Placenta
Volume 28, Supplement , Pages S4-S13, April 2007

Gabor Than Award Lecture 2006: Pre-Eclampsia and Villous Trophoblast Turnover: Perspectives and Possibilities

  • I. Crocker

      Affiliations

    • Corresponding Author InformationMaternal and Fetal Health Research Centre, St Mary's Hospital, Hathersage Road, Manchester M13 0JH, UK. Tel.: +44 (0)161 276 5462; fax: +44 (0)161 276 6134.

Division of Human Development, The Medical School, University of Manchester, UK

Accepted 26 January 2007. published online 24 March 2007.

Article Outline

Abstract 

Placental apoptosis is exaggerated in pre-eclampsia and cytotrophoblast proliferation is enhanced. This imbalance may be a primary pathogenic event, whereby excessive syncytiotrophoblast apoptosis counters cytotrophoblast fusion, promoting the liberation of syncytial material which perturbs the maternal vascular endothelium. We have previously shown that primary trophoblasts and explant cultured villous fragments from pre-eclamptic pregnancies elicit greater levels of terminal differentiation and apoptosis. This review considers current opinions in trophoblast cell turnover in normal pregnancy and pre-eclampsia. In the context of other findings, this review highlights: (i) the disparity in expression of pro-apoptotic transcription factor p53 in the syncytiotrophoblast in pre-eclampsia, (ii) the importance of reactive oxygen species and hypoxia in initiating villous trophoblast apoptosis and (iii) the concept that aberrant intervillous haemodynamics, as opposed to oxygen per se, initiates excessive syncytiotrophoblast shedding. Finally, therapeutic ways of restoring the syncytiotrophoblast in pre-eclampsia and preventing excessive placental apoptosis are considered, including a role for mitotic manipulators and growth factor replacement strategies.

Keywords: Apoptosis, Pre-eclampsia, Syncytiotrophoblast

 

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1. The placenta and pre-eclampsia 

Pre-eclampsia, a syndrome of vascular endothelial dysfunction and heightened systemic inflammatory response, is a severe disorder affecting up to 10% of primiparous women in industrialised countries [1]. The fact that pre-eclampsia occurs in molar pregnancies in the absence of a fetus, but presence of a placental mass, confirms the importance of the placenta as the crucial stimulus. Certainly this is verified in cases where the placenta is retained after delivery, as the maternal symptoms persist until the organ is removed. With respect to systemic inflammation, there is substantial evidence to support the concept that pre-eclampsia develops when the normal inflammatory response of pregnancy is exaggerated [2]. It is therefore hypothesised that the responsible factor is liberated from the placenta in moderation in normal pregnancy and to excess in pre-eclampsia. This is substantiated by an increased incidence in pregnancies of larger placental mass, i.e. those of multiple or molar pregnancies [3].

Numerous factors produced by the placenta are elevated in pre-eclampsia. Many are implicated in its pathogenesis but no factor, yet suggested, satisfactorily explains its clinical progression. When looking to identify such a factor and the basis for its release, the haemochorial nature of the placenta is undoubtedly important. Amongst other rationale, it is speculated that humans more than any species are predisposed to pre-eclampsia for the following reasons: (i) the extensive nature and depth of trophoblast invasion, (ii) the extent of trophoblast-induced uterine artery transformation, (iii) the complexity of the chorionic villous tree, and (iv) the surface area for transport and diffusion. Individually or in combination, these features may play a significant role, but the latter takes on the greatest importance when the causative factor is itself the villous trophoblast.

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2. The syncytiotrophoblast and pre-eclampsia 

