The Stages of Human Embryonic Development

Stage 2 begins with the division of the zygote into two cells and ends with the appearance of the blastocystic cavity. Stage 2 embryos have an estimated postfertilization age of two to three days and are approximately 0.1 to 0.2 mm in diameter. The zygote undergoes cleavage during this period whereby it divides mitotically into an increasing number of cells that become progressively smaller with each division. The individual cells are called blastomeres and sometimes are referred to as founder cells because they give rise to embryonic stem cells. True growth does not occur during cleavage since the volume of the entire protoplasmic mass actually diminishes, however, the DNA content increases dramatically duplicating with each cell division.

Cleavage divisions do not occur synchronously so that stage 2 embryos are comprised of anywhere from one to sixteen blastomeres which can differ in their size, staining ability and electron density. During the early cleavage period the fate of each blastomere is not yet determined. The advanced embryo of 16 blastomeres or more is frequently called a morula as it resembles a mulberry.

Stage 3 begins when the blastocystic cavity first appears in the morula and ends when the zona (capsula) pellucida is shed as the embryo makes contact with the endometrial lining of the uterus. Stage 3 embryos are called free blastocysts. They have an estimated postfertilization age in vivo of between four and five days and measure 0.1 to 0.2 mm in diameter.

During Stage 3 the embryo separates into outer trophoblast cells and inner embryoblasts (inner cell mass) that will develop into the embryo proper. The site of the inner cell mass determines the embryonic pole of the blastocyst. The opposite side of the blastocyst becomes the abembryonic pole. Two axes can be distinguished; the polar (anterior-posterior) axis through the polar body and a perpendicular (dorso-ventral) axis at a right angle to the polar axis.

In vitro studies have shown that Stage 3 embryos go through four characteristic processes: 1) cavitation, 2) collapse/ expansion 3) hatching and 4) discarded cells.

The initiation of cavitation in the morula heralds the beginning of Stage 3. Cavitation occurs in vitro between post insemination days 5 and 7. Cavitation results from the accumulation of fluid inbetween the blastomeres. Since the total volume of the embryo does not change substantially during cavity formation, initial fluid accumulation appears to be a metabolic product of the blastomeres rather than transport from the outside.

Stage 3 blastocysts in vitro typically undergo repeated collapse and expansion before they escape from the zona pellucida. Collapse is rapid, occurring in less than five minutes whereas complete re-expansion requires several hours. The phenomenon has been called blastocyst “breathing” and occurs just before hatching. It is not known whether or not it occurs in vivo .

Hatching is the process by which the expanded blastocyst breaks through and escapes from the zona pellucida. It must occur before implantation into the endometrium is possible. Hatching of healthy blastocysts in vitro usually occurs between post insemination days 7 and 8. After hatching the blastocyst enlarges substantially, the trophoblast cells thin and tight junctions form between them, and , small fimbriated trophoblast projections appear in the region of the blastocyst where attachment usually occurs.

Examination of in vitro Stage 3 blastocysts with TEM reveals two types of cells that are apparently discarded by the developing embryo, 1) sequestered cells and 2) isolated cells. Sequestered cells are groups of cells located between the zona pellucida and the trophoblast, while iIsolated cells are found mainly in the blastocystic cavity.

Stage 4 is reserved for the attaching blastocyst that is adhering to the endometrial lining of the uterus. The attaching process is called adplantation and heralds the onset of implantation. Stage 4 embryos have an estimated postfertilization age of approximately 6 days.

The outer trophoblast layer of the hatched blastocyst has multiple areas of attachment to the endometrial epithelium. In the areas of contact the syncytiotrophoblast dissolves the endometrial epithelium. The epithelial cytoplasm disappears before the nuclei, resulting in an accumulation of nuclei in these areas. Progressive differentiation of the trophoblast is taking place. The cytotrophoblast becomes distinct from the overlying syncytiotrophoblast. Amniogenic cells are separating from the trophoblast and are distinct from the inner cell mass. The hypoblast layer is a distinct, thin layer on the underside of the inner cell mass. The remaining cells constitute the epiblast.

Stage 5 embryos are characterised by various degrees of implantation into the uterine mucosa. Two distinct layers are evident in the trophoblast; 1) a thicker outer layer without cell boundaries, called the syncytiotrophoblast and 2) a thinner inner layer with cell boundaries called the cytotrophoblast. The chorion and chorionic cavity are defined with the formation of extraembryonic mesoblast but the conceptuses are previllous, i.e., they do not yet show any definitive chorionic villi. The definitive amniotic cavity appears between the embryonic disc and the chorion. Stage 5 embryos have a postfertilization age of 7 to 12 days, the diameter of the embryonic disc measures approximately 0.1 to 0.2 mm and the greatest diameter of the chorion varies from 0.3 to 1 mm.

