FERTILIZATION and IMPLANTATION of the Early Human Embryo: Accurate Scientific Resources (pp2)

IV: O'Rahilly Text -- Chapters on Stages 1 (Fertilization) to 6 (Including "Implantation")

Ronan O'Rahilly and Fabiola Muller, Human Embryology & Teratology, 3rd ed. (New York: Wiley-Liss, 2001):

Chapter 3: Gametogenesis, Ovulation, and Fertilization [Stage 1]

Human oocytes and early embryos (2-cell to the blastocysts) can be analyzed molecularly by use of the polymerase chain reaction, and cDNA "libraries" constructed as a resource for the study of early gene expression. The time of onset of specific genes can thereby be determined and so aid in investigating congenital anomalies and inherited diseases resulting from mutation of those genes. (p. 30)

Although life is a continuous process, fertilization ... is a critical landmark because, under ordinary circumstances, a new, genetically distinct human organism is formed when the chromosomes of the male and female pronuclei blend in the oocyte. ... During the embryonic period proper, milestones include fertilization, activation of embryonic from extra-embryonic cells, implantation, and the appearance of the primitive streak and bilateral symmetry. ... Fertilization is the procession of events that begins when a spermatozoon makes contact with a secondary oocyte or its investments, and ends with the intermingling of maternal and paternal chromosomes at metaphase of the first mitotic division of the zygote. ... Fertilization takes place normally in the ampulla (lateral end) of the uterine tube. (p. 31)

Chapter 4: Week 1 (Stages 2-4): Morula; (free) Blastocyst

During the first week, the EMBRYO becomes a solid mass of cells and then acquires a cavity, at which time it is known as a blastocyst. (p. 37)

Approximately 2-3 days (Stage 2) (pp. 37- 38)

The cleaving EMBRYO proceeds along the uterine tube under the influence of tubal contractions and movement of cilia. Mitotic divisions occur at a rate of about one per day, so that the number of cells (named blastomeres) increases, although the total protoplasmic volume at first does not. The blastomeres of the 2-cell human EMBRYO already become polarized: microvilli and endocytotic activity disappear where cell-to-cell contact occurs, and early cellular junctions form (J. Tesarik).

It is likely that a considerable number of human embryos become abnormal during the first cleavage division, as indicated by the presence of multinucleated blastomeres. Although spontaneous abortion may result, in other instances the presence of one normal blastomere may suffice for normal development. A progressive loss of chromosomally abnormal embryos occurs prior to implantation.

Biopsy of an embryo can be performed by removing one cell from a 4-cell, or two cells from an 8-cell, embryo. This does not seem to decrease the developmental capacity of the remaining cells. ...

The EMBRYO enters the uterine cavity after about half a week, when probably at least 8-12 cells are present and the endometrium is early in its secretory phase. Each cell (blastomere) is considered to be still totipotent (capable, on isolation, of forming a complete embryo), and separation of these early cells is believed to account for one-third of cases of monozygotic twinning.

The convenient term morula can be used for embryos when about a dozen or more cells are present and until the blastocystic cavity appears. The term is not ideal, because it was used originally for amphibians, in which it gives rise to EMBRYONIC TISSUE ONLY and not, as in mammals, to BOTH EMBRYONIC AND NON-EMBRYONIC structures (e.g., chorion, amnion).

The Embryonic Genome (pp. 38-39)

Initial development occurs independently of the embryonic genome and is under oocyt-DERIVED genetic control, which is exerted by RNA molecules synthesized during growth and maturation of the oocyte. Some indications exist, however, that the zygote already synthesizes RNA. Even the 2-cell embryo shows ultrastructural signs (modification of the cell membrane) of regionalization on the surface of the blastomeres, apparently under the control of an oocyt-coded message (perhaps through proteins of the cytoskeleton). Human embryonic gene transcription is activated from outside the nucleolus by at least the 4-cell embryo, but the level of RNA synthesis is low. In the 8-cell embryo, however, some blastomeres undergo an overall increase in transcriptional activity, both extranucleolar and nucleolar, so that by this time human embryonic genes and the already-present genetic uniqueness of the embryo are expressed in the phenotype. ...

Cells differentiate by the switching off of large portions of their genome. Future somatic cells thereby lose their totipotency and are liable to senescence, whereas germ cells regain their toipotency after meiosis and fertilization (R. F. Rosenberger).

Approximately 4-5 days (Stage 3) [free blastocyst] (pp. 39-40)

As soon as a cavity can be detected (by light microscopy) in the cellular mass of the morula, the organism is termed a blastocyst. This occurs when about 16-32 cells are present. The embryo is about 4 days in age and is not yet attached to the uterine mucosa.

