Placentation and Placental Function in Normal and Preeclamptic Pregnancies

Introduction

It is widely acknowledged that the placenta is central to the causation of preeclampsia, but its precise role in the pathophysiology is still uncertain. Ever since systematic investigation of the placental bed was enabled in the 1950s by the development of biopsy techniques, much attention has been paid to the involvement of the uteroplacental vasculature in development of the syndrome. A spectrum of pathological changes has been observed, associated not just with preeclampsia but also normotensive conditions, such as fetal growth restriction (FGR), abruption, and preterm delivery. ^, The extent of these changes correlates approximately with the severity of the clinical condition, being most extreme in cases of preeclampsia presenting with FGR. It is fitting, therefore, to consider preeclampsia as part of a spectrum of disorders related to poor placental development rather than as a separate disorder. Within that spectrum may be included different subtypes of preeclampsia. Increasingly, distinction is being made between those cases presenting prior to 34 weeks of gestation, early-onset preeclampsia, and those presenting near or at term, late-onset preeclampsia. ^, Pathological placental and maternal uteroplacental vascular changes are greatest in the former, and the often associated asymmetric growth restriction reflects an impoverished nutrient supply to the fetus. By contrast, placentation and birth weight are relatively normal in late-onset cases, indicating a different underlying pathophysiology that may be related more closely to maternal predisposition to cardiovascular disease. The placenta is an essential component of the pathophysiology of both subtypes, however, and so in this chapter we consider how it may promote the syndrome across the gestational spectrum, starting with development of the placenta postimplantation.

Early Placental Development

Placentation requires coordinated interactions between the trophoblast lineages of the placenta and the maternal endometrium. Although the delivered placenta is essentially a fetal organ with only a few remnants of maternal cells adherent to the outer surface of the basal plate, it cannot develop and function without the support of the endometrium and its vasculature. There is an increasing appreciation that defects in placentation that only manifest themselves clinically during mid-late pregnancy, such as preeclampsia, may have their pathophysiological roots in poor endometrial function preconceptionally or an aberrant dialogue between the two tissues following implantation.

Placentation starts when an implantation competent blastocyst makes contact with a receptive endometrial epithelium at around days 5–6 postfertilization. The human blastocyst is oriented such that the trophectoderm at the embryonic pole overlying the inner cell mass is apposed to the epithelium. The molecular mechanisms underpinning this orientation are not understood, but may relate to restricted gene expression due to signaling from the inner cell mass cells. For example, expression of FGFR1 becomes restricted to the trophectoderm in this area as the blastocyst develops. This orientation is important, for when the connecting stalk, the future umbilical cord, grows out from the embryo, it will normally be centered over the placental disc. Malrotation of the blastocyst at this stage is one way in which eccentric or even velamentous cord insertions can be generated.

Once the trophectoderm makes stable contact with the uterine epithelium, neighboring cells undergo lateral fusion to form localized masses of multinucleated syncytial trophoblast. Protrusions from these masses penetrate between the uterine cells and displace them, and then the masses merge to form the trophoblastic plate that is in contact with the endometrial stromal cells. The trophectoderm cells forming the rest of the blastocyst wall remain unfused and are henceforth referred to as cytotrophoblast cells. These represent a progenitor population that fuses with, and expands, the primary syncytiotrophoblast of the plate. By the time the conceptus is fully embedded within the superficial endometrium, the primary syncytiotrophoblast covers the entire gestational sac.

Although human implantation is of the interstitial type and described as invasive, there is evidence that upgrowth of the endometrial cells plays an important role in encapsulating the conceptus. Original descriptions of early implantation sites describe mitotic figures in the endometrial stromal cells close to the conceptus, and more recent experimental data support the concept of trophoblast-induced proliferation and migration contributing to formation of the decidua capsularis. This view is consistent with high-resolution ultrasound scans that show the implantation site raised above the general level of the endometrial surface and bulging into the uterine lumen.

