Pre-eclampsia is a multisystem disorder with profound implications for both mother and fetus. Its origins lie in the earliest stages of pregnancy. Abnormal interactions between fetal trophoblast and maternal decidua, including the cells of the maternal immune system, lead to inadequate placental invasion and maternal vascular remodelling. However, abnormal placentation is only one step in the cascade of events that ultimately result in maternal organ dysfunction. Pre-existing maternal conditions predisposing to inflammation and vascular pathology, fetal factors, including multiple gestations and macrosomia, and environmental exposures, including infection, may contribute to the release of placental substances, including anti-angiogenic molecules, into the maternal circulation. These may act directly or indirectly upon the endothelia of end organs, including the kidney, liver and brain. The liberation of reactive oxygen species, cytokines, and microthrombi from damaged endothelia contribute further to organ damage. In studying the normal processes that occur during human placentation and early pregnancy, we will develop a greater understanding of what may go awry in pre-eclampsia. Such research will be crucial in discovering novel biomarkers for prediction of the disorder and, eventually, in finding targets for effective interventions.
Introduction
Human placentation is a complex, tightly regulated process, which is unique even among other closely related mammals in its invasiveness and dramatic remodelling of the maternal uterine vasculature. Inadequate invasion and development of the placenta have been implicated in a growing list of pregnancy complications, including recurrent pregnancy loss, intrauterine growth restriction, preterm labour, and pre-eclampsia. Conversely, uncontrolled placental invasion may lead to life-threatening conditions, such as placenta accreta, increta and percreta, or gestational trophoblastic disease and choriocarcinoma.
Elucidating the role of abnormal placentation in the development of pre-eclampsia is of particular interest. As discussed in Chapters 1 and 3, the disorder is common (affecting 5–8% of pregnancies), and has significant consequences both for mother and fetus. In studying the cellular, molecular and immunological mechanisms of normal human placentation, the mechanisms underlying pre-eclampsia and other disorders of placentation are becoming better characterised.
Pre-eclampsia: the clinical syndrome
Clinically, pre-eclampsia is defined both by its maternal and fetal manifestations. The most common maternal manifestations are new-onset hypertension (blood pressure >140/90 mmHg) occurring after 20 weeks gestation, which normalises after delivery, and proteinuria (>0.3 g/24 h). Although hypertension and proteinuria are the hallmarks of pre-eclampsia, the disorder is in fact a complex, multi-systemic disease that results in diffuse endothelial dysfunction.
With the availability of modern anti-hypertensive drugs and magnesium sulphate, deaths resulting from cerebrovascular accidents and eclampsia have decreased, particularly in the developed world. As a consequence, multi-organ dysfunction has become the most common cause of pre-eclampsia-associated mortality. Essentially, every organ system may be affected by pre-eclampsia, and severe end-organ sequelae may occur even in the absence of severe hypertension or proteinuria.
Haemolysis, elevated liver enzymes and low platelets (HELLP) syndrome is the most profound example of this. Clinically, the kidneys, liver, brain and haemostatic systems are most affected; why this predilection occurs remains unclear, but presumably it relates to a greater ability of some vascular beds to ‘defend’ against vasoconstriction and endothelial dysfunction than others.
The maternal syndrome of pre-eclampsia, with its profound systemic inflammation and endothelial dysfunction, is similar in many respects to that seen in people with the systemic inflammatory response syndrome. However, the key difference is that in systemic inflammatory response syndrome, significant hypotension occurs, rather than the hypertension which is the hallmark of pre-eclampsia. If the placenta is considered as another end-organ, the fetal manifestations of intrauterine growth restriction (IUGR), oligohydramnios, abnormal umbilical artery Doppler flows and fetal acidaemia may be included as sequelae of the maternal syndrome.
Pre-eclampsia: the clinical syndrome
Clinically, pre-eclampsia is defined both by its maternal and fetal manifestations. The most common maternal manifestations are new-onset hypertension (blood pressure >140/90 mmHg) occurring after 20 weeks gestation, which normalises after delivery, and proteinuria (>0.3 g/24 h). Although hypertension and proteinuria are the hallmarks of pre-eclampsia, the disorder is in fact a complex, multi-systemic disease that results in diffuse endothelial dysfunction.
