IMPLANTATION

Implantation involves the initial attachment of the trophoblastic wall of the blastocyst to the endometrial luminal epithelium. The trophoblast lineage gives rise to three main cell types in the human placenta: the syncytiotrophoblast, which forms the epithelial covering of the villous tree and is the main endocrine component of the placenta; the villous cytotrophoblast cells, which represent a germinative population that proliferate throughout pregnancy and fuse to generate the syncytiotrophoblast; and extravillous trophoblast cells, which are non-proliferative and invade the maternal endometrium. The first two cell lines can be seen from stages 4 and 5 onwards. The cytotrophoblast cells that form the mural and polar trophoblast are cuboidal, and covered externally with syncytial trophoblast (syncytiotrophoblast), a multinucleated mass of cytoplasm that forms initially in areas near the inner cell mass after apposition of the blastocyst to the uterine mucosa (see Fig. 8.10).

Preimplantation embryos produce matrix metalloproteinases, which mediate penetration of the maternal subepithelial basal lamina by the syncytiotrophoblast. Trophoblast cells express L-selectin (usually seen as a mediator of neutrophil rolling and tethering in inflamed endothelium), and the maternal epithelium upregulates selectin-oligosaccharide-based ligands. Thus, differentiating cytotrophoblast cells appear to use processes that also occur in vasculogenesis and during leukocyte emigration from the blood into the tissues. Flanges of syncytial trophoblast grow between the cells of the uterine luminal epithelium towards the underlying basal lamina without apparent damage to the maternal cell membranes or disruption of the intercellular junctions. Instead, shared junctions, including tight junctions, are formed with many of the maternal uterine epithelial cells.

Implantation continues with erosion of maternal vascular endothelium and glandular epithelium, and phagocytosis of secretory products, until the blastocyst occupies an uneven implantation cavity in the stroma (interstitial implantation) (Fig. 9.1). In the early postimplantation phase, the maternal surface is resealed by re-epithelialization and the formation of a plug, which may contain fibrin. As the blastocyst burrows more deeply into the endometrium, syncytial trophoblast forms over the mural cytotrophoblast, but never achieves the thickness of the syncytial trophoblast over the embryonic pole.

Fig. 9.1 Implanting conceptus at stage 6. The embryo is composed of epiblast, with the amniotic cavity above it and hypoblast, with the secondary yolk sac below it. Both cavities are covered externally with extraembryonic mesoblast which also lines the larger chorionic cavity. Primary villi cover the outer aspect of the conceptus and extend into the maternal endometrium and in places, maternal blood fills the lacunae.

The syncytiotrophoblast secretes numerous hormones; the most well known, human chorionic gonadotrophin (hCG), can be detected in maternal urine from as early as 10 days after fertilization, and forms the basis for tests for early pregnancy. hCG prolongs the life of the corpus luteum, which continues to secrete progesterone and oestrogens during approximately the first two months of pregnancy. Thereafter, these essential hormones are produced by the placenta.

Menstruation ceases on successful implantation. The endometrium, now known as the decidua in pregnancy, thickens to form a suitable nidus for the conceptus. Decidualization of the endometrial stroma may occur without an intrauterine pregnancy, e.g. in the presence of an ectopic pregnancy, after prolonged treatment with progesterone, and in the late secretory phase of a non-conception cycle.

Decidual differentiation is not evident in the stroma at the earliest stages of implantation, and it may not be until a week later that fully differentiated cells are present. During decidualization the interglandular tissue increases in quantity. It contains a substantial population of leukocytes (large granular lymphocytes, macrophages and T cells) distributed amongst large decidual cells: the most numerous are uterine natural killer (NK) cells which accumulate in the endometrium during the secretory phase of the cycle and persist until mid-pregnancy. Decidual cells are mesenchymally derived stromal cells which contain varying amounts of glycogen, lipid, and vimentin-type intermediate filaments in their cytoplasm. They are generally rounded, but their shape may vary depending on the local packing density. They may contain one, two or sometimes three nuclei and frequently display rows of club-like cytoplasmic protrusions enclosing granules. The cells are associated with a characteristic capsular basal lamina. Decidual cells produce a range of secretory products, including insulin-like growth factor binding protein 1 (IGF-BP1) and prolactin, which may be taken up by the trophoblast. These secretions probably play a role in the maintenance and growth of the conceptus in the early part of postimplantational development, and can be detected in amniotic fluid in the first trimester of pregnancy.

Extracellular matrix, growth factors and protease inhibitors produced by the decidua all probably modulate the degradative activity of the trophoblast and support placental morphogenesis and placental accession to the maternal blood supply. Once implantation is complete, distinctive names are applied to different regions of the decidua (Fig. 9.2). The part covering the conceptus is the decidua capsularis; that between the conceptus and the uterine muscular wall is the decidua basalis (where the placenta subsequently develops); and that which lines the remainder of the body of the uterus is the decidua parietalis. There is no evidence that their respective resident maternal cell populations exhibit site-specific properties.

Fig. 9.2 The gravid uterus in the second month. A placental site precisely in the uterine fundus as indicated in the plan is rather unusual: the dorsal, ventral or lateral wall of the corpus uteri is more usual. The maternal endometrium is now termed decidua; different regions are distinguished.

