Apoptosis and its role in the trophoblast

American Journal of Obstetrics and Gynecology (2006) 195, 29–39

www.ajog.org

REVIEW ARTICLES

Apoptosis and its role in the trophoblast Berthold Huppertz, PhD,a Mamed Kadyrov, MD,a John C. P. Kingdom, MDb Department of Anatomy II, University Hospital RWTH,a Aachen, Germany; Department of Obstetrics and Gynecology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto,b Toronto, Canada Received for publication April 6, 2005; revised May 30, 2005; accepted July 6, 2005

KEY WORDS Villous and extravillous trophoblast Apoptosis Invasion Intrauterine growth Preeclampsia

During early placentation the trophoblast of the human placenta differentiates to the villous and extravillous types of trophoblast. Villous trophoblast provides the epithelial cover of the placental villous trees in direct contact to maternal blood. Extravillous trophoblast invades maternal uterine tissues thus directly contacting maternal stromal and immune cells. A subset of extravillous trophoblast, endovascular trophoblast initially occludes the lumen of spiral arteries and comes into direct contact with maternal blood. In recent years apoptosis has been described in both types of trophoblast and the importance of this cascade for the normal function of the trophoblast has become obvious. One feature of serious conditions such as preeclampsia or intrauterine growth restriction is changes in apoptosis regulation in villous and/or extravillous trophoblast resulting in altered trophoblast invasion and/ or shedding into the maternal circulation. This review summarizes recent findings on trophoblast apoptosis in normal and pathologic pregnancies. Ó 2006 Mosby, Inc. All rights reserved.

The trophoblast lineage is the first to differentiate during human development, at the transition between morula and blastocyst. Initially, at day 6 to 7 postconception, a single layer of mononucleated trophoblasts surrounds the blastocoel and the inner cell mass. At the site of attachment and direct contact to maternal tissues, trophoblast cells fuse to form a second layer of postmitotic multinucleated syncytiotrophoblast.1 Once formed, the syncytiotrophoblast grows by means of steady incorporation of new mononucleated trophoblasts from a proximal subset of stem cells.2 Only at around day 14 mononucleated cytotrophoblasts break through the syncytiotrophoblast and begin to invade the uterine stroma at sites called trophoblastic cell columns. Such cells are termed extravillous trophoblasts. Reprints not available from the author. 0002-9378/$ - see front matter Ó 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.ajog.2005.07.039

Both types of trophoblast keep a subset of cells in direct contact to the villous basement membranedthese cells retain their generative potential (Figure 1) and are able to proliferate in response to growth factors such as FGF4.3 In the extravillous compartment, cell proliferation advances rows of extravillous trophoblasts into the uterine stroma where they stop proliferating and undergo invasion as a result of differentiation. Similarly, differentiating cells resulting from mitosis in the villous compartment are postmitotic and undergo syncytial fusion directed by the transcription factor glial cell missing (GCM1).4 Somehow the physical interaction of proximal cytotrophoblasts with the basement membranes allows them to retain a proliferative phenotype. Villous cytotrophoblasts can be made to form extravillous columns, indicating that they have stem cell-like properties.3

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Figure 1 Schematic representation of the 2 types of trophoblast: villous and extravillous trophoblast. The villous trophoblast stem cells (with dark nuclei) differentiate, fuse, and maintain the multinucleated syncytiotrophoblast. Within the syncytiotrophoblast, a second differentiation pathway takes place resulting in the accumulation of aged nuclei in syncytial knots (black arrows in villus on the lower left). The extravillous trophoblast stem cells (with dark nuclei) are localized at the basement membrane of anchoring villi in the most proximal part of the cell columns. Their postproliferative daughter cells invade maternal tissues as interstitial trophoblast penetrating endometrium and the first third of the myometrium (light grey arrow). A subset of the interstitial trophoblast reaches the walls of spiral arteries and becomes endovascular trophoblast (dark grey arrow to the right).