With the availability of modern anti-hypertensive drugs and magnesium sulphate, deaths resulting from cerebrovascular accidents and eclampsia have decreased, particularly in the developed world. As a consequence, multi-organ dysfunction has become the most common cause of pre-eclampsia-associated mortality. Essentially, every organ system may be affected by pre-eclampsia, and severe end-organ sequelae may occur even in the absence of severe hypertension or proteinuria.
Haemolysis, elevated liver enzymes and low platelets (HELLP) syndrome is the most profound example of this. Clinically, the kidneys, liver, brain and haemostatic systems are most affected; why this predilection occurs remains unclear, but presumably it relates to a greater ability of some vascular beds to ‘defend’ against vasoconstriction and endothelial dysfunction than others.
The maternal syndrome of pre-eclampsia, with its profound systemic inflammation and endothelial dysfunction, is similar in many respects to that seen in people with the systemic inflammatory response syndrome. However, the key difference is that in systemic inflammatory response syndrome, significant hypotension occurs, rather than the hypertension which is the hallmark of pre-eclampsia. If the placenta is considered as another end-organ, the fetal manifestations of intrauterine growth restriction (IUGR), oligohydramnios, abnormal umbilical artery Doppler flows and fetal acidaemia may be included as sequelae of the maternal syndrome.
Abnormal placentation: setting the stage for the maternal syndrome
Pre-eclampsia may be sub-divided into early onset (<34 weeks gestation) and late-onset (>34 weeks) disease. Although this gestational age is an arbitrary designation, evidence suggests that the causes and outcomes of these two disorders are divergent. Pre-eclampsia occurring remote from term provides significant clinical challenges: in attempting to ameliorate the risks of prematurity through expectant management, severe maternal sequelae may develop. This concept will be discussed in detail in Chapter 7. Late-onset pre-eclampsia is more often associated with adequate placentation, but excessive fetal demands, as in fetal macrosomia and multiple gestations. In contrast, early onset pre-eclampsia is more often associated with abnormal placentation. Inadequate placentation is also associated with recurrent first-trimester pregnancy loss and IUGR complicating normotensive pregnancies (nIUGR). It is recognised that early onset pre-eclampsia is associated with excess maternal and perinatal sequelae and contributes disproportionately to the number of maternal deaths from this disorder worldwide. Nevertheless, late-onset pre-eclampsia is a more common event and may still be associated with poor pregnancy outcomes.
It seems that the maternal inflammatory phenotype differs between women with pre-eclampsia and women with nIUGR alone. In addition, the amount of placental debris shed into the maternal circulation (a potential trigger for the systemic inflammation and endothelial dysfunction) is greater in early onset pre-eclampsia than in matched normal pregnancies. Conversely, it is lower in nIUGR pregnancies compared with normal pregnant controls. In both pre-eclampsia and nIUGR, the clinical manifestations of abnormal placentation do not arise until later in pregnancy, when fetal demands begin to exceed placental supply. This ’uteroplacental mismatch’ may also arise in the context of normal placentation when there are excessive fetal demands, as may occur with fetal macrosomia, or multiple gestations.
Early origins of placental dysfunction
Evidence is growing that inadequate placentation may be implicated in the pathophysiology of pre-eclampsia, IUGR and preterm labour. Formation of the human maternal–fetal interface requires the co-ordinated development of both maternal and fetal cellular compartments. The appearance of maternal decidual natural killer cells (dNK cells) is a hallmark of decidual development. At the time of placentation, the decidua seems to support and restrain extravillous trophoblast (EVT) invasion, thus playing a crucial role in regulating the formation of the placenta.
During the process of implantation, mononuclear cytotrophoblasts leave the trophoblast shell in order to invade the full thickness of the endometrium into the inner third of the myometrium (interstitial invasion), as well as into the maternal uterine vasculature (endovascular invasion). Endovascular invasion places the fetal trophoblast in direct contact with the maternal circulation, and therefore establishes the definitive uteroplacental circulation.
