Tutoring at home

As the villi continue to branch and the terminal ramifications become more numerous and smaller, the volume and prominence of cytotrophoblasts decrease. As the syncytium thins, the fetal vessels become more prominent and lie closer to the surface. The stroma of the villi also exhibits changes as gestation progresses. In placentas of early pregnancy, the branching connective tissue cells are separated by an abundant loose intercellular matrix. Later, the stroma becomes denser and the cells more spindly and more closely packed.
Another change in the stroma involves the infiltration of Hofbauer cells, which represent fetal macrophages. These cells are nearly round with vesicular, often eccentric nuclei and very granular or vacuolated cytoplasm. Hofbauer cells are characterized histochemically by intracytoplasmic lipid and by phenotypic markers specific for macrophages. They increase in numbers and maturation state as pregnancy progresses. Although phagocytic, they have an immunosuppressive phenotype (Vince and Johnson, 1996). In addition, they can produce a variety of cytokines and are capable of paracrine regulation of trophoblast functions (Cervar and colleagues, 1999).
Some of the histological changes that accompany placental growth and maturation provide an increased efficiency of transport and exchange to meet increasing fetal metabolic requirements. Among these changes are a decrease in thickness of the syncytium, a significant reduction of cytotrophoblast cells, a decrease in the stroma, an increase in the number of capillaries, and an approximation of these vessels to the syncytial surface. By 4 months, the apparent continuity of the cytotrophoblasts is broken, and at term, the covering of the villi may be focally reduced to a thin layer of syncytium with minimal connective tissue where the fetal capillaries appear to abut the trophoblast. The villi become dominated by thin-walled capillaries.
Other changes in placental architecture, however, can cause a decrease in the efficiency of placental exchange if they include a substantial portion of the exchange area. These changes include thickening of the basal lamina of the trophoblast or capillaries, obliteration of certain fetal vessels, and fibrin deposition on the surface of the villi in the basal and chorionic plates as well as elsewhere in the intervillous space.

In the first trimester, growth of the placenta is more rapid than that of the fetus, but by approximately 17 weeks postmenstruation (from the last menstrual period), placental and fetal weights are approximately equal. At term, the placental weight may be roughly one sixth that of fetal weight. According to Boyd and Hamilton (1970), the average placenta at term is 185 mm in diameter and 23 mm in thickness, with a volume of 497 mL and a weight of 508 g. These measurements vary widely, and there are multiple variant forms of the human placenta and several types of umbilical cord insertions.
Viewed from the maternal surface, the number of slightly elevated convex areas, called lobes, varies from 10 to 38. These lobes are incompletely separated by grooves of variable depth, overlying the placental septa, which arise from folding of the basal plate. These grossly visible lobes have also been referred to as “cotyledons”, however, this use should be avoided, because they bear no relation to the functional units supplied by each primary villus, which are termed either lobules or cotyledons.
The total number of lobes remains the same throughout gestation, and individual lobes continue to grow, although less actively in the final weeks (Crawford, 1959). Placental weights vary considerably, depending on how the placenta is prepared. If the fetal membranes and most of the cord are left attached and the adherent maternal blood clot is not removed, the weight may be greater by nearly 50 percent (Thomson and colleagues, 1969).

Early Trophoblast Invasion
After gentle erosion between epithelial cells of the surface endometrium, the invading trophoblasts burrow deeper into the endometrium, and by the 10th day the blastocyst becomes totally encased within the endometrium. This process of erosion and invasion into the endometrium is carried out actively by the trophoblast cells. The mechanisms leading to trophoblast invasion into the endometrium are similar to the characteristics of metastasizing malignant cells. These mechanisms are discussed in more detail in Trophoblast Invasion of the Endometrium.
One of the earliest implanting blastocysts discovered by Hertig and Rock (1945). It measured only 0.36 by 0.31 mm, and it was believed to have been in the process of penetrating the endometrium, with the thin outer wall of the blastocyst still within the uterine cavity. An implanting blastocyst at a similar stage of development, 9 days after fertilizatio. It appears to have been flattened in the process of penetrating the uterine epithelium; the enlargement and multiplication of the trophoblasts in contact with the endometrium are alone responsible for the increase in size of the implanted blastocyst as compared with that of the free blastocyst.

At 9 days of development, the wall of the blastocyst that faces toward the uterine lumen is a single layer of flattened cells. The opposite, thicker wall comprises two zones, the trophoblasts and the embryo-forming inner cell mass. As early as 71/2 days after fertilization, the inner cell mass, referred to as the embryonic disc, is differentiated into a thick plate of primitive ectoderm and an underlying layer of endoderm. Some small cells appear between the embryonic disc and the trophoblast, enclosing a space that will become the amnionic cavity.

