Placental oxidative stress: From miscarriage to preeclampsia

REVIEW ARTICLES

Placental Oxidative Stress: From Miscarriage to Preeclampsia Graham J. Burton, MD, and Eric Jauniaux, MD, PhD OBJECTIVE: To review the role of oxidative stress in two common placental-related disorders of pregnancy, miscarriage and preeclampsia. METHODS: Review of published literature. RESULTS: Miscarriage and preeclampsia manifest at contrasting stages of pregnancy, yet both have their roots in deficient trophoblast invasion during early gestation. Early after implantation, endovascular trophoblast cells migrate down the lumens of spiral arteries, and are associated with their physiological conversion into flaccid conduits. Initially these cells occlude the arteries, limiting maternal blood flow into the placenta. The embryo therefore develops in a low oxygen environment, protecting differentiating cells from damaging free radicals. Once embryogenesis is complete, the maternal intervillous circulation becomes fully established, and intraplacental oxygen concentration rises threefold. Onset of the circulation is normally a progressive periphery-center phenomenon, and high levels of oxidative stress in the periphery may induce formation of the chorion laeve. If trophoblast invasion is severely impaired, plugging of the spiral arteries is incomplete, and onset of the maternal intervillous circulation is premature and widespread throughout the placenta. Syncytiotrophoblastic oxidative damage is extensive, and likely a major contributory factor to miscarriage. Between these two extremes will be found differing degrees of trophoblast invasion compatible with ongoing pregnancy but resulting in deficient conversion of the spiral arteries and an ischemia-reperfusiontype phenomenon. Placental perfusion will be impaired to a greater or lesser extent, generating commensurate placental oxidative stress that is a major contributory factor to preeclampsia. CONCLUSION: Miscarriage, missed miscarriage, and early- and late-onset preeclampsia represent a spectrum of disorders secondary to deficient trophoblast invasion. (J Soc Gynecol Investig 2004;11: 342–52) Copyright © 2004 by the Society for Gynecologic Investigation.

KEY

WORDS:

Oxidative stress, placenta, pregnancy, miscarriage, preeclampsia.

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bnormalities of human placentation are associated with disorders that are either unique to our species, such as preeclampsia, or very rare in other species, such as miscarriage. There is considerable evidence implicating placental oxidative stress in the pathogenesis of preeclampsia,1,2 and increasing evidence indicating that it may also contribute to early pregnancy failure.3,4 Our hypothesis is that these disorders, although manifesting at contrasting stages of pregnancy, represent different points on a spectrum of placental stress induced by changes in the intraplacental oxygen concentration. Underlying these changes is a common deficit in trophoblast invasion during the first and early second trimesters of pregnancy, and hence incomplete conversion of the endometrial spiral arteries. We review here the anatomic, physiologic, and pathologic evidence in support of this concept. From the Department of Anatomy, University of Cambridge, Cambridge; and the Academic Department of Obstetrics and Gynaecology Royal Free and University College London Medical School, London, United Kingdom. This work was supported by a grant from the WellBeing Charity, London, United Kingdom The authors are grateful to Dee Hughes for preparing the final version of Figure 4. Address correspondence and reprint requests to: Graham J. Burton, MD, Department of Anatomy, Downing Street, Cambridge CB2 3DY, United Kingdom. E-mail: [email protected] Copyright © 2004 by the Society for Gynecologic Investigation. Published by Elsevier Inc.

FREE RADICALS AND REACTIVE OXYGEN SPECIES Evolution from unicellular life in the oceans to multicellular life on land has been associated with remarkable metabolic changes linked to the increasing demand for energy required to live, grow, and reproduce. Energy transformation of dietary proteins, carbohydrates, and fats occurs mainly in the mitochondria of animal cells through a series of oxidation-reduction reactions, and the energy released in these reactions is used to phosphorylate adenosine diphosphate (ADP), thus generating adenosine triphosphate (ATP). The final step of this process uses oxygen (O2) as an electron recipient, and this became possible some 2 billion years ago with the accumulation of O2 in the atmosphere due to the photosynthetic activities of cyanobacteria.5 The entire process is known as oxidative phosphorylation, and ATP is pivotal as the storage form of the chemical energy required to drive many biochemical reactions in the cell, in particular, protein biosynthesis, active transport of molecules through cellular membranes, ionic homeostasis, and muscular contractions. Most of the O2 used during the oxidation of dietary organic molecules is converted into water through the integrated ac1071-5576/04/$30.00 doi:10.1016/j.jsgi.2004.03.003