The characteristic pathological lesion of pre-eclampsia is shallow cytotrophoblast invasion to varying degrees and, more consistently, restricted endovascular invasion. In both early onset pre-eclampsia and IUGR, placental invasion is limited by increased trophoblast apoptosis, resulting in narrower spiral arteries than in control pregnancies. In addition, it has been shown that in pre-eclampsia, cytotrophoblasts invading uterine vessels fail to change their adhesion molecule receptor repertoire in order to mimic an endovascular phenotype.
Progressive uteroplacental insufficiency seems to result from incomplete uterine artery remodelling. Altered patterns of vascularisation have been observed in first-trimester chorionic villous samples from pregnancies that go on to develop either early onset pre-eclampsia or IUGR, with reduced distances between peripheral vessels and intervillous spaces compared with normal control participants or pregnancies complicated by term pre-eclampsia.
Trophoblast differentiation during early placentation
The crucial fetal cells involved at the feto–maternal interface are cytotrophoblasts, which enter one of two mutually exclusive pathways at the time of implantation. Differentiation toward the villous pathway involves the terminal differentiation and fusion of mononuclear cytotrophoblasts into the multinucleate syncytiotrophoblast. The syncytiotrophoblast is in direct contact with maternal endometrium and blood, and is responsible for most of the biological functions of the placenta, including the transfer of respiratory gases, nutrients and waste products; acting as a barrier against blood-borne pathogens and the maternal immune system; and acting as an endocrine organ capable of secreting hormones, growth factors and other bioactive substances, In contrast, cytotrophoblasts entering the EVT pathway proliferate, form large cellular columns, and then develop a highly invasive phenotype. This allows EVT to detach and invade deeply into both the underlying maternal tissue and vasculature, ensuring a constant blood supply to the developing fetus.
EVT may be further categorised into two subpopulations. Interstitial cytotrophoblasts invade the decidual stroma and superficial myometrium, whereas endovascular cytotrophoblasts invade the lumen of maternal spiral arteries. The latter process converts low-flow, high-resistance vessels into high-flow, low-resistance conduits. Failure of this process results in impaired intervillous blood flow, an aberration observed in pre-eclampsia. The cellular mechanisms underlying the development of the invasive trophoblast phenotype are similar to those observed during tumour progression and metastasis. Both processes involve the proliferation, detachment and migration of cells into the underlying stroma and vasculature. In addition, at the molecular level, trophoblast and malignant cell invasion both depend upon the regulated expression of cell–cell adhesion molecules, cell-matrix adhesion molecules expressed on the surface of these cells, and extracellular matrix remodelling by matrix metalloproteinases. Aberrant expression patterns of these adhesive and proteolytic systems in human trophoblasts have been associated with pre-eclampsia.
The feto–maternal immune interface: evasion during invasion
Trophoblasts define the boundary between mother and fetus. Major histocompatibility (MHC) and MHC-like receptors expressed by trophoblast act as ligands for immune receptors on dNK cells, lymphocytes and myelomonocytic cells present at the feto–maternal interface. At this interface, trophoblast contact the maternal immune system at two areas. The first is at the level of the villous trophoblast, which interacts directly with maternal blood, and the second is at the level of the EVT, which is in direct contact with maternal uterine tissue. The syncytiotrophoblast contacts the systemic but not the uterine immune system, and expresses no MHC antigens. In contrast, EVT are characterised by their specific antigen expression profile. They lack classical HLA-A and HLA-B expression, which may prevent interaction with maternal cytotoxic T-cells, while expressing classical HLA-C. EVT also express the non-classical HLA-E and HLA-G antigens. Maternal dNK cells possess specific killer inhibitory receptors (KIR), for these Class 1 HLA molecules expressed by EVT. HLA-C, -E, and -G are critical in mediating dNK–EVT interactions through several mechanisms.
dNK are found in large numbers and in close proximity to infiltrating EVT during implantation. They comprise a unique population of CD56brightCD16- lymphocytes, phenotypically different from circulating CD56dimCD16bright NK cells (pbNK). dNK have lower cytolytic activity and express a different cytokine repertoire than pbNK cells. dNK cells express high levels of CD94–NKG2A, which bind to HLA-E and inhibits NK-cell cytotoxicity, and KIRDL4, which binds to HLA-G. The subsequent interaction results in increased expression of pro-inflammatory and pro-angiogenic cytokines by dNK cells.