Formation of the decidua capsularis reinstates the uterine epithelium at approximately day 8 postfertilization, sealing the conceptus off from the uterine lumen. At the same time a key step in placental development occurs when fluid-filled spaces appear in the primary syncytiotrophoblast. These spaces are referred to as the lacunae, and as they enlarge, they split the syncytiotrophoblast mass into two layers, one in contact with the endometrium, the future basal plate, and the other against the cytotrophoblast cells of the blastocyst, the future chorionic plate. This separation establishes the fundamental boundaries of the placenta, with strands of syncytiotrophoblast, the trabeculae, extending between the two and representing the forerunners of anchoring villi that span between the two plates. During this process, the primary syncytiotrophoblast transitions into the definitive syncytiotrophoblast of the mature placenta, forming a microvillous, polarized epithelium that lines the lacunae. It is notable that a syncytial trophoblast forms when human embryos are cultured on plastic in vitro and so is independent of factors derived from the endometrium. Transcriptomic analysis of these masses indicates evidence of intense hormone production, but also of migration and invasion. Whether these masses correspond to the primary syncytiotrophoblast in vivo is intriguing, but difficult to ascertain due to the lack of appropriate specimens. It is also not known whether the primary syncytiotrophoblast degenerates and is replaced, or whether it undergoes a gradual differentiation as further cytotrophoblast cells fuse.

Around day 12 postfertilization, the cytotrophoblast cells of the original blastocyst wall proliferate and penetrate into the trabeculae as cellular columns. Approximately 2 days later, the cells reach the tips of the trabeculae and break through the syncytiotrophoblast, spreading laterally and merging with neighbors. Consequently, a new layer is interposed between the syncytiotrophoblast and the endometrium, the cytotrophoblastic shell. Shortly after, mesenchymal cells penetrate into the cytotrophoblast columns, giving rise to the future villous stromal core. Hemangioblastic clusters differentiate within the mesenchyme, forming the first elements of the fetal vascular network. Lateral branching of these early stem villi then gives rise to generations of free-floating intermediate and terminal villi. Initially, villi form over the entire surface of the gestational sac, constituting the chorion frondosum, although they are never quite so elaborate over the superficial pole. Later, they regress over this pole to leave the definitive placenta apposed to the original endometrium, which by this stage has transformed into the decidua.

The Cytotrophoblastic Shell

The shell is a transient, but crucial, structure that encapsulates and protects the conceptus during the earliest stages of pregnancy. Its importance is often overlooked due to the current rarity of placenta in situ specimens from postimplantation stages, for it is “markedly thinned” by day 38 postfertilization onward and subsequently incorporated into the developing basal plate. Initially, the shell is composed of several layers of rounded cytotrophoblast cells containing large quantities of glycogen. The shell provides for rapid circumferential expansion of the implantation site, and in doing so will be the first fetal tissue to encounter the tips of the maternal spiral arteries within the endometrium. When this happens, cells from the shell migrate into the lumen of each artery in a cone-like fashion ( Fig. 5.1 ). Because of their location, the cells are classified as endovascular trophoblast, and the aggregates are often referred to as “plugs” for the following reason. The cells are loosely linked by desmosomal attachments, and a complex network of intercellular spaces is continuous through the shell and into the lacunae. These spaces are too small to allow the passage of maternal erythrocytes, and so communication between the arteries and the lacunae, which can now be regarded as the forerunner of the intervillous space, is restricted to the seepage of plasma. These cones or “plugs” persist for most of the first trimester, and consequently over this period the intervillous space is filled with a slow-moving, clear fluid.

Restricting the inflow of maternal erythrocytes means that the oxygen tension within the placenta and the embryo is relatively low during the first trimester compared to later pregnancy, being limited to dissolved O ₂ at ~20 mmHg. This low tension is thought to protect the embryo against the potentially teratogenic effects of oxygen free radicals during the critical period of organogenesis. ^, It also helps to maintain stem cells in a pluripotent state and promotes proliferation of cytotrophoblast cells and formation of the villous trees. The placental tissues are not energetically compromised, however, and maintain a normal ATP/ADP ratio through a high rate of glycolysis. The latter is facilitated by Warburg-type metabolism and high levels of activity in the polyol pathways that avoid excessive fermentation to lactate.

