Best Practice & Research Clinical Obstetrics and Gynaecology 25 (2011) 287–299

Contents lists available at ScienceDirect

Best Practice & Research Clinical

Oxidative stress
Graham J. Burton, MD, DSc, Professor of Reproductive Biology a, *,
Eric Jauniaux, MD, PhD, Professor in Obstetrics and Fetal Medicine b
a
Centre for Trophoblast Research, University of Cambridge, Cambridge, UK
b
Academic Department of Obstetrics and Gynaecology, EGA Institute of Women Health, Royal Free and University College
London Medical School, London, UK

Keywords:
Considerable evidence implicates oxidative stress in the patho-
placenta physiology of many complications of human pregnancy, and this
oxidative stress topic has now become a major focus of both clinical and basic
antioxidants science research. Oxidative stress arises when the production of
miscarriage reactive oxygen species overwhelms the intrinsic anti-oxidant
pre-eclampsia defences. Reactive oxygen species play important roles as second
messengers in many intracellular signalling cascades aimed at
maintaining the cell in homeostasis with its immediate environ-
ment. At higher levels, they can cause indiscriminate damage to
biological molecules, leading to loss of function and even cell
death. In this chapter, we will review how reactive oxygen species
are generated and detoxified in the human placenta, and what
roles they may play at homeostatic concentrations. We will then
consider their involvement in normal placental development, and
in complications ranging from miscarriage to pre-eclampsia and
premature rupture of the membranes.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Oxygen is often referred to as the Janus gas, as it has both positive benefits and potentially damaging
side-effects for biological systems. Reactivity allows oxygen to participate in high-energy electron
transfers, and hence support the generation of large amounts of adenosine-5-triphosphate (ATP)
through oxidative phosphorylation. This is necessary to permit the evolution of complex multicellular
organisms, but also renders it liable to attack any biological molecule, be it a protein, lipid or DNA.
Consequently, our body is under constant oxidative attack from reactive oxygen species (ROS).

* Corresponding author: Physiological Laboratory, Downing Street, Cambridge CB2 3EG, UK. Tel: þ44 1223 333856.
E-mail address: gjb2@cam.ac.uk (G.J. Burton).

1521-6934/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bpobgyn.2010.10.016
288 G.J. Burton, E. Jauniaux / Best Practice & Research Clinical Obstetrics and Gynaecology 25 (2011) 287–299

A complex system of antioxidant defences has evolved that generally holds this attack in balance. On
occasions, however, this balance can be perturbed, leading to oxidative stress. Because of the multiple
and diverse effects that oxygen toxicity can have on a cell, oxidative stress is best defined in broad
terms as an alteration in the pro-oxidant–antioxidant balance in favour of the former that leads to
potential damage.1 Oxidative stress is now recognised to play a central role in the pathophysiology of
many different disorders, including complications of pregnancy.
The concept of a pro-oxidant–antioxidant balance is central to an understanding of oxidative stress
for several reasons. Firstly, it emphasises that the disturbance may be caused through changes on
either side of the equilibrium (e.g. abnormally high generation of ROS or deficiencies in the antioxidant
defences). Secondly, it highlights the homeostatic concentrations of ROS. Although ROS first came to
the attention of biologists as potentially harmful by-products of aerobic metabolism, it is now rec-
ognised that they play important roles as secondary messengers in many intracellular signalling
pathways.2 Finally, the concept of a balance draws attention to the fact that there will be a graded
response to oxidative stress. Hence, minor disturbances in the balance are likely to lead to homeostatic
adaptations in response to changes in the immediate environment, whereas more major perturbations
may lead to irreparable damage and cell death. The boundary between normal physiological changes
and pathological insults is therefore inevitably indistinct.
The definition of oxidative stress provided above is necessarily broad because the outcome depends
in part on the cellular compartment in which the ROS are generated. There are many potential sources
of ROS, and the relative contributions of these will depend on the environmental circumstances pre-
vailing. As the reactions of ROS are often diffusion-limited, the effects on cell function depend to a large
extent on the biomolecules in the immediate vicinity. Different insults will therefore generate different
outcomes.
A further feature of oxidative stress that affects its clinical presentation is that it rarely occurs in
isolation. It is now appreciated that complex interactions take place between oxidative and other forms
of cell stress, such as endoplasmic reticulum (ER) stress. The clinical manifestation will therefore
depend on the balance of metabolic activities in a particular cell type or organ, and so may vary from
system to system.
In this review, we will consider the main reactive oxygen species and their generation, the principal
antioxidant defences, and then how oxidative stress may be manifested at the maternal–fetal interface
during human pregnancy.

2. Reactive oxygen species

The term ‘reactive oxygen species’ is applied to both free radicals and their non-radical interme-
diates. Free radicals are defined as species containing one or more unpaired electrons, and it is this
incomplete electron shell that confers their high reactivity. Free radicals can be generated from many
elements, but in biological systems it is those involving oxygen and nitrogen that are the most
important (Fig. 1).
Under physiological conditions, the most common oxygen free radical is the superoxide anion (O 2 ),
and mitochondria are considered the principal source.3 The transfer of electrons along the enzymes of
the respiratory chain is not totally efficient, and leakage of electrons on to molecular oxygen, in
particular from complexes I and III, results in the formation of O2 . The rate of formation is determined
by the number of electrons present on the chain, and so is elevated under conditions of hyperoxia and
of raised glucose, as in diabetes. Paradoxically, it is also increased under conditions of hypoxia, when
the reduced availability of oxygen to act as the final electron acceptor for complex IV causes electrons to
accumulate. Under normal conditions, 2% of oxygen consumed is converted to O 2 in the mitochondria
rather than being reduced to water. Because of its charge, O 2 is membrane impermeable, and so
remains within the mitochondrial matrix.
Similarly, superoxide can also be generated through leakage of electrons from the shorter electron
transport chain within the ER.4 The formation of disulphide bonds during protein folding is an
oxidative process, and about 25% of O2 within cells is generated within the ER. This can increase in
cells with a high secretory output, and also under conditions of ER stress when repeated attempts to
refold misfolded proteins may take place.
 G.J. Burton, E. Jauniaux / Best Practice & Research Clinical Obstetrics and Gynaecology 25 (2011) 287–299 289

Fig. 1. The principal reactive oxygen species, their potential origins and detoxification pathways. NADPH, nicotinamide adenine
dinucleotide phosphate.