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Figure 1. Diagrammatic representation of the main detoxification pathways for ROS. Excess production of superoxide anions (O2.⫺) can lead to formation of the more dangerous hydroxyl (OH.) ions through the iron-catalyzed Fenton reaction. Alternatively, O2.⫺ may react with nitric oxide to form the prooxidant peroxynitrite. The rate reaction for this combination is some ten times faster than that between O2.⫺ and SOD.

tions of the enzymes of the mitochondrial respiratory chain. However, these enzymes, in particular complex III, are not totally efficient and electrons can “leak” onto molecular oxygen to form the superoxide anion (O2.⫺).6 A significant amount (1–2%) of the O2 we consume is diverted into the production of O2.⫺ in this way, with O2.⫺ being formed at a rate dependent on the prevailing oxygen tension.7 Because it possesses an unpaired electron, the superoxide anion belongs to a class of molecules termed free radicals, which along with their nonradical intermediates fall under the umbrella term of reactive oxygen species (ROS). ROS are characterized by their high reactivity, and in order to prevent damage to biomolecules an array of antioxidant defenses has evolved. Due to its charge O2.⫺ is membrane impermeable, and remains within the mitochondrial matrix where it is detoxified by the enzyme manganese superoxide dismutase (MnSOD). SOD is present in all aerobic cells, and is found in the cytoplasm as the alternative copper/zinc isoform (Cu/Zn SOD). The enzyme converts O2.⫺ to hydrogen peroxide (H2O2), which in turn is reduced to water by the antioxidant enzymes catalase (CAT) and glutathione peroxidase (GPX) (Figure 1). In addition to the antioxidant enzymes, other molecules such as thiols, ceruloplasmin, or transferrin and dietary vitamins, for example, ascorbate (vitamin C) and ␣-tocopherol (vitamin E), play a crucial role in the defense against oxygen free radicals.5 A complex homeostatic balance is thus achieved, and at physiological levels free radicals regulate a wide variety of cell functions through their actions on redox-sensitive transcription factors.8,9 It is essential that these defenses act in concert, as an imbalance can lead to the production of other more highly reactive radical species such as the hydroxyl (OH.), peroxyl (RO2), and hydroperoxyl (HO2.) anions.5 If the generation of these radicals exceeds the cellular defenses, then indiscriminate damage can occur to proteins, lipids, and DNA, resulting in cellular oxidative stress. The consequences may

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range from the activation of stress-response proteins through disruption of signaling mechanisms, structural damage to apoptosis, or necrosis.10 Although under physiological conditions the main source of intracellular ROS is as a byproduct of aerobic respiration, they can arise from other metabolic reactions and oxidase enzymes. These include NAD(P)H oxidase, a membrane-associated enzyme that plays an important role in oxygen sensing in endothelial cells and myocytes,11 and that is also present in the placenta.12,13 Under pathological conditions, however, different mechanisms may come into play, and one potentially important source is the enzyme xanthine dehydrogenase/xanthine oxidase. In the dehydrogenase form, this enzyme converts hypoxanthine to xanthine, and xanthine to uric acid, passing the electron released on to NAD⫹. During periods of hypoxia, the enzyme can be cleaved by calcium-dependent proteases to the oxidase form, which uses O2 as the electron recipient, so generating O2.⫺. This conversion is responsible for the burst of free radical production that is associated with episodes of ischemia-reperfusion,14 and accounts for why fluctuations in O2 concentration can be particularly damaging to cells. Fluctuations in O2 concentration may occur within the human placenta due to the unique pattern of development of the maternal blood supply to the organ.

