Berthold Huppertz

Endovascular Trophoblast Invasion: Implications for the Pathogenesis of Intrauterine Growth Retardation and Preeclampsia

Abstract Maternal uteroplacental blood flow increases during pregnancy. Altered uteroplacental blood flow is a core predictor of abnormal pregnancy. Normally, the uteroplacental arteries are invaded by endovascular trophoblast and remodeled into dilated, inelastic tubes without maternal vasomotor control. Disturbed remodeling is associated with maintenance of high uteroplacental vascular resistance and intrauterine growth restriction (IUGR) and preeclampsia. Herein, we review routes, mechanisms, and control of endovascular trophoblast invasion. The reviewed data suggest that endovascular trophoblast invasion involves a side route of interstitial invasion. Failure of vascular invasion is preceded by impaired interstitial trophoblast invasion. Extravillous trophoblast synthesis of nitric oxide is discussed in relation to arterial dilation that paves the way for endovascular trophoblast. Moreover, molecular mimicry of invading trophoblast-expressing endothelial adhesion molecules is discussed in relation to replacement of endothelium by trophoblast. Also, maternal uterine endothelial cells actively prepare endovascular invasion by expression of selectins that enable trophoblast to adhere to maternal endothelium. Finally, the mother can prevent endovascular invasion by activated macrophage-induced apoptosis of trophoblast. These data are partially controversial because of methodological restrictions associated with limitations of human tissue investigations and animal studies. Animal models require special care when extrapolating data to the human due to extreme species variations regarding trophoblast invasion. Basal plates of delivered placentas or curettage specimens have been used to describe failure of trophoblast invasion associated with IUGR and preeclampsia; however, they are unsuitable for these kinds of studies, since they do not include the area of pathogenic events, i.e., the placental bed.

Placental Origins of Preeclampsia: Challenging the Current Hypothesis

Preeclampsia is a major contributor to the maternal and neonatal mortality and morbidity.1,2 It is the 2nd largest cause of maternal mortality worldwide and affects 5% to 7% of pregnant women worldwide.3,4 The precise etiopathogenesis of preeclampsia remains to be a subject of extensive research, but it is believed that it is likely to be multifactorial. Nevertheless, it is accepted that it is the presence of the placenta rather than the fetus, which is responsible for development of preeclampsia. Although the placenta plays a crucial role in the development of preeclampsia, the onset, severity, and progression is significantly affected by the maternal response to placentally derived factors and proteins. Therefore, mother and fetus should be taken into account when calculating the risk for preeclampsia. Preeclampsia is generally defined as the development of hypertension and proteinuria after 20 weeks of gestation in a previously normotensive woman,3,4 although different variations of this have been proposed by different groups and organizations. (ACOG, ISSHP, Australian college). It has also been further subdivided into mild, moderate, and severe preeclampsia as well as early and late onset preeclampsia, of which the latter is a more contemporary concept.5 It has been suggested that early (before 34+0 weeks) and late (after 34+0 weeks) onset preeclampsia have different etiologies and therefore a different clinical expression, but it is still a subject of considerable research. There are, however, some basic differences between the 2 groups:

VILLOUS CYTOTROPHOBLAST REGULATION OF THE SYNCYTIAL APOPTOTIC CASCADE IN THE HUMAN PLACENTA

Abstract Villous trophoblast in the human placenta consists of a population of proliferating stem cells which differentiate and individually fuse into the syncytiotrophoblast. We studied the apoptotic cascade in this complex epithelial layer by immunohistochemical localization of Fas, FasL, Bcl-2, Mcl-1, pro-caspase-3 and caspase-3, T-cell-restricted intracellular antigen-related protein (TIAR), poly(ADP-ribose) polymerase (PARP), lamin B, topoisomerase IIα, and transglutaminase II in cryostat and paraffin-fixed tissue sections from normal human first-trimester and term placental villi. The relationship between the apoptotic cascade and syncytial fusion was studied by coincubation of intact villi with FITC-coupled annexin-V, to detect the phosphatidylserine flip, and propidium iodide, to detect plasma membrane permeability. The final events of the apoptotic cascade were studied by the TUNEL reaction and ultrastructural appearance of the trophoblast. The phosphatidylserine flip was identified in some of the villous cytotrophoblastic cells, but the presence of both Bcl-2 and Mcl-1 proteins presumably prevented continuation of the apoptotic cascade. The syncytiotrophoblast demonstrated heterogeneous findings, suggesting variable progression along the apoptotic cascade. In some areas Bcl-2 and Mcl-1 predominated, with preservation of the nuclear proteins PARP, lamin B, and topoisomerase IIα; in other areas, especially in and around syncytial sprouts, Bcl-2 and Mcl-1 were absent, accompanied by loss of nuclear proteins, presence of phosphatidylserine flip, and TUNEL positivity. These data suggest that the apoptotic cascade is initiated in the villous cytotrophoblast, which in turn promotes syncytial fusion. Donation of anti-apoptotic proteins into the syncytium, such as Bcl-2 and Mcl-1, focally inhibits further progression along this cascade. Completion of the apoptotic cascade takes place in and around syncytial sprouts, providing further evidence that these are the sites of trophoblast shedding into the maternal circulation.