Fragments of trophoblast are liberated from the surface of the villous placenta and readily enter the maternal peripheral circulation. These trophoblast remnants are detectable in uterine veins at Caesarean section and are more prevalent in pre-eclampsia than normal pregnancy [4]. In contrast to their presence in uterine veins, very few trophoblast fragments are found in the maternal peripheral circulation, suggesting that these alone are unlikely to cause maternal leukocyte activations or vascular endothelial complications. Focus has therefore turned to sub-cellular fractions of the syncytiotrophoblast, the outer trophoblast layer of the human placenta. In this context, Redman and Sargent propose that syncytiotrophoblast microparticles, liberated from the villous, circulate freely because of their diminished size and thus avoid entrapment and removal in the lungs [5]. The presence of these micro-particles has now been defined in plasma in normal pregnancy and shown to be significantly greater in pre-eclampsia [6]. Artificially generated syncytiotrophoblast membrane fragments, termed STBMs, can inhibit lymphocyte proliferation, induce apoptosis of T-lymphocytes and can disrupt endothelial cell monolayers and proliferation-cornerstones of pre-eclamptic pathogenesis [7]. Other components of the syncytiotrophoblast, circulating in increased quantities, include cytokeratin, a cytoskeletal protein of trophoblast origin, fetal proteins (e.g. activin-A, inhibin-A, placental alkaline phosphatase) and cell-free RNA and DNA of fetal origin [8], [9], [10]. Together, these culminate in robust evidence for the excessive deportation of syncytiotrophoblast in pre-eclampsia.

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3. Syncytiotrophoblast apoptosis 

Apoptosis, programmed cell death, was first defined in the human placenta by Sakuragi and co-workers [11]. With more recent advances, this process has now been more tightly associated with cytotrophoblast differentiation (Fig. 1). Throughout gestation the functional and morphological maintenance of the multinucleated syncytiotrophoblast, including the supply of fresh mRNA, proteins and organelles, depends exclusively on the fusion of subjacent, post-mitotic cytotrophoblast cells [12]. These events are coordinated by the transcription factor GCM-1 [13] and other regulatory proteins, including synctin-1 and -2 of retroviral origin, and ADAM-12, a recognised component of myoblast cell-cell fusion [14]. Besides these regulatory factors, fusing cytotrophoblasts undergo a necessary flip of phosphatidylserine from the inner to the outer leaflet of the plasma membrane. This loss of asymmetry is attributed to the activation of the cysteine aspartase, caspase 8, an initiator of the common apoptotic pathway [15]. Once fusion has occurred, these early but reversible phases of apoptosis are abruptly halted, thus restricting indiscriminate decline of the vasculo-syncytial membrane. At this stage, the inhibition of apoptosis is attributed to the abundance of cytoplasmic anti-apoptotic oncoproteins in the syncytiotrophoblast, including Bcl-2, Mcl-1 and Mdm2, the presence of X-linked inhibitor of apoptosis protein (XIAP) and profusion of Flice-like inhibitory protein (FLIP), a blocker of caspase 8 activity [16], [17]. These focal inhibitions, which are often lost in areas of grouped and aggregated nuclei, are generally but not inevitably followed by the activation of effector caspases, including caspases 3 and 6. What confines these caspases to discrete areas of the syncytiotrophoblast is unclear, but upon restoration of caspase activity, nuclear proteins, such as PARP, lamin B and topoisomerase II, are reduced and annular chromatin condensation initiated [17]. Finally, aging nuclei accumulate within the tips of villi where they protrude from the apical membrane as syncytial knots. Although difficult to distinguish histologically, these knots are eventually shed as membrane sealed vesicles into the intervillous spaces [18].

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  • Fig. 1 

    Diagram of cell turnover of the villous trophoblast. Proliferation of stem cell cytotrophoblasts is followed by the induction of early stages of apoptosis culminating in the dissolution of their plasma membrane, fusion and incorporation into the overlying syncytiotrophoblast. Apoptotic progression leads to the accumulation of condensed nuclei into syncytial knots and the shedding of these aggregates into the intervillous spaces. CT, cytotrophoblast; FC, fetal capillary; IVS, intervillous space; MVM, microvillous membrane; SKT, syncytial knot; ST, syncytiotrophoblast. Diagram kindly provided by Dr Carolyn Jones of the Division of Human Development, University of Manchester, UK.

