Most of our knowledge about early post-implantation development was gained from an extensive research. Fixed and stained human embryos after pregnancy loss at different stages of gestation provided information about the early human post-implantation period. The Carnegie Collection of Human Development at the National Institute of Child and Human Development in Washington, DC, has made a collection of several thousand serial microscopic cross-sections. Human embryos have been classified into 23 developmental stages based on a number of morphological features, known as the “Carnegie stages.”
The aforesaid stages represent a series of events that occur during development, with overlap between the different stages. More recently, researchers have developed an in-vitro system to culture human embryos in the absence of maternal tissues from early cleavage or blastocyst stages through to post-implantation stages up to dates 12-13, and compared their observations with the Carnegie series.
Using immuno-staining for lineage-specific markers, they identified the series of events that occur after implantation:
Segregation of epiblast and hypoblast progenitors (Day 7),
Epiblast polarization and formation of the pro-amniotic cavity (Day 8-10),
Differentiation of the trophoblast into cytotrophoblast and syncytiotrophoblast (Day 8-10),
Formation of the prospective amniotic ectoderm and yolk sac and bilaminar disc (Day 11).
By extending the studies using human pluripotent stem cells, they showed that embryonic cell lineages reorganize via cellular polarization, leading to cavity formation. Unexpectedly, the human embryos were able to self-organize autonomously, without the participation of maternal tissue.
The brief overview presented below describing early embryo development that is based upon the Carnegie Collection of beautiful color three-dimensional reconstructions and animations, which can be viewed at http://virtualhumanembryo.lsuhsc.edu/.
7-12 Days Post Ovulation
Once the blastocyst has hatched and trophoblast cells have started the process of implantation, the cells of the inner cell mass start to reorganize into distinct layers, each layer destined for a different developmental fate. Cellular morphology changes, and there is active movement of individual cells and groups of cells, setting up new relationships between them.
In the human embryo, the first 14-18 days of development are concerned mainly with the differentiation of various extra-embryonic tissues, and after this time separate tissues can begin to be identified.
13-15 Days Post Ovulation (Second Week of Development)
Cells of the inner cell mass separate into two different sheets of cells to form a bilaminar embryonic disc:
“Epiblast” – dorsal germinal layer,
“Hypoblast” – Ventral germinal layer.
The flatter hypoblast cells lie on top of the epiblast and will form the yolk sac; the epiblast contains the cells of the future embryo.
The two layers of the embryonic disc divide the blastocyst into two chambers, the amnion and the yolk sac; outside the embryo, the extra-embryonic spaces (chorionic, amniotic, yolk sac) continue to develop, the endometrium is being converted into the decidua and early placentation has begun. The amnion fills with fluid that cushions the developing fetus; the yolk sac contributes to the formation of the extra-embryonic membranes, chorion and amnion.
It is also the site of early blood cell formation, and part of the yolk sac becomes incorporated into the gut later in development. The cells of the trophoblast form another chamber, the extra-embryonic coelom, around the amnion, yolk sac and developing embryo; this will later become the chorionic sac and placenta.
13-15 Days Post Ovulation (End of Second Week of Development)
Embryonic epiblast cells move along the surface, begin to pile up near the center to form a node and then move internally to create a furrowed cell mass, the primitive streak. This is a visible feature showing that cells are migrating, and the furrow begins to extend toward the cranial (head) end of the bilaminar disc. Cells near the advancing edge of the streak begin to pull apart slightly to form an open pit, and cells bordering this pit then migrate between the epiblast and hypoblast to create a new layer, the mesoderm.
Cells from the epiblast also displace the hypoblast, creating the endoderm. The cells that remain on the surface will form ectoderm, and this formation of a now tri-laminar disc is known as gastrulation. During gastrulation, cells from the mesoderm form the notochord, which will define the primitive axis of the embryo and establish cranio-caudal orientation and bilateral symmetry of the fully developed body. The three layers of cells form the primary germ cells that will evolve into the specialized cells, tissues and organs of the body:
Endoderm (inner layer): forms the lining of the primitive digestive tract and its associated glandular structures, as well as portions of the liver, pancreas, trachea and lungs,
Mesoderm (middle layer): initially forms a loose aggregate of cells, the mesenchyme; this then organizes into regions that evolve into the vertebral column, skeletal muscle, ribs, skull and the dermis of the skin. Mesenchymal cells also form tubular structures: urogenital system, heart, blood vessels, and the lining of the pericardial, pleural and peritoneal cavities,
ectoderm (outer layer): develops into skin epidermis, brain and spinal cord. Sensory receptors for vision, hearing and smell, as well as the future autonomic nervous system and adrenal medulla, also develop from ectoderm.
17-19 Days (Third Week of Development)
After gastrulation, the mesoderm starts to segment into mesenchymal tissue somites, and the notochord induces rapid growth in the ectoderm. Over the next 2-3 days, the ectodermal layer thickens to form a neural plate, which folds to form a neural groove: the nervous system is one of the first organs to develop. During this process of neurolation, the ectoderm will subdivide into neural tissue and epithelial tissue lineages (pigmented cells of the epidermis, adrenal medullary cells, skeletal and connective tissues of the head).
Somites continue to form in the mesoderm, the neural groove fuses dorsally to form a tube at the level of the fourth somite, and closes. The cranial end of the neural plate is wider, enclosing the region that will form the brain. The spinal cord will form at the caudal end, which is narrower (the notochord is eventually replaced by the vertebral column).