Villous trophoblast and the role of apoptosis In the last decade we and others have characterised the role of the apoptosis cascade in villous trophoblast turnover and syncytium formation. Observations indicate that the process of syncytial fusion is linked to the ‘‘initiator stages’’ of the apoptosis cascade within the cytotrophoblast cells, whereas the extrusion of syncytial knots from the syncytiotrophoblast is the result of the

final ‘‘execution stages’’ of the apoptosis cascade within the syncytiotrophoblast.5-9

Cytotrophoblast and initiator stages of apoptosis At present, the mechanisms through which the apoptosis cascade is initiated in cytotrophoblasts, then subsequently

Huppertz, Kadyrov, and Kingdom regulated in the syncytiotrophoblast layer, remains unclear. Clearly, the initiation stage must be confined to a subset of cells, so that a population of proliferating cells can continuously provide new cellular material for the overlying syncytiotrophoblast layer. A subset of human cytotrophoblasts expresses the DNA transcription factor GCM14 that in mice is known to arrest cell proliferation and trigger syncytial fusion.10 This pattern of asymmetric expression of a transcription factor regulates the process by which cells are selected for syncytial fusion: These same cells start the expression pathway of apoptosis-related proteins that are needed for syncytial fusion. These include effectors as well as inhibitors of the cascade such as caspases or proteins of the Bcl-2 protein family.7 The family of caspase proteins includes intracellular proteases that cleave their targets next to an aspartic acid residue, thus termed cysteine aspartases or caspases. On the basis of structural homologies and substrate preferences, the family has been subdivided into subfamilies. All the members of the subfamily of the caspase 3-like caspases (caspases 3, 6, 7, 8, 9, and 10) play central roles in the apoptosis cascade.11 This subfamily is further divided into signalling/initiator caspases 8, 9, and 10 and effector/execution caspases 3, 6, and 7.12,13 The major difference between members of the 2 groups is that initiator caspases are active during the early, or reversible, stages of the apoptosis cascade, whereas activation of the so-called effector caspases, is a subsequent transition that will ultimately lead to apoptotic cell death. Caspases are synthesized as inactive enzymes that require cleavage to exert their biologic roles. Initiator caspases 8 and 10 are activated in a subset of differentiated cytotrophoblasts, presumably destined for syncytial fusion.6 By contrast, effector caspases (3, 6, and 7) are expressed only in their inactive forms in the cytotrophoblast layer.14 Ex vivo explant and cell culture systems may be prone to execution caspase activation in cytotrophoblasts in vitro; investigators should be cautious regarding the interpretation of these findings because freshly obtained tissues do not show activity of these enzymes in the cytotrophoblast layer.6 Activation of the initiator caspase 8 is achieved by ligand-receptor interactions, eg, interactions between tumor necrosis factor-a (TNF-a) and the TNF-receptor 1 (TNF-R1)15,16 (Figure 2). The cytokine TNF-a is capable of inducing apoptosis in isolated trophoblast cells in vitro,17,18 but this may not be of physiologic relevance. The crucial role of caspase 8 in preparing cytotrophoblasts for syncytial fusion was demonstrated by Black et al9 who showed that antisense and inhibitor disruption of caspase 8 expression and activation hindered syncytial fusion in floating villous explants, resulting in an accumulation of layers of mononuclear cytotrophoblasts. Attempts to isolate cytotrophoblasts