The appearance of the blastocyst demonstrates the differentiation into (1) trophoblast (or trophectoderm), the peripherally situated cells and (under the influence of E-cadherin) the first epithelium formed, and (2) embryonic cells proper. The latter, at first few in number, form the inner cell mass (ICM). The trophoblast at the future site of attachment is sometimes termed polar, the remainder being called mural. The cells of the ICM are considered to be totipotent initially. They give rise directly to various lines of embryonic stem cells. ...

Duplication of the inner cell mass is probably the basis for most cases of monozygotic twinning, and it is possible that such divisions arise during "hatching" from the zona pellucida.

The cavity of the blastocyst is the blastocystic cavity, and it should be stressed that the cavity of the mammalian blastocyst is not a blastocoel, which is a different feature found in amphibians and birds. ... The trophoblastic cells and those of the inner cell mass are linked by desmosomes, gap junctions, and tight junctions ...

The term "hatching" refers to the escape of the blastocyst from the zona pellucida. The zona becomes thicker, the blastocyst alternately expands and contracts, and then squeezes through a hole that appears by digestion in the zona. This occurs at about 6 days, when the blastocyst consists of a hundred or more cells. After "hatching," the embryo is free to increase in mass, and it secretes hCG. ...

Approximately 6 days (Stage 4) [implanting blastocyst] (pp. 40-41)

The blastocyst now begins to become attached to the uterine lining (the endometrium)m and this is taken as the beginning of implantation. ...

The uterine wall in which the embryo is being implanted consists of three layers: endometrium (mucosa), myometrium (muscular coat), and perimetrium (peritoneal covering). Of these the most important at the time of implantation is the endometrium, which is in the secretory phase; i.e., it contains active glands, large amounts of nutrients, and coiled arteries. If implantation takes place, the endometrium is conserved by the activity of the corpus luteum (which produced progesterone); if implantation does not occur, the endometrium is discharged at the end of a uterine cycle (menstruation).

... Failure of implantation may result from rejection of the antigenic embryo by the maternal immune system.

Chapter 5: Weeks 1 - 2½ (Stages 5 and 6): Implantation; Primitive Streak; Twinning

The second week is characterized by implantation. The trophoblast, which is at first solid, soon shows lacunae, and chorionic villi begin to appear. The amniotic cavity and the umbilical vesicle (yolk sac) develop. Some dorsally situated cells of the embryonic disc move ventrally (to the interior of the embryo) by means of the primitive streak, which also makes manifest the bilateral symmetry of the embryo. Human chorionic gondadotropin (hCG) may be detectable as early as day 8. Implantation triggers the synthesis of hCG by the conceptus, and the hormone can be detected first in the maternal blood and, within a couple of days, in the urine. This is the basis of the early diagnosis of pregnancy.

From implantation onward, growth factors, some of which are produced by the embryo itself, are necessary for growth. Peptides known as insulin-like growth factors are important for mitosis and differentiation in the human during the first trimester. (pg. 43)

Approximately 7-12 days (Stage 5) (pp. 43-44)

... The trophoblast has differentiated into two chief varieties: cytotrophoblast, nearer the embryonic disc, and syncytiotrophoblast, which is derived from cytotrophoblast and is situated more peripherally. ...

The amniotic cavity appears probably within the inner cell mass, perhaps by a rearrangement of epiblastic cells. The cavity is bounded by the epiblast and, dorsally, by a layer of amniotic ectoderm. The amnion, like the chorion, is one of the developmental adnexa. On the ventral aspect of the embryonic disc, extra-embryonic endoderm grows around to enclose a cavity termed the primary umbilical vesicle (or yolk sac). The embryonic disc is still bilaminar, being composed of epiblast and endoderm, the latter being probably derived from the epiblast.

Implantation (pp. 44-45)

Implantation is the process that leads to the formation of a specialized, intimate cellular contact between the trophoblast and the endometrium (or other tissue in cases of ectopic implantation). The conceptus leaves the uterine lumen and becomes embedded in the uterine mucosa. Implantation includes dissolution of the zona pellucida and adhesion between the external surface of the trophoblastic cell membrane and the endometrial cells. Later, trophoblastic cells penetrate the uterine epithelium and its subjacent basement membrane, and thereby enter the stroma; finally multiple interactions occur between the trophoblast and the uterine cells, as well as with the extracellular matrix, so that the blastocyst comes to lie entirely within the uterine lining. Platelet-derived growth factor, fibronectin, and epidermal growth factor are important in implantation. In discussing implantation, trophoblastic penetration is preferable to the concept of trophoblastic "invasion", a term better reserved for carcinomatous processes.

