus and
mitochondria is an 8-oxoguanine glycosylase, known as 8-oxoguanine
glycosylase1(OGG1).46 When sperm DNA experiences an oxidative
attack OGG1 immediately clips the 8OHdG residues out of the DNA
generating an abasic site, releasing the oxidized base into the extracellular
space.  e next enzyme in the base excision repair(BER) pathway, APE1,
then incises DNA at the phosphate groups3’ and 5’ to the baseless site
leaving 3’-OH and 5’-phosphate termini ready for the insertion of a
new base. Spermatozoa do not possess this enzyme.46 As a result, they
carry their abasic sites into the oocyte for continuation of the repair
process(Figure1). For its part, the oocyte engages in a round of DNA
repair immediately a er fertilization and puts S-phase on hold until this
activity has been completed.47,48 If the oocyte should make a mistake
during the completion of this post-fertilization repair process, it creates
the potential for de novo mutations in the o spring which could have a
profound impact on the health and well-being of the latter(Figure1).
LIFESTYLE, AGE AND OXIDATIVE STRESS
Given this propensity for oxidative damage to sperm DNA and a
heavy reliance on OGG1 to cleave out damaged base adducts prior to
fertilization, it would not be surprising if factors that impeded OGG1
activity had a profound impact on fertility and the health of progeny.
 e classic inhibitor of OGG1 activity is cadmium and the latter has a
long history of being associated with the etiology of male infertility.49,50
Importantly, cadmium exposure has been shown to increase levels of
DNA damage in spermatozoa51 and positive correlations have been
observed between 8OHdG levels in spermatozoa and the cadmium
concentration in seminal plasma.52 Since one of the classical sources of
cadmium is cigarette smoke, it is also no surprise to learn that men who
smoke heavily exhibit signi cantly elevated levels of oxidative DNA
damage in their spermatozoa.53 Furthermore, the impact of smoking
on 8OHdG levels in human spermatozoa is signi cantly impacted by
the presence of Ser326Cys polymorphism in the OGG1 gene.54  ose
individuals with variant Cys/Cys homozygosity for OGG1 showing
higher levels of sperm 8OHdG than wildtype homozygote carriers
(Ser/Ser).53  e fact that paternal(not maternal) smoking is associated
with a signi cant increase in the risk of childhood cancer in the
o spring55 is further testimony to the lasting clinical consequences of
cigarette smoking and the power of the relationship between oxidative
DNA damage in the paternal germ line and the long-term health
trajectory of the o spring(Table1).
If the oxidative DNA damage induced in the germ line as a
consequence of smoking can impact on the incidence of cancer in the
progeny, then surely any factor capable of inducing oxidative damage in
spermatozoa is potentially capable of profoundly in uencing the health
of children. Furthermore, because there is no particular proposed
order to the nature of the DNA damage or aberrant DNA repair in the
oocyte, we might anticipate that the range of pathologies generated
as a consequence of oxidative stress in the male germ line might be
considerable. Acase in point is paternal aging. It is well-recognized that
as men get older they do not stop producing spermatozoa; however,
the quality of their gametes exhibits a progressive age-related decline
as indicated by a highly signi cant, age-dependent increase in sperm
DNA damage.56,57 Studies on the brown Norway rat indicate that this
age-dependent increase in DNA damage in spermatozoa is associated
with a concomitant down regulation of genes associated with the BER
pathway and a corresponding increase in the levels of oxidative DNA
damage in the spermatozoa.58
This relationship between paternal age and oxidative DNA
damage in spermatozoa has also been indicated by recent studies
on the senescence-accelerated mouse prone 8(SAMP8).  is mouse
strain contains a suite of naturally occurring mutations resulting in
an accelerated senescence phenotype largely mediated by oxidative
stress, which is further enhanced by a mutation in the Ogg1 gene,
greatly reducing the ability of the enzyme to excise 8OHdG adducts. An
analysis of the reproductive phenotype of the SAMP8males revealed
a high level of DNA damage in caudal epididymal spermatozoa as
detected by the alkaline Comet assay. Furthermore, these lesions were
con rmed to be oxidative in nature, as demonstrated by signi cant
increases in 8OHdG adduct formation in the SAMP8 testicular tissue
and mature spermatozoa, relative to a control strain.