DEVELOPMENT OF THE PLACENTA

Formation of the human placenta requires a developmental progression which proceeds in a specific order over time; it is specified by the trophoblast, but dependent on the maternal environment for its correct expression. Immunological rejection of the semi-allogenic conceptus does not occur because the syncytiotrophoblast expresses neither Class I nor Class II MHC antigens. By contrast, the invading extravillous trophoblast expresses human leukocyte antigen-G (HLA-G) and -C, which interact with receptors on the uterine NK cells.

As the blastocyst implants, the syncytiotrophoblast invades the uterine tissues, including the glands and walls of maternal blood vessels (Fig. 9.1), and increases rapidly in thickness over the embryonic pole (Fig. 9.3). A progressively thinner layer covers the rest of the wall towards the abembryonic pole. Microvillus-lined clefts and lacunar spaces develop in the syncytiotrophoblastic envelope (days 9–11 of pregnancy) and establish communications with one another. Initially, many of these spaces contain maternal blood derived from dilated uterine capillaries and veins, as the walls of the vessels are partially destroyed. As the conceptus grows, the lacunar spaces enlarge, and become confluent to form an intervillous space.

Fig. 9.3 Conceptus at about stage 14. The embryonic pole shows extensive villous formation at the chorion frondosum whereas the abembryonic pole is smooth and villous free at the chorion laevae.

(Photograph by courtesy of P Collins.)

The projections of syncytiotrophoblast into the maternal decidua are called primary villi. They are invaded first with cytotrophoblast and then with extraembryonic mesenchyme (days 13–15) to form secondary placental villi. Fetal capillaries develop in the mesenchymal core of the villi. The cytotrophoblast within the villi continues to grow through the invading syncytiotrophoblast and makes direct contact with the decidua basalis, forming anchoring villi. Further cytotrophoblast proliferation occurs laterally so that neighbouring outgrowths meet to form a spherical cytotrophoblastic shell around the conceptus (Fig. 9.4). Lateral projections from the main stem villus form true and terminal villi.

Fig. 9.4 Placental development is shown from left to right.

As secondary villi form, single mononuclear cells become detached from the distal cytotrophoblast and infiltrate the maternal decidua. These cells are the third line of the original trophoblastic cells. They are found both within and around the spiral arteries in the central area of the placenta and gradually extend laterally, reaching the periphery of the placenta around mid-gestation. They normally extend into the inner third of the uterine myometrium within the central region of the placental bed, but the extent of invasion is progressively shallower towards the periphery. At the same time, cytotrophoblast from the spherical shell penetrates into and migrates along the inner walls of maternal spiral arteries (endovascular extravillous trophoblast) so that by the 18th week it has reached the inner myometrial segments. The interstitially migrating cells invade the spiral arteries from their adventitia. The vessels lose their elastic lamina and consequently their responsiveness to circulating vasoactive compounds. The smooth muscle and associated elastic and collagenous matrix is replaced with non-resistive fibrinoid, an arrangement that permits an expansion of the vessels and as much as a 20-fold increase in the flow of blood into the intervillous space. In normal pregnancies the transformation of spiral arteries into utero-placental arteries is completed around mid gestation. The main aim of these vascular changes appears to be to optimize the distribution of maternal blood into a low-resistance uterine vascular network. Common pregnancy pathologies, including intrauterine growth restriction, pre-eclampsia and spontaneous abortion, are all associated with incomplete vascular remodelling, which probably reflects a failure of penetration by extravillous trophoblast.

With the onset of the embryonic heartbeat, a primitive circulation exists between the embryo and the yolk sac, succeeded later by that between the embryo and the placenta. The formed placenta is composed of a chorionic plate on its fetal aspect and a basal plate on the maternal aspect, and an intervening intervillous space containing villous stems with branches in contact with maternal blood (Fig. 9.4). Since maternal blood bathes the surfaces of the chorion which bound the intervillous space, the human placenta is defined as haemochorial. Different grades of fusion exist between the maternal and fetal tissues in many other mammals (e.g. epitheliochorial, syndesmochorial, endotheliochorial). The chorion is vascularized by the allantoic blood vessels of the body stalk, and so the human placenta is also termed chorio-allantoic (whereas in some mammals a choriovitelline placenta either exists alone or supplements the chorio-allantoic variety). In addition, the human placenta is defined as discoidal (in contrast to other shapes in other mammals) and deciduate because maternal tissue is shed with the placenta and membranes at parturition as part of the afterbirth.

Growth of the placenta

Expansion of the entire conceptus is accompanied by radial growth of the villi and, simultaneously, an integrated tangential growth and expansion of the trophoblastic shell. Eventually each villous stem forms a complex that consists of a single trunk attached by its base to the chorion, from which second and third order branches (intermediate and terminal villi) arise distally. Terminal villi are specialized for exchange between fetal and maternal circulations; each one starts as a syncytial outgrowth and is invaded by cytotrophoblastic cells, which then develop a core of fetal mesenchyme as the villus continues to grow. The core is vascularized by fetal capillaries (i.e. each villus passes through primary, secondary and tertiary grades of histological differentiation). The germinal cytotrophoblast continues to add cells that fuse with the overlying syncytium and so contribute to the expansion of the haemochorial interface. Terminal villi continue to form and branch within the confines of the definitive placenta throughout gestation, projecting in all directions into the intervillous space (Fig. 9.4).