31 inevitably results in a mixture of cytotrophoblasts together with mononucleated fragments of the syncytiotrophoblast.6,19 In such cell fractions, many markers of apoptosis can be found, including TUNEL activity, annexin V binding, ATP:ADP ratio changes, and activity measurements of caspases.20,21 These data underscore the limitation of studying apoptosis regulation in isolated cell preparations. Caspase 8 activity results in the cleavage of proteins linking the cytoskeleton to the plasma membrane such as alpha-fodrin, and in externalization of phosphatidylserine from the inner to the outer leaflet of the plasma membrane,6,16 the so-called ‘‘PS-flip’’ (Figure 2). Blockage of fodrin by microinjection of anti-alpha-fodrin antibodies into IMR-33 cells and Madin-Darby bovine kidney epithelial cells22 triggered cell-cell fusion. The flip of phosphatidylserine can be blocked by the caspase inhibitor zVAD-fmk23,24 and the flip takes place before the release of cytochrome c into the cytoplasm.25 Huppertz et al5 have demonstrated that the phosphatidylserine flip occurs in villous cytotrophoblast before syncytial fusion. This temporal sequence has also been observed during the formation of the multinucleated myotubes of the skeletal muscle.26 In general, once a cell has activated the effector caspase pathway, it will die by apoptosis within 24 hours. However, in the villous trophoblast, cytotrophoblast nuclei that enter the syncytiotrophoblast remain intact and viable for a few weeks. Cytotrophoblasts preparing for syncytial fusion initiate some of the cellular machinery needed for apoptosis, yet they in tandem produce several apoptosis inhibitors (Figure 2). One of the earliest reports of apoptosis-related proteins in the human placenta showed high concentrations of the apoptosis inhibitor Bcl-2 in the trophoblast.27 Other antiapoptotic members of the Bcl-2 protein family have been described in the cytotrophoblast as well.5,28-30 Antiapoptotic members of the Bcl-2 protein family inhibit the progress of the apoptosis cascade at the mitochondrial level between activity of initiator caspases 8 and 10 and activation of execution caspases. Therefore, they are optimally positioned to stop the cascade after the early stages characterised by the activity of the initiator caspases (Figure 2). Regulation of the number of active caspases is possible by flice-like inhibitory protein (Flip). Flip is expressed in the placenta31 and competes with caspase 8 for binding to activated death receptors such as TNFR1 or Fas.32 Such a mechanism inside villous cytotrophoblast may tightly regulate the number of activated caspase 8 proteins and reduce the degradative activity of these enzymes. Most members of the inhibitor of apoptosis proteins (IAP) family of apoptosis inhibitors have been described to be expressed in villous trophoblast throughout gestation.33 Gruslin et al34 demonstrated XIAP to be

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Figure 2 Scheme of the apoptosis cascade within the villous trophoblast. In postproliferative cytotrophoblasts initiator caspases such as caspases 8 or 10 are activated by ligand receptor interactions. Activity of initiator caspases results in cleavage of alpha-fodrin and the redistribution of phosphatidylserine within the plasma membrane (PS-flip), both are know to be prerequisites for syncytial fusion. Activation of downstream effector caspases 3, 6, or 7 is hindered by activity of inhibitors such as the Bcl-2 family of proteins. Only after fusion the downstream events of the apoptosis cascade take place. Thus, within specific sites of the syncytiotrophoblast activity of effector caspases leads to cleavage of cytokeratin 18 (formation of the neoepitope recognised by the antibody M30; 39) and cleavage of DNA (TUNEL positivity).

highest in first trimester placenta and nearly absent at term, whereas Straszewski-Chavez et al35 were able to show that XIAP confers resistance to Fas-induced apoptosis in trophoblast cells.

Syncytiotrophoblast and execution stages of apoptosis Formation, growth, and maintenance of the syncytiotrophoblast layer throughout pregnancy are achieved by continuous fusion of new cytotrophoblasts. The fusion process integrates the cytoplasmic content and nuclei from cytotrophoblasts into the syncytiotrophoblast. This process supplements the syncytiotrophoblast with

the complete machinery of the apoptosis cascade into this layer. As stated, these proteins include antiapoptosis enzymes, thus explaining why the apoptosis cascade does not immediately progress to its final events inside the syncytiotrophoblast, ie, DNA degradation and formation of syncytial knots. Our morphometric data imply that a single nucleus of a trophoblast cells stays within the syncytiotrophoblast for about 3 to 4 weeks after syncytial fusion5,8 (for a detailed calculation see ref 36, p 75 and 76). Arresting the apoptosis cascade directly after fusion may be due to the same mechanisms as in the cytotrophoblast, the activity of inhibitors at the mitochondrial level. Compared with the cytotrophoblast, antiapoptotic members of the Bcl-2 family seem to be upregulated in the syncytiotrophoblast, suggesting