If aging is associated with oxidative DNA damage to spermatozoa
then we might expect to see these lesions re ected in the incidence of
morbidity in the o spring of ageing fathers. In fact, we see three major
kinds of paternal age-mediated pathology in the o spring; miscarriage,
dominant genetic mutations and complex neurological conditions, as
set out in Table1. One of the  rst paternally-mediated pathologies
to be detected was an increase in the incidence of dominant genetic
diseases in children as an exponential function of their fathers’ age.59
 ese diseases classically include achondroplasia, Apert syndrome
and multiple endocrine neoplasias.60 The traditional explanation
given for the appearance of these conditions is that they represent the
Table 1: Summary of factors that are capable of causing oxidative DNA damage in the male germ line and their consequences for the offspring
Environmental or lifestyle factor Sperm damage Consequences for the offspring References
Smoking Oxidative damage to sperm DNA Increased incidence of childhood cancer 53-55
Age Oxidative damage to sperm DNA Increase in miscarriage
Increase in dominant genetic disease
Increased miscarriage
Increased neurological disorders such as autism, bipolar disease,
spontaneous schizophrenia and epilepsy
Increased death in offspring associated with congenital
malformations, injury and poisoning
Increased risk of cleft palate, diaphragmatic hernia, right
ventricular outfl ow tract obstruction and pulmonary valve stenosis
19,42,56,58-67
Infertility Oxidative damage to sperm DNA Unknown
Possible increase in birth defects
Possible increase in imprinting disorders
Increased hospitalization
42,70-77
Environmental toxicants,
insecticides, herbicides, heavy
metals and so on
Oxidative damage to sperm DNA Unknown 51,52,68

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Asian Journal of Andrology
consequences of replication error in the germ line. As men age, their
germ cells experience multiple rounds of pre-meiotic replication,
and with each cellular iteration, the risk of a mutation occurring as a
consequence of replication error correspondingly increases. In certain
cases, such as the FGFR2( broblast growth factor receptor 2) mutation
associated with Apert syndrome, there does indeed appear to be a
correspondence between the incidence of this mutation in spermatozoa
and the appearance of the condition in children.60 However, the
underlying cause is not just replication error.61  e mutations that
cause this condition are thought to become over-represented in
the sperm population as a consequence of age-dependent clonal
expansion; mutant spermatogonial stem cells having a proliferative
advantage over non-mutated cells. Recent studies suggest that such
mutations occur in clusters within the seminiferous tubules possibly
as a consequence of failures of asymmetrical division within the germ
line.60  is germ line selection model may also explain the origins
of achondroplasia,62 although in this case there does appear to be a
discrepancy between the incidence of the mutation in spermatozoa
and the appearance of the disease in the progeny.63
An alternative explanation for paternal age e ects may be aberrant
repair of oxidative DNA damage in the fertilized oocyte, as suggested
above in the context of smoking.64 Such a mechanism could account for
the increase in miscarriage rates observed as a function of paternal age65
and could also contribute to the etiology of a range of other complex
polygenic conditions that correlate with the age of the father at the
moment of conception.  us, paternal age is also associated with an
increase in the incidence of complex polygenic neurological conditions
in the offspring including epilepsy, spontaneous schizophrenia,
bipolar disease and autism, as well as an increased rate of death in the
F1 generation associated with congenital malformations, injury and
poisoning.19 An analysis of birth defects has also revealed signi cant
associations between paternal age with the etiology of cle palate,
diaphragmatic hernia, right ventricular out ow tract obstruction and
pulmonary valve stenosis.66 As a result of recent studies conducted
on the Icelandic population, there is now powerful incontrovertible
evidence that the mutational load carried by children is correlated with
the age of their fathers at the moment of conception and that once this
load exceeds a certain critical level, overt pathologies such as autism
appear in the o spring.67  e link between this age-dependent increase
in mutational load in children and the aberrant repair of oxidative
sperm DNA damage in the zygote has yet to be de nitively established,
however such a relationship appears probable. Furthermore, given the
range of environmental and lifestyle factors that can in uence oxidative
stress in the germ line from pesticides to electromagnetic radiation,68
the potential contribution of such mechanisms to the integrity of the
human genome is signi cant(Table1).