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4. Placental apoptosis and pre-eclampsia 

The view of human villous trophoblast as a continuously renewing epithelium, promotes the idea of a steady state between cytotrophoblast and syncytiotrophoblast compartments. A consensus on the numeric ratio between cytotrophoblast and syncytiotrophoblast has been defined in the third trimester and described as 1:9, respectively [19], [20], but uncertainty in early gestation prevents an agreement as to the maintenance and coverage of cytotrophoblasts throughout gestation [19], [20], [21]. Perturbations in trophoblast volume and thickness are induced by alterations in cytotrophoblast proliferation, fusion and/or syncytiotrophoblast loss. Investigations of placental apoptosis in pre-eclampsia have utilised morphological and biochemical features of apoptosis, typically heterochromatin condensation and oligonucleosomal DNA laddering (e.g. TUNEL) and have highlighted an increase in placental apoptosis, predominantly in the syncytiotrophoblast [22], [23], [24]. Fewer studies of placental proliferation have been conducted. In 1991, Arnholdt and colleagues showed an increase in proliferative activity of cytotrophoblasts in pre-eclampsia through BrdU incorporation and Ki-67 recognition [25]. Our own investigations, combining image analysis morphometry and E-cadherin as a marker of cytotrophoblasts, have verified cytotrophoblast hyperplasia [20]. An explanation offered by Huppertz and Kingdom is that villous trophoblast cell turnover, from proliferation through fusion to syncytiotrophoblast apoptosis, is enhanced [26]. This process would coincide with excessive trafficking of trophoblast material, but whether this is an adaptive response to the utero-placental environment or one of damage or inflammatory insult remains to be defined. In this context a number of possibilities are proposed.

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5. Placental hypoxia and pre-eclampsia 

Placental development at 8–10weeks’ gestation occurs under oxygen deprivation (<2% O2, 15–20mmHg) with endovascular trophoblastic plugs obstructing the openings of the utero-placental arteries, preventing influx of oxygenated maternal blood [27]. This restriction is considered critical for protecting against damaging haemodynamics and oxygen-dependent remodelling. Towards the end of the first trimester, progressive dislocation of these plugs initiates perfusion, with a dramatic rise in intervillous oxygen [27]. This level decreases to ∼6% O2 (40–50mmHg) towards term, with oxygen extraction by the fetus [28]. Normally various locations within the placenta are differentially perfused, with central regions (proximal to the umbilical cord) more oxygenated than peripheral sites. It is therefore predicted that the villous trophoblast encounters a range of oxygen tensions throughout gestation, depending on perfusion and spatial location.

In pre-eclampsia, typically of increased severity and early onset, intervillous hypoxia and localised ischaemia are anticipated from uterine artery Doppler velocimetry, where resistance indices and waveform notching are predictive of impedance to flow. In normal placentation, extravillous trophoblasts invade and remodel the maternal uterine spiral arteries, degrading and replacing the endothelium, smooth muscle and internal elastic lamina, increasing utero-placental blood flow and reducing vascular reactivity. In pre-eclampsia, shallow invasion by extravillous trophoblast restricts vascular remodelling to the decidua, maintaining vascular reactivity and resistance in the myometrium, potentiating vasospasm and intervillous under-perfusions [29]. The question therefore arises as to the influence of oxygen and ischaemia on villous trophoblast cell turnover and the potential for increased syncytiotrophoblast shedding.

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6. Oxygen and syncytiotrophoblast deportation 

Typically, cell-lines from extravillous trophoblasts undergo proliferation and differentiation in response to hypoxia [30], whilst those of villous origin and primary cytotrophoblasts from term placentae show enhanced proliferation and reduced syncytialisation [31]. In many, hypoxia induces both cell-cycle arrest and apoptosis. Kilani and colleagues have shown villous trophoblasts to be highly resilient to mild hypoxia, but succumb to apoptosis under more extremes of oxygen i.e. <15mmHg or >140mmHg [32]. We have identified similar effects in explanted villi, where cell-cell and cell-matrix interactions are maintained. Some of these findings are reported in this issue and show the impact of severe hypoxia on trophoblast apoptosis and syncytial knot formation.

