Implantation (embryology)

The sperm and ovum unite through fertilization, creating an embryo that over the course of 8–9 days in the human will implant in the uterine wall, where it will reside over the nine-month course of gestation.

There are two stages of migration involved in implantation, the first is the migration of the zygote, and the second is the migration of the trophoblast.[7]

Following the fertilization of the oocyte that creates the single diploid cell called the zygote, the zygote starts to migrate towards the uterus. Fertilization takes place in the ampulla of the fallopian tube, and the zygote is transported along the tube to the uterus. The fallopian tube is lined with ciliated epithelium, and it is the cilia that move the zygote.[7]

During this migration the zygote undergoes a number of cell divisions that creates a ball of 16 compacted blastomeres called a morula.[8] The morula enters the uterus after three or four days, and as it does a cavity called the blastocoel is formed in the morula to produce the blastocyst. The blastocyst contains the inner cell mass that will go on to develop into the embryo proper, and an outer cell layer of trophoblasts that will develop into the extraembryonic membranes (fetal membranes).[9]

The blastocyst is still enclosed in the egg-coat known as the zona pellucida, and for it to be able to implant into the uterine wall it must rid itself of this covering. This stage is known as zona hatching, and when there is sufficient dissolution the blastocyst is able to initiate the apposition stage of implantation. Lytic factors in the uterine cavity, as well as factors from the blastocyst itself are essential for the breakdown of the egg-coat. Mechanisms in the latter are indicated by the fact that the zona pellucida remains intact if an unfertilized egg is placed in the uterus under the same conditions.[10]

Among the known molecular regulators that promote hatching are predominantly proteases that are stimulated by various growth factors.[11] The blastocyst also produces cytokines, both pro-inflammatory and anti-inflammatory, that have crucial roles during implantation and other stages of pregnancy. Both types of cytokines modulate the activity of proteases, including MMPs, plasminogen activators, and cathepsins.[11] It is unknown whether the cytokines involved in hatching are pro-inflammatory or anti-inflammatory, or which proteases are involved. However, it is well accepted that the pro-inflammatory cytokines are dominant during implantation. Cytokines are also present in the uterine milk which might regulate the development and function of the blastocyst but there is no evidence to support their involvement in hatching. Leukemia inhibitory factor (LIF) is a pro-inflammatory cytokine expressed in the endometrium during the luteal phase of the menstrual cycle, with the highest expression seen during the window of implantation. LIF plays a role in adhesion and invasion. [11]

Assisted zona hatching may take place in assisted reproduction, where the zona pellucida may be artificially pierced to facilitate hatching.[12]

Following zona hatching, the very first loose connection or contact between the blastocyst and the endometrium is called the apposition. The apposition is usually made where there is a small crypt in the endometrium, and also where there has been enough breakdown of the zona pellucida to allow the blastocyst trophoblast to directly contact the underlying endometrium. Ultimately, the inner cell mass (also embryoblast), inside the trophoblast layer, is aligned closest to the decidua. If the inner cell mass is not aligned with the decidua at apposition, it has the ability to freely rotate within the trophoblast and achieve this alignment.[13]

The identity of the molecules on the trophoblast and the endometrial epithelia that mediate the initial interaction between the two remain unidentified. However, a number of research groups have proposed that MUC1, a member of the mucin family of glycosylated proteins, is involved.[14] MUC1 is a transmembrane glycoprotein expressed at the apical surface of endometrial epithelial cells during the window of implantation in humans and has been shown to be differentially expressed between fertile and infertile subjects during this time.[14] MUC1 displays carbohydrate moieties on its extracellular domain that are ligands of L-selectin, a cell adhesion molecule on the surface of trophoblast cells.[15][16] An in vitro model of implantation gave evidence to support the hypothesis that L-selectin mediates apposition of the blastocyst to the uterine epithelium by interacting with its ligands.[17]

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.