ONSET OF THE MATERNAL ARTERIAL BLOOD SUPPLY TO THE PLACENTA IN NORMAL PREGNANCIES The definitive human placenta consists of the elaborately branched fetal villous tree bathed by the maternal blood circulating within the intervillous space, and so is classified histologically as being of the hemochorial type (Figure 2). During implantation the invading trophoblast erodes into capillaries and small veins within the superficial endometrium,15 and shortly after maternal erythrocytes can be observed within the precursors of the placental intervillous space. Traditionally, therefore, it has been widely assumed that the maternal intraplacental circulation is established soon after implantation,16 and the precocious supply of nutrients this provides for has been considered a key evolutionary advantage of the invasive form of implantation displayed by the great apes and humans. However, the presence of maternal erythrocytes does not necessarily signify an effective circulation, and in the classic texts describing placental development doubt was expressed as to when connections between the maternal arteries and the intervillous space are first observed.17,18 Although this remains a somewhat controversial subject, recent studies employing diverse techniques, including hysteroscopy, perfusion of hysterectomy specimens with the placenta in situ, and Doppler ultrasound studies of the early placenta in vivo, have presented compelling evidence that the maternal intraplacental circulation is only fully established at 10 to 12 weeks of pregnancy.19 –23 Until then, the intervillous space is filled with a clear fluid, most likely a maternal plasma filtrate supplemented by secretions from the uterine glands.24,25 The human placenta

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Figure 2. Photomicrograph of a placenta-in-situ associated with a 60-mm fetus (Boyd Collection) showing the definitive placenta (large arrow). P ⫽ placental tissue; D ⫽ decidua; M ⫽ myometrium. Note the regression of villi over the nonembryonic pole to form the chorion laeve, and the irregularity of the decidua with the presence of maternal vessels in a septum (small arrow). *Fixation artifact.

cannot therefore be considered truly hemochorial until the end of the first trimester. Fundamental to this new understanding is the process of physiological conversion of the endometrial spiral arteries. In the nonpregnant state, the spiral arteries are small-caliber, vasoreactive vessels that arise from the radial arteries and feed

Burton and Jauniaux into the superficial capillary plexus within the endometrium. During pregnancy they undergo conversion into distended, flaccid uteroplacental vessels, capable of accommodating the increase in uterine arterial blood flow from just a few milliliters per minute in the nonpregnant state to approximately 700 mL/min at term.18,23 The architecture of their decidual and myometrial segments is disrupted during this process by the loss of myocytes from the media and the internal elastic lamina, which are progressively replaced by fibrinoid.26,27 The physiological change only occurs in the presence of extravillous trophoblast cells that arise from the tips of the cytotrophoblast cell columns of anchoring villi.28 Where these columns abut the endometrium, the cytotrophoblast cells spread laterally to form a continuous layer several cells deep, termed the cytotrophoblastic shell. Cells migrate from the deep surface of this shell, invading between the uterine glands as interstitial trophoblast, and down the lumens of the vessels as endovascular trophoblast.26,28,29 The process begins soon after the blastocyst has implanted and gradually extends laterally, reaching the periphery of the placenta around mid-gestation. Depth-wise the changes normally extend as far as the inner third of the myometrium within the central region of the placental bed, but the extent of invasion is progressively shallower towards the periphery.30 Therefore, even in normal pregnancies not all the arteries are completely transformed.31 In the early stages the volume of endovascular trophoblast cells migrating down the arterial lumens is sufficient to occlude or “plug” the tips of the maternal vessels.22,32 Free flow of maternal blood into the intervillous space is therefore not possible, although slow seepage of plasma through the network of intercellular clefts may occur. At about the 10th week these plugs begin to dissipate, establishing free communications between the spiral arteries and the placenta (Figure 3). The greater trophoblast invasion that occurs in the central region of the placental bed30 means that the plugs are more extensive and complete in this region, and so it might be predicted that dissipation of the plugs will occur first at the periphery of the

Figure 3. Doppler mapping and spectral analysis of uteroplacental blood flows at 12 weeks’ gestation. A) Intervillous blood flow showing a typical venous-like nonpulsatile flow. B) Spiral artery blood flow at the level of the placental bed.