Other sources of superoxide under physiological conditions include the enzymes nicotinamide
adenine dinucleotide phosphate (NADPH) oxidase, which generates substantial quantities throughout
pregnancy but particularly in early pregnancy,5 cytochrome P450, and other oxido-reductases. Hence,
various growth factors, drugs and toxins cause increased generation of ROS.2 Under pathological
conditions, the enzyme xanthine dehydrogenase becomes an important contributor. This enzyme
degrades purines, xanthine and hypoxanthine to uric acid and, under normal conditions, uses NADþ as
the electron recipient. However, under hypoxic conditions it is proteolytically cleaved to the oxidase
form, which donates electrons to molecular oxygen. This enzyme plays a key role in the reperfusion
phase of ischaemia–reperfusion injury, when its action is augmented by the build up of hypoxanthine
as a result of ATP breakdown during the hypoxic period.
Superoxide is detoxified by the superoxide dismutase enzymes, which convert it to hydrogen
peroxide. Hydrogen peroxide is not a free radical, and so is less reactive than O2. However, it comes
under the term of ROS as it is intimately involved in the generation and detoxification of free radicals.
As it is non-polar, it is able to diffuse through cell and organelle membranes, and hence acts widely as
a second messenger in signal transduction pathways. Hydrogen peroxide is in turn detoxified to water
by the enzymes catalase and glutathione peroxidase. It is important that the antioxidant enzymes act
in concert, as an imbalance in the concentrations of O2 and hydrogen peroxide can result in the
formation of the much more dangerous hydroxyl ion (OH). This reaction is catalysed by free ferrous
ions in the Fenton reaction. The hydroxyl ion has an estimated life of 109 s,1 and reacts with any
biological molecule in its immediate vicinity in a diffusion-limited manner. Because it is so highly
reactive there is no known scavenger of OH.
Excessive generation of superoxide can also lead to interactions with nitric oxide (NO) to form
peroxynitrite (ONOO). Peroxynitrite is a powerful pro-oxidant. As it is capable of diffusing up to 5 mm,
it may affect neighbouring cells.6

3. Antioxidant defences

Enzymatic and non-enzymatic defences inhibit oxidant attack. The enzymatic defences all have
a transition metal at their core, capable of taking on different valences as they transfer electrons during
290 G.J. Burton, E. Jauniaux / Best Practice & Research Clinical Obstetrics and Gynaecology 25 (2011) 287–299

the detoxification process. Two isoforms of superoxide dismutase convert O2 to hydrogen peroxide,
the manganese form that is restricted to the mitochondria, and the copper and zinc form that is located
in the cytosol. The hydrogen peroxide is then broken down to water by the actions of catalase or
glutathione peroxidase, a tetrameric selenoprotein.
The activity of glutathione peroxidase depends on the presence of reduced glutathione (GSH) as
a hydrogen donor. Glutathione is the major cellular thiol redox buffer in cells, and is synthesised in
the cytosol from L-glutamate, L-cysteine and glycine. GSH participates in a large number of detoxifying
reactions forming glutathione disulfide, which is converted back to GSH by the action of glutathione
reductase at the expense of NADPH. The latter is generated through the pentose phosphate pathway,
of which glucose-6-phosphate dehydrogenase is the first enzyme. This enzyme is subject to
common polymorphisms, and decreased activity may compromise GSH concentrations and lead to
embryopathy.7
The non-enzymatic defences include ascorbate (vitamin C) and a-tocopherol (vitamin E). These
again act in concert, with ascorbate being necessary to regenerate reduced a-tocopherol. In addition,
thiol compounds, such a thioredoxin, are capable of detoxifying hydrogen peroxide, but in turn require
converting back to the reduced form by thioredoxin reductase. Ceruloplasmin and transferrin also play
important roles by sequestering free iron ions and so inhibiting the Fenton reaction and production of
OH.
Polymorphisms in the antioxidant enzymes7,8 or dietary restriction of micronutrients, such as
selenium, can thus play an important role in predisposing to oxidative stress and complications of
pregnancy.9

4. Biological actions of reactive oxygen species

At homeostatic levels, ROS have diverse actions on cell function,2 including activation of redox-
sensitive transcription factors and activation of protein kinases. These are described below.

4.1. Activation of redox-sensitive transcription factors

Activation of redox-sensitive transcription factors, such as AP-1, p53 and NF-kB10 regulate the
expression of pro-inflammatory and other cytokines, cell differentiation and apoptosis. Under normal
conditions NF-kB is held inactive by the binding of its inhibitory sub-unit IkB. However, under
conditions of stress, IkB becomes phosphorylated and dissociates from NF-kB, which then translocates
to the nucleus and activates expression of pro-inflammatory and other cytokines. Increased phos-
phorylation of IkB is observed in term placental explants subjected to hypoxia-reoxygenation in vitro,
which provides a model for malperfusion of the placenta in vivo.11 Activation of the pathway is asso-
ciated with increased tissue levels of the proinflammatory enzyme COX-2, interleukin 1ß, increased
secretion of TNF-a, and activation of the apoptotic cascade as evinced by cleavage of caspase 3.12,13 All
these effects can be blocked by the addition of vitamins C and E or sulfasalazine, an inhibitor of NF-kB
activation.

4.2. Activation of protein kinases

With activation of protein kinases, cells respond to a variety of extracellular signals and stress
through a family of mitogen-activated protein kinases (MAPK). Of this family, ROS-induced activation
of extracellular regulated kinases (ERK1/2) generally promotes cell survival and proliferation, whereas
stimulation of p38MAPK (p38) and stress-activated protein kinase–c-Jun amino terminal kinases
(SAPK–JNK) mostly induces apoptosis. p38 and SAPK–JNK are activated by phosphorylation through an
upstream kinase, apoptosis-regulating signal kinase 1 (ASK1). Under normal conditions ASK1 is held
inactive by binding to thioredoxin, but O2 is capable of oxidising the thiol groups in the latter, leading
to a conformational change and its release. Increased phosphorylation of p38, but not SAPK, is observed
in the term placenta after labour compared with control participants delivered by caesarean section.13
ASK1 is also activated in explants exposed to either hypoxia-reoxygenation or hydrogen peroxide, and
is inhibited by addition of vitamins C and E.13 Activation is associated with increased levels of the
 G.J. Burton, E. Jauniaux / Best Practice & Research Clinical Obstetrics and Gynaecology 25 (2011) 287–299 291

