Introduction

The transformation of a single cell into a complex multicellular organism is an intricate, fascinating process, entailing a series of rapid cell divisions and differentiation. The second and third weeks of embryological development are crucial, involving the implantation of the blastocyst into the uterine wall; the establishment of three distinct germ layers - the mesoderm, endoderm and ectoderm - through gastrulation; and encompass the beginnings of the neurulation process, resulting in central nervous system (CNS) development and neural tube formation. Abnormal blastocyst implantation may cause in ectopic pregnancy, hydatidiform mole or gestational trophoblastic diseases, while defects in primary neurulation are implicated in anencephaly, spina bifida, and craniorachischisis.

Development

Cleavage Divisions

The fusion of the male and female haploid pronuclei following fertilization produces a single diploid nucleus capable of beginning its first mitotic cycle almost immediately. However, cell division at this early stage does not also grow; rather, the embryo divides every 12 to 24 hours to create smaller individual cells known as blastomeres. These embryonic cell divisions not accompanied by growth are known as cleavage divisions.  

At the 16 cell stage, otherwise known as a morula, the individual blastomeres are spherical and undifferentiated; however, as cell division proceeds, they undergo a process called compaction whereby the alteration in blastomere shape and alignment generates a small internal population of cells with no direct contact with the outside surface of the embryo. This arrangement of cells creates a central fluid-filled cavity called the blastocoel, with the embryo now referred to as a blastocyst. The blastocyst comprises of an outer layer of trophoblast cells which will form the placenta and the inner cell mass which will ultimately form the fetus, Heuser’s membrane, amniotic membrane, and the extraembryonic vasculature.

Implantation

The second week of development is predominately associated with implantation of the blastocyst into the uterine wall to establish a source of nutrition. As it migrates towards the uterus, the zona pellucida surrounding the blastocyst prevents direct contact with the epithelial lining of the fallopian tubes, ensuring implantation in approximately 99% of pregnancies will occur within the uterus.[1] Once the blastocyst enters the uterus, the zona pellucida degenerates to expose the underlying trophoblast layer, thus enabling it to attach to the endometrium. The subsequent release of mitogens stimulates the trophoblast to differentiate into two layers, the syncytiotrophoblast, and they cytotrophoblast. As the syncytiotrophoblast is acellular, mitosis is not possible, hence proliferation creates a syncytium composed of many nuclei. Conversely, the cytotrophoblast undergoes mitosis to generate a single layer of cells lining the blastocoel and the inner cell mass. The enzymes released by the syncytiotrophoblast digest the extracellular matrix, enabling the blastocyst to penetrate the uterine wall until implantation is complete.  

Lacunae are cavities that form in the syncytiotrophoblast layer which soon extend to form a lacunar network. As the blastocyst implants, uterine capillaries nearby dilate to form sinusoids, which enables maternal blood to fill the lacunae as the blastocyst invades the uterine wall, thus establishing the beginning of the uteroplacental circulation.

Human Chorionic Gonadotropin   

An additional consequence of implantation is the synthesis of the hormone human chorionic gonadotropin (hCG) by the syncytiotrophoblast layer. In early pregnancy, the corpus luteum requires hCG to sustain progesterone production and subsequently, the uterine endometrium. The placenta eventually assumes the role of producing progesterone later in the pregnancy. Other hormones produced by the trophoblast layer include:

• Chorionic somatomammotropin: maintains placental growth
• Chorionic thyrotropin: regulates maternal metabolism
• Chorionic corticotropin: the growth of mammary glands

Bilaminar Disc Formation

Similar to the outer trophoblast layer, the inner cell mass also differentiates to generate a bilaminar disc composed of the hypoblast and epiblast. The hypoblast spreads along the cytotrophoblast lining the blastocoel to form an extraembryonic coelom, the yolk sac, enclosed by a layer of extraembryonic endodermal cells known as Heuser’s membrane. The yolk sac is continuous the endodermal lining of the gastrointestinal tract and is essential in forming the vitelline circulation; branches of the dorsal aorta, the vitelline arteries, deliver blood to the wall of the yolk sac before it gets returned to the blastocyst via vitelline veins. The epiblast will later differentiate into embryonic germ layers, the amniotic membrane and extraembryonic mesoderm. The extraembryonic mesodermal cells migrate to line various parts of the embryo; extraembryonic somatic mesoderm covers the amnion and also forms the connecting stalk. The yolk sac covering is a layer of extraembryonic visceral mesodermal cells.