Placental Oxidative Stress placenta. Our recent Doppler studies have confirmed that is the case in the majority of normal pregnancies.3

INTRAPLACENTAL OXYGEN CONCENTRATIONS DURING EARLY PREGNANCY The human fetoplacental unit is therefore exposed to major fluctuations in O2 concentration during pregnancy. The O2 tension in the oviduct and uterus of most mammalian species at the time of implantation has been found to range between 11 and 60 mmHg, which corresponds to approximately 1–9% O2.33–35 The partial pressure of oxygen (PO2) measured within the human placenta in vivo is less than 20 mmHg at 7 to 10 weeks gestation, and is therefore equivalent.36,37 Maintaining a low oxygen concentration during the embryonic period appears to favor blastulation and normal cell differentiation, and may protect from the damaging effects of oxygen free radical species.38 Once this process is complete at 11 to 14 weeks, the intraplacental oxygen tension rises to greater than 50 mmHg as the maternal circulation becomes fully established.36,37 Despite this rise values remain low within the fetus as the diffusional characteristics of the placenta are limited at this stage of gestation.39,40 At 13 to 16 weeks, the PO2 in the fetal blood is 24 mmHg, whereas during the second half of pregnancy that in the umbilical vein ranges between 35 and 55 mmHg. All of these values are relatively low compared to the PO2 values found in the maternal circulation,41 suggesting that there is a significant O2 gradient between the maternal and fetal tissues throughout pregnancy.

PHYSIOLOGICAL OXIDATIVE STRESS IN EARLY PLACENTAL TISSUES The sharp increase in O2 tension experienced by the placenta in vivo when the maternal circulation is fully established is associated with a burst of oxidative stress within the placental tissues.37 This is particularly marked in the syncytiotrophoblast, where we detected immunohistochemically the presence of nitrotyrosine residues indicating the formation of peroxynitrite (ONOO2⫺) from nitric oxide (NO.) and O2.⫺ (Figure 1), hydroxynonenal adducts (HNE) indicating oxidation of lipids, and expression of heat shock protein (Hsp) 70.37 The latter is recognized as a sensitive marker of oxidative stress in other systems.42 The expression of these markers can be induced in vitro by exposing villi to 21% O2, and is associated with increased generation of ROS as detected by the fluorescent dye dichlorofluorescein diacetate (DCFH-DA).43,44 The cellular source of the ROS is not known, but the fact we observed swelling of the mitochondrial intracristal space both in vivo and in vitro suggests that mitochondria are a significant contributor. This is supported by the finding that addition of 10 ␮m diazoxide, a mitochondrial ATP-dependent K⫹ channel opener, partially reduces both the generation of ROS and the expression of markers of oxidative stress.45 Generation of ROS occurs within minutes of exposure to 21% O2, but if villi are maintained for longer periods then mitochondrial membrane potential is lost after 1 hour, and degeneration of the syncytiotrophoblast occurs after 4 hours.46 The tissue rapidly

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undergoes vacuolation, dilation of the mitochondrial intracristal space, loss of the microvillous covering, and blebbing of the apical membrane. However, the underlying cytotrophoblast and stromal cells remain viable, reflecting the greater concentrations of the principal antioxidant enzymes within these cell types.47,48 Indeed, the cytotrophoblast cells differentiate and fuse to form a new syncytiotrophoblastic layer that is morphologically equivalent to the original.46 Degeneration and regeneration of the syncytiotrophoblast have subsequently been reported for term villi maintained under 21% O2.49,50 The physiological role of this burst of oxidative stress is only beginning to be elucidated, but a number of cell functions within the placenta are now recognized as being influenced by the prevailing oxygen concentration. These include matrix remodeling,51 angiogenesis,52 cytotrophoblast proliferation and migration,53–55 cytotrophoblast fusion,56,57 endocrine secretion,58,59 and cytokine production.60 A further and more dramatic effect may be villous regression over the superficial pole of the chorionic sac to form the smooth chorion or chorion laeve (Figures 2 and 4). We recently reported that the degree of oxidative stress is greatest in the peripheral regions of early placentas, correlating with the pattern of onset of the maternal blood flow.3 Although measurements of the O2 concentration within different regions of the early placenta are not available, it is not unreasonable to assume on the basis of the Doppler evidence of maternal blood flow that the PO2 will be greatest in the peripheral region. Examination of placentae-in situ specimens has revealed that the villi over the superficial pole are less extensive than their central counterparts at 8 weeks gestational age.3 They are surrounded by maternal erythrocytes but are themselves conspicuously avascular, consistent with down-regulation of vascular endothelial growth factor (VEGF), a hypoxically regulated gene.52 They also have a thin trophoblastic covering and a virtually acellular stromal core. Hence, onset of the maternal circulation may have a profound impact on villous function and integrity, and it is essential for a successful pregnancy that it happens in a carefully coordinated periphery-central manner.