soluble receptor for vascular endothelial growth factor (sFlt-1), which has been implicated in the
pathogenesis of pre-eclampsia. Levels of sFlt-1 can be reduced by the addition of vitamins C and E, or
inhibitors of the p38 pathway. They can also be reduced by the addition of sulfasalazine, which indi-
cates considerable interactions and mutual reinforcement between the NF-kB and MAPK signalling
pathways in the placenta.13
The above responses may be considered as physiological adaptive changes to alterations in the
environment aimed at restoring homeostasis. More severe attack by ROS may lead to more extensive
and irreparable cell damage, resulting ultimately in cell death through necrosis or apoptosis. These
more pathological effects are mediated by opening of ion channels, lipid peroxidation, protein modi-
fications and DNA oxidation. These are discussed below.

4.3. Opening of ion channels

Imbalances of ROS lead to loss of intracellular Ca2þ homeostasis, with release of Ca2þ ions from the
endoplasmic reticulum and other stores. The calcium concentration within the ER lumen is much
higher than in the cytosol, reaching millimolar levels. This concentration is maintained by pumps
belonging to the sarco and endoplasmic reticulum calcium ATPase family, and is necessary for the
correct functioning of the protein-folding machinery. ROS are able to activate calcium release channels
in the ER membrane, which include the inositol-1,4,5,triphosphate receptor (IP3R) and the ryanodine
receptor.14
The resultant release of Ca2þ from the ER will activate diverse Ca2þ-sensitive processes within the
cell,14,15 including many of the signalling pathways above. It also has a profound effect on function. The
loss of chaperone activity results in the accumulation of misfolded proteins within the lumen, leading
to further generation of ROS as attempts are made to refold them.4 The accumulation will also stim-
ulate the unfolded protein response (UPR), a highly conserved set of signalling pathways that aim to
restore homeostasis, but, if this fails, will stimulate apoptosis.16,17 The relationship between oxidative
and ER stress will be considered in greater detail later.
The rise in cytosolic Ca2þ ion concentration will also adversely affect mitochondrial function,
including an increase in their own production of ROS and opening of the permeability transition
pore. Opening of the membrane permeability transition is promoted synergistically by increased
Ca2þ ions and oxidation of the thiol groups on proteins in the inner mitochondrial membrane.18 As
a result, the mitochondrial membrane potential and ATP synthesis collapse. If mitochondria
throughout the cell are affected, ATP concentrations fall precipitously, ionic homeostasis is lost and
the cell undergoes primary necrosis. Involvement of a more limited number of organelles, or tran-
sient opening of the pore, may allow ATP to be maintained at levels sufficient to permit apoptosis to
occur instead.19

4.4. Lipid peroxidation

Hydroxyl radicals are capable of causing lipid peroxidation in the plasma membrane or that of any
organelle that contains large quantities of polyunsaturated fatty acid side chains. By abstracting
hydrogen from the hydrocarbon side-chain of a fatty acid, they create a carbon-centred radical, C. If
oxygen is present, this may react to form a peroxyl radical (–C–O–O), which in turn is capable of
abstracting hydrogen from an adjacent fatty acid, so propagating the reaction.
Because vitamin E is lipid-soluble and possesses a hydrophobic tail, it tends to accumulate
within the interior of lipid membranes. Here, it acts as the most important chain-breaker, as it
reacts with lipid peroxyl radicals about four times faster than they can react with adjacent fatty
acid side chains.1
Evidence of lipid peroxidation can be detected using antibodies directed against one of the principal
products, 4-hydroxynonenal. It can be efficiently detoxified in cells by the glutathione S-transferase
group of enzymes, but high levels are associated with loss of membrane fluidity and function, and
activation of the apoptotic cascade.
292 G.J. Burton, E. Jauniaux / Best Practice & Research Clinical Obstetrics and Gynaecology 25 (2011) 287–299

4.5. Protein modifications

Amino acids, both free and in proteins, are a target for oxidative damage. Direct oxidation of the side
chains leads to the formation of carbonyl groups (aldehydes and ketones), and proline, argenine, lysine
and threonine are particularly vulnerable to attack.20 The carbonyl products are stable, and their
detection using enzyme-linked immunosorbent assay or western blotting is the most commonly used
method to assay protein oxidation.
Abstraction of hydrogen ions from the thiol group of cysteine can lead to the formation of disulfide
bonds and abnormal protein folding, in a manner analagous to the activation of ASK1. Abnormal folding
can lead to loss of function, but also protein aggregation and cell death.
Finally, peroxynitrite will react with tyrosine residues to form 3-nitrotyrosine, which can again be
detected immunohistochemically. At physiological levels, protein nitration is thought to be a selective
and reversible process that leads to activation in a manner analogous to phosphorylation, but at higher
levels can be detrimental. Protein nitration in the placenta can therefore have diverse effects, with both
gain and loss of function.21

4.6. DNA oxidation

DNA is attacked principally by OH radicals, and a variety of products can be generated through
reactions with either the DNA bases or the deoxyribose sugars.1 For example, OH can add on to
guanine to produce 8-hydroxy-2’-deoxyguanosine, which may be measured biochemically and
detected immunohistochemically. Attacks on the sugar moieties may cause strand breakages, whereas
those on histone proteins may lead to cross-linkages that interfere with chromatin folding, DNA repair
and transcription. Mutation or aberrant gene expression may therefore result.
Mitochondrial DNA is particularly vulnerable to ROS attack owing to its proximity to the site of O2
generation from the electron transport chain, the lack of histone protection, and the minimal repair
mechanisms that exist. Consequently, damage to mitochondrial DNA is extensive even under normal
conditions, and mutations occur at five to 10 times the rate seen in nuclear DNA.22 As mitochondrial
DNA encodes several proteins, including enzymes of the electron transport chain, mutations may lead
to impaired energy production and the risk of further electron leakage, compounding the original
stress.