This adhering activity is by microvilli that are on the trophoblast. The trophoblast have binding fiber connections, laminin, collagen type IV, and integrins that assist in this adhesion process.[18]

Mucin-16 is a transmembrane mucin expressed at the apical surface of uterine epithelia. This mucin prevents the blastocyst from implanting in an undesired located on the epithelium. Thus, MUC-16 inhibits cell-cell adhesion. Its removal during pinopode formation has been shown to facilitate trophoblast invasion in vitro.[19]

Illustration of an implanting embryo

Invasion is the further establishment of the blastocyst into the endometrium. The protrusions of trophoblast cells that adhere into the endometrium continue to proliferate and penetrate into the endometrium. As these trophoblasts penetrate, they terminally differentiate into a multinucleated tissue, a syncytium known as the syncytiotrophoblast. Between this layer and the blastocyst lies the cytotrophoblast.[20] When the syncytiotrophoblast reaches the basal membrane beneath the decidual cells, it penetrates it and further invades into the uterine stroma. Finally, the whole embryo is embedded in the endometrium. Eventually, the syncytiotrophoblast comes into contact with maternal blood and forms chorionic villi. This is the beginning of placentation.

The penetration of the trophoblast to the endometrium is demonstrated through metalloproteinase MMP-2 and MMP-9.[21] Trophoblasts invade the uterus attempting to reach maternal blood supply, for setting up the foundation for fetal blood flow [22]

Extravillous trophoblasts are cells from the invading villi that migrate into the myometrium of the mother’s uterus. These cells remodel the spiral arteries to improve and secure maternal blood flow to the growing embryo. There is also evidence that this process occurs with the uterine veins, stabilizing them to improve drainage of fetal blood and metabolic wastes.[23] Trophoblasts have also been documented to migrate into various tissues in the mother. Due to this trophoblasts have been implicated in a phenomenon known as fetomaternal microchimerism where fetal cells establish cell lines in maternal tissues.[24]

The blastocyst secretes factors for a multitude of purposes during invasion. It secretes several autocrine factors, targeting itself and stimulating it to further invade the endometrium.[25] Furthermore, secretions loosen decidual cells from each other, prevent the embryo from being rejected by the mother, trigger the final decidualization and prevent menstruation.

Human chorionic gonadotropin is an autocrine growth factor for the blastocyst.[25] Insulin-like growth factor 2,[25] 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.[25] Cadherins are cell adhesion molecules, and beta-catenin helps to anchor them 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.[25] These collagenases digest Type-I collagen, Type-II collagen, Type-III collagen, Type-VII collagen and Type-X collagen.[25] The gelatinases exist in two forms; one digesting Type-IV collagen and one digesting gelatin.[25]

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 did not secrete immunosuppressive agents. Such agents include platelet-activating factor, human chorionic gonadotropin, early pregnancy factor, prostaglandin E2, interleukin-1 alpha, interleukin 6, interferon-alpha, leukemia inhibitory factor and colony-stimulating factor.

Human chorionic gonadotropin (hCG) not only acts as an immunosuppressive,[25] but also signals to the mother that she is pregnant, preventing luteolysis of the corpus luteum and menstruation by sustaining the function of the corpus luteum.

Other factors secreted by the blastocyst are;

• Embryo-derived histamine-releasing factor
• Tissue plasminogen activator as well as its inhibitors
• Estradiol
• β1-integrins
• Fibroblast growth factor
• CYTL1[26]
• Transforming growth factor alpha
• Inhibin
• Preimplantation factor

The window of implantation is a limited timeframe for the successful attachment of the blastocyst. [33] In humans uterine receptivity is optimum on days 20-24 of the secretory phase of the menstrual cycle when luteinizing hormone levels are at their peak. [3][34][29] In humans the window of implantation is only available for 24-36 hours.[35]

The endometrial microbiome has been indicated as having an important role in successful implantation in controlling endometrial cell function, and the function of the local immunity system that prevents pathogen growth. This is associated with the secretion of protective substances.[36][37]

Pinopodes are formed at the beginning of the window of implantation, and are found in many species.[38][33] They are mushroom-like protrusions from the apical cell membrane of uterine epithelial cells.[33] Pinopodes are formed by the swelling of these epithelial cells, and the fusing together of a number of microvilli, to reach a maximum size.[38] They appear between day 19 and day 21 of gestational age, and are fully formed at day 20.[33] This corresponds to a fertilization age of approximately five to seven days, which corresponds well with the time of implantation. Pinopodes only persist for a maximum of two days, and are seen as the ultrastructural markers of receptivity. [38]

