Human embryogenesis is the process of cell division and cellular differentiation of the human embryo during early prenatal development. It spans from the moment of fertilization to the end of the 8th week of gestational age, where after it is called a fetus.
From One Cell to Blastocyst:
A human develops from a single cell called a zygote, which results from an ovum (egg) being fertilized by a single spermatozoan (sperm). The cell is surrounded by a strong membrane of glycoproteins called the zona pellucida which the successful sperm has managed to penetrate.
The zygote undergoes cleavage, increasing the number of cells within the zona pellucida. After the 8-cell stage, embryos undergo what is called compactation, where the cells bind tightly to each other, forming a compact sphere. After compactation, the embryo is in the morula stage (16 cells).
Cavitation occurs next; where the outermost layer of cells (the trophoblast) secretes water into the morula. As a consequence of this when the number of cells reaches 40 to 150, a central, fluid-filled cavity (blastocoel) has been formed.
The zona pellucida begins to degenerate, allowing the embryo to increase its volume. This stage in the developing embryo, reached after four to six days, is the blastocyst (related to the blastula stage), and lasts approximately until the implantation in the uterus.
The blastocyst is characterised by a group of cells, called the inner cell mass (also called embryoblast) and the mentioned trophoblast (the outer cells).
The inner cell mass gives rise to the embryo proper, the amnion, yolk sac and allantois, while the trophoblast will eventually form the placenta. The blastocyst can be thought of as a ball of a layer of trophoblast cells, with the inner cell mass attached to this ball’s inner wall.
The embryo plus its membranes is called the conceptus. By this stage the conceptus is in the uterus. The zona pellucida ultimately disappears completely, allowing the blastocyst to invade the endometrium, performing implantation.
The trophoblast then differentiates into two distinct layers- the inner is the cytotrophoblast consisting of cuboidal cells that are the source of dividing cells, and the outer is the syncytiotrophoblast. The syncytiotrophoblast implants the blastocyst in the endometrium (innermost epithelial lining) of the uterus by forming finger-like projections called chorionic villi that make their way into the uterus, and spaces called lacunae that fill up with the mother’s blood.
This is assisted by hydrolytic enzymes that erode the epithelium. The syncytiotrophoblast also produces human chorionic gonadotropin (hCG), a hormone that “notifies” the mother’s body that she is pregnant, preventing menstruation by sustaining the function of the corpus luteum. The villi begin to branch, and contain blood vessels of the fetus that allow gas exchange between mother and child.
i. Implantation Window:
There are many conditions that must be satisfied for a successful implantation to take place. There is only a specific period of time during which implantation is possible; this is the “implantation window”. A reason for this window is that if implantation does not occur at a certain time, then it signifies that something is wrong. And when there is a risk that something is wrong, there will most likely be a miscarriage rather than the continued gestation of a malformed fetus.
The implantation window is started by preparations in the endometrium of the uterus, both structurally and in the composition of its secretions.
ii. Adaption of Uterus:
To enable implantation, the uterus goes through changes in order to be able to receive the embryo.
Predecidualisation is a preparation of the endometrium of the uterus, prior to implantation, to facilitate it. The endometrium increases in thickness, becomes more vascularised and its glands grow to be tortuous and boosted in their secretions. These changes reach their maximum about 7 days after ovulation.
Furthermore, the surface of the endometrium produces a kind of rounded cells, which cover the whole area towards the uterine cavity. This happens about 9 to 10 days after ovulation.
These cells are called decidual cells, which emphasises that the whole layer of them is shed off in every menstruation if no pregnancy occurs, just as leaves of deciduous trees. The uterine glands, on the other hand, decrease in activity and degenerate already 8 to 9 days after ovulation in absence of pregnancy.
The stromal cells originate from the stromal cells that are always present in the endometrium. However, the decidual cells make up a new layer, the decidua. The rest of the endometrium, in addition, expresses differences between the luminal and the basal sides. The luminal cells form the zona compacta of the endometrium, in contrast to the basalolateral zona spongiosa, which consists of the rather spongy stromal cells.