Expression of HLA-G has been proposed as a signal that protects EVT against dNK lysis. Its main role, however, is thought to be to stimulate the production of cytokines, chemokines and angiogenic factors by dNK cells, thus facilitating trophoblast invasion. Normally, EVT invading the decidua express and upregulate HLA-G ; evidence exists that this does not occur in pre-eclampsia.
HLA-C is recognised by members of the KIR gene family, which are expressed on dNK cells. The interaction of HLA-C and KIRs expressed on dNK cells have been shown to influence the development of the human placenta. HLA-C is upregulated by interferon gamma (IFN-γ), a cytokine produced in large quantities by dNK cells. On a population level, specific combinations of maternal dNK KIR and fetal HLA-C genes seem to alter the balance between risk of pre-eclampsia and reproductive success.
The maternal innate immune system in normal and abnormal placentation
dNK cells are involved in successful trophoblast invasion of maternal spiral arteries during placentation. The differentiation of trophoblasts to the invasive extravillous phenotype is seen as a crucial part of placental angiogenesis, and has been given the term ‘pseudovasculogenesis’. dNK cells produce a number of cytokines involved in the regulation of trophoblast invasion, including granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage CSF (GM-CSF), macrophage CSF (M-CSF), leukemia inhibitory factor, tumour necrosis factor alpha (TNF-α), and IFN-γ.
IFN-γ’s role in placentation has been extensively investigated using mouse models, in which it is crucial in the initiation of uterine vascular modification and decidual integrity. Evidence is emerging that dNK-secreted IFN-γ also has crucial regulatory roles in human placentation, including limiting EVT migration from villous tips, and promoting the formation and maintenance of vascular tube-like projections from trophoblasts in culture. Although dNK cells have been shown to effect trophoblast migration and invasion both in vivo and in vitro , the mechanisms behind this effect is still largely unknown. dNK cells express high levels of vascular endothelial growth factor (VEGF)-C, placental growth factor (PlGF), and angiopoietin 2 during the secretory phase of the menstrual cycle. dNK cells, but not pbNK subsets, are thought to regulate trophoblast invasion both in vitro and in vivo through the production of the pro-angiogenic chemokine IL-8, and the anti-angiogenic chemokine, IP-10. Thus, dNK cells may play an important role in the induction of vascular growth during the process of placentation, the balance of pro- and anti-angiogenic factors produced by dNK cells serving as regulators of angiogenesis and maternal vascular remodelling.
It has been theorised that inappropriately high maternal levels of inflammatory cytokines or an imbalance of TH1/TH2 immunity has a role in poor placentation and resultant pre-eclampsia. However, this putative TH1/TH2 imbalance is only one component of a much more complex immune process that involves subsets of T cells and other types of immune cells, such as dNK cells, decidual dendritic cells (DC) and macrophages. Dysregulation of the maternal complement system, with elevations in complement split products, has also been implicated in the pathogenesis of pre-eclampsia. Complement activation is closely tied with the coagulation cascade, the recruitment of leukocytes, and the upregulation of inflammatory cytokines. Recently, it has been argued that the adaptive immune system is less crucial in the pathogenesis of pre-eclampsia than is innate immunity.
Macrophages, which make up 20–25% of decidual leukocytes, are key antigen-presenting cells at the maternal–fetal interface, and are responsible for ingesting apoptotic debris during normal placentation. Macrophages become activated through ingestion of apoptotic or aponecrotic trophoblasts; the presence of activated macrophages, which produce inflammatory cytokines such as TNF-α, may contribute to inadequate trophoblast invasion. Higher numbers of activated macrophages have been found in the placentas of women with pre-eclampsia.