Stage 5 is divided into three substages based on the condition of the trophoblast and its relationship to the maternal vasculature.

Stage 5a specimens have a postfertilization age of 7 to 8 days and are characterized by a trophoblast that is still mostly solid. The greatest diameter of the trophoblastic shell is less than 0.5 mm. The endometrial stroma is edematous. The blastocystic cavity is usually flattened because of the collapse of the conceptus during implantation. Endoblast formation begins along the inner side of the trophoblast. The embryonic disc is approximately 0.1 mm in diameter and is composed of two layers, a thick layer called the epiblast and a thin layer called the hypoblast. The amniotic cavity is apparent for the first time and is formed by the curved epiblast of the embryonic disc.

The distinguishing characteristic of Stage 5b specimens is the presence of numerous irregular, slit like lacunae within the cytoplasm of the syncytiotrophoblast. Most of these lacunae communicate with each other and with the endometrial sinusoids but they contain relatively little maternal blood. The endometrial stroma shows an early decidual reaction and future villi begin as cytotrophoblastic clumps that project into the syncytiotrophoblast. There are few extraembryonic mesoblasts lining the inner surface of the trophoblastic shell. The umbilical vesicle (yolk sac) appears for the first time and becomes limited by a layer named the exocoelomic membrane. The bilaminar embryonic disc is slightly oval with an epiblast composed of pseudostratified, columnar epithelium and a hypoblast that is a single layer of cuboidal or polyhedral cells.

The distinguishing characteristic of Stage 5c embryos is the presence of large, irregular, intercommunicating lacunar spaces that contain enough blood to form a discontinuous red circle that is visible on the endometrial surface. The bilaminar embryonic disc is oval in shape when viewed from above. Extraembryonic mesoblasts are concentrated at the caudal end of the embryonic disc.

Stage 6 embryos are characterized by the first appearance of chorionic villi. The amniotic cavity and secondary umbilical vesicle (yolk sac) are well defined by this stage. The embryo is in the presomite, disc period and axial differentiation is present for the first time. During this stage the embryonic endoderm and the gastrulation (primitive) streak first appear. The embryonic disc varies from 0.15 to 0.5 mm in maximum diameter. Initially, stage 6 embryos were believed to be about 13 days postfertilization, however, more recently, O’Rahilly & Muller (1996) have reexamined the ages assigned to the Carnegie stages and have estimated that stage 6 is ca. 17 days postfertilization.

Stage 7 embryos are in the presomite period and have a well defined embryonic disc that typically has a greatest diameter between 0.3 and 0.7 mm. The embryonic disc can vary in shape from oval to pyriform but most specimens have a circular disc. The disc is symmetrical and slightly convex in the plane of its longitudinal axis. The age of stage 7 embryos was initially believed to be about 16 days postfertilization, however, O’Rahilly and Müller (2001) have reassigned the ages of the Carnegie stages and have placed stage 7 at ca. 18 – 21 days. The stage is characterized by the appearance of the notochordal process and the gastrulation (primitive) node.

Stage 8 embryos are characterized by the appearance the neural groove; a notochordal canal, and gastrulation (primitive) node and pit are also typically present. The embryonic disc can vary considerably in shape and size, it can be ovoid, circular or pyriform. Ovoid embryos can have their greatest diameter along either the rostral-caudal or transverse axis while pyriform discs taper caudally. The embryos are still in the presomite period although the embryonic discs are completely trilaminar with mesoblastic cells extending to the edges and joining with extra-embryonic mesoblast. Stage 8 embryos have a maximum chorionic diameter of between 9 – 15 mm and an embryonic disc greatest diameter of 0.5 to 2 mm. They have an estimated postfertilization age of 23 days.

Stage 9 is characterized by the first appearance of somites and prominent neural folds. The rapid growth of the rostral neural folds dominates this stage. It leads to formation of a distinct ‘head’, the initial appearance of the foregut and the rotation and translocation of the primordial heart from a rostro-dorsal location to a more ventral-caudal location.

Stage 9 embryos have a greatest length of 1.5 to 2.5 mm and an estimated postfertilization age of 24 – 26 days.

Stage 10 embryos have a greatest length of 1.5 to 3.6 mm and an estimated postfertilization age of 28 to 30 days. There are 4 to 12 pairs of somites along the body axis. The neural tube begins to form and has rostral and caudal neuropores. The five major subdivisions of the brain can be identified in the neural folds. The optic sulcus is present with a chiasmatic plate in the midline. The first pharyngeal pouch is close to the relatively large otic placode. Presumptive left and right atria and ventricles of the heart can be identified by their relative locations. The epimyocardium and the bulbotruncus region are present. The stomodeum, thyroid gland primordium, laryngotracheal sulcus and hepatic plate are all present. The nephrogenic cord or intermediate mesoderm can be identified lateral to the somites. There is no evidence of limb buds.