Decidualisation succeeds predecidualisation if pregnancy occurs. This is an expansion of it, further developing the uterine glands, the zona compacta and the epithelium of decidual cells lining it. The decidual cells become filled with lipids and glycogen and take the polyhedral shape characteristic for decidual cells.
It is likely that the blastocyst itself makes the main contribution to this additional growing and sustaining of the decidua. An indication of this is that decidualisation occurs at a higher degree in conception cycles than in non-conception cycles. Furthermore, similar changes are observed when giving stimuli mimicking the natural invasion of the embryo.
b. Parts of Decidua:
The decidua can be organised into separate sections, although they have the same composition.
1. Decidua Basalis:
This is the part of the decidua which is located basalolateral to the embryo after implantation.
2. Decidua Capsularis:
Decidua capsularis grows over the embryo on the luminal side, enclosing it into the endometrium. It surrounds the embryo together with decidua basalis.
3. Decidua Parietalis:
All other decidua on the uterine surface belongs to decidua parietalis.
4. Decidua throughout Pregnancy:
After implantation the decidua remains, at least the first trimester. However, its most prominent time is during the early stages of pregnancy, meanwhile as implantation. Its function as a surrounding tissue is replaced by the definitive placenta. However, some elements of the decidualisation remain throughout pregnancy.
The compacta and spongiosa layers are still observable beneath the decidua in pregnancy. The glands of the spongiosa layer continue to secrete during the first trimester, when they degenerate. However, before the disappearance, some glands secrete unequally much. This phenomenon of hypersecretion is called the Arias-Stella phenomenon, after the pathologist Javier Arias-Stella.
Pinopodes are small, finger-like protrusions from the endometrium. They appear between day 19 and day 21 of gestational age. This corresponds to a fertilization age of approximately 5 to 7 days, which corresponds well with the time of implantation. They only persist for 2 to 3 days. The development of them is enhanced by progesterone but inhibited by estrogens.
a. Function in Implantation:
Pinopodes endocytose uterine fluid and macromolecules in it. By doing so, the volume of the uterus decreases, taking the walls closer to the embryoblast floating in it. Thus, the period of active pinocytes might also limit the implantation window.
b. Function During Implantation:
Pinopodes continue to absorb fluid, and removes most of it during the early stages of implantation.
c. Adaption of Secretions:
Proteins, glycoproteins and peptides secreted by the endometrial glands.
4. Type-IV collagen
5. Heparan sulfate
4. Placental protein 14 (PP14) or glycodelin
5. Pregnancy-associated endometrial alpha-2-globulin (alpha-2- PEG) endometrial protein 15
9. Fibroblast growth factor 1
10. Fibroblast growth factor 2
11. Pregnancy-associated plasma protein A (PAPP-A)
12. Stress response protein 27 (SRP-27)
16. Diamine oxidase
17. Tissue plasminogen activator
Not only the lining of the uterus transforms, but also the secretion from its epithelial glands changes. This change is induced by increased levels of progesterone from the corpus luteum. The target of the secretions is the embryoblast, and has several functions on it.
The embryoblast spends approximately 72 hours in the uterine cavity before implanting. In that time, it cannot receive nourishment directly from the blood of the mother, and must rely on secreted nutrients into the uterine cavity, e.g., iron and fat-soluble vitamins.
vi. Growth and Implantation:
In addition to nourishment, the endometrium secretes several steroid- dependent proteins, important for growth and implantation. Cholesterol and steroids are also secreted. Implantation is further facilitated by synthesis of matrix substances, adhesion molecules and surface receptors for the matrix substances.
Implantation occurs approximately 7 days after fertilization, and is initiated when the blastocyst comes into contact with the uterine wall.
a. Zona Hatching:
To be able to perform implantation, the blastocyst first needs to get rid of its zona pellucida. This process is called “hatching”.
Lytic factors in the uterine cavity, as well as factors from the blastocyst itself are essential for this process. Mechanisms in the latter are indicated by that the zona pellucida remains intact if an unfertilized egg is placed in the uterus under the same conditions.