The embryonic mesenchyme first appears as isolated cells within the cavity of the blastocyst. When the cavity is completely lined with mesoderm, it is termed the chorionic vesicle, and its membrane, now called the chorion, is composed of trophoblasts and mesenchyme. The amnion and yolk sac, with both epithelial and mesenchymal components. The mesenchymal cells within the cavity are the most numerous and eventually will condense to form the body stalk, which serves to join the embryo to the nutrient chorion and later develops into the umbilical cord. The body stalk can be recognized at an early stage at the caudal end of the embryonic disc.

Lacunae Formation Within the Syncytiotrophoblast

About 12 days after conception, the syncytiotrophoblast of the trophoblast shell is permeated by a system of intercommunicating channels of trophoblastic lacunae, or small cavities. As the embryo enlarges, more maternal tissue (decidua basalis) is invaded by the basal syncytiotrophoblast, including the walls of the superficial decidual capillaries, and these lacunae become filled with maternal blood. At the same time, the decidual reaction intensifies in the surrounding stroma, which is characterized by enlargement of the decidual stromal cells and glycogen storage.

Development of Primary Villous Stalks

With deeper blastocyst invasion into the decidua, the extravillous cytotrophoblasts give rise to the solid primary villi composed of a cytotrophoblast core covered by syncytium. As the lacunae join, a complicated labyrinth is formed that is partitioned by solid cytotrophoblastic columns, which arise from buds of cytotrophoblast that begin to protrude into the primitive syncytium before 12 days postfertilization. The trophoblast-lined labyrinthine channels and the solid cellular columns form the intervillous space and the primary villous stalks, respectively. The villi initially are located over the entire blastocyst surface, but later disappear except over the most deeply implanted portion, the site destined to form the placenta.