The apoptosis cascade--morphological and immunohistochemical methods for its visualization.

 Apoptosis is involved in morphogenesis of embryonic tissues as well as in homeostasis of adult organs and tissues. It is the main process by which organs maintain cell mass and at the same time eliminate excess and aged cells that have lost their functional importance. The typical morphological signs of apoptosis (cellular shrinkage, membrane blebbing, nuclear condensation and fragmentation) are the final results of a complex biochemical cascade of events, some of which are inextricably linked to the process of differentiation. Studies that analyze all stages of this cascade, rather than the final morphological stages of apoptotic death, are essential in order that specific link(s) between differentiation and apoptosis are appreciated. This review outlines the main stages of the apoptosis cascade together with current methods for their morphological visualization. Starting with (a) receptors and ligands known to induce apoptosis, we continue with (b) early initiator stages of apoptosis, and (c) proteins regulating and potentially inhibiting further progression of the cascade, into (d) irreversible execution stages of the cascade, and finally (d) the morphological events of apoptotic death. For each stage we present those aspects of the biochemical background that are morphologically relevant, together with proven methods for their visualization. We offer technical advice at each stage based upon our experience of studying differentiation and apoptosis in human placental trophoblast.

Macrophage-Induced Apoptosis Limits Endovascular Trophoblast Invasion in the Uterine Wall of Preeclamptic Women

Impaired invasion of uteroplacental arteries by extravillous trophoblast cells is a key pathogenic mechanism of preeclampsia. We previously demonstrated that reduced trophoblast invasion into uteroplacental spiral arteries was associated with an excess of macrophages in and around these arteries. To explore the significance of these observations, we correlated the extent of extravillous trophoblast apoptosis in placental bed biopsy specimens with macrophage distribution and studied the effect of macrophages upon trophoblast apoptosis in vitro. Extravillous trophoblast hybrid cells were cocultured with activated macrophages exposed to exogenous tumor necrosis factor α (TNFα), anti-tumor necrosis factor receptor I (TNF-RI), and tryptophan depletion, and the rates of trophoblast apoptosis were measured. Extravillous trophoblast hybrid cells showed increased rates of apoptosis following exposure to exogenous TNFα, with tryptophan depletion, and when cocultured with activated macrophages. The proapoptotic effects of macrophages in vitro were completely inhibited only by simultaneous addition of tryptophan and anti–TNF-RI. Our data indicate that macrophages, residing in excess in the placental bed of preeclamptic women, are able to limit extravillous trophoblast invasion of spiral arterial segments through apoptosis mediated by the combination of TNFα secretion and tryptophan depletion. The mechanisms by which macrophages are activated and recruited to the placental bed are presently unknown but are likely central to the pathogenesis of preeclampsia.

Apoptosis in the Trophoblast—Role of Apoptosis in Placental Morphogenesis

Villous trophoblast is the epithelial cover of the placental villous tree and comes in direct contact with maternal blood. The turnover of villous trophoblast includes proliferation and differentiation of cytotrophoblast, syncytial fusion of cytotrophoblast with the overlying syncytiotrophoblast, differentiation in the syncytiotrophoblast, and finally extrusion of apoptotic material into the maternal circulation. In recent years, it has become clear that apoptosis is a normal constituent of trophoblast turnover and the release of apoptotic material does not lead to an inflammatory response of the mother. During preeclampsia there seems to be an altered balance between proliferation and apoptosis of villous trophoblast leading to a dysregulation of the release from the syncytiotrophoblast. The normal apoptotic release may be reduced in favor of a necrotic release. Since apoptosis is still ongoing in the syncytiotrophoblast, a necrotic release of intrasyncytial and partly apoptotic material lead us to call this type of release “aponecrotic shedding.” In this situation, cell-free components such as G-actin and DNA freely floating in maternal blood may trigger damage to the maternal endothelium, thereby triggering preeclampsia. This review highlights the importance of the apoptosis cascade in permitting normal physiologic turnover of villous trophoblast. It will demonstrate the participation of initial stages of this cascade within the cytotrophoblast and of the execution stages within the syncytiotrophoblast. Moreover, this review presents hypotheses of how dysregulation of the apoptosis cascade may be linked to endothelial dysfunction of the maternal vasculature in preeclampsia.