A unique population of DC has been characterised in the decidua and in early pregnancy. The human endometrium and early pregnancy decidua harbour mature CD83+ DC similar to those found in other mucosal surfaces. Decidual DC precursors express dendritic cell specific ICAM-3 grabbing non-integrin, CD 209 (DC-SIGN). The number of DC-SIGN+ cells increase dramatically in early pregnancy, suggesting a role for these cells in immune modulation during implantation and placentation. Decidual DCs demonstrate a reduced capacity to produce IL-12, a potent inducer of T cell cytoloytic activity. By preventing T-cell activation, decidual DCs may promote a TH2-dominant state, which supports the maintenance of pregnancy. Because DC-SIGN+ DCs and dNK cells are the predominant leukocyte populations in first-trimester decidua, it is likely that interactions between them contribute to the development of maternal immune tolerance to the invading trophoblast.
Placental hypoxia, inflammation and oxidative stress
Although pre-eclampsia and IUGR are both associated with placental hypoxia, the role of hypoxaemia in inadequate trophoblast invasion and placental ischemia remains controversial. Evidence shows that oxygen (O2) tension at the feto–maternal interface may have a role in EVT proliferation and invasion. Before the tenth week of gestation, there is no true blood flow into the intervillous space; trophoblast ‘plugs’ prevent blood flow under physiological conditions. After spiral artery modelling is complete, however, O2 tension increases rapidly within the placental bed. In-vitro , O2 tension has been shown to alter the phenotype of both EVT and villous explants; those cultured under hypoxic conditions remain proliferative, non-invasive and immature, failing to undergo the physiological integrin switch and mimicking the phenotype observed in pre-eclamptic villi. Other responses of EVT to low O2 include syncytialisation, increased production of inflammatory cytokines, VEGF, and PAI-1. The effects of O2 are mediated by transcription factors such as hypoxia inducible factor-1α (HIF-1α) and -2α (HIF-2α) that activates gene transcription in response to low O2. Both HIF-1α and HIF-2α are over-expressed in pre-eclampsia. In contrast, HIF upregulation is not observed in pregnancies complicated by nIUGR. However, hypoxia is not the only trigger for HIF upregulation that may contribute to the pathogenesis of pre-eclampsia. Under normoxic conditions, HIF-1α is also responsive to a variety of stimuli, including thrombin, growth factors, vasoactive peptides, insulin, lipopolysaccharide and cytokines, including TNF-α.
It is still uncertain whether the placental oxidative stress implicated in pre-eclampsia is due to chronic hypoxia secondary to improperly remodelled ‘narrow’ spiral arteries, or due to intermittent high-velocity flow secondary to the persistence of vascular smooth muscle in improperly remodelled maternal vasculature. In the latter model, ischaemia-reperfusion leads to oxidative stress and, thus, the formation of reactive oxygen species.
A number of epidemiologic studies have explored the relationship between infection, inflammation and pre-eclampsia. Pathogens such as Chlamydia pneumoniae and cytomegalovirus, which have also been implicated in the development of atherosclerosis, may contribute to placental dysfunction and the enhancement of the maternal inflammatory state through activation of toll-like receptors (TLR). Urinary tract infections and periodontal disease during pregnancy also seem to increase the risk of pre-eclampsia, probably through similar mechanisms. In observational studies, placental malaria infection has also been associated with an increased risk of pre-eclampsia, particularly in primigravid women.
The role of placental anti-angiogenic proteins in pre-eclampsia
The key research question that remains unexplained is how ‘inadequate’ placentation results in the maternal syndrome of pre-eclampsia. Most explanations postulate that placental insufficiency results in the shedding of placental-derived substances, into the maternal circulation. A multitude of syncytiotrophoblast-secreted factors are present in differing quantities in the circulation of women with pre-eclampsia compared with normal pregnant controls, including PlGF, corticotrophin-releasing hormone, activin A, inhibin A, leptin, and syncytiotrophoblast microparticles. These factors may act directly or indirectly on the endothelia of vulnerable maternal organs, resulting in the pathophysiologic triad of vasoconstriction, enhanced capillary leak with plasma volume reduction, and platelet activation.
Within the last decade, the shedding of anti-angiogenic molecules from the placenta into the maternal bloodstream has been of great interest as both a marker for, and as a cause of, the maternal syndrome of pre-eclampsia. An imbalance of soluble angiogenic receptors and ligands has been shown in animal models and clinical studies.