5. Relationship between oxidative stress and endoplasmic reticulum stress

Because ROS can activate so many cell processes, it is not surprising that oxidative stress rarely
occurs in isolation, but is usually accompanied by other forms of cell stress. As explained above, close
interactions take place between ROS, mitochondrial and ER function, mediated through Ca2þ release
that potentially constitute a feed–forward system.23
The ER has recently become recognised as a major centre for the co-ordination of cellular responses
to a variety of stressors. In part, this is because protein synthesis accounts for about 30% of the energy
expenditure of a cell, and so needs to be finely tuned to oxygen and nutrient availability. Under
conditions of stress, the UPR aims to restore homeostasis by a co-ordinated set of responses that reduce
the burden of misfolded proteins. Firstly, it blocks the influx of new proteins through phosphorylation
and inhibition of the eukaryotic initiation factor eIF2a, which regulates the initiation of translation.
Secondly, it increases the expression of the ER chaperone proteins GRP78 and 94 in an attempt to
sequester or refold the misfolded proteins. Thirdly, it increases the synthesis of ER cisternae, and finally
it stimulates the ER-associated degradation machinery.16,17 If these attempts fail and ER stress persists,
then the UPR will activate the apoptotic cascade through increased expression of the C/EBP homolo-
gous protein in order to eliminate the cell.
A further aspect of ER stress that is of particular relevance to pregnancy is its relationship to
pro-inflammatory pathways. Activation of these pathways can occur through at least two mech-
anisms. The first involves the NF-kB pathway. One of the three signalling transducers activated
during initiation of the UPR, the inositol-requiring protein-1 (Ire1) pathway, has dual actions. Ire1
contains an endoribonuclease domain, which when activated splices XBP-1 pre-mRNA to produce
 G.J. Burton, E. Jauniaux / Best Practice & Research Clinical Obstetrics and Gynaecology 25 (2011) 287–299 293

the transcription factor, XBP-1, that stimulates transcription of genes regulating the breakdown of
misfolded proteins and ER biogenesis. Ire1 also contains a Ser/Thr kinase domain that is capable of
activating the NF-kB pathway through phosphorylation of IkB, and the p38 and SAPK–JNK path-
ways through ASK1. In addition, activation of NF-kB can also occur as a consequence of inhibition
of protein translation secondary to phosphorylation of eIF2a, as the half-life of IkB is shorter than
that of NF-kB.24
The second mechanism is not related directly to activation of the UPR, but involves proteins
whose predicted structure is similar to that of one of the other signal transducing proteins, acti-
vating transcription factor.17 In the liver, cyclic AMP response element binding protein hepatocyte
is thought to reside in the ER membrane but is released upon ER stress to activate acute-phase
genes. This leads to the increased secretion of acute-phase response proteins, such as C-reactive
protein.
Of the two stresses, oxidative and ER stress, it is likely that latter will be detected at lower levels of
insults as the UPR is a homeostatic mechanism, whereas the commonly used markers of oxidative
stress reflect cell injury.

6. Oxidative stress at the maternal–fetal interface

From the preceding descriptions, it can be seen that oxidative stress may induce a range of cellular
responses depending upon the severity of the insult and the compartment in which the ROS are
generated. Some of the more major signalling pathways involved and potential outcomes are pre-
sented in Fig. 2. The close interaction between oxidative stress and ER stress is important when
considering potential therapeutic interventions, as there will be little benefit in addressing one of the
stresses without addressing the other.
Oxidative stress is manifested at the maternal–fetal interface from early pregnancy onwards. It
plays a role in both the normal development of the placenta as well as in the pathophysiology of
complications such as miscarriage, pre-eclampsia, intrauterine growth restriction (IUGR), and
premature rupture of the membranes.25,26 The more important aspects will be considered in turn.

Fig. 2. How reactive oxygen species may be generated within the syncytiotrophoblast, and the principal consequences for the
function of the tissue. CHOP, C/EBP homologous protein; NADPH, nicotinamide adenine dinucleotide phosphate; ROS, reactive
oxygen species; UPR, unfolded protein response.
294 G.J. Burton, E. Jauniaux / Best Practice & Research Clinical Obstetrics and Gynaecology 25 (2011) 287–299

6.1. Oxidative stress and placental remodelling

The human placenta is unique because villi form initially over the entire surface of the chorionic
sac. Starting towards the end of the first trimester, however, the villi over the superficial pole
regress to leave the definitive discoid placenta. It is now recognised that oxidative stress plays
a central role in this process, and as regression occurs in all pregnancies this can be considered
physiological.
Our appreciation of the intrauterine environment during human early pregnancy has undergone
a radical revision over the last two decades. It is now accepted that placental development occurs in
a relatively low oxygen concentration, supported by secretions from the endometrial glands rather
than the maternal circulation 27,28 We have speculated that this environment protects the developing
embryo from oxygen free radical mediated teratogenesis.29 Maternal arterial blood is prevented from
entering the intervillous space of the placenta by plugs of extravillous cytotrophoblast cells that
invade down the mouths of the uterine spiral arteries as part of the process of physiological
conversion.30,31 The maternal intraplacental circulation is only fully established towards the end of
the first trimester, when these plugs dislocate through a mechanism that is currently unknown.
Ultrasonographic evidence has shown that the circulation starts preferentially in the periphery of the
placenta, where trophoblast invasion is the least extensive, and progressively extends into the central
region.32
Onset of the circulation is associated with a three-fold rise in the oxygen concentration within the
placenta.27 This will stimulate higher rates of generation of ROS, particularly in the critical syncytio-
trophoblastic layer, which contains low concentrations of the principal antioxidant enzymatic
defences, copper and zinc superoxide dismutase and catalase.33,34 Consequently, villi sampled from the
peripheral region of the placenta demonstrate elevated levels of the chaperone protein HSP70,
nitrotyrosine residues indicative of peroxynitrite formation, and morphological evidence of degener-
ative changes within the syncytiotrophoblast compared with those from the central region.32
Molecular evidence confirms that the apoptotic cascade is activated in the peripheral villi and, over
an extended period, this would be sufficient to account for their regression.

