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Publication
Developmental regulation and decisions in the human preimplantation embryo
Journal Contribution - Journal Article Conference Contribution
Introduction:
The zygote is the ultimate totipotent cell. Totipotency is lost during preimplantation development, however it is unknown when and in which cells. According to the inside-outside hypothesis of Tarkowski et al. (1967), the outer cells of a morula become trophectoderm (TE) cells, whereas the inner cells become inner cell mass (ICM) cells. The first morphologically visible sign of differentiation becomes apparent at the fifth day of preimplantation development during blastulation when the two lineages can be distinguished. Blastomeres of 2-cell, 4-cell and 8-cell stage human embryos are thought but not proven to be totipotent. In order to allow the embryo to overcome perturbations in its organization such as cell loss due to fragmentation, cryodamage and biopsy for preimplantation genetic diagnosis or disturbance of the cleavage planes, the blastomeres should be flexible and capable to develop into ICM and TE. This has been called regulative development. The developmental decision to become ICM or TE is crucial. We aimed (1) to investigate regulative development and (2) to determine when the decision to develop into ICM or TE becomes irreversible.
Material and Methods:
Embryos were created in vitro with the approval of the Belgian Federal Committee on medical and scientific research on embryos in vitro and the Local Ethical Committee using gametes from consenting donors. In addition, embryos which were diagnosed to carry a genetic disease were used after informed consent. Only top quality embryos were used. Embryos were biopsied in Ca2 + /Mg2 + free medium using laser biopsy. Blastomeres were aspirated using a pipette with an inner diameter of 60-80 µm and put into an empty zona pellucida (ZP). Reconstituted embryos were cultured individually in sequential medium in microdroplets under oil up to day 6 of preimplantation development. They were analysed by immunocytochemistry and confocal microscopy for the expression of NANOG (ICM) and HLA-G (TE) (data not discussed).
Results:
In order to investigate regulative development in vitro, we disturbed the organization of the embryo by changing the position of the blastomeres at the 4-, 8- and compacting/compacted stage. Seven tetrahedron 4-cell stage embryos (day 2), eight 8-cell stage embryos (day 3) and eight compacting/compacted embryos (14-28 cells on day 4) were disaggregated and the blastomeres were randomly transferred into an empty ZP. Six, eight and seven reconstituted embryos respectively developed into good quality blastocysts with TE and ICM.
In order to determine at which stage the decision to become ICM or TE is irreversibly taken, we investigated the potency of the outer cells to develop into blastocysts with TE and ICM. Seven compacting/compacted embryos were manipulated by removing a limited number of outer cells (5-7) in order not to confuse with inner cells and these cells were transferred into an empty ZP. Six out of these ZPs with outer cells only developed into blastocysts with TE but none of them appeared to have an ICM. Six out of the seven original embryos (few remaining outer + all inner cells) developed into good quality blastocysts. In order to exclude the possibility that 5-7 outer cells is not enough mass for the development of an inner cell population within the embryo, outer cells of two distinct embryos were transferred into an empty ZP (12-14 cells). Four embryos reconstituted with outer cells only developed into good quality blastocyst with TE and ICM, as well as the eight original embryos.
Conclusions:
Interference with the localisation at the 4-cell, 8-cell and compacting/compacted stage does not interfere with blastocyst formation, indicating that human preimplantation development is highly regulative. The decision by the outer cells to become TE is not irreversibly taken at the compacting/compaction stage of human preimplantation development.
The zygote is the ultimate totipotent cell. Totipotency is lost during preimplantation development, however it is unknown when and in which cells. According to the inside-outside hypothesis of Tarkowski et al. (1967), the outer cells of a morula become trophectoderm (TE) cells, whereas the inner cells become inner cell mass (ICM) cells. The first morphologically visible sign of differentiation becomes apparent at the fifth day of preimplantation development during blastulation when the two lineages can be distinguished. Blastomeres of 2-cell, 4-cell and 8-cell stage human embryos are thought but not proven to be totipotent. In order to allow the embryo to overcome perturbations in its organization such as cell loss due to fragmentation, cryodamage and biopsy for preimplantation genetic diagnosis or disturbance of the cleavage planes, the blastomeres should be flexible and capable to develop into ICM and TE. This has been called regulative development. The developmental decision to become ICM or TE is crucial. We aimed (1) to investigate regulative development and (2) to determine when the decision to develop into ICM or TE becomes irreversible.
Material and Methods:
Embryos were created in vitro with the approval of the Belgian Federal Committee on medical and scientific research on embryos in vitro and the Local Ethical Committee using gametes from consenting donors. In addition, embryos which were diagnosed to carry a genetic disease were used after informed consent. Only top quality embryos were used. Embryos were biopsied in Ca2 + /Mg2 + free medium using laser biopsy. Blastomeres were aspirated using a pipette with an inner diameter of 60-80 µm and put into an empty zona pellucida (ZP). Reconstituted embryos were cultured individually in sequential medium in microdroplets under oil up to day 6 of preimplantation development. They were analysed by immunocytochemistry and confocal microscopy for the expression of NANOG (ICM) and HLA-G (TE) (data not discussed).
Results:
In order to investigate regulative development in vitro, we disturbed the organization of the embryo by changing the position of the blastomeres at the 4-, 8- and compacting/compacted stage. Seven tetrahedron 4-cell stage embryos (day 2), eight 8-cell stage embryos (day 3) and eight compacting/compacted embryos (14-28 cells on day 4) were disaggregated and the blastomeres were randomly transferred into an empty ZP. Six, eight and seven reconstituted embryos respectively developed into good quality blastocysts with TE and ICM.
In order to determine at which stage the decision to become ICM or TE is irreversibly taken, we investigated the potency of the outer cells to develop into blastocysts with TE and ICM. Seven compacting/compacted embryos were manipulated by removing a limited number of outer cells (5-7) in order not to confuse with inner cells and these cells were transferred into an empty ZP. Six out of these ZPs with outer cells only developed into blastocysts with TE but none of them appeared to have an ICM. Six out of the seven original embryos (few remaining outer + all inner cells) developed into good quality blastocysts. In order to exclude the possibility that 5-7 outer cells is not enough mass for the development of an inner cell population within the embryo, outer cells of two distinct embryos were transferred into an empty ZP (12-14 cells). Four embryos reconstituted with outer cells only developed into good quality blastocyst with TE and ICM, as well as the eight original embryos.
Conclusions:
Interference with the localisation at the 4-cell, 8-cell and compacting/compacted stage does not interfere with blastocyst formation, indicating that human preimplantation development is highly regulative. The decision by the outer cells to become TE is not irreversibly taken at the compacting/compaction stage of human preimplantation development.
Journal: Hum Reprod
ISSN: 0268-1161
Volume: 25
Pages: 175
Publication year:2010
Keywords:PGD