Huppertz, Kadyrov, and Kingdom a role for these proteins in preventing ongoing apoptosis following syncytial fusion.27,28 The apoptosis cascade somehow is reactivated after several weeks but the trigger mechanisms are presently unknown. The syncytiotrophoblast may exist in a latent stage of apoptosis, because aging of syncytiotrophoblast nuclei from fusion to packing into syncytial knots is reflected by changes of the nuclear shape. Syncytial nuclei that are freshly incorporated after syncytial fusion are large, ovoid, and rich in euchromatin. During their passage through the syncytium the nuclei become smaller and denser. Finally, they display dense aggregations of chromatin underneath the nuclear membrane.5,37,38 Parallel to nuclear aging the cytoplasmic organelles such as rough endoplasmic reticulum, polysomes, and mitochondria are reduced, ending with the degranulation of the endoplasmic reticulum into a smooth endoplasmic reticulum.36 Finally, effector caspases such as caspases 3 and 6 are active in specific sites of the syncytiotrophoblast, leading to cleavage and degradation of proteins and nucleic acids, thus representing the final stages of the apoptosis cascade.5,6 These execution stages within the syncytiotrophoblast are tightly regulated both temporally and spatially. For example, despite entry of cytotrophoblast into a borderless syncytium, activation of caspase 3, and progression toward the nuclear component of apoptosis, is confined to discrete areas that ultimately develop into syncytial knots. Under normal conditions the degradative effects, such as DNA degradation (TUNEL positivity) and degradation of the cytokeratin filaments (effector caspase-dependent cytokeratin 18 neo-epitope formationdM30 reactivity) are restricted to certain sites and do not distribute to larger compartments9,39,40 (Figure 2). A morphologic proof for DNA degradation is the use of an electron microscope, which is regarded as the gold standard to detect nuclear changes during late apoptosis even today.14 The degradation of the cytoskeleton in parts of the syncytiotrophoblast may be responsible for the impaired anchorage of older syncytial nuclei. The aged nuclei seem to move toward the villous tips and accumulate in tightly packed clumps known as syncytial knots. Whether the driving force for the nuclear accumulation is provided by shear stress caused by maternal blood flow in the intervillous space still remains open. This hypothesis is supported by the fact that nuclear accumulations do not take place at sites with arrested maternal circulation in vivo. The final event of the apoptosis cascade inside the syncytiotrophoblast is the package of old and late apoptotic nuclei into protrusions of the apical membrane of the syncytiotrophoblast, called syncytial knots. The knots morphologically correlate to apoptotic bodies found in late apoptotic mononucleated cells. In the multinucleated syncytiotrophoblast formation of syncytial knots is the

33 classical morphologic feature of the final stages of apoptosis in this layer.5,37,38 The syncytial knots are then released from the apical plasma membrane of the syncytiotrophoblast and extruded as tightly sealed corpuscular structures into the maternal circulation (Figure 3, A). Inside the maternal blood stream they have been detected in maternal uterine vein blood41 as well as in pulmonary vessels,42,43 sometimes leading to trophoblast embolism inside the lung.44-46 These large corpuscular and apoptotic syncytial fragments are found in uterine venous, but not arterial, blood of pregnant women.41,47 This difference is due to engulfment by pulmonary macrophages,42,48 thereby dramatically reducing the number of syncytial knots in peripheral blood of the mother. In contrast, subcellular membrane fragments and cell-free molecules may circulate freely and pass the capillaries of the lung, eg, in preeclampsia.47 Occasionally, larger trophoblast structures may be found in peripheral venous blood of healthy pregnant women.49 Calculation of villous cytotrophoblast proliferation, syncytial fusion, and villous growth over gestation leads to the conclusion that several grams of material are shed per day as apoptotic syncytial knots into the maternal circulation by term.5,8,36 It is important to note that the released material is packed into tightly sealed syncytial knots hence preventing an inflammatory response by the mother’s blood vessels and organs.

Extravillous trophoblast and the role of apoptosis At least 2 main subpopulations of extravillous trophoblast exist: (1) interstitial trophoblast, comprising all those extravillous trophoblasts that invade uterine tissues and that are not located inside vessel walls and lumina; and (2) endovascular trophoblast, located inside the media or lining the spiral artery lumina and partly occluding those (sometimes this subtypes is further subdivided into intramural and endovascular trophoblast). There is recent evidence that endovascular trophoblasts are derived from a side route of differentiation from interstitial trophoblast.36,50

Regulation of extravillous trophoblast invasion Eventually, during early placentation the invading extravillous cytotrophoblasts come into contact with the tips of endometrial spiral arteries. These cells penetrate the vessel walls, reach the lumen of the arteries and block the transfer of blood cells to the placenta51 by generating aggregates inside the lumen and thus plugging the distal ends of the vessels.52,53 Hence, free communication between spiral arteries and the placenta