6.2. Oxidative stress and spontaneous miscarriage

In cases of miscarriage, onset of the maternal intraplacental circulation is both precocious and
disorganised compared with normal ongoing pregnancies.27,32 Thus it starts at an earlier stage, and
occurs randomly throughout the placenta. This is probably because, in 70% of these cases, extravillous
trophoblast invasion is superficial, and consequently plugging of the spiral arteries is less
complete.35,36 As might be expected, levels of HSP70 and nitrotyrosine are increased in villi sampled
from the central region of these placentas as well as in the periphery.32,37 The apoptotic index is also
increased compared with control placentas of a similar gestational age, and there is morphological
evidence of degenerate syncytiotrophoblast sloughing off in some areas. In these cases, it seems that
overwhelming oxidative stress causes widespread destruction of the trophoblast, incompatible with an
ongoing pregnancy.
In confirmation of these findings, lipid peroxides have been shown to be increased in villous and
decidual tissues, and the serum of women undergoing pregnancy loss.7,38–40 Also, elevated
concentrations of protein carbonyls and DNA damage have been reported in cases of complete
hydatidiform mole, where plugging of the spiral arteries by extravillous trophoblast is equally
deficient.41
Onset of the maternal circulation clearly represents an oxidative challenge to the human placenta.
Even in normal pregnancies, a burst of oxidative stress in the placenta takes place at 10–12 weeks of
gestation, which resolves as the placental tissues adapt to their new oxygen environment. That
adaptation involves an increase in the expression and activity of the principal antioxidant enzymes.27
The efficacy of the antioxidant defences provides the other half of the pro-oxidant-antioxidant balance
that determines the oxidative status of a tissue. Thus, polymorphisms in the enzymes detoxifying ROS
have been linked to an increased risk of miscarriage.8,42,43 Equally, some evidence shows that selenium
deficiency, which will reduce the efficacy of glutathione peroxidase, is associated with miscarriage.9,44
 G.J. Burton, E. Jauniaux / Best Practice & Research Clinical Obstetrics and Gynaecology 25 (2011) 287–299 295

Conversely, it has been reported that administration of the antioxidant N-acetyl cysteine to women
suffering recurrent pregnancy loss improves the take-home baby rate.45
Further research is therefore required to test prospectively whether dietary supplementation with
micronutrients or antioxidants can reduce the rate of spontaneous miscarriage.

6.3. Placental oxidative stress in pre-eclampsia

Normal pregnancy is said to be a condition of oxidative stress, as circulating levels of oxidised low-
density lipoproteins increase and the total antioxidant capacity in pregnant women decreases
compared with non-pregnant women.46 Pregnancy is also associated with a systemic inflammatory
response, as evinced by activation of peripheral granulocytes, monocytes and lymphocytes during the
third trimester, all of which produce ROS.47 These states are therefore obviously interlinked, and
capable of forming dangerous feed–forward systems.
Oxidative stress and the systemic inflammatory response are observed to a much greater degree in
pre-eclampsia.48 There is irrefutable evidence of placental oxidative stress in cases of early onset pre-
eclampsia, including increased concentrations of protein carbonyls, lipid peroxides, nitrotryosine resi-
dues and DNA oxidation.49,50 The cause for the oxidative stress is thought to be vascular, because early
onset pre-eclampsia is associated with deficient conversion of the spiral arteries. In particular, the
myometrial segments of the arteries are adversely affected.51–53 As the myometrial segment contains
a highly contractile portion of the artery, we have proposed that failure to convert this section results in
intermittent perfusion of the placenta, and a low-grade ischaemia-reperfusion type injury.11,25 In support
of this hypothesis, we have shown that hypoxia-reoxygenation in vitro is a potent inducer of oxidative
stress in term placental explants, much more than hypoxia alone.11 Exposure of explants to changes in
oxygenation causes generation of ROS within the trophoblast and endothelial cells, as shown by fluo-
rescent markers and the formation of nitrotyrosine residues in a pattern matching closely to that seen in
pre-eclamptic placentas. Furthermore, labour, in which the placenta is exposed to repeated episodes of
ischaemia–reperfusion, induces high levels of oxidative stress.54 This is associated with increased
xanthine oxidase activity,55 and changes in gene expression that mimic those seen in pre-eclampsia.54
Another potential source of oxidative stress in pre-eclampsia is autoantibodies against the angiotensin
AT1 receptor.56 These antibodies stimulate NADPH oxidase, leading to an increase in ROS production.
In the classic two-stage model of pre-eclampsia, oxidative stress induced in the placenta is thought
to cause the release of factors into the maternal circulation that stimulate the inflammatory response
and activation of the maternal endothelial cells.48 Many placental factors have been implicated,
including microparticulate apoptotic debris, pro-inflammatory cytokines and angiogenic factors.13,48,57
To date, no single factor has been indentified that can account for all cases of pre-eclampsia. This may
indicate that the true causation has yet to be discovered, or that the syndrome is capable of being
initiated by a variety of different stimuli.
Early onset pre-eclampsia is almost invariably associated with IUGR, and we have recently reported
morphological and molecular evidence of high levels of ER stress in these placentas.58 Strong phos-
phorylation of eIF2a will cause suppression of protein synthesis. Consequently, the level of cyclin D1,
a kinase that plays a central role in the regulation of cell proliferation, is significantly reduced.
Induction of similar stress in trophoblast-like cell lines causes a reduction in their proliferation rate.
Increased expression of the pro-apoptotic C/EBP homologous protein also takes place. This combina-
tion would provide a sufficient cause for the placental growth restriction observed. In addition, the
high levels of ER stress may contribute to the inflammatory response by stimulating the p38 and NF-kB
pathways. Hence, we speculate that both ER stress and oxidative stress contribute to the placental
pathophysiology in pre-eclampsia in a mutually reinforcing fashion.50
This weight of evidence provides a strong rationale for considering antioxidants as a potential
therapy for pre-eclampsia. Unfortunately, recent trials of vitamins C and E in a number of different
settings have not proved successful.59–61 This failure contrasts strikingly to the beneficial effects
observed on specific signal transduction pathways and placental outcomes to oxidative challenge in
vitro.12 The difference may result from the ability of the vitamins to access the relevant trophoblast cell
compartment in the necessary concentration in vivo. Alternatively, in the clinical trials, the antioxidants
are only given once pregnancy is established, by which time the feed–forward cycles may already be
296 G.J. Burton, E. Jauniaux / Best Practice & Research Clinical Obstetrics and Gynaecology 25 (2011) 287–299

established. It is notable that multivitamin usage during the periconceptional period is associated with
a reduced risk of pre-eclampsia among lean or normal weight women.62,63 Conversely, women with
a low dietary intake of vitamin C have been reported to have a trend towards increased risk.64 These
data suggest that more attention needs to be focused on ensuring optimal health and diet in women
planning to conceive.