Stage 11 embryos have estimated greatest lengths within the range of 2.2 to 5 mm, although most are probably between 3.0 to 4.5 mm before sectioning. They have an estimated postfertilization age between 26 and 30 days. The characteristic feature of stage 11 is the presence of between 13 and 20 pairs of somites, or body segments, along the body axis. Sclerotomes are forming in the ventromedial part of the somites as the somite remnants move dorsolaterally. The rostral neuropore closes during this stage.

Also during stage 11 the optic vesicle forms in the wall of the diencephalon and the otic placode, or pit, forms as a region of thickened ectoderm lateral to the rhombencephalon. The adenohypophyseal primordium is forming just rostral to the oropharyngeal membrane that is beginning to disappear, the right and left horns of the sinus venosus are continuous with their respective atria, and the hepatocystic diverticulum can be identified in the endoderm at the fore- mid-gut junction. Mesonephric vesicles and duct appear in the nephrogenic cord.

Stage 12 embryos have a greatest length similar to embryos from stage 11 with most embryos being in the 3 to 4 mm range. This is due to the fact that much of the growth during this stage is in the thoracolumbar region which leads to an increasing curvature of the embryos rather than an increase in greatest length. Stage 12 embryos have an estimated postfertilization age of 29 to 31 days.

The characteristic feature of stage 12 is the presence of 21 to 29 body segments (somites). In the older specimens the caudal neuropore is closed and secondary neurulation has begun. Most of the cephalic neural crests can be identified. The otic vesicle forms but may remain connected to the overlying ectoderm. The sinu-atrial openings and atrioventricular canal are present in the heart. Trabeculation has begun in the ventricular walls. The aortic arches are present and the dorsal aortae are fused. Four pharyngeal arches, grooves and pouches are present. Future hepatic sinusoids are occupied by hematopoetic tissue and the mesonephric duct terminates caudally in the cloaca.

Stage 13 embryos have 30 or more somites, although by this stage it is getting increasingly difficult to distinguish individual somites and make accurate counts. Stage 13 is the first stage in which all four limb bubs can be recognized. The upper limb bud , which may be just visible in some stage 12 embryos, is now a definitive elevated ridge, and the start of stage 13 is marked by the first appearance of the lower limb buds.

Other features of this stage are the first appearance of the lens disc and the closure of the otic vesicle (pit). Internally, the lung buds appear, a distinct trachea begins to develop and the septum primum and foramen primum appear in the heart.

Most stage 13 embryos have a greatest length of 4 to 5 mm, although they can range from 3 to 6 mm. If greatest length is taken as the only measure of embryo growth it would appear that growth slows between stages 11 and 13, however, it should be noted that during these stages the embryos are also growing in the other two dimensions and increasing in bulk not just length.

Stage 13 embryos have an estimated postfertilization age of about 32 days

Stage 14 embryos are characterized by the invagination of the lens disc and the presence of projecting upper limb buds that curve ventrally but do not yet display a distinct hand plate.

The greatest length of stage 14 embryos is in the range of 5 to 8 mm but most are in the 6 to 7 mm range. They have an estimated postfertilization age of around 33 days

Stage 14 embryos are characterized by the invagination of the lens disc and the presence of projecting upper limb buds that curve ventrally but do not yet display a distinct hand plate.

The greatest length of stage 14 embryos is in the range of 5 to 8 mm but most are in the 6 to 7 mm range. They have an estimated postfertilization age of around 33 days

Stage 16 embryos have a greatest length of 8 to 11 mm and an estimated postfertilization age between 37 and 40 days. The lower limb bud develops into three distinct regions, corresponding to thigh, leg and foot while in the upper limb bud the digital plates develops. More advanced stage 16 specimens have visible retinal pigmentation.

Stage 17 embryos have a greatest length of 11 to 14 mm and an estimated post-fertilization age of 41 days. Embryos are becoming less ‘C’-shaped as the main axis of the trunk becomes straighter. In some stage 17 embryos a slight lordotic curvature may be seen in the lumbar region. Other characteristic external features include the presence of all the auricular hillocks, definite digital rays in the hand plate and a rounded digital foot plate.

Internally, the palate is beginning to develop, auditory ossicles are apparent, and the olfactory bulb begins to appear. Chondrification starts in some of the vertebral bodies, the humerus and the radius. The dorsal and ventral parts of the pancreas are fused and the vermiform appendix is visible for the first time.

Stage 18 embryos have a greatest length between 13 and 17 mm and an estimated postfertilization age of approximately 44 days. Many structures become apparent for the first time during this stage. In the head region the tip of the nose becomes discernable as well as the volmeronasal organ, eyelids, and nasolacrimal ducts. The phallus and genital tubercle (glans penis or glans clitoris) are evident for the first time.