Reflecting the relatively low oxygen concentration, the syncytiotrophoblast in particular has low levels of the principal antioxidant enzymes, copper-zinc superoxide dismutase, and catalase. ^, Consequently, it is vulnerable to oxidative stress when the oxygen concentration rises threefold as the “plugs” loosen and the maternal arterial flow to the placenta becomes established. ^, The extent of the “plugs” varies across the placental bed, being best developed in the central region under the original implantation site and least in the periphery. Consequently, onset of the circulation is seen preferentially in the peripheral regions and gradually extends centripetally with advancing gestational age. Onset is associated with locally high levels of oxidative stress in the peripheral villi that initially form over the entire gestational sac. We speculate that this promotes their regression and the formation of the discoid definitive placenta and the smooth membranes. ^,

Correct formation of the cytotrophoblastic shell is therefore essential to provide an adequate source of endovascular trophoblast to initially restrict flow of maternal arterial blood into the placenta. As will be considered later, endovascular trophoblast cells also play a key role in remodeling of the spiral arteries to provide an optimal blood supply later in pregnancy. In cases of miscarriage, onset of the maternal circulation is premature and disorganized throughout the whole placenta, causing overwhelming oxidative stress and loss of function. ^, Development of the shell is severely deficient in 70% of these cases, irrespective of the fetal karyotype, and will result is poor “plugging” of the arteries. ^, This deficiency most likely represents one end of a spectrum of development of the shell, with normal pregnancy at the opposite end. We have speculated that less severe examples of deficient development are associated with other problems seen at the maternal–fetal interface at the time of onset of the maternal circulation, such as intrauterine hematomas. Intrauterine or subchorionic hematomas are well defined on ultrasonic examination as crescentic hypoechogenic areas between the placental membranes and the endometrium/decidua. If the hematoma expands under the basal plate of the definitive placenta, it can lead to detachment and miscarriage, which is observed in around 10% of the cases within 48 h of the first bleeding episode. In the 90% of pregnancies that continue, there is a fourfold [95% CI: 2.3–7.0] increased risk of preeclampsia and of other complications such as abruption and preterm delivery. Intrauterine hematomas are thus an example of how events taking place early in placental development may lay the foundation for complications that manifest clinically later in gestation.

In the case of preeclampsia, it might be expected that if development of the shell is sufficiently deficient to predispose to intrauterine hematomas, onset of the maternal circulation might be abnormal due to incomplete “plugging” of the spiral arteries. While no studies have yet focused on this question, advances in high-resolution imaging toward the end of the first trimester may resolve the issue. Circumstantial support comes from the fact that placentas from cases of early-onset preeclampsia and growth restriction are often small and have irregular margins, indicative of excessive villous regression at the end of the first trimester. ^,

If the cytotrophoblastic shell is critical to a successful pregnancy, what then determines its development? Mounting evidence suggests this must be dependent on histotrophic nutrition from the endometrial glands.

The Trophoblast–Endometrial Gland Dialogue During Early Pregnancy

In all mammals, nutrition of the conceptus is histotrophic as it moves down the Fallopian tube and into the uterus. Initially, the secretions are from the oviduct and then from the endometrial glands lining the uterus. The duration of this form of nutrition varies according to the mode of implantation, being relatively long in those species such as the sheep and horse that have noninvasive placentation. There, the secretions have long been referred to as “uterine milk.” In humans, it was always assumed that the period was brief due to the formation of the decidua capsularis isolating the conceptus from the uterine lumen. However, it is now appreciated that histotrophic nutrition supports development of the placenta throughout the first trimester. ^,

The importance of histotroph for early placental and embryo development has been shown conclusively in the sheep, and more recently the mouse, by suppression of development of the glands in neonatal females. The females are capable of ovulation and fertilization, but the embryo fails to develop once it enters the uterus. ^, Moreover, there is strong evidence that activity of the glands is upregulated during early pregnancy by lactogenic hormones secreted from the trophoblast. Thus, transcripts encoding epidermal growth factor (EGF) increase in the gland epithelial cells, coincident with upregulation of the prolactin receptor. ^, This interaction has been referred to as a servomechanism and is a way by which the placenta is able to stimulate its own development in advance of that of the embryo.

In the human, implantation is thought to occur equidistant between the openings of endometrial glands. Once implanted, the enlarging primary syncytial trophoblast soon encounters the glands within the superficial endometrium. The gland epithelium shows local degenerative changes and loss, and by days 11–12 postfertilization, the trophoblast is in contact with the secretions. These communications with the glands persist as the placenta develops, ^, and remnants of the glands can even be observed in the delivered placenta, although their functional significance at this stage is not known. We have shown that glycoproteins secreted uniquely by the glands are taken up by the syncytiotrophoblast during the first trimester and that they immunolocalize with the lysosomal pathway suggesting subsequent breakdown into amino acids for use in anabolic pathways. Amino acids and other proteins derived from the decidua accumulate within the coelomic cavity of the gestational sac, providing a reservoir of nutrients for the embryo.

