The knowledge of early human embryonic development is crucial to develop novel treatments for infertility and improve in vitro fertilisation procedures. Despite tremendous clinical significance, owing to limited availability of human embryos, mechanisms underlying early human embryogenesis are poorly understood.
A faithful in vitro model of human embryos, as reported in our recently published study, allows for the first time to perform high-throughput and complex experimental manipulations to unravel previously unknown mechanisms and to develop novel therapeutic strategies for women’s reproductive health.
Initiation of pregnancy in humans is surprisingly inefficient. While it is difficult to determine the precise number, the conservative estimates suggest that 40-60 per cent of the fertilised eggs fail to contribute to live birth.1 The majority of these losses occur in the first weeks of pregnancy, especially during implantation, a process in which a free-floating embryo at the blastocyst stage interacts with the uterus in order to nest into it and develop further. A thorough understanding of the mechanisms governing early embryo development and implantation is essential for learning about the underlying causes of pregnancy loss and developing novel prevention strategies.
Historically, we have relied on model organisms to understand human embryonic development. While this has been largely beneficial, over the years we have learnt the divergence in the mechanisms between humans and other model organisms. Such differences in developmental mechanisms are especially striking at the early stages of embryonic development. For example, mammalians, including humans, form by nesting into the mother’s uterus that supports its growth and development. To do so, in embryos of most of the mammalian species, a few cells from the outermost layer of the blastocyst, called trophectoderm, become “sticky” so as to attach to the lining of the uterus. However, the mechanisms by which the subpopulation in the trophectoderm layer acquires stickiness is fundamentally different in humans from that of mice and therefore, remains unknown.
Purely from a scientific perspective, in an ideal case, human embryos are necessary to understand human embryonic development. Indeed, some countries allow scientists to use surplus embryos from in vitro fertilisation (IVF) clinics for research purposes upon obtaining patient consent and fulfilling all the necessary regulatory guidelines. However, these embryos come in small numbers and can only be used under strict ethical oversight. It is thus extremely challenging to perform experiments (e.g. genetic manipulations, genetic and chemical screenings, etc.) that lie at the heart of scientific discoveries.
In the recent study published in the journal Nature, we have reported the development of a robust, efficient, and faithful model of early human embryos from stem cells.2 Deep characterisation of these structures using cutting edge technologies such as single-cell transcriptome sequencing demonstrated their remarkable similarity to the embryos at the stage of a blastocyst. These self-organised structures are formed with the same developmental sequence and pace as that of the human blastocyst. For all these reasons, we refer to them as blastoids. Moreover, several previously known developmental mechanisms underlying human blastocyst development are conserved in the blastoids. For example, treatment of previously reported drugs has the same impact at the molecular levels on the blastoids as described in the literature for human blastocysts.
Overall, the formation of blastoids mimics blastocyst development occurring between days five and seven of embryogenesis. At the end of their formation, blastoids contain analogues of all the cell types present in the blastocysts, namely, epiblast (forms the whole organism), trophectoderm (forms the placenta), and the primitive endoderm (forms extraembryonic tissue called yolk sac). Importantly, blastoids have a negligible number of “off-target” cells, i.e. the cells that are not found in blastocyst stage embryos (for example, cells corresponding to the earlier or the later developmental stages). The analysis is now independently verified by a consortium of scientists.3
The striking similarity of blastoids to the blastocyst at the molecular level prompted us to test the conservation of functions in the blastoids. Because of our long-standing interest, we wanted to test if blastoids could replicate features of implantation. However, since the formation of mouse blastoids by our group, in 2018, we have participated in the establishment of ethical guidelines by the International Society for Stem Cell Research, recommending that it would be ethically unacceptable to transfer human blastoids into the uterus of humans or any other animals for the purpose of research or otherwise. Therefore, to test the implantation capacity of blastoids, we developed a novel in vitro assay using cells from human endometrial organoids. These are the 3-dimensional microstructures that are derived from the outer lining of the uterus from healthy individuals and can be expanded in the lab indefinitely.
Previous studies from various groups have shown that endometrial organoids respond to reproductive hormones such as Estrogen and Progesterone in a similar way as that of the uterus undergoing the menstruation cycle. We thus deposited the cell from the endometrium organoid on a 2D cell culture dish and had a Eureka moment when we observed that blastoids transferred on these cells specifically attached to the endometrial cells. To our surprise, similar to the “window of implantation” of the uterus, only the hormone-stimulated endometrial cells became receptive to interacting with the blastoids. Also, the attachment of blastoids was always mediated by the trophectoderm cell analogues lying adjacent to the inner cell cluster as has been observed for human blastocysts. Blastoids thus mimic in a dish several key aspects of the first step of implantation.