From the third week until about the second month of pregnancy, the entire chorion is covered with villous stems. They are thus continuous peripherally with the trophoblastic shell, which is in close apposition with both the decidua capsularis and the decidua basalis. The villi adjacent to the decidua basalis are stouter, longer and show a greater profusion of terminal villi. As the conceptus continues to expand, the decidua capsularis is progressively compressed and thinned, the circulation through it is gradually reduced, and adjacent villi slowly atrophy and disappear. This process starts at the abembryonic pole, and by the end of the third month, the abembryonic hemisphere of the conceptus is largely denuded. Eventually the whole chorion apposed to the decidua capsularis is smooth and is now termed the chorion laeve. In contrast, the villous stems of the disc-shaped region of chorion apposed to the decidua basalis increase greatly in size and complexity and the region is now termed the chorion frondosum (Fig. 9.3). The chorion frondosum and the decidua basalis constitute the definitive placental site (Fig. 9.2). Abnormalities in this process may account for the persistence of villi at abnormal sites on the chorionic sac and hence the presence of accessory or succenturiate lobes. At term, the placental diameter varies from 200 to 220 mm, the mean placental weight is 470 g, its mean thickness is 25 mm and the total villous surface area is 12–14 m².

Coincidentally with the growth of the embryo and the expansion of the amnion, the decidua capsularis is thinned and distended and the space between it and the decidua parietalis is gradually obliterated. By the second month of pregnancy the three endometrial strata recognizable in the premenstrual phase, i.e. compactum, spongiosum and basale, are better differentiated and easily distinguished. The glands in the spongiosum are compressed and appear as oblique slit-like fissures lined by low cuboidal cells. By the beginning of the third month of pregnancy the decidua capsularis and decidua parietalis are in contact, while by the fifth month the decidua capsularis is greatly thinned, and during the succeeding months it virtually disappears.

Chorionic plate

The chorionic plate is covered on its fetal aspect by the amniotic epithelium, on the stromal side of which is a connective tissue layer carrying the main branches of the umbilical vessels (Fig. 9.4 and Fig. 9.5). Subjacent to this is a diminishing layer of cytotrophoblast and then the inner syncytial wall of the intervillous space. The connective tissue layer is formed by fusion between the mesenchyme-covered surfaces of amnion and chorion: it is more fibrous and less cellular than Wharton’s jelly (of the umbilical cord), except near the larger vessels. The umbilical vessels radiate and branch from the cord attachment, with variations in the branching pattern, until they reach the bases of the trunks of the villous stems and then arborize within the intermediate and terminal villi. There are no anastomoses between vascular trees of adjacent stems. The two umbilical arteries are normally joined at, or just before they enter, the chorionic plate, by some form of substantial transverse anastomosis (Hyrtl’s anastomosis).

Fig. 9.5 The arrangement of the placental tissues from the chorionic plate (fetal side) to the basal plate or decidua basalis (maternal side).

Basal plate

The basal plate, from fetal to maternal aspect, forms the outer wall of the intervillous space. The trophoblast and adjacent decidua are enmeshed in layers of fibrinoid and basement membrane-like extracellular matrix to form a complex junctional zone. In different places the basal plate may contain syncytium, cytotrophoblast or fibrinoid matrix, remnants of the cytotrophoblastic shell, and, at the site of implantation, areas of necrotic maternal decidua (the so-called Nitabuch’s stria) (Figs 9.4 and 9.5). Nitabuch’s stria and the decidua basalis contain cytotrophoblast and multinucleate trophoblast giant cells derived from the mononuclear cytotrophoblast population, which infiltrate the decidua basalis during the first 18 weeks of pregnancy. These cells penetrate as far as the inner one-third of the myometrium, but can often be observed at or near the decidual–myometrial junction. They are not found in the decidua parietalis or the adjacent myometrium, from which it may be inferred that the placental-bed giant cell represents a differentiative end stage in the extravillous trophoblast lineage. The striae of fibrinoid are irregularly interconnected and variable in prominence. Strands pass from Nitabuch’s stria into the adjacent decidua which contains basal remnants of the endometrial glands and large and small decidual cells scattered in a connective tissue framework that supports an extensive venous plexus.

From the third month onwards the basal plate develops placental or cotyledonary septa, which are ingrowths of the cytotrophoblast covered with syncytium that grow toward but do not fuse with the chorionic plate (Fig. 9.4). The septa circumscribe the maternal surface of the placenta into 15–30 lobes, often termed cotyledons. Each cotyledon surrounds a limited portion of the intervillous space associated with a villous trunk from the chorionic plate. From the fourth month these septa are supported by tissue from the decidua basalis. Throughout the second half of pregnancy the basal plate becomes thinned and progressively modified: there is a relative diminution of the decidual elements, increasing deposition of fibrinoid, and admixture of fetal and maternal derivatives.

Intervillous space

The intervillous space contains the main trunks of the villous stems and their arborizations into intermediate and terminal villi (Figs 9.4 and 9.5). A villous trunk and its branches may be regarded as the essential structural, functional and growth unit of the developing placenta.