Extraembryonic Coeloms Formation

In addition to the yolk sac, two further extraembryonic coeloms form during the second week, the amniotic cavity and the chorionic cavity. Development of the amniotic membrane begins when a small space forms in the epiblast layer. The epiblast cells then differentiate into amnioblasts, which will form the amnion, a layer of flattened epithelial cells lining the cavity. Later on in development, amniotic fluid will fill this cavity to cushion the fetus and allow movement. The chorion is a membrane enclosing the chorionic cavity and forms from trophoblast cells and extraembryonic somatic mesoderm. The connecting stalk connects the embryo and yolk sac to the chorion and suspends them in the chorionic sac.

Maternal-Fetal Circulation

Small projections of cytotrophoblast surrounded by syncytiotrophoblast grow into the lacunae to form primary chorionic villi in the second week of development. As the second week progresses, the villi continue to grow and now contain a core of extraembryonic mesoderm; vascularisation occurs when branches of the umbilical artery and vein invade the mesoderm during the third week of development. Chorionic villi are essential maximizing the contact with maternal blood to facilitate the exchange of nutrients and waste with the fetus.

The cytotrophoblast containing tips of the chorionic villi can later further differentiate to form extravillous trophoblasts. These extend from the placenta to anchor it to the uterine wall. Moreover, extravillous trophoblasts play an essential role in ensuring that adequate perfusion to the fetus gets sustained throughout pregnancy by altering the vasculature in the uterus. Specialized extravillous trophoblasts can invade and remodel the uterine spiral arteries emptying into the lacunae to ensure they remain open, regardless of maternal vasoconstriction, thus maximizing the oxygen and blood supply to the fetus.

Gastrulation

Gastrulation is a complex process involving the embryo reorganizing itself to generate the three embryonic germ layers, endoderm, mesoderm, and ectoderm. On day 14, mesenchymal cells appear along the midline of the caudal half of the epiblast layer to form the primitive streak; this marks the anteroposterior axis of the embryo and the beginning of gastrulation. At the rostrum of the primitive streak, a group of cells forms the primitive node which establishes the left/right axis of the embryo. Epiblast cells undergo epithelial to mesenchymal transition whereby the cells enter the primitive streak and break away from the epithelium, thus enabling the migration of individual cells in a process known as ingression.[2] The first cells to ingress through the streak invade the hypoblast layer to form embryonic endoderm.  Cells continue ingress the following day between the epiblast and endoderm layers to form mesoderm. The remaining layer of cells creates embryonic ectoderm. The newly formed mesodermal cells further differentiate into new structures.

Mesoderm cells that migrate through the node form the prechordal plate and notochord, whereas those from the primitive streak will condense adjacent to the notochord to form paraxial, intermediate and lateral plate mesoderm. Paraxial mesoderm develops closest to the notochord and forms somites. Somites are masses of tissue flanking the notochord which develop into three cell populations: sclerotome, dermatome, and myotome. Cells situated ventrally in each somite de-epithelise to form sclerotome, which migrates around the notochord and neural tube to differentiate into the bones of the axial skeleton.[3] Similarly, the myotome also de-epithelializes to form skeletal muscle, with the remaining dermatome forming the dermis of the skin.  Intermediate mesoderm will later go on to form the urinary and reproductive systems. Lateral plate mesoderm forms two layers, somatopleuric and splanchnopleuric mesoderm; the somatopleuric mesoderm will form the connective tissue of the body wall and limbs whereas the splanchnopleuric mesoderm will form the connective tissue and smooth muscle layers of the gut, cardiac muscle and the circulatory system.

Neurulation

Although neurulation begins in the third week, the process does not complete until the fourth week of development. Signals from the notochord and prechordal plate in the mesoderm induces ectoderm cells rostral to the primitive node to form the neural plate. Neural plate cells are columnar and will later go on to form the CNS. In addition to the neural plate, the remaining ectoderm cells will differentiate to form the epidermis. Next, apical constriction of neural plate cells along the midline creates wedge-shaped cells; this fold is the medial hinge point.[4] There are further hinge points along the rostral-caudal axis of the embryo which will assist in the folding and fusion of the neural plate in the fourth week to create the neural tube which gives rise to the CNS. Moreover, during neurulation, ectoderm also forms the so-called neural crest involved in the formation of structures of the face and brain.

Cellular

The three embryonic germ layers, developed through the process of gastrulation, form the basis for all the tissues and organs in the body.

• The endoderm will form the epithelium of the digestive tract, lungs, liver, pancreas, and thyroid
• The mesoderm will give rise to muscle, cartilage and bone, heart and circulatory system, gonads, and the urogenital system
• The ectoderm will give rise to outer components of the body, such as skin, hair, and mammary glands and part of the nervous system (see neurulation).