DEFECTIVE PLACENTATION, OXIDATIVE STRESS, AND EARLY PREGNANCY FAILURE In approximately two thirds of early pregnancy failures there is anatomical evidence of defective placentation, which is mainly characterized by a thinner and fragmented trophoblast shell and reduced cytotrophoblast invasion of the lumen at the tips of the spiral arteries.61 This is associated with both premature onset of the maternal circulation and loss of the peripherycenter coordination in most cases of miscarriage, with blood flow occurring throughout the placenta.3,62,63 These defects are similar in euploid and most aneuploid miscarriages,64 but they are more pronounced in triploid partial moles. In complete hydatidiform mole the extravillous trophoblast invasion into the decidua and superficial myometrium is almost entirely absent.65,66 A recent histological investigation of the products of conception from miscarriage associated with primary an-

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Figure 4. Diagrammatic representation of the proposed relationship between the degree of oxidative stress and placental development in normal pregnancies, late-onset preeclampsia, early-onset preeclampsia, and miscarriage. In normal pregnancies trophoblast invasion is extensive and aggregates of cytotrophoblast cells derived from the cytotrophoblastic shell effectively plug the tips of the spiral arteries. Onset of the maternal circulation (arrows) starts in the periphery, and causes local oxidative stress (depicted by shading), villous regression and formation of the chorion laeve. In miscarriage, trophoblast invasion is severely deficient, leading to a thin cytotrophoblastic shell, incomplete plugging of the spiral arteries, premature and disorganized onset of blood flow, and overwhelming oxidative stress throughout the placenta. The situation is intermediate in preeclampsia. In early-onset cases, the onset of the maternal circulation may be abnormal due to poor development of the cytotrophoblastic shell. Early oxidative stress may lead to impaired villous growth, while secondary atherotic changes (depicted by shading) in the spiral arteries will cause placental hypoxia and intrauterine growth restriction. In late-onset cases, the situation is less severe, and placental oxidative stress only develops towards the end of gestation.

tiphospholipid (aPL) antibody syndrome has also confirmed defective decidual endovascular trophoblast invasion in these cases.67 The data show that by contrast to what has been found in other organs, in aPL syndrome the frequency of placental thrombosis is not increased compared to aneuploid early pregnancy failure. In vitro, aPL antibodies induce VE-cadherin down-regulation and E-cadherin up-regulation at both the protein and mRNA levels.68 The abnormal trophoblast adhesion molecules expression found in aPL antibody syndrome, and in particular the decrease in alpha-1 integrin expression, is similar to that reported in preeclampsia. As might be expected, the excessive entry of maternal blood into the intervillous space has a direct mechanical effect on the villous tissue, and an indirect oxidative stress effect, which contributes to cellular dysfunction and/or damage. Levels of oxidative stress are considerably higher within the whole placenta than in normal cases3,4 (Figure 4). Indeed, the extent of syncytiotrophoblast degeneration on central villi from cases of missed miscarriage is almost identical to that seen within the peripheral regions of normal pregnancies (healthy syncytiotrophoblast represents 45.9 ⫾ 20.8% versus 46.4 ⫾ 19.6% of the villous surface, P ⫽ .964). Large areas of degenerate syncytiotrophoblast may be seen sloughing from the villous surface, accompanied by increased apoptosis and reduced proliferation

among the underlying cytotrophoblast cells.4,69 Despite this, some of the cytotrophoblast cells differentiate and fuse to form a new syncytiotrophoblast layer that is immunoreactive for human chorionic gonadotrophin. As a result, serum concentrations are within the normal range, maintaining the pregnancy as a missed miscarriage.64 With time, however, the fetal capillaries within the villi regress, presumably due to downregulation of VEGF in response to the increased oxygen tension prevailing, and the villous cores become virtually acellular.4,70 Villous atrophy follows, so that the placenta becomes only a thin shell, and finally the pregnancy is lost. Overall, the placental histology closely reflects that seen within the peripheral regions of the normal placenta, suggesting that regression of the villi over the nonembryonic pole of the chorionic sac to form the chorion leave and that seen in missed miscarriage are manifestations of the same process induced by an elevated O2 concentration. The former might be considered physiological and the latter pathological, although it is arguable that miscarriage may be a physiological mechanism for the removal of genetically abnormal conceptuses. Maternal screening of the migrating extravillous trophoblast cells and limitation of invasion may be the fundamental process, with abnormal onset of the maternal blood flow providing the mechanism.