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Huppertz, Kadyrov, and Kingdom is not established until the end of the first trimester. Plugging hinders the entrance of maternal blood cells to the inside of the placenta, the implication being that development of the conceptus and the placenta takes place in a low-oxygen environment, less than 20 mm Hg, until 10 to 12 weeks of gestation.54,55 During differentiation of the principal embryonic organs and the placenta, development is highly vulnerable to disturbances by free radicals.56 Hence, plugging of spiral arteries may serve as a mechanism to protect the fetus and the placenta from free radical mediated distress.57,58 At the end of the first trimester, the luminal plugs of the trophoblast cells begin to disintegrate and first arterial inflow from the mother to the intervillous space is observed.59 Studies that used Doppler ultrasound have revealed that arterial inflow normally starts in the periphery of the placenta, extending progressively toward the center.60 The depth of vessel erosion is greatest under the center of the placenta.61 This onset of intervillous blood flow is associated with a clear rise in oxygen concentration within the placenta,54,55 providing an important stimulus for placental oxidative stress and possibly trophoblast damage.55,62

The apoptosis cascade in extravillous trophoblast

Figure 3 Modes of releasing syncytial material into the maternal circulation. A, Normal apoptotic shedding. Turnover of villous trophoblast comprises cytotrophoblast (CT) proliferation and differentiation, syncytial fusion of CT with the syncytiotrophoblast (ST), further differentiation inside the ST and package of old material into apoptotic syncytial knots. These corpuscular structures are surrounded by a sealed plasma membrane when they are extruded into the maternal circulation not inducing any inflammatory response of the mother. B, Aponecrotic shedding. Finalization of the apoptosis cascade may fail because of energetic or other problems. At these sites necrotic breakdown of the plasma membrane may take place. Thus, the already apoptotically cleaved material is released by necrosis (aponecrosis) and may induce inflammation in the mother. C, Necrotic shedding. Necrotic breakdown of syncytial sites releases cell-free trophoblast material into the maternal circulation, thus inducing an inflammatory reaction of the mother.

Regulation of the apoptosis cascade in extravillous trophoblast is poorly understood in part because of the lack of access to relevant cell types and tissues, such as placental bed biopsy specimens. Mostly, end stages of the cascade have been quantified, eg, by detecting M30 positive or TUNEL positive cells. Thus far, isolation of pure primary extravillous trophoblast has not been possible without altering the phenotype. Further, all cell lines tested so far only partly resemble the characteristics of extravillous trophoblast. Therefore, most of the studies that have been performed have made use of fixed tissues from placental bed biopsy specimens63 or hysterectomy specimens.64,65 Present information can be summarized as follows: Inducers of the cascade such as Fas/CD95 and its ligand FasL/CD95L have been detected in extravillous trophoblasts. Fas and FasL have been shown to be present on extravillous trophoblasts of first-trimester tissues as well as of term basal plates.66-68 Bcl-2, one of the inhibitors of apoptosis, has been described to be expressed mostly in the upper parts of cell columns (representing the proliferative cells and early postproliferative daughter cells).66,68,69 Nothing is known about presence and/or activity of caspases and presence of other inhibitors or inducers of apoptosis. We have investigated the apoptosis cascade along the invasive pathway in early pregnancy but only published it in an abstract70: We showed that the

Huppertz, Kadyrov, and Kingdom proliferative stem cells do not show any signs of apoptosis with a start of expression of death receptors (Fas and TNF-R1) as well as apoptosis inhibitors (Bcl-2 and Mcl-1) in the early postproliferative daughter cells as described previously. Slightly deeper both groups of proteins disappear again being replaced by the expression of the effector caspase 3. In the depth of the junctional zone, single extravillous trophoblasts appear to be TUNEL positive, thus showing signs of end stages of apoptosis. Late stages of apoptosis are mostly detected in the lower part of the endometrium or in the myometrium.64,66,68,71 Although only few data are available, the apoptosis cascade in the extravillous trophoblast can be depicted as follows: Along the invasive pathway there is a shift from being vulnerable to induction (expression of death receptors) and blockage of apoptosis (expression of Bcl2 family proteins) to start of the execution program (expression of caspase 3) and finally death of the cells (TUNEL and M30 positivity) deep in the placental bed. Similar to the villous trophoblast it seems as if the extravillous trophoblast contains 2 parallel pathways of differentiation and apoptosisdor simply 1 pathway that uses events from both cascades. Regulation of the apoptosis pathway is still a mystery but it seems as if decidual cells and local maternal immune cells present in the uterus and placental bed play crucial roles in the balance of life and death of extravillous trophoblasts.67,72 This interplay between trophoblast and an array of maternal cells increases the complexity of cell death regulation in the placental bed.73