Over the past half-century, many attempts to explain the survival of the semiallogenic fetal graft have been proposed. One of the earliest explanations was based on the theory of antigenic immaturity of the embryo-fetus. This explanation was disproved by Billingham (1964), who showed that transplantation (HLA) antigens are demonstrable very early in embryonic life. Another explanation was based on diminished immunological responsiveness of the pregnant woman. There is, however, no evidence for this to be other than an ancillary factor. In a third explanation, the uterus (decidua) is proposed as an immunologically privileged tissue site. This would preclude well-documented advanced ectopic pregnancies, discussed in Chapter 10.
Clearly, the lack of transplantation immunity manifest in the uterus is unique compared with that of other tissues. Therefore, the acceptance and the survival of the conceptus in the maternal uterus must be attributed to an immunological peculiarity of the trophoblasts, not the decidua. The trophoblasts are the only cells of the conceptus in direct contact with maternal tissues or blood, and these tissues are genetically identical with fetal tissues. Several features of trophoblast cells likely contribute to the survival of these cells in an immunologically hostile environment (Thellin and colleagues, 2000). The most important may be the novel aspects of the expression of the HLA system in trophoblasts, coupled with a unique set of uterine lymphocytes.
Immunogenicity of the Trophoblasts
Over 50 years ago, Sir Peter Medawar (1953) suggested that the solution to the riddle of the fetal semiallograft might be explained by immunological neutrality. The placenta was considered immunologically inert and therefore unable to cause a maternal immune response. Subsequently, many researchers focused on defining the expression of the major histocompatibility complex (MHC) antigens on trophoblasts. Human leukocyte antigens (HLA) are the human analogue of the major histocompatibility complex. MHC class I and II antigens are absent from villous trophoblasts at all stages of gestation (Weetman, 1999). Thus, these cells do appear to be immunologically inert with regard to fetal–maternal interactions. However, the invasive cytotrophoblasts do express MHC class I molecules and these have been the focus of considerable study.
Trophoblast HLA (MHC) Class I Expression
The HLA genes are the products of multiple genetic loci of the MHC located within the short arm of chromosome 6 (Hunt and Orr, 1992). There are 17 HLA class I genes, including three classical genes, HLA-A, -B, and -C, that encode the major class I (class Ia) transplantation antigens. Three other class I genes, designated HLA-E, -F, and -G, encode class Ib HLA antigens. The remaining DNA sequences appear to be pseudogenes or partial gene fragments.
Moffett-King (2002) reasoned that normal implantation is dependent on controlled trophoblastic invasion of maternal endometrium–decidua and the spiral arteries. Trophoblast invasion must proceed far enough to provide for normal fetal growth and development, and a mechanism for regulating the depth of trophoblast invasion must exist. She suggested that the uterine large granular lymphocytes (LGLs) and the unique expression of three specific HLA class I genes in extravillous cytotrophoblasts act to permit and subsequently to limit the process of trophoblast invasion.
Class I antigens in extravillous cytotrophoblasts are accounted for by the expression of the classical HLA-C and the nonclassical class Ib molecules of HLA-E and HLA-G. To elucidate the importance of HLA-C, HLA-E and HLA-G expression, it is important to understand the nature of the unusual lymphocyte population of the human decidua.
Uterine Large Granular Lymphocytes (LGLs)
These distinctive lymphocytes are believed to originate in bone marrow and belong to the natural killer cell lineage. They are by far the predominant population of leukocytes present in midluteal phase endometrium at the expected time of implantation (Johnson and colleagues, 1999). These LGLs have a distinct phenotype characterized by a high surface density of CD56 or neural cell adhesion molecule (Loke and King, 1995; Moffett-King, 2002). The infiltration of LGLs is increased by progesterone as well as stromal cell production of IL-15 and prolactin (Dunn and colleagues, 2002; Gubbay and colleagues, 2002).
Near the end of the luteal phase of nonfertile ovulatory cycles, the nuclei of the uterine LGL begin to disintegrate. With blastocyst implantation, however, these cells persist in large numbers in the decidua during the early weeks of pregnancy. At term, however, there are relatively few LGLs in the decidua. In first-trimester decidua, many LGLs are in close proximity to the extravillous trophoblast. It is speculated that LGLs are involved in the regulation of trophoblast invasion. They secrete large amounts of granulocyte-macrophage–colony-stimulating factor (GM-CSF), suggestive that the LGLs in first-trimester decidua are in an activated state. This has led Jokhi and co-workers (1999) to speculate that GM-CSF may function primarily not to promote trophoblast replication but rather to forestall trophoblast apoptosis. Moreover, expression of angiogenic factors by uterine natural killer cells is suggestive of a potential role for these cells in decidual vascular remodeling (Li and colleagues, 2001). According to this theory, LGLs rather than the T lymphocytes, would bear the primary responsibility for immunosurveillance in decidua.
HLA-G Expression in Human Trophoblasts
HLA-G is expressed only in humans and is distinguished from the HLA class Ia products by a highly restricted tissue distribution. Indeed, HLA-G antigen expression is identified only in extravillous cytotrophoblasts in the decidua basalis and in the chorion laeve (McMaster and colleagues, 1995). HLA-G is not present in villous trophoblasts, either in the syncytium or in the cytotrophoblasts. HLA-G is expressed, however, in cytotrophoblasts that are contiguous with maternal tissues, viz., decidual cells and LGLs. Interestingly the HLA-G gene produces several mRNA transcripts as a result of alternative splicing to yield at least seven isoforms, some soluble and some membrane bound (Carosella and colleagues, 2000). There are few individual DNA variations or polymorphisms in the HLA-G gene sequence, further suggesting an important role for this factor. It has been shown that a soluble major isoform, HLA-G2, is increased during pregnancy (Hunt and colleagues, 2000a, 2000b). Additionally, a recent study of embryos being used for in vitro fertilization showed that pregnancy did not occur in any of the embryos that did not produce the soluble form of HLA-G (Fuzzi and colleagues, 2002). It may be that HLA-G is immunologically permissive of the antigen mismatch between mother and fetus (LeBouteiller and colleagues, 1999). One hypothesis is that its expression may be stimulated by hypoxia, leading to developmental modifications in HLA-G class I antigen expression on trophoblasts (Kilburn and colleagues, 2000). In that regard, Goldman-Wohl and associates (2000) have provided evidence for abnormal HLA-G expression in extravillous trophoblasts from women with preeclampsia.