The auricular hillocks begin to blend to form parts of the definitive external ear. Two or three semicircular ducts form from the otic vesicle in the otic capsule. The oronasal membrane breaks down leaving behind the choanae through which the nasal cavity will communicate with the pharynx. The subarachnoid space surrounds the entire central nervous system and the choroid plexus is forming in the roof of the 4th ventricle. The adenohypophyseal pouch is closed ending below as a stem.

Mammary glands appears in the surface ectoderm on the side of the thorax. Metacarpals are evident as rays of precartilage condensations, or cartilage, in the hand plate and intermetacarpal notches are present at the periphery of the plate. Similarly, metatarsals appear as rays of precartilage condensations or cartilage in the foot plate with intermetatarsal notches at the periphery of the foot plate. Many individual muscles and cartilages that were earlier blastema can now be identified. Internally, the second interatrial septum and foramen ovale can be identified in the heart and the left postcardinal vein near the heart has disappeared with its distal portion draining into the newly formed inferior vena cava. Tertiary segmental bronchi are distinct in the lung and the muscular part of the diaphragm is forming at the periphery of the septum transversum. The paramesonephric duct begins to form along the lateral side of the cephalic part of the mesonephros.

Stage 19 embryos have a greatest length of 16 to 18 mm and an estimated postfertilization age of approximately 45 to 47 days. The trunk is elongating and straightening. The head no longer forms a right angle with the back and is beginning to elevate. The hand and footplates contain distinct metacarpal and metatarsal condensations, respectively. The auricular hillocks are no longer evident as they have blended to form definitive parts of the external ear. The cochlear duct begins its 2 1/2 turn spiral with the tip turned upward. The mesonephric duct and ureter join close to the urinary bladder and continue to the horn of the bladder as a common excretory duct.

Stage 20 embryos have a greatest length of 18 to 22 mm and an estimated post-fertilization age of approximately 49 days. At this stage the upper limbs are slightly bent at the elbow. The hands are still far apart and the fingers are short, stubby and slightly curved over the cardiac prominence. The interorbital groove is conspicuous.

Stage 21 embryos have a greatest length of 20 to 26.4 mm and an estimated post-fertilization age of approximately 49 to 52 days. At this stage the fingers become more elongated and the hands and feet approach each other. For the first time the cortical plate is present in the wall of the cerebral vesicle (hemisphere).

Stage 22 embryos have a greatest length of 23.4 to 27.5 mm and an estimated postfertilization age of 52 to 55 days. At this stage the eyelids cover approximately half of the exposed part of the eye. The fingers approach those on the other side and sometimes touch and overlap. Laminations in the tectum of the mesencephalon represent the presumptive superior colliculi. Many nuclei, fiber tracts and commissures are evident in the brain. The cochlear duct has formed a complete circle with the tip upturned for the second time. The palatine shelves have moved to the horizontal position above the tongue and make contact with the lower edge of the nasal septum. A lumen is present in a portion of the submandibular duct. All of the vertebral segments have formed and exhibit normal spina bifida. All of the joints of the extremities can be identified. Ossification has begun in the clavicle and long bones. A few large glomeruli are present in the kidney and the paramesonephric ducts fuse with each other near the dorsal side of the bladder trigone.

Stage 23 embryos have a greatest length of 23 to 32.2 mm and an estimated postfertilization age of 53 to 58 days. Over 90{a886d2509afb02fdbd678c9c9cbef29e9b4ac8f1454580a0bf53ee67e764b753} of the more than 4,500 named structures in the adult body are present. All named muscles can be identified. The limbs are quite elongated and the hands and feet can overlap each other. The external genitalia are prominent but not yet sexually distinct. The insula and subdivisions of the lateral ventricle can be identified in the cerebral vesicle (hemisphere), the superior and inferior colliculi are evident in the tectum of the mesencephalon and the external granular layer of the cerebellum has formed in the alar plate of the metencephalon. Many nuclei, fiber tracts, decussations and commissures are evident in the brainstem and all four parasympathetic ganglia in the head are present. The upper and lower eyelids are beginning to fuse with each other both laterally and medially. The spiral cochlear duct attains its definitive arrangement of 2 ½ turns. The laryngeal cartilages are present, the palatine shelves are fusing with each other and the nasal septum, and the salivary gland ducts have begun secondary branching. Ossification has begun in the skull and the long bones. Most of the named arteries to the brain can be identified and the left superior vena cava is no longer present. The kidney exhibits 4 to 5 orders of tubules, and tubules can be identified in the testis but are not yet fused with the rete testis and mesonephric duct.