It is predicted that syncytial fusion of cytotrophoblasts facilitates the delivery of the molecular machinery necessary for controlled apoptosis in the syncytiotrophoblast. It is therefore suggested that in explant cultures of term placental normoxia or hyperoxia, represented by 6% O2 (∼50mmHg) and higher, the efficient transfer of molecules crucial for the timely initiation and execution of syncytiotrophoblast apoptosis is maintained. Under restricted oxygen, typically 0–3% O2, two possibilities may explain the untimely demise of the syncytiotrophoblast, as observed in pre-eclampsia. In mild hypoxia, where cytotrophoblast proliferation predominates and syncytial fusion is inhibited, Huppertz and co-workers predict that the insufficient transfer of mRNA and protein prevents the syncytiotrophoblast from completing its extended program of apoptotic decline [33]. As a consequence, apoptotic events are interrupted and the aging syncytiotrophoblast is alternatively shed by necrosis, in a combination termed ‘aponecrosis’. Alternatively, under severe hypoxia, mitochondria could shift from being ATP producers to consumers, with anaerobic glycolysis significantly altering pH and stimulating the intracellular release of calcium, leading to lysosomal release and the loss of membrane and organelle integrity. In both situations, the liberation of necrotic syncytiotrophoblast material, in addition to, or in excess of, apoptotic extrusion, may be important.

In alterative cell systems, apoptotic material is generally phagocytosed in the absence of inflammation and may even promote active immunosuppression. In contrast, phagocytosis of necrotic cells encourages inflammation, a feature attributable to pre-eclampsia, but absent in other placental complications. Abumaree and colleagues recently demonstrated active phagocytosis of deported apoptotic trophoblasts by a monocytic cell-line and showed enhanced secretion of the anti-inflammatory cytokine, interleukin-10, and increase in the immunosuppressive enzyme, indoleamine 2,3-dioxygenase [34]. In addition to professional phagocytes, endothelial cells also engulf elements of shed and dying trophoblast in culture. In this respect, apoptotic trophoblast, generated again from villous explant cultures, failed to stimulate the expression of the endothelial activation marker intracellular adhesion molecule 1 (ICAM-1), but necrotic material enhanced its upregulation [35]. Together these findings imply that the mechanism of cell death (apoptotic or necrotic) could have a major influence on maternal leukocyte and vascular responses to deported trophoblast material in vivo.

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7. Oxidative stress and syncytiotrophoblast liberation 

In addition to utero-placental under-perfusion, the incomplete conversion of myometrial spiral arteries results in the retention of smooth muscle in the arterial media. Vasoactivity could persist, leading to intermittent perfusions, transient hypoxia and the potential for chronic low-grade ischaemia-reperfusion. Reported oxidative stress in the placenta is typified in pre-eclampsia by an oxidant-antioxidant imbalance, leaving the syncytiotrophoblast vulnerable to free radical attack [36]. Like hypoxia, oxidative stress is a powerful inducer of apoptosis and necrosis. During reperfusion, reactive oxygen species (ROS) are generated by the mitochondria and through the actions of xanthine oxidase and NAD(P)H oxidase; all are notably attenuated in pre-eclampsia [37].

Hung et al showed that hypoxia-reoxygenation of placenta villous explants stimulates syncytiotrophoblast apoptosis in vitro, supporting the involvement of oxidative stress in syncytiotrophoblast liberation [38]. Further investigations have correlated stress-induced apoptosis with the release of STBMs and modulation by the antioxidants vitamins C and E. Similar outcomes are noted for the liberation of cell-free feto-placental DNA and tumour necrosis factor (TNF)α [39], [40]. It is of note that circulating fetal DNA is 5-fold higher in pre-eclampsia [41], whilst TNFα is significantly elevated in maternal plasma and has the potential to upregulate molecules with the capacity to elicit endothelial activations, e.g. endothelin-1.

In a similar way to hypoxia, the path from reperfusion to syncytiotrophoblast degeneration potentially combines necrotic and apoptotic events. An excess of ROS can liberate intracellular Ca2+, opening mitochondrial permeability transition pores, releasing apoptogenic components such as cytochrome c and Apaf-1 (activators of the executioner caspases). The associated loss of membrane potential could also reduce ATP production to an extent where ionic homeostasis is lost and primary necrosis ensues. Thus, in addition to apoptosis, placental oxidative stress may also encourage the more detrimental form of syncytiotrophoblast necrosis.

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8. Is oxygen really involved? 