The formation of the human placenta begins with the trophectoderm, which is the first tissue to differentiate at the morula stage of development, giving rise to a layer of trophoblast cells encircling the blastocyst. From the early blastocyst to term placenta, the trophoblast plays critical roles at the fetal–maternal interface. The trophoblast exhibits the most variable structure, function, and developmental pattern of all placental components. Its invasiveness provides for attachment of the blastocyst to the decidua; its role in nutrition of the conceptus is reflected in its name; and its function as an endocrine organ in human pregnancy is essential to maternal physiological adaptations and to the maintenance of pregnancy.
Trophoblast Differentiation
By the eighth day postfertilization, after initial implantation of the blastocyst, the trophoblast has differentiated into an outer multinucleated syncytium, the primitive syncytiotrophoblast, and an inner layer of primitive mononuclear cytotrophoblasts. The cytotrophoblasts are the germinal cells for the syncytium; the latter acts as the primary secretory component within the placenta. Although the ability to undergo DNA synthesis and mitosis, a well-demarcated cell border, and a single nucleus characterize each cytotrophoblast, these characteristics are lacking in the syncytium covering the chorionic villi (Arnholdt and colleagues, 1991). The syncytium has no individual cells, only a continuous syncytial lining. Therefore, the cellular term used is syncytiotrophoblast, in which the cytoplasm is amorphous, without cell borders, and the nuclei are multiple and diverse in size and shape. The absence of cell borders facilitates transport across the syncytiotrophoblast, because the control of transport is not dependent on the participation of individual cells.
After implantation is complete, the trophoblast further differentiates along two main pathways, giving rise to villous and extravillous trophoblast. Both pathways give rise to populations of trophoblast cells with distinct functions, which come into contact with maternal tissues (Loke and King, 1995). The villous trophoblast, as its name suggests, gives rise to the chorionic villi of the placenta, and primarily functions in the transport of oxygen and nutrients between the fetus and mother. The extravillous trophoblast migrates into the decidua and myometrium and also penetrates maternal vasculature, thus coming into contact with a variety of maternal cell types (Pijnenborg, 1994). The extravillous trophoblast is thus further classified as interstitial trophoblast and endovascular trophoblast. The interstitial trophoblast both invades the maternal decidua, eventually penetrating the myometrium to form placental bed giant cells, and surrounds the maternal spiral arteries. The endovascular trophoblast penetrates the lumen of the spiral arteries (Pijnenborg and colleagues, 1983). Both the formation of chorionic villi and the remarkable process of invasion of maternal tissues by extravillous trophoblast are discussed separately in greater detail in the sections that follow.

Ovum Fertilization and Zygote Cleavage
The union of egg and sperm at fertilization represents one of the most important processes in biology. Ovulation frees the secondary oocyte and the adhering cells of the cumulus oophorus from the ovary. Although technically this mass of cells is released into the peritoneal cavity, the oocyte is quickly engulfed by the infundibulum of the fallopian tube. Transport of the oocyte through the fallopian tube toward the uterus is accomplished by directional movement of ciliary action as well as peristalsis. Fertilization occurs in the fallopian tube, and it is generally agreed that fertilization of the ovum must occur a few hours and no more than a day after ovulation. Consequently, spermatozoa must be present in the fallopian tube at the time of oocyte arrival. Almost all pregnancies occur when intercourse occurs during the 2 days preceding or on the day of ovulation. Thus the postovulatory and postfertilization developmental ages are similar. The steps involved to achieve fertilization are highly complex and have been the topic of much research. The molecular mechanisms that allow passage of spermatozoa between the follicular cells, through the zona pellucida, and into the oocyte cytoplasm leading to the formation of the zygote continue to be unraveled and recently have been reviewed (Primakoff and Myles, 2002).
In this chapter, the timing of events in early human development is described as days or weeks postfertilization, viz., postconceptional, rather than using the clinical pregnancy dating convention of weeks from the start of the last menstrual period. As discussed in Proliferative (Preovulatory) Phase of the Endometrium, the length of the follicular phase of the menstrual cycle is subject to more variability than that of the luteal phase. Thus, 1 week postfertilization corresponds to approximately 3 weeks from the last menstrual period in women with regular 28-day cycles.
After fertilization in the fallopian tube, the mature ovum becomes a zygote—a diploid cell with 46 chromosomes—that then undergoes cleavage into blastomeres. In the two-cell zygote, the blastomeres and the polar body are free in the perivitelline fluid and are surrounded by a thick zona pellucida. The zygote undergoes slow cleavage for 3 days while still within the fallopian tube. As the blastomeres continue to divide, a solid mulberry-like ball of cells, referred to as the morula, is produced. The morula enters the uterine cavity about 3 days after fertilization. The gradual accumulation of fluid between the cells of the morula results in the formation of the early blastocyst.