6.4. Placental oxidative stress in intra-uterine growth restriction

IUGR can have many causes, but most cases that have no genetic or infectious cause are thought to
arise from compromise of the maternal circulation to the placenta. This conclusion is based on the
common association with high resistance uterine arterial waveforms, but also on earlier morphological
studies of the spiral arteries. The investigators reported deficient physiological conversion of the
arteries as in pre-eclampsia, but to a lesser degree, especially in the myometrial segment.52 Indeed,
a positive correlation was observed in one study between the birth weight and the degree of
conversion.51 One might predict, therefore, that the vascular insult is less severe in cases of IUGR alone,
but this requires confirmation.
In contrast to pre-eclampsia, surprisingly little attention has been paid to the role of placental
oxidative stress in cases of IUGR alone. The scanty evidence available indicates that the level of stress is
either similar to the normal control or intermediate between a normal control and an early onset pre-
eclamptic placenta,50,65 These findings are in keeping with the predicted vascular insult above.
However, we do still observe morphological and molecular evidence of ER stress in these placentas.58
The level of stress is lower than in pre-eclampsia, with no evidence of activation of the apoptotic
cascade. But it is sufficient to cause phosphorylation of eIF2a and suppression of protein synthesis, with
a reduction in the level of cyclin D1. ER stress can be induced by malperfusion through disturbances of
Ca2þ homeostasis. Hence, we speculate that chronic low-level ER stress may occur from the time of
onset of the maternal circulation onwards, and leads to a lower growth trajectory of the placenta. This
is consistent with the reduced rate of growth of the placenta observed with serial ultrasound scans in
these cases66

6.5. Preterm premature rupture of the membranes

Oxidative stress has been implicated in preterm premature rupture of the membranes, a condition
associated with proteolytic degradation of the collagen fibres in the chorio-amnion. In this condition,
increase in the generation of ROS may arise from infection and inflammation, cigarette smoking, vaginal
bleeding and the release of free iron, and cocaine abuse which leads to ischaemia–reperfusion.67 It has
also been shown that deficient conversion of the spiral arteries predisposes to this condition.68 Exposure
of the chorio-amnion to O2 in vitro results in up-regulation of matrix metalloproteinase-9, which can
be suppressed by the antioxidant N-acetylcysteine.67 Intriguingly, pre-treatment with vitamins C and E
protects the membranes against oxidative attack in vitro, but whether they can be effective in vivo
remains to be determined.

7. Conclusion

The role of ROS in human placentation and complications of pregnancy is a relatively recent field,
but one that is expanding rapidly. It is increasingly apparent that they play a central role in many signal
transduction pathways, and it is important to recognise that homeostatic concentrations are present in
all tissues. Excessive antioxidant intake could potentially threaten and inactivate these vital signalling
responses, with adverse consequences. At excessive concentrations, whether caused by increased
generation or reduced detoxification, ROS can cause widespread and indiscriminate damage to cells
and tissues. ROS can be generated through many pathways within cells, but the mitochondria, ER and
enzymes such as NADPH oxidase are the most important sources. These pathways can respond to
a variety of stimuli, but arguably the most important ones for pregnancy are perturbations in the
maternal blood supply to the placenta and inflammation. Because of interactions between downstream
signalling pathways, oxidative stress rarely occurs in isolation, but usually interacts with ER and other
 G.J. Burton, E. Jauniaux / Best Practice & Research Clinical Obstetrics and Gynaecology 25 (2011) 287–299 297

forms of cell stress. We propose that spontaneous miscarriage, early onset pre-eclampsia and IUGR
represent a spectrum of placental oxidative stress-related disorders, secondary to deficient trophoblast
invasion and physiological conversion of the spiral arteries. Although trials of antioxidant interventions
have not proved successful in pre-eclampsia, the rationale for this treatment remains strong, and
further research of the benefits of periconceptional supplementation is required.

Practice points

 The maternal circulation to the placenta is not fully established until towards the end of the
first trimester in normal human pregnancies.
 The establishment of the maternal circulation is a progressive phenomenon that modulates
the development of the definitive placenta.
 That deficient conversion of the maternal spiral arteries underlies placental-related
complications of pregnancy, such as miscarriage and pre-eclampsia.
 Fluctuations in maternal blood flow to the placenta may be of greater pathological conse-
quence than hypoxia alone.
 The pathophysiology of miscarriage is linked to a premature and overwhelming entry of
maternal blood inside the placenta, whereas that of pre-eclampsia is linked to an ischaemia-
reperfusion phenomenon.

Research agenda

To establish the following:

 Factors that regulate spiral arterial remodelling and unplugging of the spiral arteries
 Importance of polymorphisms in the antioxidant pathways for complications of pregnancy
 Factors released from the placenta that lead to the maternal inflammatory response
 Importance of preconceptional nutrition to the risk of miscarriage and pre-eclampsia
 Why antioxidants vitamins taken during pregnancy have no effect on rates of pre-eclampsia
or miscarriage
 The role of environmental oxidative stress on placental-related disorders of pregnancy.

Acknowledgements

We thank all our colleagues and collaborators who have contributed to these studies over the years,
in particular our research associates Drs T Cindrova-Davies, J Hempstock, J Johns, A Watson and H-W
Yung. We are grateful to the MRC, Tommy’s the baby Charity, WellBeing of Women and the Wellcome
Trust for funding.