At term, from the inner myometrium to the intervillous space, the walls of most spiral arteries consist of fibrinoid matrix within which cytotrophoblast is embedded. This arrangement allows expansion of the arterial diameter (and so slows the rate of arterial inflow and reduces the perfusion pressure) independent of the local action of vasoconstrictive agents. Endothelial cells, where present, are often hypertrophic. The veins that drain the blood away from the intervillous space pierce the basal plate and join tributaries of the uterine veins. The presence of a marginal venous sinus, which hitherto has been described as a constant feature occupying the peripheral margin of the placenta and communicating freely with the intervillous space, has not been confirmed.

Recent anatomic and in vivo studies have shown that human placentation is in fact not truly haemochorial in early pregnancy (Jauniaux et al 2003). From the time of implantation, the extravillous trophoblast not only invades the uterine tissues but also forms a continuous shell at the level of the decidua. The cells of this shell not only anchor the placenta to the maternal tissue but also form plugs in the tips of the utero-placental arteries (Burton et al 1999). The shell and the plugs act like a labyrinthine interface that filters maternal blood, permitting a slow seepage of plasma but no true blood flow into the intervillous space. This is supplemented by secretions from the uterine glands, which are discharged into the intervillous space until at least 10 weeks (Burton et al 2002). The comparison of these anatomical features with physiological data obtained in utero reveals that the architecture of the human first trimester gestational sac is designed to limit fetal exposure to oxygen to that which is strictly necessary for its development This creates a physiological placental hypoxia which may protect the developing embryo against the deleterious and teratogenic effects of oxygen, and also a uterine O₂ gradient which exerts a regulatory effect on placental tissue development and function. In particular it influences cytotrophoblast proliferation and differentiation along the invasive pathway and villous vasculogenesis. At the end of the first trimester the trophoblastic plugs are progressively dislocated, allowing maternal blood to flow progressively more freely and continuously within the intervillous space. During the transitional phase of 10–14 weeks gestation, two-thirds of the primitive placenta disappears, the chorionic cavity is obliterated by the growth of the amniotic sac, and maternal blood flows progressively throughout the entire placenta (Jauniaux et al 2003). These events bring the maternal blood closer to the fetal tissues, facilitating nutrient and gaseous exchange between the maternal and fetal circulations.

Structure of a placental villus

Chorionic villi are the essential structures involved in exchanges between mother and fetus. The villous tissues separating fetal and maternal blood are therefore of crucial functional importance. From the chorionic plate, progressive branching occurs into the villous tree, as stem villi give way to intermediate and terminal villi. Each villus has a core of connective tissue containing collagen types I, III, V and VI, as well as fibronectin. Cross-banded fibres (30–35 nm) of type I collagen often occur in bundles, whereas type III collagen is present as thinner (10–15 nm) beaded fibres, which form a meshwork that often encases the larger fibres. Collagens V and VI are present as 6–10 nm fibres closely associated with collagens I and III. Laminin and collagen type IV are present in the stroma associated with the basal laminae that surround fetal vessels and also in the trophoblast basal lamina. Overlying this matrix are ensheathing cyto- and syncytiotrophoblast cells bathed by the maternal blood in the intervillous space (Figs 9.4, 9.5 and 9.6). Cohesion between the cells of the cytotrophoblast and also between the cytotrophoblast and the syncytium is provided by numerous desmosomes between the apposed plasma membranes.

Fig. 9.6 A, Chorionic villus and its arterio–capillary–venous system carrying fetal blood. The artery carries deoxygenated blood and waste products from the fetus and the vein carries oxygenated blood and nutrients to the fetus. Sections through a chorionic villus at 10 weeks, B, and at full term, C. The villi would be within the intervillous space bathed externally in maternal blood. The placental membrane, composed of fetal tissues, separates the maternal blood from the fetal blood.

In earlier stages, the cytotrophoblast forms an almost continuous layer on the basal lamina. After the fourth month it gradually expends itself producing syncytium, which comes to lie on the basal lamina over an increasingly large area (80% at term), and becomes progressively thinner. Cytotrophoblastic cells persist until term, but because the increase in villous surface area outstrips their proliferation they are usually disposed singly. In the first and second trimester cytotrophoblastic sprouts, covered in syncytium, are present and represent a stage in the development of new villi. Cytotrophoblast columns at the tips of anchoring villi extend from the villous basal lamina to the maternal decidual stroma.

The cells of the villous cytotrophoblast (Langhans cells) are pale-staining with a slight basophilia. Ultrastructurally, they have a rather electron-translucent cytoplasm, and relatively few organelles. They contain intermediate filaments, particularly in association with desmosomes. Between the desmosomes, the membranes of adjacent cells are separated by approximately 20 nm. Sometimes the intercellular gap widens to accommodate microvillous projections from the cell surfaces, and it occasionally contains patches of fibrinoid. A smaller population of intermediate cytotrophoblast may also be found in the chorionic villi. This postmitotic population represents a state of partial differentiation between the cytotrophoblast stem cell and the overlying syncytium.

The syncytium is an intensely active tissue layer across which most transplacental transport must occur. It is a selectively permeable barrier that allows water, oxygen and other nutritive substances and hormones to pass from mother to fetus, and some of the products of excretion to pass from fetus to mother. It secretes a range of placental hormones into the maternal circulation. Syncytial cytoplasm is more strongly basophilic than that of the cytotrophoblastic cells and is packed with organelles consistent with its secretory phenotype. Where the plasma membrane adjoins basal lamina it is often infolded into the cytoplasm, whereas the surface bordering the intervillous space is set with numerous long microvilli, which constitute the brush border seen by light microscopy.