Pertinent to this debate is the global increase in the use of assisted
reproductive technology(ART) to solve human infertility. In advanced
western countries such as Australia, nearly 4% of newborn children
are the product of assisted conception therapy.69 Since many of these
conceptions will have been triggered by male factor infertility and the
latter involves a high incidence of oxidative DNA damage in the germ
line, it is inevitable that conceptions are being achieved invitro with
severely DNA damaged spermatozoa, that could never have occurred
in vivo.70 One of the consequences of this trend is that we might
anticipate an increase in disease incidence in children conceived using
ART.  e emerging data on this point is suggestive but unsubstantiated.
 us, the incidence of birth defects following ART is approximately
double the background rate and there is also evidence that imprinting
disorders are more frequent in children conceived invitro.71,72 Infants
produced by ART are also signi cantly more likely to be admitted to a
neonatal intensive care unit, to be hospitalized and to stay in hospital
longer than their naturally conceived counterparts.19 Recent studies
have also shown an increase in the hospitalization of ART o spring in
infancy and early childhood compared with spontaneously conceived
children, as well as abnormal patterns of retinal vascularization and an
increase in the incidence of undescended testicles in boys conceived
by ICSI.73–77
Similarly, there are many environmental toxicants(herbicides,
pesticides and so on) that will induce oxidative DNA damage in the
male germ line and are therefore potential contributors to disease
in the offspring.68 Notwithstanding their possible impact, such
transgenerational relationships still remain largely unexplored(Table1 ).
DNA REPAIR IN THE GERM LINE DURING SPERMATOGENESIS
Most of the above discussion has focused on the impact of oxidative
stress at the level of gamete. However, if the oxidative insult is earlier
in spermatogenesis, what are the likely consequences for fertility and
the health and well-being of the o spring? Under these circumstances,
severe oxidative DNA damage in germ cells entering meiosis will
simply precipitate an i ncrease in apoptosis.78 However, milder levels of
oxidative stress might induce compensatory mechanisms on the part
of the germ line that will favor survival of the o spring. An example
of such an e ect might be the impact of paternal ageing on telomere
length. As discussed above, ageing is associated with oxidative stress
in the germ line. One of the ways in which the germ line responds to
the stresses associated with ageing is to upregulate telomerase activity
and increase the length of telomeres in spermatozoa.79 Importantly
telomere length is a paternally inherited trait and so the o spring of
ageing fathers also have longer telomeres.80 Because telomere length
is associated with longevity,81 one of few positive consequences of
having an older father is that he may confer upon his children the
molecular basis for a long life. By contrast, if the paternal germ line
has experienced an oxidative stress post-meiotically, when telomerase
can no longer increase(as is typically the case in infertile patients)
then telomere length in the spermatozoa will be abnormally short and
the implications for the health of ART o spring, potentially serious.82
QUESTIONS FROM THE PANEL
Q1: Which lifestyle factors may cause oxidative stress?
A1:  e factors that we know can cause oxidative stress in the
male germ line are age, subfertility and smoking. However, because
mitochondrial free radical generation is an early feature of apoptosis
in spermatozoa, it is probable that any factor capable of compromising
the vitality of male germ cells will initiate a state of oxidative stress.
Alist of potential factors has been compiled68 and includes exposure
to industrial pollutants such as bisphenol A, insecticides, pesticides,
nonionizing electromagnetic radiation, heavy metals and a variety of
small molecular mass toxicants, all of which are potentially in uenced
by interindividual di erences in occupation and lifestyle.
Q2: What is known about oxidative stress in the mitochondria of
male germ cells including spermatozoa, in response to di erent types
of environmental chemicals(e.g.,phthalates, dioxins and so on)? Is
there any speci city in such responses?
A2: Any factor that causes oxidative stress in the germ line will
automatically trigger mitochondrial ROS generation. It is a central
feature of the intrinsic apoptotic cascade. In addition, exposure to
free unsaturated fatty acids will trigger this activity by impeding
the  ow of electrons along the mitochondrial electron transport
chain.  e physiological signi cance of this association is indicated

Oxidative stress
RJ Aitken et al
34
Asian Journal of Andrology
by the correlation observed between the spontaneous levels of
mitochondrial ROS generation by human spermatozoa and their
cellular content of free arachidonic and decosahexaenoic acids.16,17
A variety of synthetic and natural electrophiles are also capable of
triggering superoxide release from the sperm mitochondria. In this
context, the ability of electrophilic aldehydes(e.g.,4HNE, acrolein and
malondialdehyde) generated as a consequence of lipid peroxidation to
trigger mitochondrial ROS generation is particularly signi cant.18 As
a consequence of this pathway, any environmental factor that triggers
oxidative stress in the germ line will potentiate the generation of further
oxidative stress as a direct result of lipid peroxidation. Environmental
factors such as dioxins are certainly capable of eliciting ROS generation
from sperm mitochondria in an experimental situation.84,85 However,
whether such toxicants contribute signi cantly towards the oxidative
stress observed in association with male infertility and sperm DNA
damage is not currently understood.