Implantation begins by adhesion (a cell-to-cell interaction) between the trophoblast and the uterine epithelium of a receptive endometrium (Thie et al., 1997, 1998). Implantation is highly unusual in that it (1) resembles invasion by malignant neoplasms, (2) involves the toleration of antigenically different cells (the trophoblastic "graft") by the endometrium (the "host"), and (3) begins by a paradoxical attachment of trophoblast and endometrium by their apical cell membranes (Denker, 1993, 1994), whereas usually the apically situated cell membrane of an epithelium is not adhesive, in contrast to the basally and laterally location membrane. In other words, the trophoblast acquires some mesenchymal characteristics.

Implantation of the blastocyst begins during the secretory (progestational) phase of the uterine cycle. The endometrium is conditioned primarily by estradiol, but secretory changes depend on progesterone, which blocks further menstruation. It is believed that a blastocyst can become attached to the uterine epithelium during only a limited receptive phase (of about 4 days, days 20-24 of the cycle) that is controlled by ovarian steroidal hormones. The period during which the endometrium is receptive for implantation is sometimes termed the "implantation window". At that time the uterine epithelium partly loses its epithelial character of apicobasal polarity, becoming restructured from a polarized to a non-polarized phenotype, which is essential for apical adhesiveness (Thie and Denker, 1997). Genomic imprinting (i.e., gene expression based on the gamete of origin) is believed to be important for implantation, and paternally expressed genes are linked to placental proliferation.

The Decidua (p. 45)

As soon as the blastocyst begins to penetrate the uterine epithelium, the endometrium is termed decidua (Latin, a falling off: the mucous membrane shed after birth). Decidualization (the decidual reaction) is the series of endometrial changes that occur in response to the blastocyst. Decidualization, which is under the influence of progesterone, begins late in the luteal phase and takes several days. It is characterized by enlargement of endometrial stromal cells, which accumulate glycogen and on cross section become epithelioid in appearance. The changes indicate increased synthetic and secretory activity. Although implantation begins in an endometrium that is not yet decidualized, stromal fibroblasts soon become large and polygonal, and contain abundant glycogen and lipid. They are then known as decidual cells. These cells produce prolactin and contain insulin-like growth factor. Large granular leukocytes are also found in the decidua. The superficial region of the endometrium that undergoes decidualization is the stratum compactum, whereas the deeper region of dilated glands and scant stroma is the stratum spongiosum.

Primitive Streak (p. 48)

The primitive streak probably appears between day 12 and day 17. The streak is believed to be the result of interaction between the endoderm and the pluripotent epiblast. The remaining epiblastic cells on the dorsal aspect of the embryo constitute the embryonic ectoderm. The primitive streak is a means of entrance whereby cells ingress, proliferate, and migrate to form embryonic mesoderm and endoderm. ... As the embryo elongates, the primitive streak regresses [disappears] along the rostrocaudal apix and leaves behind a cellular trail that forms the notochord. Caudally it becomes replaced by the caudal eminence.

V: Carnegie Stages of Early Human Embryonic Development: Stages 1-6 Online

A. National Museum of Health and Medicine, Developmental Anatomy Center:

URLs for 23 Stages of the human embryo:

Chart of 23 Stages:

Stage One (a, b, and c):

Approximately 0.1-0. 15 mm in diameter
Approximately 1 postovulatory day
Characteristic feature: unicellularity

"Embryonic life commences with fertilization, and hence the beginning of that process may be taken as the point de depart of stage 1. Despite the small size and weight of the organism at fertilization, the embryo is "schon ein individual-spezifischer Mensch" [definitely and specifically a human person] (Blechschmidt, 1972). ... Fertilization is the procession of events that begins when a spermatozoon makes contact with an oocyte or its investments and ends with the intermingling of maternal and paternal chromosomes at metaphase of the first mitotic division of the zygote (Brackett et al, 1972). ... Fertilization, which takes place normally in the ampulla of the uterine tube (i.e., fallopian tube - not the uterus], includes (a) contact of spermatozoa with the zona pellucida of an oocyte, penetration of one or more spermatozoa through the zona pellucida and the ooplasm, swelling of the spermatozoal head and extrusion of the second polar body, (b) the formation of the male and female pronuclei, and (c) the beginning of the first mitotic division, or cleavage, of the zygote. ... The three phases (a, b, and c) referred to above will be included here under stage 1, the characteristic feature of which is unicellularity. ... The term "ovum", which has been used for such disparate structures as an oocyte and a 3-week embryo, has no scientific usefulness and is not used here. Indeed, strictly speaking, "the existence of the ovum ... is impossible (Franchi, 1970). The term "egg" is best reserved for a nutritive object frequently seen on the breakfast table. ... Pronuclear fusion does not occur. Rather, the two pronuclear envelopes break down ("post-apposition envelope vesiculation," Szabo and O'Day, 1983), and the two groups of chromosomes move together and assume positions on the first cleavage spindle. Thus the zygote lacks a nucleus.