Glycogen is thought to be present in both layers of the trophoblast at all stages, although it is not always possible to demonstrate its presence histochemically. Lipid droplets occur in both layers and are free in the core of the villus. In the trophoblast they are found principally within the cytoplasm, but they also occur in the extracellular space between cytotrophoblast and syncytium, and between the individual cells of the cytotrophoblast. The droplets diminish in number with advancing age and may represent fat in transit from mother to fetus, and/or a pool of precursors for steroid synthesis. Membrane-bound granular bodies of moderate electron density occur in the cytoplasm, particularly in the syncytium, some of which are probably secretion granules. Other membrane-bound bodies, lysosomes and phagosomes, are involved in the degradation of materials engulfed from the intervillous space.

In the immature placenta, syncytial sprouts represent the first stages in the development of new terminal villi, which later become invaded by cytotrophoblast and villous mesenchyme. Occasionally, adjacent syncytial sprouts make contact and fuse to form slender syncytial bridges. The sprouts may become detached, forming maternal syncytial emboli, which pass to the lungs. It has been computed that some 100,000 sprouts pass daily into the maternal circulation. In the lungs they provoke little local reaction and apparently disappear by lysis. However, they may occasionally form foci for neoplastic growth. Syncytial sprouts are present in the term placenta, but are usually degenerating.

Syncytial knots are aggregates of degenerating nuclei, and may represent a sequestration phenomenon by which senescent nuclear material is removed from adjacent metabolically active areas of syncytium. Fibrinoid deposits are frequently found on the villous surface in areas lacking syncytiotrophoblast. They may constitute a repair mechanism in which the fibrinoid forms a wound surface that is subsequently re-epithelialized by trophoblast. The extracellular matrix glycoprotein tenascin has been localized in the stroma adjacent to these sites.

The core of a villus contains small and large reticulum cells, fibroblasts, and macrophages (Hofbauer cells). Early mesenchymal cells probably differentiate into small reticulum cells, which in turn produce fibroblasts or large reticulum cells. The small reticulum cells appear to delimit a collagen-free stromal channel system through which Hofbauer cells migrate. Mesenchymal collagen increases from a network of fine fibres in early mesenchymal villi to a densely fibrous stroma within stem villi in the second and third trimester. After approximately 14 weeks, the stromal channels found in immature intermediate villi are infilled by collagen to give the fibrous stroma characteristic of the stem villus.

Fetal vessels include arterioles and capillaries. Pericytes may be found in close association with the capillary endothelium, and from late first trimester the vessels are surrounded externally by a basal lamina. From the second trimester (and a little later in terminal villi), dilated thin-walled capillaries are found immediately adjacent to the villous trophoblast; their respective basal laminae apparently fuse to produce a vasculo-syncytial interface.

Transport across placental villi

The mechanism of transfer of substances across the placental barrier (membrane) is complex. The volume of maternal blood circulating through the intervillous space has been assessed at 500 ml per minute. Simple diffusion suffices to explain gaseous exchange. Transfer of ions and other water-soluble solutes is by paracellular and transcellular diffusion and transport: the relative importance of each of these for most individual solutes is unknown, and the paracellular pathway is morphologically undefined. Glucose transfer involves facilitated diffusion, while active transport mechanisms carry calcium and at least some amino acids. The fat-soluble and water-soluble vitamins are likely to pass the placental barrier with different degrees of facility. The water-soluble vitamins B and C pass readily. Water is interchanged between fetus and mother (in both directions) at approximately 3.5 litres per hour. The transfer of substances of high molecular weight, such as complex sugars, some lipids and hormonal and non-hormonal proteins, varies greatly in rate and degree, and is not so well understood: energy-dependent selective transport mechanisms including receptor-mediated transcytosis are likely to be involved.

Lipids may be transported unchanged through and between the cells of the trophoblast to the core of the villus. The passage of maternal antibodies (immunoglobulins) across the placental barrier confers some degree of passive immunity on the fetus: it is widely accepted that transfer is by micropinocytosis. Investigation of transplacental mechanisms is complicated by the fact that the trophoblast itself is the site of synthesis and storage of certain substances, e.g. glycogen.

The placenta is an important endocrine organ. Some steroid hormones, various oestrogens, β-endorphins, progesterone, hCG and human chorionic somatomammotropin (hCS), which is also known as placental lactogen (hPL), are synthesized and secreted by the syncytium. The trophoblast also contains enzyme systems that are associated with the synthesis of steroid hormones.

It has been suggested that leukocytes may migrate from the maternal blood through the placental barrier into the fetal capillaries. It has also been shown that some fetal and maternal red blood cells may cross the barrier. The former may have important consequences, e.g. in Rhesus incompatibility.

The majority of drugs are small molecules and are sufficiently lipophilic to pass the placental barrier. Many are tolerated by the fetus, but some may exert grave teratogenic effects on the developing embryo (e.g. thalidomide). A well-documented association exists between maternal alcohol ingestion and fetal abnormalities. Addiction of the fetus can occur to substances of maternal abuse such as cocaine and heroin.