Trophoblast apoptosis in intrauterine growth and preeclampsia Apoptosis of villous trophoblast is upregulated in both of the common pregnancy diseases related to the placenta, namely, intrauterine growth (IUGR) and preeclampsia.74-77 Because apoptosis does not incite an inflammatory response, it seems unlikely that an apoptotic release of trophoblast material in 1 case has no effect (IUGR), whereas it should have a detrimental effect in the other case (preeclampsia). Another explanation may include the following: First, the whole turnover of villous trophoblast is increased in preeclampsia, commencing with increased proliferation of cytotrophoblast.78 This alone could produce increased end stages of apoptosis in the syncytiotrophoblast.7 However, if apoptosis is temporally regulated, an increased input of material caused by the increased proliferation and fusion may not permit sufficient time for the apoptosis cascade to be completed before shedding takes place. In this hypothesis, a direct consequence of this imbalance is that necrotic breaks occur in the syncytio-

35 trophoblast, leading to the necrotic release of syncytiotrophoblast that has only partly completed the apoptosis cascade (Figure 3, B). This is termed aponecrosis79,80 and refers to the disruption of an energy-dependent programmed process (apoptosis) in favor of a chaotic energy-independent process (necrosis) that disrupts cellular contents, including activated enzymes, into the surrounding local environment to cause local tissue damage. Increased necrotic release of trophoblast (Figure 3, C) may incite both local placental damage (focal excessive perivillous fibrin deposition) as well as the systemic manifestations of preeclampsia. Upregulated apoptosis alone cannot directly induce these changes. Apoptosis of extravillous trophoblast may be upregulated in both IUGR and early-onset preeclampsia though no consensus exists at present in the literature. It is generally accepted that several placental diseases are associated with reduced trophoblast invasion of uteroplacental arteries. Spontaneous abortion may be accompanied by a complete absence of trophoblast invasion,60,81 whereas less severe impairments are associated with early-onset IUGR, with or without coexistent preeclampsia.64,82,83 More than 85% of such cases have abnormal uterine artery Doppler, which in turn correlates with the extent of decidual vasculopathy in the placental bed18,84 and membranes.84 Basic research suggests that this reduced invasion of uteroplacental arteries in the placental bed results from intrinsic (trophoblastic) factors in combination with extrinsic (maternal uterine) factors, such as impaired remodeling of the deciduas,85,86 macrophage-originated defense mechanisms,18,87 impaired function of uterine natural killer cells,88 and failure of the maternal endothelium to express selectins.89,90 Interpretation of histopathologic data is therefore challenging, especially as apoptosis is temporally regulated. All the above factors may interact and/or depend on each other generating a cascade of events finally resulting to the malinvasion observed in IUGR.50

Apoptosis of interstitial trophoblast An old but never proven hypothesis claims that the sole cause for preeclampsia is a generalized impaired trophoblast invasion, that is, both interstitial and endovascular trophoblast invasion are reduced because of increased apoptosis of all extravillous trophoblasts. Statements such as ‘‘.preeclampsia is associated with abnormally shallow placentation.,’’50 ‘‘.hypoinvasive placental phenotype characteristic of preeclampsia.,’’91 and ‘‘.shallow trophoblast invasion.predisposing the pregnancy to preeclampsia.,’’92 reinforce the notion that impaired/shallow invasion of interstitial trophoblast results in reduced endovascular trophoblast invasion, leads to maladaptation of uteroplacental arteries and that this