Although implied in vitro, a survey of the impact of oxygen on placental development and structure indicates that the concept of chronic placental hypoxia is over simplistic in establishing the aetiology of pre-eclampsia. For example, an examination of placental metabolism fails to reveal a reduction in energy utilisation in pre-eclampsia, as would be expected with chronic hypoxia [42]. Moreover, it is well accepted that the placenta is capable of considerable metabolic adaptations in vivo, so that its structural and functional integrity is maintained, even under stable low oxygen [43]. Despite numerous claims of intervillous hypoxia in late pregnancy, no direct in vivo measurements of dissolved oxygen have ever been reported. Failed trophoblast invasion may lead to placental ischaemia, but this is not specific to pre-eclampsia. Furthermore, in pregnancies at high altitude, i.e. those where oxygen is naturally reduced, placental structures are remarkably consistent and levels of infarction and lesions not typically raised.

Further confirmation for an alterative to oxygen-induced syncytiotrophoblast decline is presented in animal models which fail to show evidence of spontaneous pre-eclampsia, despite induced placental ischaemia. Moreover, in these situations, placental tissues show high levels of resistance to oxygen deprivation, often at the expense of the developing fetus [44]. In humans, ATP levels in the perfused placenta remain stable during periods of anoxia and even return to their pre-anoxic state following reperfusion, suggesting a general recovery in metabolic arrest [45]. It is predicted that such adaptations rely partly on suppression of protein synthesis and partly on the capacity for anaerobic glycolysis. Under physiological conditions, it is known that glycolysis in the placenta is partially uncoupled from the synthesis of ATP, due to mitochondrial deficiencies in respiration [46]. This partial uncoupling explains a general tolerance to low oxygen. Similarly, high levels of Hypoxia-inducible factor (HIF), an oxygen responsive transcription factor, may also provide an additional genetic basis for metabolic adjustments to oxygen restriction [47].

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9. Trophoblast predisposition to apoptosis 

When given the placenta's capacity to accommodate hypoxia in vivo and in vitro, alterative mechanisms for excessive syncytiotrophoblast shedding must be considered. Dependent upon stimulation, apoptosis is initiated by one of two pathways: the mitochondrial or intrinsic pathway, such as that induced by hypoxia, and the death receptor mediated or extrinsic pathway, initiated by ligand binding to a member of the superfamily of TNF-receptors. In observing both pathways in isolated cytotrophoblast and in vitro generated syncytiotrophoblast from pre-eclamptic pregnancies, we found apoptotic rates were significantly elevated in response to hypoxia and TNFα [48], a finding confirmed and extended in villous explant cultures [49]. These results suggest that anti-apoptotic elements of the trophoblast, particularly syncytiotrophoblast, are down-regulated in pre-eclampsia, conferring greater susceptibility and impaired resistance to apoptosis in vivo. In support, although not confirmed, a decline in anti-apoptotic Bcl-2 has been previously reported in the syncytiotrophoblast [21], [23] and, more recently, we have shown a fundamental imbalance in p53 and Mdm2, one predisposing both cytotrophoblasts and syncytiotrophoblast to early apoptosis in pre-eclampsia, particularly in response to exogenous stimuli [50].

Under normal circumstances, p53, an intracellular transcription factor, is present at low levels in the cytoplasm and is tightly controlled by Mdm2 [51], [52]. Through negative feedback, p53 promotes Mdm2 transcription and the balance between each determines cellular fate i.e. proliferation, differentiation or apoptosis. In mouse fibroblasts, the absence of Mdm2 permits p53 to act unopposed, thereby inducing apoptosis. In mouse knockout studies, Mdm2 deficient mice are embryonic lethal with signs of placental abnormalities. When superimposed with the simultaneous knockout of p53, the viability of offspring is restored, thus confirming the importance of p53 in both placental and fetal development [53].

