Deciphering the mechanisms facilitating rapid uterine invasion of implanting human embryos

Most of what we understand about mammalian development has been garnered by studying mouse embryos. However, although the early stages, prior to implantation in the uterus, appear to be quite similar between species, the method of implantation can vary enormously. Following fertilisation, all mammalian embryos undergo several rounds of cell division to form a spherical structure. This comprises trophectoderm on the outside, an 'inner cell mass' (ICM) that will segregate into epiblast, the founder of the foetus, and hypoblast that will form the yolk sac on the inside. The ICM is displaced to one side by an expanding cavity (the 'blastocoel'), that defines this stage as the 'blastocyst'. The whole structure is surrounded by a protective 'zona pellucida' to allow it to travel along the oviduct to the uterus. Subtle differences in the details of how blastocysts of different mammals form have been reported, but the subsequent process of implantation can vary enormously. After hatching from the zona pellucida, mouse embryos become encased in decidual tissue secreted by the uterus which persists throughout the early stages of tissue specification and they do not make direct contact with maternal tissue until after the first trimester. In contrast, human blastocysts implant directly into the uterine wall via rapid invasion by the trophectoderm that overlies the ICM. This invasion is essential to secure the embryo within the womb and establish the connection to the mother for exchange of nutrients and waste for development of the foetus. During our studies with human blastocysts under our HFEA licence we have noticed that the trophectoderm overlying the ICM (known as the 'polar' trophectoderm) becomes several layers thick as the embryos mature in preparation for implantation. The mechanism by which polar trophectoderm expands has not been studied, so we have developed methods for sequential labelling of the outside cells to determine whether the rapid expansion of the human trophectoderm occurs by replication of trophectoderm cells or by recruitment and conversion of cells from the underlying ICM, to satisfy the demand for implanting trophectoderm tissue. We suspect that expansion of the human trophectoderm is prone to become out of control, leading to abnormal development, since we observe trophectoderm overgrowth at the expense of derivatives of the ICM in around 1/3 of embryos left over from IVF treatment from multiple clinics, donated to our project with informed consent. We hypothesise that this aberrant overgrowth of the human trophectoderm may be a consequence of the evolutionary need for rapid attachment and invasion into the uterus, which is not the case in the mouse. Furthermore, some of the known problems arising during early human pregnancies, such as ectopic implantation, early post-implantation failure, or formation of a hydatidiform mole composed entirely of trophectoderm tissue, may be an abnormal downstream consequence attributable to the rapid trophectoderm expansion required for human implantation. These malfunctions rarely, if ever, occur during implantation of mouse embryos. We will use various molecular analyses to investigate trophectoderm formation and growth. Not only will the output from this project further our understanding of how embryos from some non-rodent mammals prepare for implantation, it will also provide a discrete and tractable system with which to investigate how multiple layers can form from a single epithelium, which may share features with other systems in the body. The blastocyst stage of development can be modelled using stem cell lines that can be induced to assemble into tissues closely resembling trophectoderm, epiblast and hypoblast. We will use our knowledge of the instructive signals required to specify each lineage to build models of trophectoderm overgrowth and thereby scrutinise the mechanisms by which it occurs and identify supplements for the culture medium that may restrain it.

In vitro fertilisation is currently responsible for 4.2% of babies born in the UK. Many embryos are left over and can be donated for research, providing a rich resource for studies of development and mechanisms of early lineage specification and regulation. The blastocyst comprises an outer layer of trophectoderm, source of the placenta, and inner cell mass comprising epiblast and hypoblast that will form foetus and yolk sac, respectively. Published single cell RNA sequencing revealed that, in contrast to mouse embryos, cells taken from inside human blastocysts often exhibit trophectoderm identity. We found that the trophectoderm overlying the inner cell mass of expanded human blastocysts consists of more than one layer. We will investigate the mechanism of this phenomenon via sequential labelling of outside cells before culture and after 24 hours during blastocyst expansion using fluorescent labels of contrasting colours to determine the origin of cells contributing to the thickening polar trophectoderm. These novel experiments will uncover mechanisms of human development potentially applicable to additional non-rodent mammals as well as other epithelial systems in the body. We have noticed that ~35% of embryos donated to our project do not expand, but collapse to form a solid ball of cells. Immunofluorescence for markers of the founder lineages revealed an excessive amount of trophectoderm dominating the structure. We will use single cell multi-omics of embryos at various stages and analyse secretions in the medium to characterise aberrant embryos and thereby determine mechanisms associated with this phenotype. Molecular causes of trophectoderm overgrowth will be tested using 'blastoids' (blastocyst-like structures made from human stem cell lines) by manipulating the signalling milieu, for induction or inhibition of trophectoderm overgrowth. Optimal inhibiting conditions will also be tested on donated embryos to find out if the aberrant phenotype can be prevented.