A wide variety of bacteria, spirochaetes, protozoa and viruses, including human immunodeficiency virus (HIV), are known to pass the placental barrier from mother to fetus, although the mechanism of transfer is uncertain. The presence of maternal rubella in the early months of pregnancy is of especial importance in relation to the production of congenital anomalies.

FETAL MEMBRANES

The implanting conceptus consists initially of three cavities and their surrounding epithelia. The original blastocyst cavity, surrounded by trophoblast, is now termed the chorionic cavity (synonymous with extraembryonic coelom). It is a large cavity containing the much smaller amniotic cavity and secondary yolk sac (see Fig. 9.1). The apposition of the latter two cavities delineates the extent of the early embryo. The chorionic cavity becomes lined with extraembryonic mesoblast which is also reflected over the outer surface of the amnion and yolk sac. A fourth cavity, the allantois, develops later as a caudal hypoblastic diverticulum that becomes embedded within the extraembryonic mesenchyme, forming the connecting stalk of the embryo. It does not have a direct mesothelial covering.

Chorion

The chorion consists of developing trophoblast and extraembryonic mesothelium. It varies in thickness during development both temporally and spatially. It is thickest at the implantation site throughout gestation as the chorion frondosum and then the placenta, and thinner as gestation progresses over the remainder of its surface as the chorion laevae (Fig. 9.3). At term the chorion consists of an inner cellular layer containing fibroblasts and a reticular layer of fibroblasts and Hofbauer cells, which resembles the mesenchyme of an intermediate villus. The outer layer consists of cytotrophoblast 3 to 10 cells deep, resting on a pseudo-basement membrane, which extends beneath and between the cells. Occasional obliterated villi within the trophoblast layer are the remnants of villi present in the chorion frondosum of the first trimester. Although the interface between the trophoblast and decidua parietalis is uneven, no trophoblast infiltration of the decidua parietalis occurs.

Yolk sac

As the secondary yolk sac forms it delineates a cavity lined with parietal, and perhaps visceral, hypoblast, which is continuous with the developing endoderm from the primitive streak (Ch. 10). The secondary yolk sac is the first structure that can be detected ultrasonographically within the chorionic cavity (Jauniaux et al 1991). Its diameter increases slightly between 6 and 10 weeks of gestation, reaching a maximum of 6–7 mm after which its size decreases.

The inner cells of the yolk sac (denoted endoderm in many studies, although this layer is restricted to the embryo itself) display a few short microvilli and are linked by juxtaluminal tight junctions (Jones & Jauniaux 1995). Their cytoplasm contains numerous mitochondria, whorls of rough endoplasmic reticulum, Golgi bodies and secretory droplets, giving them the appearance of being highly active synthetic cells. With further development the epithelium becomes folded to form a series of cyst-like structures or tubules, only some of which communicate with the central cavity. The cells synthesize several serum proteins in common with the fetal liver, such as alpha-fetoprotein (AFP), alpha-1-antitrypsin, albumin, pre-albumin and transferrin (Jauniaux & Gulbis 2000). With rare exceptions, the secretion of most of these proteins is confined to the embryonic compartments.

The yolk sac becomes coated with extraembryonic mesenchyme, which forms mesenchymal and mesothelial layers. A diffuse capillary plexus develops between the mesothelial layer and the underlying secondary yolk sac wall, and subsequently drains through vitelline veins to the developing liver. The mesothelial layer bears a dense covering of microvilli: the presence of numerous coated pits and pinocytotic vesicles gives it the appearance of an absorptive epithelium (Jones & Jauniaux 1995).

The secondary yolk sac plays a major role in the early embryonic development of all mammals. In laboratory rodents it has been demonstrated as one of the initial sites of haematopoiesis. Recent human data indicate that it has an absorptive role for molecules of maternal and placental origin found in the chorionic cavity (Gulbis et al 1998) and mediates the main movement of molecules passing from the chorionic cavity to the yolk sac and subsequently to the embryonic gut and circulation.

After week 9 the cellular components of the wall of the secondary yolk sac start to degenerate, and their function is subsumed into exchanges at the placental chorionic plate. With embryonic development of the midgut, the connection of the yolk sac to the embryo becomes attenuated to a slender and elongated vitelline intestinal duct. Both the yolk sac and its duct remain within the extraembryonic coelom (chorionic cavity) throughout gestation, located between the amnion and chorion as they fuse, near the placental attachment of the umbilical cord.

Allantois

The allantoenteric diverticulum (see Fig. 10.1) arises early in the third week as a solid, endodermal outgrowth from the dorsocaudal part of the yolk sac into the mesenchyme of the connecting stalk. It soon becomes canalized. When the hindgut is developed, the proximal (enteric) part of the diverticulum is incorporated in its ventral wall. The distal (allantoic) part remains as the allantoic duct and is carried ventrally to open into the ventral aspect of the cloaca or terminal part of the hindgut (Fig. 9.7A). The allantois is a site of angiogenesis, giving rise to the umbilical vessels which connect to the placental circulation. The extraembryonic mesenchyme around the allantois forms the connecting stalk, which is later incorporated into the umbilical cord.