The conservation of shape, size, composition, pace, sequence of development, previously reported molecular mechanisms and function, as demonstrated by their ability to mimic implantation, between blastoids and human blastocysts indicate that blastoids can be reliably used to understand human embryonic development. At least two different drugs with known effects on human blastocyst have been tested on the blastoids and have been found to have the same effect on the blastoids, thus testifying to the predictivity of the blastoids. Conversely, the drugs developed on blastoids are also likely to show similar effects on human embryos.
Blastoids are also formed at remarkably high efficiency. More than 70 per cent of the aggregates of stem cells with appropriate induction consistently form blastoids, making it an easily scalable model. Taken together, blastoids are ideal models to perform high-throughput drug screenings to identify new drug candidates affecting human embryonic development and implantation. In its current state, blastoids, along with the in vitro implantation assay, can be readily used to identify novel drug candidates to improve IVF outcomes, among several other applications.
Ever since the first IVF baby was born in 1978, the technology has rapidly evolved. Because of societal change towards delayed childbearing wishes among couples, the number of patients opting for IVF procedures has steeply increased. The ever increasing demand has resulted in several innovations in the treatment pipeline. Despite this, currently, the efficiency of live birth per IVF cycle is around 20 per cent, causing severe emotional stress and financial burden on the patient, thus leading to a high dropout rate of the patients.
Currently, there are two major roadblocks to the success of IVF––high-quality embryo formation and the implantation rate. Upon fertilisation of sperm and egg in the lab, the embryo is cultured in the lab for up to five days to reach the stage of blastocyst before it is transferred into the uterus of patients. However, according to experts in the clinic, currently, only around 40 per cent of the fertilised eggs reach the blastocyst stage with sufficient quality for the transfer. Among this small number of “high-quality” embryos, upon transfer into the uterus, only around 40 per cent of them successfully implant to initiate pregnancy. Improving these two steps of blastocyst development and implantation is crucial to ensure a high success rate of IVF.
Blastoids now provide a unique opportunity to screen for large numbers of molecules to identify drug candidates that could be included in the IVF embryo culture media to improve the efficiency of high-quality blastocysts with a high potential to undergo implantation. Using blastoids, we have already identified at least two drug candidates to improve the formation of high-quality blastocysts. We are currently teaming up with IVF clinics in Europe to verify the effects of these drug candidates on human embryos.
With these candidates and several more molecules in our drug screening pipelines, we are confident that the blastoids can be readily used as a faithful drug discovery tool to improve IVF procedures. In the long term, blastoids combined with in vitro implantation assay also provides an opportunity to identify molecular pathways associated with reproductive diseases causing infertility (e.g., endometriosis, repeated implantation failure) and thus, identify new drug targets and drug candidates for their treatment. While the discoveries from blastoids may need validations using a small number of human embryos, before translating it into the clinical settings, it largely reduces the number of embryos required for the research. Overall, blastoids provide an ethical alternative to complement human embryo research and to develop new drugs and treatment strategies in an extremely robust and accelerated manner. It has the potential to disrupt the drug discovery pipelines in women’s reproductive health. [APBN]
- Jarvis, G. E. (2016). Early embryo mortality in natural human reproduction: What the data say. F1000Research, 5, 2765.
- Kagawa, H., Javali, A., Khoei, H. H., Sommer, T. M., Sestini, G., Novatchkova, M., Scholte Op Reimer, Y., Castel, G., Bruneau, A., Maenhoudt, N., Lammers, J., Loubersac, S., Freour, T., Vankelecom, H., David, L., & Rivron, N. (2022). Human blastoids model blastocyst development and implantation. Nature, 601(7894), 600–605.
- Zhao, C., Reyes, A. P., Schell, J. P., Weltner, J., Ortega, N. M., Zheng, Y., Björklund, Å. K., Rossant, J., Fu, J., Petropoulos, S., & Lanner, F. (n.d.). Reprogrammed blastoids contain amnion-like cells but not trophectoderm. DOI: https://doi.org/10.1101/2021.05.07.442980.
About the Authors
Alok Javali, postdoctoral researcher, IMBA-Institute of Molecular Biotechnology, Vienna, Austria
AJ is a postdoctoral researcher in the lab of Nicolas Rivron. He is one of the lead authors of the publication that reported the human blastoid technology.
Nicolas Rivron, group leader, IMBA-Institute of Molecular Biotechnology, Vienna, Austria
NR is a group leader at IMBA-Institute of Molecular Biotechnology, Vienna, Austria. His lab has extensively contributed to the understanding of early mammalian embryonic development and implantation using cutting edge technologies including blastoids.