Stage Two:

Approximately 0.1-0.2 mm in diameter
Approximately 1½ -3 postovulatory days
Characteristic feature: more than 1 cell but no blastocystic
cavity seen by light microscopy

Stage 2 comprises specimens from 2 cells up to the appearance of the blastocystic (or segmentation) cavity. The more advanced examples (from about 12 cells on) of stage 2 are frequently called morulae (L., morus, a mulberry). The term morula is not historically appropriate for mammals, however, because the amphibian morula gives rise to embryonic tissues only, whereas in mammals non-embryonic structures (such as the chorion and the amnion) are also derived from the initial mass of cells. ... In vitro, 2 cells may be found at 1½ days, 4 cells at 2 days, and 8 cells by about 2½ days. ... The organism proceeds along the uterine tube by means not entirely understood (reviewed by Adams, 1960). It leaves the tube and enters the uterine cavity during the third or fourth day after ovulation, when probably 8-12 cells are present, and when the endometrium is early in the secretory phase. ... It has been shown experimentally that a blastomere isolated from the mammalian 2-cell organism is capable of forming a complete embryo. Separation of the early blastomeres is believed to account for about one-third of all cases of monozygotic twinning in the human (Corner, 1955). ... There is reason to believe, however, that the blastomeres are not determined very early in development. For example, it has been shown experimentally in the mouse that the ability to develop into trophoblastic cells is inherent in all blastomeres of the first two stages, Up to 16 cells, none of the blastomeres is yet determined to give rise to cells of the inner mass. It may be that the primary factor responsible for the determination of one of the two alternative routes of differentiation (trophoblast or inner cell mass) is simply the position (peripheral or internal) that a given cell occupies. According to the "inside/outside hypothesis," micro-environmental differences influence the determination of blastomeres (between 8 and 16 cells in the mouse) so that those on the outside become more likely to form trophoblast (with more restricted potential) whereas those enclosed by other cells become more likely to form the inner cell mass. ...

Furthermore, it has been possible in the mouse to unite two 16-cell organisms and obtain from them one giant, but otherwise perfectly normal, blastocyst. Fusion of mouse organisms with close to 32 cells each has also resulted in a single blastocyst.

Stage Three: free blastocyst

Approximately 0.1-0.2 mm in diameter
Approximately 4 postovulatory days
Characteristic feature: free blastocyst

Stage 3 consists of the free (that is, unattached) blastocyst, a term used as soon as a cavity (the blastocystic, or segmentation, cavity) can be recognized by light microscopy. ... The blastocyst is the hollow mass of cells from the initial appearance of the cavity (stage 3) to immediately before the completion of implantation at a subsequent stage. The blastocystic cavity, under the light microscope, begins by the coalescence of intercellular spaces when the organism has acquired about 32 cells. In in vitro studies, a cavity formed in some human embryos at 16-20 cells (Edwards, 1972). ... Embryos of stage 3 are believed to be about 4 days in age. in vitro embryos of stage 1 have been recorded at 9-32 hours after insemination; stage 2 at 22-40 hours (2 cells), 32-45 hours (4 cells), and 48 hours (8 cells); stage 3 at 100 hours, and extruding from the zona pellucida at 140-160 hours, at which time they show differentiation into trophoblast, epiblast, and hypoblast (Mohr and Trounson, 1984). ... As the blastocyst develops, it undergoes expansions and contractions. When contracted, a "pseudomorula" of about 100 cells in the mouse can be seen. ... Duplication of the inner cell mass probably accounts for most instances of monozygotic twinning (Corner, 1955; Bulmer, 1970).

