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G.1 The term “embryo” and other terms used to describe early stages of development

Blastocyst: The stage of preimplantation embryo development that, for humans, occurs around day 5–6 after fertilization or intracytoplasmic sperm injection. The blastocyst contains a fluid filled central cavity (blastocoele), an outer layer of cells (trophectoderm) and an inner cell mass (ICM). The trophectoderm cells attach the embryo to the uterine wall, and the ICM forms the embryo proper. The human blastocyst hatches from the zona pellucida (a surrounding glycoprotein shell) around days six-seven after fertilization. Thereafter, and coupled to implantation, the ICM of the blastocyst begins to organize itself into a flattened embryonic disc and associated amnion.

Cleavage stage embryo (preimplantation stage): The embryonic stage that follows the first division of the zygote and ends upon morula compaction; precise stages include the two-cell, four-cell, eight-cell and 16-cell embryo. In humans, each cleavage division takes around 18-24 hours.

Embryo: The term “embryo” has been defined and used differently in various biological contexts as discussed below.

In this document, the term “embryo” is used generically to describe all stages of development from the first cleavage of the fertilized ovum to nine weeks post fertilization in the human, including the placenta and other extraembryonic membranes. 

More precise terms have been used to describe specific stages of embryogenesis; for example, the two, four and eight cell stages, the compacting morula and the blastocyst all describe particular stages of preimplantation embryonic development.
Prior to implantation, the embryo represents a simple cellular structure with minimal cellular specialization, but soon after implantation a defined structure called the primitive streak begins to form and marks the future posterior region of the embryo. After this time twinning of the embryo can no longer occur as there is irreversible commitment to the development of more complex and specialized tissues and organs.

Classical embryology used the term embryo to connote different stages of post-implantation development (for example, the primitive streak and onwards to fetal stages). Indeed, Dorland’s Illustrated Medical Dictionary (27th edition,1988 edition, W. B. Saunders Company) provides the definition “in animals, those derivatives of the fertilized ovum that eventually become the offspring, during their period of most rapid development, i.e., after the long axis appears until all major structures are represented. In man, the developing organism is an embryo from about two weeks after fertilization to the end of seventh or eighth week.” An entry in Random House Webster’s College Dictionary reads “in humans, the stage approximately from attachment of the fertilized ovum (egg or MII oocyte) to the uterine wall until about the eighth week of pregnancy.” However, the nomenclature is often extended by modern embryologists for the human to include the stages from first cleavage of the fertilized ovum onwards to seven to nine weeks post fertilization, after which the term fetus is used.

Fetus: In this document, the term “fetus” is used to describe post-embryonic stages of human prenatal development, after major structures have formed. In humans, this period is from eight to nine weeks after fertilization until birth. The term is often not used in animals where “embryo” is used for any stage from fertilization to term. 

Stem cell-based embryo models: Advances in cellular engineering make possible the assembly, differentiation, aggregation, or re-association of cell populations in a manner that models or recapitulates key stages of embryonic development. Such experimental systems can provide essential insights into embryo and tissue development but raise concerns when such structures achieve complexity to the point where they might realistically manifest the ability to undergo further integrated development if cultured for additional time in vitro. There are two types of stem cell-based embryo models.

Non-integrated stem cell-based embryo models: These stem cell-based embryo models will experimentally recapitulate some, but not all aspects of the peri-implantation embryo, for example differentiation of the embryonic sac or embryonic disc in the absence of extraembryonic cells. These stem cell-based embryo models do not have any reasonable expectations of specifying additional cell types that would result in formation of an integrated embryo model. Gastruloids are an example of a non-integrated stem cell-based embryo model.

Integrated stem cell-based embryo models: These stem cell-based embryo models contain the relevant embryonic and extra-embryonic structures and could potentially achieve the complexity where they might realistically manifest the ability to undergo further integrated development if cultured for additional time in vitro. Integrated stem cell-based embryo models could be generated from a single source of cells, for example expanded potential human pluripotent stem cells capable of coordinately differentiating into embryonic and extraembryonic structures. Alternatively, integrated stem cell-based embryo models could also be generated through the formation of cellular aggregates where extraembryonic/embryonic cells from one source are combined with embryonic/extraembryonic cells from different sources to achieve integrated human development.  This might include using non-human primate cells as one of the sources. Previous restrictions on preimplantation human embryo culture (the “14-day/primitive streak rule”) were not written to apply to integrated stem cell-based embryo models. Thus, these guidelines specify the imperative for specialized review when such research is designed to model the integrated development of the entire embryo including its extraembryonic membranes. A guiding principle of review should be that the integrated stem cell-based embryo models should be used to address a scientific question deemed highly meritorious by a rigorous review process. Blastoids are an example of an integrated stem cell model.

Morula: The compacting grape-like cluster of 16 cells, typically formed by the human embryo four days after fertilization.

Nuclear Transfer: This process involves the insertion of a nucleus of a cell into an ovum from which the nuclear material (chromosomes) has been removed. The ovum will reprogram (incompletely) the cell nucleus to begin development again. Embryos created by nuclear transfer are typically abnormal and often die during development, but rarely are capable of development to term. ICMs from blastocysts derived by nuclear transfer can form apparently normal embryonic stem cells.

Organoid: A tissue culture-derived structure growing in 3D and derived from stem cells that recapitulate the cell composition and a subset of the physiological functions of an organ through principles of self-organization.

Parthenogenetic embryo: Activation of the unfertilized mammalian ovum (usually accompanied by duplication of the haploid genome) can result in embryonic development, and embryonic stem cells can be derived from the ICMs of parthenogenetic blastocysts. After uterine transfer, parthenogenetic embryos of non-human animals have been observed to progress through early post-implantation development but further development is compromised by an underdeveloped placental system that prevents normal gestation. Gynogenesis is a particular form of parthenogenesis in which an embryo is created from the genetic contributions (female pronuclei) of two different zygotes. Androgenesis entails creation of an embryo that incorporates the male pronuclei from two different zygotes.

Zygote: The fertilized single cell pronuclear ovum (egg), typically observed in humans between 20-35 hours after insemination with sperm.