A substance probably involved is plasmin. Plasminogen, the plasmin precursor, is found in the uterine cavity, and blastocyst factors contribute to its conversion to active plasmin. Furthermore, plasmin inhibitors also inhibit the entire zona hatching in rat experiments.
The very first, albeit loose, connection between the blastocyst and the endometrium is called the apposition.
On the endometrium, the apposition is usually made where there is a small crypt in it, perhaps because it increases the area of contact with the rather spherical blastocyst.
On the blastocyst, on the other hand, it occurs at a location where there has been enough lysis of the zona pellucida to have created a rupture to enable direct contact between the underlying trophoblast and the decidua of the endometrium. However, ultimately, the inner cell mass, inside the trophoblast layer, is aligned closest to the decidua.
Nevertheless, the apposition on the blastocyst is not dependent on if it is on the same side of the blastocyst as the inner cell mass. Rather, the inner cell mass rotates inside the trophoblast to align to the apposition. In short, the entire surface of the blastocyst has a potential to form the apposition in the decidua.
Adhesion is a much stronger attachment to the endometrium than the loose apposition. The trophoblasts adhere by penetrating the endometrium, with protrusions of trophoblast cells.
There is massive communication between the blastocyst and the endometrium at this stage. The blastocyst signals to the endometrium to adapt further to its presence, e.g., changes in the cytoskeleton of decidual cells. This, in turn, dislodges the decidual cells from their connection to the underlying basal lamina, which enables the blastocyst to perform the succeeding invasion.
This communication is conveyed by receptor-ligand- interactions, both integrin-matrix and proteoglycan ones.
Integrins are cell-membrane-spanning receptors with the ability to react with extracellular matrix-proteins, e.g., collagen, laminin, fibronectin and vitronectin.
In this case, integrins are found on the surface of the trophoblast- cells of the blastocyst, as well as on the decidual cells on the uterine wall. The integrins on the trophoblast reacts with collagen, laminin and fibronectin surrounding decidual cells. It is probably fibronectin that guides the blastocyst in between the decidual cells down to the basal lamina.
On the other hand, integrins are also found on the decidual cells, reacting with matrix proteins around decidual cells, also in this case fibronectin for instance. Experimentally, implantation is blocked when small peptides with sequences similar to fibronectin is present, because they occupy the integrins of the decidua, making them unable to attach to blastocyst fibronectins.
However, the integrins are only present on the decidua for a limited period of time, more specifically between days 20 to 24 of gestational age, contributing to the implantation window- phenomenon.
c. Proteoglycan Receptors:
Another ligand-receptor system involved in adhesion is proteoglycan receptors, found on the surface of the decidua of the uterus. Their counterparts, the proteoglycans, are found around the trophoblast cells of the blastocyst. This ligand-receptor system is also present just at the implantation window.
Invasion is an even further establishment of the blastocyst in the endometrium.
The protrusions of trophoblast cells that adhere into the endometrium continue to proliferate and penetrate into the endometrium. These penetrating cells differentiate to become a new type of cells, syncytiotrophoblast. The prefix syn- refers to that the boundaries between these cells disappears, forming a single mass of a multitude of cell nuclei. The rest of the trophoblasts, surrounding the inner cell mass, are hereafter called cytotrophoblasts.
Invasion continues with the syncytiotrophoblasts reaching the basal membrane beneath the decidual cells, penetrating it and further invading into the uterine stroma. Finally, the whole embryo is embedded in the endometrium. Eventually, the syncytiotrophoblasts come into contact with maternal blood and form chorionic villi. This is the initiation of forming the placenta.
The blastocyst secretes factors for a multitude of purposes during invasion. It secretes several autocrine factors, targeting it and stimulating it to further invade the endometrium. Furthermore, secretions loosen decidual cells from each other, prevent the embryo from being rejected by the mother, trigger the final decidualisation and prevent menstruation.
Human chorionic gonadotropin is an autocrine growth factor for the blastocyst. Insulin-like growth factor type 2, on the other hand, stimulates the invasiveness of it.