19-21 Days (End of Third Week of Development)
Rapid cellular growth elongates the embryo and expands the yolk sac. Primordial germ cells can be identified at the root of the allantois. A head fold rises on either side of the primitive streak, and endocardial cells begin to fuse and form two endocardial tubes which will develop into the heart. Pairs of mesodermal somites can be seen on either side of the neural groove, appearing first in the caudal region. The neural folds rise and fuse along the length of the neural tube, along with budding somites that close the neural tube (like a “zipper”). The neural tube begins to close in the middle of the embryo (cervical region), and then spreads in both the cranial and caudal directions.
Failure of the neural tube to close correctly at this stage can lead to anomalies such as spina bifida (caudal) or anencephaly (cranial). Masses of cells detach themselves from the side of the neural plate and form the neural crest, precursor cells of numerous differentiated cells of the nervous and glandular systems. The heart tube becomes S-shaped, with the beginning of cardiac muscle contraction. Secondary blood vessels appear in the chorion/placenta, and hematopoietic cells appear in the yolk sac.
23-27 Days (Fourth Week)
A primitive S-shaped tubal heart begins to beat, and the developing neural tube curves the embryo into a C-shape. The forebrain is closed when 20 somites are present, eyes and ears begin to form, and pharyngeal arches are present. Valves and septa begin to appear in the heart, and a blood circulatory system continues to develop.
28-35 Days (Fifth Week)
Somites organize themselves into myotomes, the groups of tissues that will develop into the musculoskeletal structure of the body wall, and rudiments of the ribs and limbs begin to appear. Sclerotome give rise to the axial skeleton, myotomes to striated muscle, and dermatomes to subcutaneous tissue and skin. Arches that form the face and neck can be seen under the enlarging forebrain. The digestive epithelial layer begins to differentiate into future places of the liver, lungs, stomach, and pancreas.
35-42 Days (Sixth Week)
By Day 35 (fifth week of development = 6 weeks from last menstrual period), when clinical pregnancy after IVF can be confirmed by ultrasound visualization of gestational sac and fetal heartbeat, the three primary germ layers have expanded and undergone a dramatic process of differentiation and transformation, to form a clearly recognizable fetus. The beating heart has chambers, and all limbs and body systems are under development. Medial thickening of the coelomic epithelium represents the formation of primordial gonads. The size of the fetus is now approximately 7.0-9.0 mm, and it will continue to grow at a rate of around 1 mm per day during the first trimester. By the beginning of the second trimester, the basic brain structure is complete, and genitalia begins to show signs of gender characteristics.
Ultrasound Confirmation of Clinical Pregnancy
Gestational sac: can be seen at around four weeks’ gestation but may not be visible until the end of the fifth week. It is characteristic of early pregnancy, but does not correspond to anatomical features of the embryo. Gestational sacs are also found in ectopic pregnancies.
Yolk sac: visible during the fifth week and grows to 6 mm. Larger yolk sacs usually indicate an abnormal pregnancy; yolk sacs that are misshapen are “floating” within the gestational sac and contain echogenic (instead of sono-lucent) material are ominous findings for the pregnancy.
Fetal heartbeat (FH): using transvaginal ultrasound, fetal cardiac activity can sometimes be seen along the edge of the yolk sac before a fetal cell mass is identifiable. In normal pregnancies, the fetal heartbeat may not be seen until the fetal pole is around 4 mm in size. Failure to identify a FH in a fetus >4 mm in size is an ominous sign.
Fetal pole: the fetus in its somite stage, first visible separation from the yolk sac by transvaginal scan (TVS) just after 6 weeks’ gestation (Day 35 post ovulation).
Crown-rump length (CRL): single most accurate measure of gestational age up to 12 weeks’ gestation.
A hatched blastocyst can sometimes implant in sites outside the uterus, most commonly in the fallopian tube. Other sites include the utero-cervical isthmus (cervical pregnancy) and the utero-tubal junction; more rarely, implantation can take place in the ovary, or in the peritoneal or abdominal cavity.
Occasionally two embryos will implant at different sites: one in each tube (bilateral tubal pregnancy) or one in the uterus and one at an ectopic site (heterotopic pregnancy).
An embryo can continue to grow at an ectopic site for several weeks, with signs and symptoms of early pregnancy in the mother. If undetected, ectopic pregnancy can result in a life-threatening crisis due to tubal rupture or erosion into a blood vessel.
Miscarriage: spontaneous abortion prior to 20 weeks’ gestation.
Biochemical pregnancy: early pregnancy loss, prior to six weeks from last menstrual period.
Blighted ovum: an embryonic gestation. Sac appears normal on TVS, but an embryo never develops; probably due to early embryonic death with continued trophoblast development.
Missed abortion: nonviable intrauterine pregnancy that has not yet aborted. TVS shows gestational sac with no FH; can be due to a blighted ovum, or early demise of an embryo after detection of FH. The cervical os is closed.
Threatened abortion: vaginal bleeding and (or) abdominal/pelvic pain during early pregnancy; the cervical os is closed, and no tissue has been passed. FH is still present on TVS.
Inevitable abortion: vaginal bleeding, usually with abdominal pain and cramps. FH is absent, cervical os is open, but no tissue has been passed. Usually progresses to complete abortion.
Incomplete abortion: Heavily vaginal bleeding, with tissue having been passed, but some remaining in utero.
Complete abortion: bleeding, abdominal pain; all products of conception have been passed; empty uterus must be confirmed by TVS.
Recurrent abortion: history of more than three spontaneous abortions.