36 somehow causes preeclampsia. This view has recently been challenged by our quantitative and comparative study on the invasion of interstitial trophoblast in full thickness uterine walls comprising the placental bed from patients with IUGR and preeclampsia and control cases.64 We were able to show that interstitial trophoblast apoptosis within the placental bed from cases with IUGR and preeclampsia was reduced in contrast to previous studies on basal plates from delivered placentas.93-95 These contrasting reports on extravillous trophoblast apoptosis give room for new and more appropriate hypotheses to explain impaired adaptation of uteroplacental arteries during pregnancy. In this context it is important to remind the new and highly important observations of Jauniaux et al and Burton et al.14,55,60 These authors described the adverse effects of the premature onset of maternal blood supply to the placenta during the first trimester of pregnancy. They mostly focused on increased oxidative stress in the chorionic villi. Another aspect of the premature rise of placental oxygen is the damage of the stem cell pool that will subsequently contribute to both the villous and the extravillous compartments of differentiated trophoblast.3 These cells, resting on the basement membranes of anchoring villi (Figure 1) show high rates of proliferation during the first trimesterdunder low oxygen. A premature rise in oxygen may dramatically reduce the generative potency of these cells impairing the amount of cells used to invade maternal tissues. For example, the density and proliferative activity of villous cytotrophoblasts is dramatically reduced in severe early-onset IUGR, with or without preeclampsia. This was interpreted as being caused by ‘‘placental hyperoxia’’ at the time of delivery,96 although a revision of this concept would state that the hyperoxia had occurred inappropriately at the end of the first trimester. Finally, inadequate endovascular trophoblast occlusion of spiral arterioles during the first trimester, producing elevated local oxygen tension, may arrest expansion of interstitial trophoblast progenitor cells at the base of anchoring columns, leading to reduced interstitial invasion as the second trimester proceeds.64

Apoptosis of endovascular/intramural trophoblast When activated in vitro, macrophages are capable of inducing apoptosis in an immortalized extravillous trophoblast cell line.18 Induction of apoptosis was achieved by the concerted action of the secretion of TNF-a that binds to the trophoblastic TNF-R1, and the secretion of indolamine 2, 3 dioxygenase (IDO) that catabolizes and depletes local levels of tryptophan. These in vitro data can explain the inverse relation between the number of macrophages and intramural trophoblast in the walls of

Huppertz, Kadyrov, and Kingdom uteroplacental arteries.87 In normal pregnancy, the walls of uteroplacental arteries become invaded by trophoblast and are largely devoid of macrophages. In contrast, in IUGR with preeclampsia, reduced trophoblast invasion of uteroplacental vessels is combined with an accumulation of apoptotic trophoblast in direct vicinity of the arterial walls.18,87 A quantitative study on the rates of apoptosis and invasion of intramural and endovascular trophoblast was recently published by Kadyrov et al.65 These authors present evidence for a significantly reduced invasion of spiral arteries in cases with early-onset IUGR and preeclampsia. This pathologic syndrome is characterized by a reduced number of trophoblast cells present in the arterial walls, an increased rate of apoptosis of these cells, and a lack of vasodilation. In early-onset IUGR with preeclampsia, the endovascular trophoblast exhibits an increased rate of apoptosis in the walls of maternal spiral arteries, thus pointing to apoptosis-promoting signals derived from maternal cells. Activated maternal macrophages may induce trophoblast apoptosis in close vicinity to spiral arteries.15,87 The data of Huppertz et al65 strengthen the concept of excessive apoptosis as a mechanism to limit endovascular trophoblast invasion into the walls of spiral arteries.50 Taking together, both pathways of trophoblast invasion (interstitial and endovascular) are affected by maternal factors.55,58,60,97 Interstitial invasion is affected by the premature rise in oxygen within the placenta and reduced proliferation, whereas the endovascular pathway is affected by macrophage-induced apoptosis of perivascular and intramural trophoblast. Finally, both events limit the number and the extent of adaptation of spiral arteries to the needs of the growing fetus.

Conclusion In both trophoblast compartments, namely, the villous trophoblast and the invading extravillous cytotrophoblast, apoptosis is a normal constituent of cell turnover. The phenomenon of apoptosis ensures that cells no longer functional can be eliminated without a local inflammatory reaction by the maternal host. By contrast, abnormalities in trophoblast cell proliferation and/or differentiation may render either trophoblast compartment susceptible to abortive forms of necrosis, termed aponecrosis. Because extravillous trophoblast cells are trapped in the decidua and myometrium, the response (decidual vasculopathy) is local. By contrast, escape of aponecrotic villous trophoblast via the uterine veins into the systemic venous circulation means that the host inflammatory reaction becomes systemic, and is manifest normally as hypertension and proteinuria.

Huppertz, Kadyrov, and Kingdom Placental research is thus posed to clarify the molecular pathology of the common placental complications of pregnancy.

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