Fig. 9.7 A, Longitudinal section of a conceptus showing the cavities associated with development. The amniotic cavity and yolk sac are both covered with extraembryonic mesoblast. They are contained within the larger chorionic cavity which is lined with extraembryonic mesoblast. The embryo is attached to the chorion frondosum by the connecting stalk into which the allantois projects. B, Longitudinal section of conceptus at a later stage showing the diminution of the chorionic cavity, expansion of the amniotic cavity, relative attenuation of the yolk sac and the structures which give rise to the umbilical cord.

In the fetus, the allantoic duct, which is confined to the proximal end of the umbilical cord, elongates and thins. However, it may persist as an interrupted series of epithelial strands at term, in which case the proximal strand is often continuous at the umbilicus with the median intra-abdominal urachus, and this in turn continues into the apex of the bladder.

Amnion (chorio-amnion)

The original amniotic cells develop from the edges of the epiblast of the embryonic disc which ultimately form the interface with the skin at the umbilical region. Between the 10th and 12th weeks of pregnancy the amniotic cavity expands until it makes contact with the chorion to form the chorio-amnion, an avascular membrane which persists to term. The amniotic membrane extends along the connecting stalk and forms the outer covering of the umbilical cord. After birth, the site of this embryonic/extraembryonic junction is important, because the extraembryonic cell lines will die, causing the umbilical cord to degenerate and detach from the body wall. In cases of anomalous development of the ventral body wall, e.g. gastroschisis and exomphalos, the reflections of the amnion along the forming umbilical cord may be incomplete (see below).

The inner surface of the amnion consists of a simple cuboidal epithelium. It has a microvillous apical surface beneath which is a cortical web of intermediate filaments and microfilaments. There are no tight junctional complexes between adjacent cells and cationic dyes penetrate between the cells as far as the basal lamina. The intercellular clefts present scattered desmosomes, but elsewhere the clefts widen and contain interlacing microvilli. These features are consistent with selective permeability properties. The epithelium synthesizes and deposits extracellular matrix into the compact layer of acellular stroma located beneath the basal lamina, as well as the basal lamina itself.

Towards the end of gestation increasing numbers of amniotic cells undergo apoptosis. Apoptotic cells become detached from the amnion and are found in the amniotic cavity at term. The highest incidence is in weeks 40–41, independent of the onset of labour. Apoptosis may play a role in the fragility and rupture of the fetal membranes at term.

Human amniotic epithelial cells are thought to be pluripotent because they arise so early from the conceptus. They can be distinguished from the epiblast cells from day 8. Amniotic cells lack the major histocompatibility complex antigen and so the amnion can be exposed to the maternal immune system without eliciting a maternal immune response. Cultured human amniotic epithelial cells express a range of neural and glial markers, including glial fibrillary acidic protein, myelin basic protein, vimentin and neurofilament proteins, suggesting that these cells may supply neurotrophic factors to the amniotic fluid. They also appear to have a hepatocyte gene expression profile, showing albumin production, glycogen storage and albumin secretion in culture. In organ culture they have been shown to secrete 30-fold larger amounts of albumin than in monolayer culture, and to secrete alpha-1-antitrypsin (Takashima et al 2004). Amnion is used in the repair of corneas after trauma and as a graft material for reconstructing vaginas in women with cloacal abnormalities.

AMNIOTIC FLUID

The amniotic fluid, or liquor amnii, is derived from multiple sources throughout gestation. These include secretions from amniotic epithelium, filtration of fluid from maternal vessels via the parietal decidua and amniochorion, filtration from the fetal vessels via the chorionic plate or the umbilical cord, and fetal urine and fetal lung secretions. In early pregnancy, diffusion from intracorporeal vessels via fetal skin provides another source. Once the gut is formed, fetal swallowing of amniotic fluid is a normal occurrence: the fluid is absorbed into the fetal circulation and passes via the placental barrier into the maternal circulation. There is rapid exchange between the amniotic fluid and maternal and fetal circulations via the placenta and fetal kidneys.

In the early stages amniotic fluid resembles blood plasma in composition and is probably formed largely by transport across the amniotic membrane. As pregnancy advances, it becomes progressively more dilute, partly by the addition of fetal urine. It contains less than 2% of solids, including urea, inorganic salts, a small amount of protein and frequently a trace of sugar. Glycoprotein secretions from amniotic epithelium include fibronectin. Secretory products of the maternal decidua, including prolactin and insulin-like growth factor binding protein 1 (IGF-BP1), are present in the liquor. There is experimental evidence of a considerable and rapid flux of water across the amniotic membrane.

The amount of amniotic fluid increases in quantity up to the sixth or seventh month and then diminishes slightly. At the end of pregnancy it is usually somewhat less than a litre. It provides a buoyant medium which supports the delicate tissues of the young embryo and allows free movement of the fetus during the later stages of pregnancy. It also diminishes the risk to the fetus of injury from without.