Placental Oxidative Stress There are, of course, other causes of oxidative stress that may also contribute to pregnancy failure. For example, among diabetic women, poor glycemic control is associated with an increased risk of spontaneous miscarriage.71 There is also increasing evidence showing an association between miscarriage and an anomaly of one of the enzymes involved in the metabolism of ROS.72,73 In addition, our recent data on the role of uterine glands in early fetal nutrition and the transport of vitamin E suggest that insufficient decidualization could have an impact on placentation.25,74 These glands remain active until at least the 10th week of pregnancy, and their secretions are delivered freely into the placental intervillous space. An endometrial thickness of 8 mm or more is considered to be favorable for embryo implantation.75 Both adequate endometrial thickness and vascularization are needed for implantation, and women with a good endometrial thickness on ultrasound but a poor intra-endometrial blood flow tend to have a poor reproductive outcome.76 Furthermore, uterine perfusion appears to regulate endometrial receptivity, and a high uterine resistance to blood flow is associated with recurrent miscarriages.77 Decreased expression of SOD and increased levels of lipid peroxidation have also been reported in the decidua of women undergoing early pregnancy loss,78 although whether these changes are a primary cause or a secondary event is not clear as they are only observed in those cases with associated vaginal bleeding. The causes of early pregnancy failure may therefore be divided into three main anatomic categories: disorders affecting primarily the villous development such as in aneuploidy and/or lethal fetal anomalies, disorders affecting mainly the cytotrophoblast invasion such as in the aPL antibody syndrome, and disorders of the uteroplacental interface such as in luteal phase deficiency or chronic inflammatory reaction. Some factors, particularly toxins contained within active or passive cigarette smoke, radiation, or viral infection, can have an impact at all three levels, whereas some fetoplacental anomalies, such as paternally inherited triploidy, can have an impact on both villous development and cytotrophoblast invasiveness. Whatever the initial factor, a major defect of the placentation process will lead to an incomplete development of the placento-decidual interface and subsequent premature and widespread onset of the intervillous circulation. This will result in severe oxidative damage to the villous trophoblast, which inevitably leads to a complete placental development arrest.

PLACENTAL OXIDATIVE STRESS IN LATER PREGNANCY AND IN PREECLAMPSIA Measurements of the O2 concentration within the intervillous space indicate that there is a gradual decline from approximately 60 mmHg at 16 weeks to about 40 mmHg at term.79 Despite this decline, and a supposed switch to a less oxidative environment, we have found evidence of oxidative stress in otherwise normal placentas delivered at term by cesarean section.80 The cause of this stress is not certain, but extrapolating our findings from the first trimester suggests that hypoxia alone is not a sufficient cause. The O2 concentration at term is still

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twice that during the first trimester, when the trophoblast shows no evidence of oxidative stress. Equally, if villi from term placentas are maintained in vitro under constant conditions of low O2 (12–16 mmHg), they display no increase in oxidative stress.80 However, reintroducing O2 after a period of hypoxia causes rapid production of ROS, and evidence of tissue oxidative stress in terms of the formation of nitrotryosine residues and lipid peroxidation. We have therefore proposed that the constancy of the O2 concentration may be a more important factor in the generation of placental oxidative stress than the absolute value.81 Oxygen concentrations may fluctuate within the intervillous space during the second and trimesters through three principal mechanisms: intrinsic contraction of the spiral arteries, external compression of the arteries, and redistribution of maternal blood flow. First, angiographic studies on the macaque, which displays a similar maternal vascular anatomy to the human, demonstrated that during normal pregnancy flow from spiral arteries into the intervillous space is often intermittent.18,82 As these studies were performed during periods of uterine relaxation, the investigators concluded that the effect was due to vasoconstriction within the unconverted segments of individual arteries rather than external compression. This was supported by the observation that an injection of L-epinephrine into the maternal circulation caused a dramatic reduction in the number of arteries that discharged into the intervillous space. Second, towards term the strength of the uterine contractions increases, and leads to transient compression of the myometrial arteries, either blocking or severely impairing inflow into the intervillous space.18,83,84 Third, major alterations in uterine perfusion can occur as part of the general redistribution of blood flow in response to factors such as maternal exercise and changes in posture.85 Since the placenta and fetus continually extract oxygen from the maternal blood within the intervillous space it is expected that transient hypoxia will result during the periods of relative stasis. Some degree of hypoxia-reoxygenation type injury is therefore likely be a feature of the normal human pregnancy, especially towards term when the fetal and placental extraction of oxygen are at their highest.81 This would certainly seem to be the case during vaginal delivery when the contractions are at their strongest, and there is evidence both of increased xanthine oxidase activity within the placenta86 and depletion of maternal serum vitamin C.87 There is now compelling evidence that placental oxidative stress plays a pivotal role in the pathogenesis of preeclampsia, although the precise mechanism remains elusive.1,2,88 Qualitatively, the situation within the placenta in preeclampsia appears to be an extension of that seen towards the end of normal gestation, as there is evidence of increased nitrotyrosine formation, increased lipid peroxidation, increased trophoblast apoptosis, decreased activity of the principal antioxidant enzymes, and reduced tissue concentrations of nonenzymatic antioxidant molecules such as vitamin E.89 –94 Again the cause for the oxidative stress is not known, although it is widely assumed to be secondary to placental hypoxia consequent to