In trophoblasts, villous p53 is upregulated in vitro and in vivo when levels of apoptosis are elevated [54], [55], [56]. Likewise, the expression of Bax and Bak, two pro-apoptotic onco-proteins upregulated and activated by p53, are associated with sites of fibrin-type fibrinoid deposition and villous damage [57], [58]. Further evidence to support the assumption that exaggerated p53 may trigger excessive apoptosis of trophoblasts comes from the observation that cultured cytotrophoblasts exhibit higher levels of constitutive apoptosis compared to their syncytialised counterparts [59]. In this context, Hu and colleagues attributed increased susceptibly to elevated basal p53 and an inverse but proportional decline in Mdm2 [60]. Within their observations, co-immunoprecipitations showed that Bak interacts more avidly with p53 in cytotrophoblasts than in syncytiotrophoblast. It could therefore be postulated that excessive trophoblast apoptosis in pre-eclampsia and consequently enhanced syncytiotrophoblast deportation could be a direct response to depleted Mdm2 and a concomitant increases in p53 and its interactions with Bak. Recent evidence suggests that elevations in N-myc down-regulated gene (NDRG1) can reduce the p60 form of Mdm2 in trophoblast in response to hypoxia [61]. Studies to confirm this and to identify other causes of this pivotal p53-Mdm2 imbalance in pre-eclampsia are currently under way.

An alternative, but perhaps allied explanation for excessive syncytiotrophoblast shedding has been presented by Soleymanlou and co-workers [62]. This group identified a novel Mtd/Bok splice isoform in the human placenta, termed Mtd-P, whose overexpression is related to trophoblast apoptosis. This pro-apoptotic oncoprotein of the Bcl-2 family appears unique to severe early onset pre-eclampsia, as tissues obtained from normotensive age-matched and term control subjects, as well as those with term pre-eclampsia, IUGR and essential hypertension, failed to exhibit elevated levels. The exact role of Mtd-P in regulating cytotrophoblast apoptosis is still unclear. In villous explant cultures, Mtd-P is increased under low oxygen, whilst in anti-sense experiments trophoblast Mtd-P is implicated in caspase activity, particularly in response to oxidative stress [62]. Whether increased Mtd-P expression is regulated directly by HIF-1α or other hypoxia-induced factors is unknown, but it is intriguing that p53 is a transcriptional regulator of Mtd in other cell systems [63].

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10. Haemodynamics and syncytiotrophoblast deportation 

Two previous structural comparisons of the human placenta investigated the combined consequences of pre-eclampsia and IUGR. The first by Teasdale showed villous composition in pre-eclampsia to be similar to that of gestationally matched controls and that changes in pre-eclampsia with IUGR, i.e. diminished villous volumes, were comparable to those with IUGR alone [64]. The second by Mayhew et al. showed that pre-eclampsia with superimposed IUGR and idiopathic IUGR had an equal impact upon trophoblast volume, both showing increased trophoblast epithelial thickness [65]. Our own investigations, using image analysis morphometry, refute the idea that differences in trophoblast content are common to growth-restricted cases; instead we identified structural similarities in the placentae of all pre-eclamptics, in the presence and absence of growth restriction [66]. These differences of reduced syncytial thickness and increased syncytial denudations are in accordance with excessive syncytiotrophoblast deportation. Moreover, by correlating these changes with birthweight at delivery (irrespective of placental weight), we provided additional testament to the importance of syncytiotrophoblast in optimising placental capabilities (Fig. 2).

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  • Fig. 2 

    Morphometric analysis of the villous placenta in pre-eclampsia. (A–C) Image manipulations of placental sections. (A) Monochrome image of villous tissue. (B) Image is pseudocoloured using morphometric software. (C) A threshold is applied (red background) and increased sequentially to highlight the syncytiotrophoblast. (D) Pre-eclampsia alone (PE) and pre-eclampsia with superimposed growth restriction (PE x IUGR) showing a reduction in syncytiotrophoblast, corrected for villous area. Idiopathic IUGR had no effect. (E) Irrespective of placental weight, syncytiotrophoblast cover was significantly correlated with birthweight. (Modified from Daayana et al. [66]; reproduced by kind permission of Elsevier Inc.)

This study, like all morphological and stereological observations, provides a global description of the villous placenta and fails to identify more discrete changes in tissue content. In pre-eclampsia, scanning EMs of the villous surface, particularly in severe cases, show signs of focal syncytiotrophoblast ulcerations, localised denudations, shedding, reduced microvilli and fibrinoid necrosis [67], [68]. To our shame as placentologists, this preferential targeting of the villous has received scant consideration, warranting considerable reassessment in light of our current thoughts on syncytiotrophoblast deportation. In this context, we have recently looked to haemodynamic forces as an alterative route to syncytiotrophoblast shedding, particularly in view of the utero-placental irregularities defined in placental-bed biopsies in pre-eclampsia, i.e. the inadequate transformation of uterine spiral arteries.