A volume of amniotic fluid in excess of 2 litres is generally considered to be abnormal and constitutes polyhydramnios. A deficiency is termed oligohydramnios and absent amniotic fluid is anhydramnios. (For information about amniotic fluid volume and ranges in gestation consult Brace & Wolf 1989.) Oligohydramnios in the second or third trimester is usually the result of premature rupture of the membranes, uteroplacental insufficiency or urinary tract malformations, e.g. bilateral renal agenesis or obstruction of the lower urinary tract. The major concern with oligohydramnios at less than 20 weeks is the significant risk of pulmonary hypoplasia and neonatal death. The mechanism for the development of pulmonary hypoplasia is poorly understood but loss of lung fluid, and chest compression are contributing factors. Conversely, increased amniotic fluid volume (polyhydramnios) is found essentially in two major circumstances: reduced fetal swallowing or absorption of amniotic fluid and increased fetal urination. Reduced fetal swallowing may be due to congenital malformations e.g. anencephaly, upper intestinal tract obstruction (oesophageal atresia), compressive pulmonary disorders (congenital diaphragmatic hernia) and neuromuscular impairment of swallowing. The cause for abnormal amniotic fluid volume can often be elucidated with detailed prenatal ultrasound examination.

UMBILICAL CORD

The formation of the connecting stalk is described in Chapter 10, and the early formation of the umbilical cord is described on p. 1209. The umbilical cord ultimately consists of an outer covering of flattened amniotic epithelial cells and an interior mass of mesenchyme of diverse origins (Fig. 9.7). It contains two tubes of hypoblastic origin, the vitelline-intestinal and allantoic ducts, and their associated vitelline and allantoic (umbilical) blood vessels. The yolk stalk and continuing duct extend the length of the cord whereas the allantoic duct extends only into its proximal part.

The mesenchymal core is derived from the somatopleuric extraembryonic mesenchyme covering the amniotic folds, splanchnopleuric extraembryonic mesenchyme of the yolk stalk (which carries the vitelline vessels and clothes the yolk duct), and similar allantoic mesenchyme of the connecting stalk (which clothes the allantoic duct and initially carries two umbilical arteries and two umbilical veins). These various mesenchymal compartments fuse and are gradually transformed into the loose connective tissue (Wharton’s jelly) that characterizes the more mature cord. The tissue consists of widely spaced elongated fibroblasts separated by a delicate three-dimensional meshwork of fine collagen fibres, which contains a variety of hydrated glycosaminoglycans, and is particularly rich in hyaluronic acid.

The vitelline and allantoic (umbilical) vessels, which are initially symmetrical, become modified as a result of changes in the circulation. The vitelline vessels involute, whereas most of the allantoic (umbilical) vessels persist. The right umbilical vein disappears but the two umbilical arteries normally remain. Occasionally one umbilical artery may disappear; there is some correlation within structural anomalies, most often cardiac, in such cases. The vessels of the umbilical cord are rarely straight, and are usually twisted into either a right- or left-handed cylindrical helix. The number of turns involved ranges from a few to over 300. This conformation may be produced by unequal growth of the vessels, or by torsional forces imposed by fetal movements. Its functional significance is obscure: perhaps the pulsations and contractions of the helical vessels assist the venous return to the fetus in the umbilical vein.

Anomalies of the fetal anterior abdominal wall such as exomphalos and gastroschisis may affect the arrangement of the outer covering of amnion cells along the proximal end of the umbilical cord. Exomphalos arises from a failure of the lateral folds along the ventral surface of the embryo resulting in failure of the normal embryonic regression of the mid-gut from the umbilical stalk into the abdominal cavity. The abdominal contents, including intestines and liver or spleen covered by a sac of parietal peritoneum and amnion, are herniated into the base of the umbilical cord. In gastroschisis, the insertion of the umbilical cord is intact and there is evisceration of the intestine through a small abdominal wall defect usually located to the right of the umbilical cord: this results in free loops of bowel in the amniotic cavity. Theories concerning the aetiology of this defect include abnormal involution of the right umbilical vein or disruption of the omphalo-mesenteric artery by ischaemia.

Mature umbilical vessels, particularly the arteries, have a strong muscular coat which contracts readily in response to mechanical stimuli. The outermost bundles pursue an interlacing spiral course, and when they contract they produce shortening of the vessel and thickening of the media, with folding of the interna and considerable narrowing of the lumen. This action may account for the periodic sharp constrictions of contour, the so-called valves of Hoboken, which often characterize these vessels.

The fully developed umbilical cord is on average some 50 cm long and 1–2 cm in diameter. Its length varies from 20–120 cm: exceptionally short or long cords are associated with fetal problems and complications during labour. A long umbilical cord may prolapse through the cervix into the vagina once the fetal membranes rupture and this may be exacerbated by conditions that prevent the fetal head from fully occupying the maternal pelvis e.g. pelvic tumours (fibroids), ovarian cysts, placenta praevia and prematurity. Compression of the cord by the presenting part of the fetus, or an umbilical artery spasm will lead to fetal hypoxia and death if untreated. The risk of perinatal death rises as the interval from diagnosis to delivery increases. The treatment is either funic replacement (pushing the cord back above the fetal head) or more commonly immediate caesarean section, depending on factors such as the fetal viability.

The distal end of the umbilical cord usually attaches in the central portion of the placenta, but in a minority of cases velamentous insertion is observed (i.e. into the membranes) and this may be associated with vulnerability to injury and fetal haemorrhage. This is especially important if the placenta is low lying and may be associated with vasa praevia in which case fetal blood vessels run across the internal os. Inadvertent rupture of the fetal vessels in spontaneous labour or at the time of amniotomy (artificial rupture of membranes to induce labour) will cause fetal haemorrhage and may prove fatal (see placental variations).