References

1. Halliwell B & Gutteridge JMC. Free radicals in biology and medicine. 3rd ed. Oxford: Oxford Science Publications, 1999.
*2. Droge W. Free radicals in the physiological control of cell function. Physiol Rev 2002; 82: 47–95.
3. Cadenas E & Davies KJA. Mitochondrial free radical generation, oxidative stress, and aging. Free Rad Biol Med 2000; 29:
222–230.
4. Tu BP & Weissman JS. Oxidative protein folding in eukaryotes: mechanisms and consequences. J Cell Biol 2004; 164:
341–346.
5. Raijmakers MT, Burton GJ, Jauniaux E et al. Placental NAD(P)H oxidase mediated superoxide generation in early pregnancy.
Placenta 2006; 27: 158–163.
6. Pacher P, Beckman JS & Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev 2007; 87: 315–424.
*7. Nicol CJ, Zielenski J, Tsui L-C et al. An embryoprotective role for glucose-6-phosphate dehydrogenase in developmental
oxidative stress and chemical teratogenesis. FASEB J 2000; 14: 111–127.
8. Tempfer C, Unfried G, Zeillinger R et al. Endothelial nitric oxide synthase gene polymorphism in women with idiopathic
recurrent miscarriage. Hum Reprod 2001; 16: 1644–1647.
298 G.J. Burton, E. Jauniaux / Best Practice & Research Clinical Obstetrics and Gynaecology 25 (2011) 287–299

9. Al-Kunani AS, Knight R, Haswell SJ et al. The selenium status of women with a history of recurrent miscarriage. BJOG 2001;
108: 1094–1097.
10. Arrigo AP & Kretz-Remy C. Regulation of mammalian gene expression by free radicals. In Aruoma OI & Halliwell B (eds.).
Molecular biology of free radicals in human diseases. Saint Lucia: OICA International, 1998, pp. 183–223.
11. Hung TH, Skepper JN & Burton GJ. In vitro ischemia-reperfusion injury in term human placenta as a model for oxidative
stress in pathological pregnancies. Am J Pathol 2001; 159: 1031–1043.
12. Cindrova-Davies T, Spasic-Boskovic O, Jauniaux E et al. Nuclear factor-kappa B, p38, and stress-activated protein kinase
mitogen-activated protein kinase signaling pathways regulate proinflammatory cytokines and apoptosis in human
placental explants in response to oxidative stress: effects of antioxidant vitamins. Am J Pathol 2007; 170: 1511–1520.
*13. Cindrova-Davies T. From placental oxidative stress to maternal endothelial dysfunction. Placenta Suppl A 2009; 30:
S55–S65.
14. Hool LC & Corry B. Redox control of calcium channels: from mechanisms to therapeutic opportunities. Antioxid Redox
Signal 2007; 9: 409–435.
15. Berridge MJ, Bootman MD & Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell
Biol 2003; 4: 517–529.
16. Xu C, Bailly-Maitre B & Reed JC. Endoplasmic reticulum stress: cell life and death decisions. J Clin Invest 2005; 115: 2656–2664.
17. Ron D & Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 2007; 8:
519–529.
18. Kowaltowski AJ, Castilho RF & Vercesi AE. Mitochondrial permeability transition and oxidative stress. FEBS Lett 2001; 495:
12–15.
19. Leist M, Single B, Castoldi AF et al. Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision
between apoptosis and necrosis. J Exp Med 1997; 185: 1481–1486.
20. Dalle-Donne I, Rossi R, Giustarini D et al. Protein carbonyl groups as biomarkers of oxidative stress. Clin Chim Acta 2003;
329: 23–38.
*21. Myatt L. Review: reactive oxygen and nitrogen species and functional adaptation of the placenta. Placenta 2010; 31(suppl):
S66–S69.
22. Richter C, Park J-W & Ames BN. Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc Nat Acad Sci
USA 1988; 85: 6465–6467.
*23. Zhang K. Integration of ER stress, oxidative stress and the inflammatory response in health and disease. Int J Clin Exp Med
2010; 3: 33–40.
24. Zhang K & Kaufman RJ. From endoplasmic-reticulum stress to the inflammatory response. Nature 2008; 454:
455–462.
25. Burton GJ & Jauniaux E. Placental oxidative stress; from miscarriage to preeclampsia. J Soc Gynecol Invest 2004; 11: 342–352.
*26. Jauniaux E, Poston L & Burton GJ. Placental-related diseases of pregnancy: involvement of oxidative stress and implications
in human evolution. Hum Reprod Update 2006; 12: 747–755.
*27. Jauniaux E, Watson AL, Hempstock J et al. Onset of maternal arterial bloodflow and placental oxidative stress; a possible
factor in human early pregnancy failure. Am J Pathol 2000; 157: 2111–2122.
28. Burton GJ, Watson AL, Hempstock J et al. Uterine glands provide histiotrophic nutrition for the human fetus during the
first trimester of pregnancy. J Clin Endocrinol Metab 2002; 87: 2954–2959.
29. Burton GJ, Hempstock J & Jauniaux E. Oxygen, early embryonic metabolism and free radical-mediated embryopathies.
Reprod BioMed Online 2003; 6: 84–96.
30. Hustin J & Schaaps JP. Echographic and anatomic studies of the maternotrophoblastic border during the first trimester of
pregnancy. Am J Obstet Gynecol 1987; 157: 162–168.
31. Burton GJ, Jauniaux E & Watson AL. Maternal arterial connections to the placental intervillous space during the first
trimester of human pregnancy; the Boyd Collection revisited. Am J Obstet Gynecol 1999; 181: 718–724.
32. Jauniaux E, Hempstock J, Greenwold N et al. Trophoblastic oxidative stress in relation to temporal and regional differences
in maternal placental blood flow in normal and abnormal early pregnancies. Am J Pathol 2003; 162: 115–125.
33. Watson AL, Palmer ME, Jauniaux E et al. Variations in expression of copper/zinc superoxide dismutase in villous
trophoblast of the human placenta with gestational age. Placenta 1997; 18: 295–299.
34. Watson AL, Skepper JN, Jauniaux E et al. Changes in the concentration, localisation and activity of catalase within the
human placenta during early gestation. Placenta 1998; 19: 27–34.
35. Hustin J, Jauniaux E & Schaaps JP. Histological study of the materno-embryonic interface in spontaneous abortion. Placenta
1990; 11: 477–486.
36. Jauniaux E, Zaidi J, Jurkovic D et al. Comparison of colour doppler features and pathologic findings in complicated early
pregnancy. Hum Reprod 1994; 9: 243–247.
37. Hempstock J, Jauniaux E, Greenwold N et al. The contribution of placental oxidative stress to early pregnancy failure. Hum
Pathol 2003; 34: 1265–1275.
38. Sugino N, Kakata M, Kashida S et al. Decreased superoxide dismutase expression and increased concentrations of lipid
peroxide and prostaglandin F(2alpha) in the decidua of failed pregnancy. Mol Hum Reprod 2000; 6: 642–647.
39. Biri A, Kavutcu M, Bozkurt N et al. Investigation of free radical scavenging enzyme activities and lipid peroxidation in
human placental tissues with miscarriage. J Soc Gynecol Investig 2006; 13: 384–388.
40. Toy H, Camuzcuoglu H, Camuzcuoglu A et al. Decreased serum prolidase activity and increased oxidative stress in early
pregnancy loss. Gynecol Obstet Invest 2010; 69: 122–127.
41. Harma M. Defective placentation and resultant oxidative stress play a similar role in complete hydatidiform mole to that in
preeclampsia and early pregnancy loss. Med Hypotheses 2006; 66: 100–102.
42. Sata F, Yamada H, Kondo T et al. Glutathione S-transferase M1 and T1 polymorphisms and the risk of recurrent pregnancy
loss. Mol Hum Reprod 2003; 9: 165–169.
43. Sata F, Yamada H, Yamada A et al. A polymorphism in the CYP17 gene relates to the risk of recurrent pregnancy loss. Mol
Hum Reprod 2003; 9: 725–728.
 G.J. Burton, E. Jauniaux / Best Practice & Research Clinical Obstetrics and Gynaecology 25 (2011) 287–299 299