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the deficient trophoblast invasion that typifies the majority of cases.30,31 The defect involves both the number of vessels that are converted, and the extent of the transformation within individual arteries. The cause for the incomplete conversion is not known, but it appears that only the endovascular component of trophoblast invasion is impaired. Interstitial invasion extends as far as normal, and so there may be a primary defect of the endovascular cytotrophoblast or an abnormality in the uterine endometrium that these cells are attempting to invade.27 It has also been reported that macrophages, which are found in excess in the placental bed of preeclamptic women, are also able to limit the extravillous trophoblastic invasion,95 although their influence on the endovascular population is not clear. The end result is a failure to convert the distal portions of the spiral arteries into large-caliber flaccid conduits. We suggest that by itself this is likely to have relatively little impact on the volume of intervillous blood flow and O2 concentrations as these sections of the arteries are not flow-limiting.96,97 The unconverted segment of the spiral artery where it arises from the radial artery will always be of smaller caliber, and so serve that function. By contrast, dilatation of the vessel distal to this section will have a major impact on the rate and the pressure with which the maternal blood enters the intervillous space, reducing both. In doing so it will benefit materno-fetal exchange by ensuring an appropriate passage time through the placenta and prevent collapse of the fetal villous capillaries. There is also no conclusive evidence that the placenta is hypoxic in preeclampsia, particularly in late-onset cases, or that the placental changes seen in preeclampsia are typical of hypoxia.81,98 An alternative explanation may lie in the fact that due to incomplete conversion the majority of spiral arteries within the placental bed retain considerable smooth muscle within their walls.99 A greater degree of vasoreactivity will therefore persist, exacerbating the intermittent perfusion seen in normal pregnancies. Modeling these effects in vitro has shown that hypoxia-reoxygenation is a much more potent stimulus for the generation of placental oxidative stress than hypoxia alone,80,100 and so likely to be the causative insult under physiological conditions.

THE SPECTRUM OF PLACENTAL OXIDATIVE STRESS Preeclampsia is not an all or nothing phenomenon and there are major differences in the clinical manifestations and outcomes between the early-onset and late-onset forms of the syndrome. In particular, early-onset preeclampsia is almost universally associated with intrauterine growth restriction, whereas this is rarely the case with late onset. Whether the two forms represent different disorders or different maternal susceptibilities to the products of the stressed placenta is not known. We propose the two forms represent a spectrum of oxidative stress induced by differing degrees of impairment of trophoblast invasion. Recent longitudinal studies of placental growth by ultrasound have revealed that in cases of early-onset pre-