In a 58-cell blastocyst, the outer cells, called the trophectoderm, can be distinguished from the inner cell mass that forms the embryo (Fig. 3–8E). In the earliest stages of the human blastocyst, the wall of the primitive blastodermic vesicle is characterized as consisting of a single layer of ectoderm. As early as 4 to 5 days after fertilization, the 58-cell blastula differentiates into five embryo-producing cells known as the inner cell mass, and 53 cells destined to form trophoblasts (Hertig, 1962).
Interestingly, the 107-cell blastocyst is found to be no larger than the earlier cleavage stages, despite the accumulated fluid (Fig. 3–8F). It measured 0.155 mm in diameter, which is similar to the size of the initial postfertilization zygote. At this stage the eight formative, or embryo-producing, cells are surrounded by 99 trophoblastic cells. It is at this stage that the blastocyst is released from the zona pellucida as a result of secretion of specific proteases from the secretory-phase endometrial glands (O’Sullivan and colleagues, 2002).
Release from the zona pellucida allows blastocyst-produced cytokines and hormones to directly influence the receptivity of the endometrium (Lindhard and colleagues, 2002). Evidence has accumulated that IL-1 and IL-1 are secreted by the blastocyst and that these cytokines can directly influence the endometrium. Embryos also have been shown to secrete human chorionic gonadotropin (hCG), which may influence endometrial receptivity (Licht and colleagues, 2001; Lobo and colleagues, 2001). The receptive endometrium is thought to respond by producing leukemia inhibitory factor and colony-stimulating factor-1, which increase trophoblast production of proteases that degrade selected endometrial extracellular matrix proteins and allow trophoblast invasion. Thus, embryo “hatching” is a critical step toward successful pregnancy as it allows association of the trophoblasts with the epithelial cells of the endometrium and the release of trophoblast-produced hormones into the uterine cavity.
Implantation of the Blastocyst
Implantation of the embryo into the wall of the uterus is a common feature of all mammals and in humans occurs six or seven days after fertilization. Successful implantation requires a receptive endometrium that has been appropriately primed with estrogen and progesterone. uterine receptivity toward the blastocyst is limited to days 20 to 24 of the ovarian–endometrial cycle (Bergh and Navot, 1992). The ability of the blastocyst to adhere to the epithelium is mediated by cell surface receptors at the implantation site that interact with receptors on the blastocyst (Carson, 2002; Lessey and Castelbaum, 2002; Lindhard and colleagues, 2002; Paria and colleagues, 2002). The development of the receptive epithelia results from the postovulatory production of estrogen and progesterone by the corpus luteum. If the blastocyst approaches the endometrium after cycle day 24, the potential for adhesion is diminished because of the synthesis of antiadhesive glycoproteins, which prevent receptor interactions (Navot and Bergh, 1991).
At the time of its interaction with the endometrium, the blastocyst is composed of 100 to 250 cells. The blastocyst loosely adheres to the endometrial epithelium, a process called apposition, which most commonly occurs on the endometrium of the upper posterior wall of the uterus. Although as subsequently discussed, syncytiotrophoblast has not been distinguished prior to human blastocyst implantation, syncytiotrophoblast has been observed in the earliest implanted blastocyst of the macaque monkey (Boyd and Hamilton, 1970). The attachment of the trophectoderm of the blastocyst to the endometrial surface by apposition and adherence appears to be closely regulated by paracrine interactions between these two tissues.
Successful adhesion of the blastocyst to the endometrium also involves modification in the expression of cellular adhesion molecules. The integrins, one of four families of cell adhesion molecules, are cell-surface receptors that mediate the adhesion of cells to extracellular matrix proteins (Lessey and Castelbaum, 2002). Great diversity of cell binding to a host of different extracellular matrix proteins is possible by differential regulation of the integrin system of receptors. An alteration of integrin subunit expression on the endometrial epithelial cells is considered a marker of receptivity for blastocyst attachment.

The development of the human placenta is as uniquely intriguing as the embryology of the fetus. During its brief intrauterine existence, the fetus is dependent on the placenta for pulmonary, hepatic, and renal functions. The placenta accomplishes these functions through its unique anatomical association with the mother. The placenta links the mother and fetus by indirect interaction with the maternal blood that spurts out of the uteroplacental vessels. This blood bathes the outer syncytiotrophoblast, allowing exchange of gases and nutrients with fetal capillary blood within the connective tissue at the villous core. Fetal and maternal blood are not mixed in this hemochorial type of placenta. There is also a paracrine system that links the mother and fetus through the anatomical and biochemical juxtaposition of extraembryonic chorion laeve of fetal origin and maternal uterine decidua parietalis tissue. This is an extraordinarily important arrangement for communication between fetus and mother, for maternal immunological acceptance of the conceptus, and possibly for controlling the timing of parturition.