Their development is enhanced by progesterone, and inhibited by estrogens. During the window of implantation, cell to cell adhesion is inhibited by MUC1 a cell surface glycoprotein. The most important attribute of pinopodes is the removal of this glycoprotein from the cell surfaces of the uterine epithelial cells.[3] MUC16 has also been shown to disappear from the cell surfaces with the development of the pinopodes. Some studies have reported that pinopodes entrap cilia, which prevents embryo movement, and during implantation allows close contact and adherence of the embryo.[33]

Pinopodes bring uterine fluid and its macromolecules into the cells by the process of endocytosis. This decreases the volume of the uterus, taking the walls closer to the blastocyst floating in it. Thus, the period of active pinopodes might limit the implantation window.[25] Pinopodes continue to absorb fluid, removing most of it during the early stages of implantation.

The endometrium increases thickness, becomes vascularized and its glands grow to be tortuous and boosted in their secretions. These changes reach their maximum about seven days after ovulation.

Furthermore, the surface of the endometrium produces a kind of rounded cells, which cover the whole area toward the uterine cavity. This happens about 9 to 10 days after ovulation.[25] 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 around 8 to 9 days[25] after ovulation in absence of pregnancy.

The decidual cells originate from the stromal cells that are always present in the endometrium, and 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 stratum compactum of the endometrium, in contrast to the basalolateral stratum spongiosum, which consists of the rather spongy stromal cells.[25]

Decidualization expands if pregnancy occurs, 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 of decidual cells. 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.[25]

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 decidualization occurs at a higher degree in conception cycles than in nonconception cycles.[25] Furthermore, similar changes are observed when giving stimuli mimicking the natural invasion of the embryo.[25]

The embryo releases serine proteases which causes the epithelial cell membrane to depolarize and activates the epithelial sodium channel. This triggers a Ca²⁺ influx and phosphorylation of CREB. Phosphorylation of CREB upregulates the expression of COX2, which leads to the release of prostaglandin E2 (PGE2) from epithelial cells. PGE2 acts on the stroma cells activating cAMP-related pathways in stromal cell leading to decidualization.[39]

The decidua can be organized into separate sections, although they have the same composition.

• Decidua basalis – This is the part of the decidua which is located basalolateral to the embryo after implantation.
• 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.
• Decidua parietalis – All other decidua on the uterine surface belongs to decidua parietalis.

After implantation the decidua remains, at least through the first trimester.[25] However, its most prominent time is during the early stages of pregnancy, during implantation. Its function as a surrounding tissue is replaced by the definitive placenta. However, some elements of the decidualization remain throughout pregnancy.[25]

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 that disappearance, some glands secrete unequally much. This phenomenon of hypersecretion is called the Arias-Stella phenomenon,[25] after the pathologist Javier Arias-Stella.

The embryo 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.[25]

In addition to nourishment, the endometrium secretes several steroid-dependent proteins, important for growth and implantation. Cholesterol, and steroids are also secreted.[25] Implantation is further facilitated by synthesis of matrix substances, adhesion molecules and surface receptors for the matrix substances.

Zinc is crucial in pre-conception, (and successful pregnancy), and its deficiciency can lead to incompetent blastocyst development. Once an egg is fertilized zinc is released in a zinc spark which promotes changes that include the hardening of the zona pellucida preventing polyspermy.[47]

Implantation bleeding is a small amount of light vaginal bleeding or spotting that can occur in early pregnancy due to the fertilized egg implanting in the uterus.[48][49] Bleeding and spotting are common during the luteal phase of the menstrual cycle, and early stages of pregnancy, and are unrelated to implantation. Implantation bleeding occurs between 7 to 14 days after fertilization,[50] and may be accompanied by symptoms such as cramping, nausea, breast tenderness, and headaches.[51] Implantation bleeding can be distinguished from period bleeding by color, clotting, strength and duration of flow.[52][53]

• Boron W, Boulpaep E (2004). Medical Physiology: A Cellular And Molecular Approaoch. Oxford: Elsevier. ISBN 1-4160-2328-3. OCLC 61527528.