Burton and Jauniaux eclampsia associated with intrauterine growth restriction, placental volume is reduced from as early as 12 weeks of gestation.101 This suggests the placental pathology is initiated at the time of onset of the maternal circulation. Because endovascular trophoblast invasion is associated with both plugging of the spiral arteries during the first trimester and their physiological conversion, it is likely that onset of the maternal circulation will be perturbed in the most serious cases of preeclampsia. We speculate that this will lead to high levels of oxidative stress, although insufficient to cause pregnancy loss (Figure 4). Nonetheless, there may be increased apoptosis and decreased proliferation, as we have seen in the missed miscarriage specimens,4 leading to a reduced villous development at term.102 The altered hemodynamics of maternal blood entry into the intervillous space, with entry being at too great a rate as result of the failure of the distal segments of the spiral arteries to dilate, may also cause the blood-lakes and increased thrombosis that typify these cases.103,104 In addition, early-onset preeclampsia is commonly associated with acute atherotic changes in the spiral arteries in later pregnancy, which restrict their caliber still further.31,105 Whether these are primary effects or secondary insults is not known. However, we speculate that the distal segments of the arteries in which these changes occur will themselves be involved in any hypoxia-reoxygenation resulting from increased vasoconstriction of the unconverted segments. We consider it most likely, therefore, that these are secondary changes, and that as they develop there will be an increasing impairment of placental perfusion. Placental hypoxia will ensue, and this, along with reduced placental function as result of increased oxidative stress, will contribute to the fetal growth restriction observed. Similar atherotic changes have also been reported in cases of intrauterine growth restriction without hypertension, and so they are not in themselves causative of preeclampsia.106 Whether the placentas from these cases display equivalent oxidative stress to that seen in preeclampsia is not known. By contrast, in cases of late-onset preeclampsia, placental volume is slightly larger than normal at 12 weeks, and grows rapidly until 22 weeks.101 The placental insult is clearly less severe as villous growth is normal,102,107 and the maternal vascular changes are less extensive.31 We propose that these cases represent an exaggeration of the situation that normally develops towards term, and that there is insufficient time for atherotic changes in the spiral arteries to become manifest (Figure 4). As a result, birth weight is normal, and there will be considerable overlap in the values of clinical parameters between these cases and normal pregnancies.

CONCLUSION Taken together, these findings emphasize the critical importance of the maternal circulation to the well-being of the placenta. Although confirmatory physiologic data are not available, the human placenta is probably unique in undergoing a transition in oxygenation at the end of the first trimester. In other species, that part of the placenta involved in hemotrophic exchange appears to develop under a more constant O2

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Figure 5. Schematic representation of the relationship between the extent of trophoblast invasion, placental oxidative stress and pregnancy outcome.

concentration. For example, the labyrinth of the mouse is vascularized by a maternal circulation almost from its origin. The difference is that in the mouse labyrinth development commences about half-way through gestation, by which time embryogenesis is almost complete, whereas in humans development of the placenta occurs much earlier. Maternal metabolic disorders, for example, diabetes, which are associated with an increased production of ROS, are also known to be associated with a higher incidence of fetal structural defects.108 Furthermore, the teratogenicity of drugs such as thalidomide has recently been shown to involve ROS-mediated oxidative damage,109 indicating that the human fetus can be irreversibly damaged by oxidative stress. These findings suggest fetal development is highly sensitive to perturbation by ROS, and hence maintaining a low O2 environment inside the human uterus during early pregnancy may confer protection. By contrast, a profuse and steady maternal blood flow to the placenta is clearly required to support fetal growth during the second and third trimesters. What we observe, however, is that the transition in the maternal placental circulation at 10 to 12 weeks is a potentially dangerous one. It must be carefully orchestrated in a periphery-center fashion to prevent overwhelming oxidative stress to the placenta, which may contribute to pregnancy failure. Plugging/unplugging of the spiral arteries appears to be related to successful invasion of the extravillous trophoblast cells, a process that is also linked to conversion of the spiral arteries. Hence, we speculate that in normal pregnancies trophoblast invasion is complete, unplugging of the vessels is orderly, and the arteries are fully converted. If trophoblast invasion is less complete, the vessels may retain some of their vasoreactivity, leading to a greater degree of intermittent perfusion of the intervillous space and placental oxidative stress and predisposing the mother to preeclampsia. If trophoblast invasion is particularly shallow, then unplugging of the vessels may be premature and disorganized, resulting in overwhelming placental oxidative stress and pregnancy failure (Figure 5). Miscarriage and preeclampsia may therefore be part

of a continuum of placental oxidative stress, which although being heavily influenced by the depth of trophoblast invasion, may also integrate other factors such as polymorphisms in enzymes either generating or scavenging ROS, metabolic disorders, drug intake, dietary micronutrients, and maternal susceptibility to the products of placental oxidative stress.

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