Immunohistochemistry of matrix metalloproteinases (MMP), their substrates, and their inhibitors (TIMP) during trophoblast invasion in the human placenta.

Abstract The invasion of extravillous trophoblast cells into the maternal endometrium is one of the key events in human placentation. The ability of these cells to infiltrate the uterine wall and to anchor the placenta to it as well as their ability to infiltrate and to adjust utero-placental vessels to pregnancy depends, among other things, on their ability to secrete enzymes that degrade the extracellular matrix. Most of the latter enzymes belong to the family of matrix metalloproteinases. Their activity is regulated by the tissue inhibitors of matrix metalloproteinases. We have studied the distribution patterns of matrix metalloproteinases-1, -2, -3, and -9 and their inhibitors TIMP-1 and TIMP-2 as compared to the distribution of their substrates along the invasive pathway of extravillous trophoblast of 1st, 2nd, and 3rd trimester placentas by means of light microscopy on paraffin and cryostat sections as well as at the ultrastructural level (only 3rd trimester placenta). The comparison of different methods proved to be necessary, since the immunohistochemical distribution patterns of these soluble enzymes are considerably influenced by the pretreatment of tissues. All three methods revealed immunoreactivities of both, proteinases and their inhibitors, not only intracellularly in the extravillous trophoblast but also extracellularly in its surrounding matrix, the distribution patterns depending on the stage of pregnancy and on the degree of differentiation of trophoblast cells along their invasive pathway. Within the extracellular matrix, immunolocalization of matrix metalloproteinases as well as their inhibitors showed a specific relation to certain extracellular matrix molecules.

Syncytial fusion of human trophoblast depends on caspase 8

AbstractDifferentiation of human placental villous trophoblast includes syncytial fusion of cytotrophoblast forming syncytiotrophoblast. Early stages of the apoptosis cascade were described to be involved in this differentiation process. We investigated the role of the initiator caspase 8 in syncytial fusion in vitro, cultivating placental villous explants with or without caspase 8 antisense oligonucleotides or peptide inhibitors for up to 120 h. Trophoblast fusion and differentiation were assessed by confocal microscopy, immunohistochemistry and Western blot analysis. Culture with caspase 8 antisense oligonucleotides or peptide inhibitors reduced the fusion of cytotrophoblast with the syncytiotrophoblast, and resulted in multilayered cytotrophoblast. Caspase 8 expression was suppressed by antisense oligonucleotides and caspase 8 activities were reduced by peptide inhibitors. The organic anion-transporter hOAT-4 normally expressed in the cytotrophoblast and transferred into the syncytiotrophoblast by syncytial fusion was retained in the cytotrophoblast due to lack of fusion. We conclude that expression and activity of caspase 8 is a prerequisite for differentiation and syncytial fusion of cytotrophoblast cells.

The anatomy of the normal placenta

The placenta is the fetal organ providing the interchange between mother and fetus. This organ needs to provide its function such as transport and secretion even during its development and thus all developmental changes need to be in accordance with its function. This review describes development of the placenta during the first few weeks of pregnancy until the villous trees with their vasculature are established. The macroscopic anatomy of the delivered placenta as well as the microscopic anatomy and histology of this organ are also described. This includes the different types of villi and the most important cellular components of the villi such as villous trophoblast, Hofbauer cells, mesenchymal cells and endothelium. Fibrinoid and its localisation is also described.

CHAPTER 5 – Anatomy and Genesis of the Placenta

The main function of placentas is to supply nutrients to the fetus, but the pathways involved may be very different. Placental types may be classified at several complementary levels, reflecting both gross and microscopic structure, the histological nature of the maternal–fetal interface, or the relative directions of the maternal and fetal blood flows. The most fundamental level relates to the origin of the fetal vessels that vascularizes the chorion. The chorion must develop a functional circulation to take part in maternal–fetal exchange. This may be derived from vessels running in the extraembryonic mesodermal covering of either the yolk sac or the allantois. Breakdown of overlying chorion and of the outer wall of the yolk sac exposes the endodermal lining to the secretions of the uterine glands. This represents an important pathway in the absorption of proteins and the transport of maternal immunoglobulins, which continues to function until term. The second level of classification to be considered relates to the gross morphology of the placenta. Four main types are recognized, and the basis of the classification is whether physical interaction between the maternal and fetal tissues occurs over all the available surfaces of the chorionic sac or whether it is restricted to specialized regions.