Until recently, spectral (velocity-based) Doppler of the uterine arteries has described increased resistance and pulsatility indices in early pre-eclampsia, and has suggested a reduction in intervillous perfusion [69]. Nevertheless, the tortuous nature and multidirectional flow within the placenta makes this approach inadequate for the true investigation of intra-placental blood flow and more specifically the measurement of haemodynamics within the intervillous compartments. Undoubtedly 3D power Doppler ultrasound will eventually readdress this problem and provide genuine real-time investigation of placental pathologies [70], but until then only a single complex analysis of intra-placental perfusions in complicated pregnancies has been reported and this unexpectedly illustrated localised intervillous hyperperfusions, not under-perfusion in pre-eclampsia [71].

This finding of exaggerated haemodynamics, although difficult to synchronise with increased uterine artery resistance, is explained using a rudimentary model of blood flow (Fig. 3). A review of the literature shows internal luminal diameters of spiral arteries at the decidual-myometrial zone are increased on average to 289±76μm in normal pregnancy [72], [73], [74], maintained at 107±12μm in pre-eclampsia [72], [75], [76], and further reduced to 34±3μm with atherosclerotic plaques (a frequent confounder of the syndrome) [72], [77]. In a simple vessel system, a reduction in luminal diameter encourages a disproportionate increase in blood flow velocity. As brilliantly demonstrated in the pioneering work of Elizabeth Ramsey, using radioangiographic techniques to visualise the maternal circulation in the primate haemochorial placenta, entry of the arterial blood into the intervillous spaces does so intermittently as characteristic ‘jets’ or ‘spurts’, quickly dispersed by the chorionic villi [78]. In normal pregnancy, utero-placental blood flow increases tenfold across gestation. It may therefore be assumed that small bore vessels, like those in pre-eclampsia, would ‘inject’ maternal blood over the villous surface under raised velocity and turbulence. Therefore, unlike the languid blood flow required for optimal oxygen transfer, the branch-like villi of the placenta in pre-eclampsia will be exposed to spurts of blood, with greater damaging capabilities. These more aggressive flow rates could emerge in pulses, still influenced by the maternal cardiac cycle and may perpetuate the physical disruption of the syncytiotrophoblast, particularly at points of spiral artery incursion.

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  • Fig. 3 

    Schematic showing the generation of intervillous haemodynamic velocities and turbulences. (A) A reduction in internal luminal diameter (or radius, r) leads to a disproportionate increase in flow velocity (v) under constant flow. (B) The potential outcome of adequate and inadequate transformations of uterine spiral arteries in normal pregnancy and pre-eclampsia. Intra-luminal obstructions such as atherosclerotic plaques may further exaggerate disruptive blood flow entering the intervillous space.

Although preliminary, our investigations of this hypothesis have been encouraging. We have used a placental perfusion system in which lobules of placentae from healthy term human pregnancies were dually perfused in open-circuit [79]. Initial maternal perfusions of the intervillous spaces were conducted at 14ml/min and then at 55ml/min; mimicking the potential blood flow and intervillous turbulences in healthy pregnancy and pre-eclampsia. Notably the villi exposed to low rate perfusions showed minimal damage, whilst those under higher pressures showed signs of tissue disruption, typically syncytiotrophoblast shedding and oedema, localised to cannulae insertions [79]. In line with this haemodynamic concept of syncytiotrophoblast liberation, perfusates recovered under higher flow conditions significantly diminished the survival of endothelial cells in culture [79]. In these experiments the liberated syncytiotrophoblast would be necrotic and therefore have greater propensity for pre-eclamptic initiation. We are currently defining the active factors within these perfusates and adapting this model to better reflect the known and predicted features of the utero-placenta in pre-eclampsia.

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11. Placental apoptosis: a therapeutic target? 