Stage Four: attaching blastocyst

Probably approximately 0.1-0.2 mm in diameter
Approximately 5-6 postovulatory days
Characteristic feature: attaching blastocyst

Stage 4, the onset of implantation, is reserved for the attaching blastocyst, which is probably 5-6 days old. ... Implantation is the specific process that leads to the formation of a specialized, intimate cellular contact between the trophoblast and the endometrium, or other tissue in the case of ectopic implantation (Denker, 1983).

Implantation is a highly complicated and ill-understood phenomenon "by which the conceptus is transported to its site of attachment, held there, oriented properly, and then attached by adhesion, trophoblastic penetration, spread, proliferation, envelopment of vessels, and other developments of the placenta, both conceptal and maternal parts" (Boving, 1963). In this broad sense, implantation includes at least stages 4 and 5.

Implantation, then, includes (1) dissolution of the zona pellucida, and contact and attachment (adhesion) between the blastocyst and the endometrium, (2) penetration, and (3) migration of the blastocyst through the endometrium. ... Human (but not macaque) implantation is interstitial in type: i.e., the blastocyst comes to lie entirely within the substance of the endometrium. In the human (as also in the macaque), implantation occurs into an edematous, non-deciduous endometrium. In other words, decidualization takes place at the end of implantation. ... The mammalian stage 2 organism and the early blastocyst are surrounded by an intact zona pellucida, which disappears at the beginning of implantation. Hence, implantation "is taken as beginning when the zona pellucida is lost and the trophoblast is in contact with the uterine epithelium throughout its circumference" (Young, Whicher, and Potts, 1968). ... The implantation site has been studied by electron microscopy in several mammals, such as the mouse.

The cell membranes of the trophoblast and uterine epithelium become intimately related, and large cytoplasmic inclusions are found in the trophoblastic cells. The ultrastructural changes taking place at implantation suggest that there may be a high degree of permeability between maternal and embryonic cells. In addition, there may be an exchange of cellular material between uterus and embryo. After the zona pellucida has become dissolved, the surface membranes of the trophoblast and uterine epithelium are separated by a very narrow interval (in the mouse). This first morphological sign of implantation can be detected only by electron microscopy.

Stage Five: [embryo still implanting]

Characteristic features: implanted but previllous; solid trophoblast in 5a;
trophoblastic lacunae, cytotrophoblastic clumps, and primary
umbilical vesicle in 5b; lacunar vascular circle and some mesoblastic
crests in cytotrophoblastic clumps in 5c

Stage 5 comprises embryos that are implanted to a varying degree but are previllous, i.e., that do not yet show definite chorionic villi. Such embryos are believed to be 7-12 days old. ... Implantation, which began in stage 4, is the characteristic feature of stage 5. It should be appreciated that both maternal and embryonic tissues are involved in the complex process of implantation: "in the normal process they are mutually supporting and neither can be regarded as chiefly responsible" (Boyd and Hamilton, 1970). ... An amniotic cavity is found by stage 5. If duplication of the embryo occurs after the differentiation of the amnion, the resulting monozygotic twins should be monochorial and monoamniotic (fig. 5-2). It has been estimated that the frequency of monoamniotic twins among monozygotic twins is about 4 percent (Bulmer, 1970). About once in every 400 monozygotic twin pregnancies, the duplication is incomplete and conjoined ("Siamese") twins (e.g., the second specimen of Shaw, 1932) result.

Stage Six

Approximately 0.2 mm in size
Approximately 13 postovulatory days
Characteristic features: chorionic villi and secondary
umbilical vesicle in 6a; primitive streak in 6b

In the human, the primitive streak appears first during stage 6. ... Although it may be possible, at least in some instances, to ascertain the rostrocaudal axis of the embryo at stages 5c and 6a, unequivocal manifestation awaits the initial appearance of the primitive streak during stage 6b (O'Rahilly, 1970). With the establishment of bilateral symmetry, the embryonic disc, in addition to its dorsal and ventral surfaces, now has rostral and caudal ends and right and left sides.

Stage Eleven

Approximately 2.5-4.5 mm
Approximately 24 ± 1 postovulatory days
Characteristic feature: 13-20 pairs of somites

Primitive streak. The caudal eminence, which lies between the cloacal membrane and the site of the neurenteric canal, represents the region of the former primitive streak. http://www.medicalmuseum.mil/assets/documents/collections/hdac/stage11.pdf

B. Louisiana State University Health Science Center (LSUHSC); The Virtual Human Embryo

Dr. Raymond Gasser; http://www.ehd.org/virtual-human-embryo/about.php?stage=1

Stage One:


Stage Two:


Stage Three:


Stage Four:


Stage Five:


Stage Six:


VI. "Regulation"

1, 2,