The soluble receptor for VEGF-1 and PlGF (sFlt-1) is of particular interest. Women with pre-eclampsia have been shown to have higher circulating serum levels of this protein than normal pregnant controls. Elevated serum levels of sFlt-1 seem to contribute to endothelial dysfunction, hypertension and proteinuria in pre-eclampsia through antagonism of VEGF, particularly at the level of the kidney. Women with both mild and severe pre-eclampsia have been shown to have reduced circulating levels of PlGF, and in severe pre-eclampsia these levels are significantly lower. In both groups, levels of sFlt-1 are elevated compared with normal pregnant controls. An elegant animal model showed that administration of sFlt-1 via an adenovirus vector to pregnant and non-pregnant rats results in hypertension and glomerular injury, which is similar to that found in pre-eclampsia.
Another anti-angiogenic factor implicated in the pathogenesis of pre-eclampsia is soluble endoglin (sEng), a TGF-β co-receptor. . Maternal serum levels of sEng have been shown to mirror the severity of the clinical state. sEng is capable of regulating EVT differentiation along the invasive pathway in human placental villous explants. In animal models, it seems to have a role in the pathogenesis of HELLP syndrome, although this has not been shown definitively in humans. In clinical studies, elevations in sEng and the sFlt-1:PlGF ratio are present in women who later develop pre-eclampsia weeks before the condition becomes clinically apparent.
Understanding the end-organ consequences of pre-eclampsia
Pre-eclampsia is a multifactorial disorder that begins with inadequate placental function and, consequently, a mismatch of fetal demands compared with placental blood supply. Why it occurs early in the minority of affected pregnancies, and close to term in most affected pregnancies, is still uncertain. Maternal factors predisposing to a pro-inflammatory state, such as obesity, chronic infection and autoimmune disease, most likely contribute to early onset or severe pre-eclampsia. Whether the shedding of placental proteins, vasoactive and inflammatory mediators is the consequence of chronic placental ischaemia, ischaemia-reperfusion injury, oxidative stress, or a combination of all of these factors, the ultimate result is diffuse maternal end-organ damage. Women with pre-existing hypertension and renal disease are at particularly high risk of developing superimposed pre-eclampsia.
The common pathophysiology of pre-eclampsia results from the following: (1) vasoconstriction with enhanced responses to vasoactive substances such as angiotensin II (AII) and endothelin ; (2) plasma volume reduction due to capillary leakage and redistribution of the total extracellular fluid volume ECFV from intravascular to interstitial compartments ; and (3) platelet activation, triggered by endothelial activation, which leads to intravascular thrombosis. This triad sets the scene for reduced blood flow to the brain, liver, and kidneys, and may further decrease utero-placental perfusion.
Renal dysfunction: not just proteinuria
The kidney seems to be one of the most vulnerable organs in pre-eclampsia. The characteristic renal lesion in women with pre-eclampsia is ‘glomerular endotheliosis’. This lesion is considered to be a variant of thrombotic microangiopathy; it is characterised by endothelial cell swelling, obliteration of endothelial fenestrae, and occlusion of capillary lumens. However, this lesion may be present in the absence of proteinuria and even in some normal pregnant women. Proteinuria, one of the diagnostic hallmarks of the disorder, proabably results from VEGF deficiency at the level of the podocytes. The presence of excess amounts of sFlt-1, in addition to decreased VEGF production by podocytes may contribute to this process. This relative VEGF deficiency disrupts the normal function of the glomerular endothelium, thus inducing proteinuria.
Clinically, oliguria and rising creatinine are markers of severe disease. Oliguria is caused by renal vasoconstriction and sodium retention, secondary to reduced plasma volume and vasoconstriction. Increased uric acid is another marker of pre-eclampsia, which generally follows sodium reabsorption; reabsorption is increased in response to reduced renal blood flow and AII. Another source of uric acid may be increased production by placental trophoblasts. Evidence shows that uric acid accelerates endothelial cell apoptosis and senescence via the production of reactive oxygen species. Rising uric acid levels may therefore be a clinically relevant marker of disease severity at both renal and placental levels.
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