The syncytiotrophoblasts dislodges decidual cells in their way, both by degradation of cell adhesion molecules linking the decidual cells together as well as degradation of the extracellular matrix between them. Cell adhesion molecules are degraded by syncytiotrophoblast secretion of Tumor necrosis factor-alpha.
This inhibits the expression of cadherins and beta-catenin. Cadherins is a cell adhesion molecule and beta-catenin helps anchoring it to the cell membrane. Inhibited expression of these molecules thus loosens the connection between decidual cells, permitting the syncytiotrophoblasts and the whole embryo with them to invade into the endometrium.
The extracellular matrix is degraded by serine endopeptidases and metalloproteinases. Examples of such metalloproteinases are collagenases, gelatinases and stromelysins. These collagenases digest Type-I collagen, Type-Il collagen, Type-Ill collagen, Type-VII collagen and Type-X collagen. The gelatinases exist in two forms; one digesting Type-IV collagen and one digesting gelatin.
The embryo differs from the cells of the mother, and would be rejected as a parasite by the immune system of the mother if it didn’t secrete immunosuppressive agents. Such agents are Platelet-activating factor, human chorionic gonadotropin, early pregnancy factor, immunosuppressive factor, Prostaglandin E2, Interleukin 1-alpha, Interleukin 6, interferon-alpha, leukemia inhibitory factor and Colony- Stimulating Factor.
Factors from the blastocyst also trigger the final formation of decidual cells into their proper form. In contrast, some decidual cells in the proximity of the blastocyst degenerate, providing nutrients for it.
g. Prevention of Menstruation:
Human chorionic gonadotropin (hCG) notjk only acts as an immunosuppressive, but also “notifies” the mother’s body that she is pregnant, preventing menstruation by sustaining the function of the corpus luteum.
Inner Cell Mass Differentiation:
While the syncytiotrophoblast starts to penetrate into the wall of the uterus, the inner cell mass (embryoblast) also develops. The embryoblast forms a bilaminar (two layered) embryo, composed of the epiblast and the hypoblast. The epiblast is adjacent to the trophoblast and made of columnar cells; the hypoblast is closest to the blastocyst cavity, and made of cuboidal cells.
The epiblast, now called primitive ectoderm will perform gastrulation, approximately at day 16 after fertilization. In this process, it gives rise to all three germ layers of the embryo- ectoderm, mesoderm, and endoderm. The hypoblast, or primitive endoderm, will give rise to extraembryonic structures only, such as the lining of the primary yolk sac.
By separating from the trophoblast, the epiblast forms a new cavity, the amniotic cavity. This is lined by the amnionic membrane, with cells that come from the epiblast (called amnioblasts). Some hypoblast cells migrate along the inner cytotrophoblast lining of the blastocoel, secreting an extracellular matrix along the way.
These hypoblast cells and extracellular matrix are called Heuser’s membrane (or exocoelomic membrane), and the blastocoel is now called the primary yolk sac (or exocoelomic cavity).
Cytotrophoblast cells and cells of Heuser’s membrane continue secreting extracellular matrix between them. This matrix is called the extraembryonic reticulum. Cells of the epiblast migrate along the outer edges of this reticulum and form the extraembryonic mesoderm, which makes it difficult to maintain the extraembryonic reticulum. Soon pockets will form in the reticulum, which ultimately coalesce to form the chorionic cavity or extraembryonic coelom.
Another layer of cells leaves the hypoblast and migrates along the inside of the primary yolk sac. The primary yolk sac is pushed to the opposite side of the embryo (the abembryonic pole), while a new cavity forms, the secondary or definitive yolk sac. The remnants of the primary yolk sac are called exocoelomic vesicles.
Toxic exposures during the first two weeks following fertilization (second and third weeks of gestational age) may cause prenatal death but do not cause developmental defects. Instead, the body performs a miscarriage. On the other hand, subsequent toxic exposures in the embryonic period often cause major congenital malformations, since the precursors of the major organ systems are developing.