Testing

Preimplantation genetic diagnosis (PGD), also known as preimplantation genetic testing (PGT), is used to screen embryos for genetic defects before implantation. This technique is a common method used in couples who are at high risk of producing children with genetic disease. In vitro fertilization (IVF) is used to produce the embryos, and a single blastomere gets removed during the cleavage stages targeted for testing. After a biopsy, the fresh or frozen-thawed blastomere then gets transferred into the uterus based on the results of genetic testing.

PGD is commonly used to screen for monogenic disorders such as cystic fibrosis, Huntington disease, and Duchenne muscular dystrophy. However, it can also be used to determine embryo quality in IVF and for HLA matching of the embryo to a sick sibling. Structural chromosomal abnormality including translocation in embryos of a couple with a balanced translocation, or deletion/duplication and de novo aneuploidy (e.g., subchromosomal deletions and duplications) in embryos of couples presumed to be chromosomally normal are other PGD objectives.

Pathophysiology

Hydatidiform mole, or molar pregnancy, is the most common of the pathologies that come under gestational trophoblastic disease. It categorizes by a non-viable fertilized egg implanting into the uterine wall, causing abnormal and excessive proliferation of placental tissue. The moles can be either complete or partial. A complete mole forms when an oocyte with no female chromosomes is fertilized by single sperm, which then duplicates its chromosomes via endoreplication to form a diploid embryo composed only of paternal chromosomes. This mechanism occurs in approximately 80% of complete molar pregnancies, with the remaining 20% formed when the empty oocyte becomes fertilized by two sperm [5]. As the embryo is unable to develop due to the lack of maternal chromosomes, it leads to the uncontrolled proliferation of the syncytiotrophoblast creating an abnormal placental mass with distended chorionic villi. Partial molar pregnancies are less common than complete molar pregnancies and occur when a normal egg is fertilized by two sperm, thus creating an embryo with 69 chromosomes. The cytotrophoblast layer undergoes proliferation in partial moles which will form both normal and distended chorionic villi. Unlike complete moles, a fetus will begin to develop. However, there will be multiple defects and abnormalities, leading to a spontaneous abortion usually within the first trimester. Although both complete and partial moles will usually miscarry without intervention, there is a risk of the masses developing into choriocarcinoma if they do not get removed.

Neural tube defects are the second commonest group of birth abnormalities worldwide following congenital heart diseases and exist on a vast clinical severity spectrum. Open lesions, such as anencephaly and craniorachischisis, are generally incompatible with life and are caused by a failure of the neural tube to close properly, thereby exposing the neural folds to amniotic fluid, while the least severe stem from secondary neuralation abnormalities, and have the name of spinal dysraphism. In these instances, there is no evidence of failed neural tube closure; instead, such pathologies are attributable to abnormal development of the cranial mesoderm.

Clinical Significance

An ectopic pregnancy results when a blastocyst implants in a site other than the uterus and accounts for approximately 2% of all pregnancies.[6] Although the blastocyst cannot implant properly in these abnormal sites, it still can attract a large maternal blood supply for nutrition; if it breaks away from these tissues, extensive bleeding may occur posing a serious threat to the mother. Most commonly, the blastocyst implants in a fallopian tube, known as a tubal pregnancy, however other common sites include the cervix, ovary, and abdominal cavity. Seventy-five percent of ectopic pregnancies are diagnosed using transvaginal ultrasounds; however, serum beta hCG can also be used to confirm the diagnosis.[7] Patients typically present with sharp abdominal pain, amenorrhea, and vaginal bleeding. These pregnancies are considered non-viable and are usually terminated, with laparoscopic removal of the blastocyst considered the gold standard.[8] The other treatment option is to give methotrexate to prevent further development of the blastocyst.

hCG is a glycoprotein comprised of alpha and beta subunits and is the principal marker used in home kits to test for pregnancy. It gets produced by the syncytiotrophoblast layer of the developing embryo; however, sufficient levels are not reached until at least two weeks after conception to be detectable via urine testing.[9] Quantitative testing can be carried out via a blood sample to test for the amount of hCG beta present to confirm a positive result and calculate gestational age. In the majority of normal pregnancies, hCG levels double every 48 hours, with levels peaking around 8 to 11 weeks after conception before declining for the remainder of the pregnancy.[10] Levels of hCG can be monitored to check for abnormalities, with low levels of hCG suggesting ectopic pregnancy or miscarriage, and higher levels possibly indicating molar or multiple pregnancies. In either case, the levels should be rechecked regularly and closely monitored.