During early pregnancy, the gland cells adopt a hypersecretory phenotype indicative of increased activity, referred to eponymously as the Arias-Stella reaction. They contain large quantities of glycogen that may either be released from the apical surface through an apocrine mechanism or converted to glucose by glycogen phosphorylase. Either way, glycogen accumulates within the syncytiotrophoblast of villi adjacent to gland openings during early pregnancy. ^, The secretions are also rich in lipid droplets, and in glycoproteins such as glycodelin-A (PP14), osteopontin and uteroglobin, which may have many functions at the maternal–fetal interface ( Fig. 5.2 ). Glycodelin-A has been implicated in the regulation of maternal immunotolerance and extravillous trophoblast invasion, for example. ^, Osteopontin has been shown to have both pro- and antiinflammatory effects ^, and may also be involved in remodeling of the spiral arteries, as will be described later, for it promotes migration of smooth muscle cells. ^, Finally, the epithelial cells of the glands are immunopositive for a number of growth factors, including EGF and vascular endothelial growth factor (VEGF). EGF is a powerful stimulant of cytotrophoblast proliferation during early pregnancy and may promote formation of both the cytotrophoblastic shell and the villous trees. Deficient functioning of the glands may therefore have many consequences for placental development and interactions at the maternal–fetal interface.

Determining whether a servomechanism operates in the human in an equivalent fashion to the sheep and horse is difficult for obvious ethical reasons. The glands express high levels of the luteinizing hormone/human chorionic gonadotropin (hCG) receptor and are also equipped with the prolactin receptor. Application of hCG to isolated endometrial epithelial cells induces the secretion of fibroblast growth factor 2 (FGF2), VEGF, leukemia inhibitory factor (LIF), and other cytokines. Further support comes from the finding that organoids derived from the endometrial glands show upregulation of glycodelin-A and osteopontin in response to hCG, prolactin, and other early pregnancy hormones.

One important difference to domestic species is that prolactin, which seems to be a particularly important stimulant of gland activity, is not secreted by the trophoblast but by the decidual cells. Consequently, correct decidualization of the endometrial stromal cells is an essential component in the placental–endometrial gland dialogue during early pregnancy ( Fig. 5.2 ). Hence, poor decidualization may contribute to impaired gland function, which may in turn lead to deficient trophoblast proliferation and invasion and complications such as preeclampsia. It is known from IVF data that there is a positive association between endometrial thickness and the live birth rate, with a minimum of 6 mm being required. Pilot data from clinical studies has implicated low levels of prolactin and glycodelin-A in cases of miscarriage. Furthermore, transcriptomic evidence from chorionic villous samples suggests that decidualization may be impaired in women who go on to develop preeclampsia. ^, Hence, it is possible that a predisposition to preeclampsia may be present preconceptionally if endometrial function is suboptimal.

The Switch to Hemotrophic Nutrition

Histotrophic nutrition can supply all the necessary nutrients for early embryonic development through the maternal plasma seeping through the “plugs” and the secretions from the endometrial glands. However, eventually a richer supply of oxygen is necessary, requiring an effective maternal circulation. Onset of this circulation is associated with the formation of channels within the “plugs” of endovascular trophoblast occluding the mouths of the arteries toward the end of the first trimester. Recent analyses have also implicated changes in more proximal elements in the uteroplacental vasculature, in particular the arcuate and radial arteries, as these correlate more closely with the flux through the intervillous space than the channels. More research is required to elucidate the mechanisms involved.

The hemochorial form of human placentation poses special hemodynamic challenges not encountered in the noninvasive forms of placentation. In essence, there has to be a high flow through the placenta but at a low pressure and velocity to avoid damage to the delicate villous trees. Reconciling these requirements with the intrinsically high pressures within the maternal circulation requires considerable remodeling of the uteroplacental vasculature. Failure of this remodeling is associated with pre-eclampsia, but also other complications of pregnancy.