44. Zachara BA, Dobrzynski W, Trafikowska U et al. Blood selenium and glutathione peroxidases in miscarriage. BJOG 2001;
108: 244–247.
45. Amin AF, Shaaban OM & Bediawy MA. N-acetyl cysteine for treatment of recurrent unexplained pregnancy loss. Reprod
Biomed Online 2008; 17: 722–726.
46. Belo L, Caslake M, Santos-Silva A et al. LDL size, total antioxidant status and oxidised LDL in normal human pregnancy:
a longitudinal study. Atherosclerosis 2004; 177: 391–399.
47. Redman CW & Sargent IL. Pre-eclampsia, the placenta and the maternal systemic inflammatory response–a review.
Placenta 2003; 24(Suppl. A): S21–S27.
*48. Redman CW & Sargent IL. Placental stress and pre-eclampsia: a revised view. Placenta 2009; 30(Suppl. A): S38–S42.
49. Myatt L & Cui X. Oxidative stress in the placenta. Histochem Cell Biol 2004; 122: 369–382.
*50. Burton GJ, Yung HW, Cindrova-Davies T et al. Placental endoplasmic reticulum stress and oxidative stress in the patho-
physiology of unexplained intrauterine growth restriction and early onset preeclampsia. Placenta 2009; 30(Suppl. A):
S43–S48.
51. Gerretsen G, Huisjes HJ & Elema JD. Morphological changes of the spiral arteries in the placental bed in relation to pre-
eclampsia and fetal growth retardation. Br J Obstet Gynaecol 1981; 88: 876–881.
52. Khong TY, De Wolf F, Robertson WB et al. Inadequate maternal vascular response to placentation in pregnancies
complicated by pre-eclampsia and by small-for-gestational age infants. Br J Obstet Gynaecol 1986; 93: 1049–1059.
*53. Meekins JW, Pijnenborg R, Hanssens M et al. A study of placental bed spiral arteries and trophoblast invasion in normal
and severe pre-eclamptic pregnancies. Br J Obstet Gynaecol 1994; 101: 669–674.
54. Cindrova-Davies T, Yung HW, Johns J et al. Oxidative stress, gene expression, and protein changes induced in the human
placenta during labor. Am J Pathol 2007; 171: 1168–1179.
55. Many A & Roberts JM. Increased xanthine oxidase during labour-implications for oxidative stress. Placenta 1997; 18:
725–726.
56. Dechend R, Viedt C, Muller DN et al. AT1 receptor agonistic antibodies from preeclamptic patients stimulate NADPH
oxidase. Circulation 2003; 107: 1632–1639.
57. Redman CW & Sargent IL. Latest advances in understanding preeclampsia. Science 2005; 308: 1592–1594.
58. Yung HW, Calabrese S, Hynx D et al. Evidence of placental translation inhibition and endoplasmic reticulum stress in the
etiology of human intrauterine growth restriction. Am J Pathol 2008; 173: 451–462.
59. Poston L, Briley AL, Seed PT et al. Vitamin C and vitamin E in pregnant women at risk for pre-eclampsia (VIP trial):
randomised placebo-controlled trial. Lancet 2006; 367: 1145–1154.
60. Roberts JM, Myatt L, Spong CY et al. Vitamins C and E to prevent complications of pregnancy-associated hypertension. N
Engl J Med 2010; 362: 1282–1291.
61. Xu H, Perez-Cuevas R, Xiong X et al. An international trial of antioxidants in the prevention of preeclampsia (INTAPP). Am
J Obstet Gynecol 2010; 202: 239.e1–239.e10.
62. Bodnar LM, Tang G, Ness RB et al. Periconceptional multivitamin use reduces the risk of preeclampsia. Am J Epidemiol
2006; 164: 470–477.
63. Catov JM, Nohr EA, Bodnar LM et al. Association of periconceptional multivitamin use with reduced risk of preeclampsia
among normal-weight women in the Danish National Birth Cohort. Am J Epidemiol 2009; 169: 1304–1311.
64. Klemmensen A, Tabor A, Osterdal ML et al. Intake of vitamin C and E in pregnancy and risk of pre-eclampsia: prospective
study among 57 346 women. BJOG 2009; 116: 964–974.
65. Wikstrom AK, Nash P, Eriksson UJ et al. Evidence of increased oxidative stress and a change in the plasminogen activator
inhibitor (PAI)-1 to PAI-2 ratio in early-onset but not late-onset preeclampsia. Am J Obstet Gynecol 2009; 201:
597.e1–597.e8.
66. Hafner E, Metzenbauer M, Hofinger D et al. Placental growth from the first to the second trimester of pregnancy in SGA-
foetuses and pre-eclamptic pregnancies compared to normal foetuses. Placenta 2003; 24: 336–342.
67. Woods Jr. JR. Reactive oxygen species and preterm premature rupture of membranes-a review. Placenta 2000; 22(Suppl.
A): 38–44.
68. Kim YM, Chaiworapongsa T, Gomez R et al. Failure of the physiologic transformation of the spiral arteries in the placental
bed in preterm premature rupture of membranes. Am J Obstet Gynecol 2002; 187: 1137–1142.