With current limitations in defining the factors responsible for pre-eclampsia, an alterative may be to directly prevent syncytiotrophoblast shedding from the villous placenta, either as a result of necrosis or apoptosis. It could be hypothesised that by suppressing trophoblast cell turnover, syncytiotrophoblast integrity could be maintained, placental capacity optimised and fetal growth encouraged. As a concept this approach has received little attention, but our recent studies have shown a number of therapeutic possibilities. In this context, we have examined the effects of mitotic manipulators on trophoblast cell turnover, explored the idea of placental gene therapy and we are currently investigating the inhibition of placental p53, as a means of suppressing syncytiotrophoblast apoptosis.

Traditionally, proliferation and differentiation are considered mutually exclusive, with cell cycle withdrawal acknowledged as a prerequisite for terminal differentiation. We have explored this concept in syncytiotrophoblast formation in vitro using a series of highly characterised mitotic blockers [80]. In these experiments, two agents successfully encouraged syncytiotrophoblast formation, Ara-c and l-mimosine, inhibitors of DNA synthesis and replication, respectively. Although we would not advocate the use of these agents in human pregnancy, particularly Ara-c which is a known teratogen [81], the potential for influencing mitotic activity in the human placenta in late gestation should not be overlooked. The suggestion that syncytiotrophoblast is an additional determinant of neonatal birthweight in humans [66], implies that a mechanism for encouraging syncytial formation, even for a short period, may be beneficial. We would therefore anticipate that a window of opportunity exists at the clinical onset of complications, such as pre-eclampsia, when syncytiotrophoblast consolidation may be advantageous.

This approach may also apply to endogenous growth factors, which are known to influence placental development and trophoblast survival. Past studies have focused on insulin-like growth factor (IGF) and epidermal growth factor (EGF), both of placental origin. IGF is a natural product of the utero-placenta I interface, shown to regulate fetal growth and placental development [82], [83], [84]. EGF is present in high concentrations in maternal and fetal serum and has multiple effects on placental trophoblasts, including the inhibition of TNFα and hypoxic-induced apoptosis through phosphatidylinositol 3-kinase/protein kinase B (Akt) survival pathways and the enhancement of differentiation and syncytiotrophoblast formation [54], [85], [86], [87]. Given this weight of evidence, it could be envisaged that the overexpression of placental IGF or EGF in vivo may have the capacity to normalise syncytiotrophoblast depletion, thereby restoring placental efficiency.

To encourage EGF expression in complicated pregnancies, we are currently considering placental gene therapy, introducing novel genetic material into placental trophoblasts. This technique, which is already exploited in the treatment of adult cancers, diabetes, neurological disorders and cardiovascular disease, is a realistic proposal in utero, with potential benefits for many conditions of placental dysfunction, not just pre-eclampsia. In terms of gene therapy, the placenta is an ideal target. Although transferring genes directly to the fetus may cause problems with immune tolerance, stability of transgene expression, tissue targeting and fetal survival, the targeting of genes to the placenta, an immunologically privileged site, means that active proteins may be delivered without modifying the maternal or fetal genome. Indeed, novel genes have already been transferred to the rodent placenta by ex vivo gene transfer techniques [88], [89]. Fibroblasts of placental origin are shown to be amenable to adenoviral transfection with IGF and this exerts a positive autocrine and paracrine effect [90], enhancing their proliferation, migration and survival in vitro. In extending these studies, we are presently over-expressing EGF in placental villous tissues, particularly in villous trophoblasts, as a target for suppressing syncytiotrophoblast shedding. If successful, these studies will progress to animal models and more physiological systems.

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12. Conclusion 

Villous trophoblast cell turnover, both in normal pregnancy and pre-eclampsia, has been an intense area of research in recent years. Fundamental questions regarding the cause of elevated placental cell death in pre-eclampsia remain, but it is increasingly acknowledged that cellular material from the villous surface contributes to the maternal syndrome. Non-invasive techniques, such as high power ultrasound, will be increasingly important in informing future studies. Moreover, greater emphasis will be placed on addressing aberrant trophoblast regulation, as a means of combating this and other pregnancy complications.

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Acknowledgements 

I would like to acknowledge the support of Tommy's—The Baby Charity, The Castang Foundation and Department of Health, and the guidance and mentoring of Professors Philip Baker and John Aplin.

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PII: S0143-4004(07)00030-6

doi:10.1016/j.placenta.2007.01.016

Placenta
Volume 28, Supplement , Pages S4-S13, April 2007

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