Q3: Are earlier stages of spermatogenesis sensitive to ROS, and
if so, does oxidative stress during fetal development play a role in the
decline in sperm quality?
A3: Whether maternal exposure to reproductive toxicants
during pregnancy can cause permanent changes in the germ line that
might subsequently impact the fertility of the F1 generation, and the
health trajectory of their o spring, is another fascinating question to
which we do not yet have a de nitive answer. Much will depend on
the nature and intensity of the oxidative stress. In general, DNA proof
reading and DNA repair in the spermatogonial stem cell population is
excellent as indicated by the low risk of birth defects in the children
of men with a history of cancer treatment.86 However, the stability
of the sperm epigenome may be less certain. Studies involving the
maternal administration of the antiandrogenic endocrine disruptor
vinclozolin, have revealed a transgenerational impact on male fertility
that is mediated by a long-lasting epigenetic change in the male germ
line.87  at epigenetic changes in the germ line might be associated
with impaired semen quality is therefore feasible. Furthermore,
oxidative distress is known to alter the pattern of DNA methylation in
spermatozoa.88 However, whether the creation of oxidative stress in the
male germ line during fetal life can subsequently in uence the fertility
of the male o spring, remains an interesting but unresolved possibility.
CONCLUSIONS
Oxidative stress is a major pathological mechanism responsible for
both male infertility and DNA damage in the germ line. When the
oxidative stress occurs in the mature gamete then 8OHdG adducts are
created that are excised by OGG1; however, the remainder of the BER
pathway is completed in the female germ line. Aberrant or ine cient
repair on the part of the oocyte has the potential to create mutations in
the o spring that will impact upon the latter’s health trajectory.  ere
is strong circumstantial evidence to support such a mechanism in that
high levels of oxidative stress in spermatozoa, due to age or smoking, are
known to increase the burden-of-disease subsequently carried by the
o spring. Mutations in the OGG1 gene are also important contributors
in this respect. Direct evidence for this causative mechanism whereby
the male and female germ lines collude to increase the mutational load
carried by the o spring(oxidative DNA lesions being acquired in the
spermatozoa being followed by imperfect or incomplete repair in the
oocyte) is currently lacking. Furthermore, we do not yet know whether
the range of environmental and lifestyle factors capable of increasing
oxidative DNA damage in human spermatozoa(e.g.,infertility, obesity,
exposure to electromagnetic radiation or environmental toxicants) have
the same degree of impact on the mutation rates in the progeny.  e role
played by the assisted conception industry in facilitating the transfer
of damaged DNA to the oocyte as a consequence of the widespread
use of ICSI is also worthy of detailed scrutiny.
Finally, we do not know whether oxidative insults during fetal or
prepubertal life can have a lasting impact on the genetic integrity of the
germ line with implications for the health trajectory of any o spring.
Studies addressing the impact of ageing on telomere length in the
germ line suggest that early in spermatogenesis, germ cells are capable
of exhibiting adaptive responses that may have a positive impact on
o spring health. As ever, the impact of oxidative stress on reproduction
is a balance of bene t and risk; quantifying the two sides of this delicate
equation will be an important task for the future.
COMPETING INTERESTS
 e authors declare that they have no competing interests.
ACKNOWLEDGMENTS
We are grateful to the Australian Research Council, National Health and Medical
Research Council, the University of Newcastle and the Hunter Medical Research
Council for  nancial support.
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How to cite this article: Aitken RJ, Smith TB, Jobling MS, Baker
MA, De Iuliis GN. Oxidative stress and male reproductive health.
Asian J Androl 2013 Dec 16. doi: 10.4103/1008-682X.122203. [Epub
ahead of print]