Regulators of cell fate decisions in human pre-implantation embryos

Human fecundity is remarkably low and lies around 30% per ovulatory cycle. Medically assisted reproductive technologies such as in vitro fertilization (IVF) aim to treat couples that fail to achieve a successful pregnancy. However, the success rates of these techniques have also remained relatively low, typically ranging between 20-30%. It is well understood that 30% of miscarriages are lost during human pre-implantation development and another 30% directly after implantation of the embryo in the endometrium. Until today researchers can only poorly explain the underlying mechanisms behind early pregnancy loss. Furthermore, most regulators involved in various cell fate decisions during the first week of human pre-implantation development remain poorly understood; most knowledge relies on model systems such as the mouse embryo. This lack of knowledge is due to ethical and legal restrictions concerning research on human embryos in many countries coupled with technical limitations that restrict functional studies. Therefore, in this thesis we aimed to study several different cell fate decisions during human pre-implantation development.

Our first study (chapter 2) focused on the regulators of lineage segregation events during which first the trophectoderm (TE) and inner cell mass (ICM) are formed from which in a second step the epiblast (EPI) and primitive endoderm (PrE) arise. The EPI will develop into the embryo proper and amnion, TE will give rise to the placenta and the PrE to the yolk sac. In the mouse, YAP1 and the co- activators TEAD1-4 regulate differentiation towardsTE and EPI and their expression is controlled by cell polarity and the Hippo-signaling pathway. Inner/apolar cells exclude YAP1 from the nucleus through phosphorylation which upregulates Sox2 and downregulates Cdx2 while the apical domain of polar outer cells promotes YAP1/TEAD4 activity which upregulates Cdx2 and Gata3. YAP1/TEAD1 activity, however, drives the formation of EPI through cell competition. We analyzed protein localization of Hippo-signaling pathway components and lineage specifiers in human embryos from 3-6 days postfertilization, covering compaction, polarization, cell positioning, and specification events. Our main finding is that, in contrast to the mouse, YAP1/TEAD1 specify the TE and PrE and that differentiation towards TE occurs independently from polarity.

One of the main reasons for reduced fecundity and low IVF success rates is the high incidence of chromosomally abnormal or aneuploid cells in human embryos. While most aneuploidies that affect every cell of the embryo are detrimental, transfer of embryos containing cells with both a normal and abnormal (mosaic) karyotype can lead to healthy live-births. Moreover, the proportion of aneuploid cells decreases from 80% at the cleavage stages to 20% at the blastocyst stage. Thus, our second study (chapter 3) focused on the cellular consequences of aneuploidy in human embryonic cells. We found that aneuploid cells are cleared from the embryo by proteotoxic stress with subsequent activation of p53-mediated apoptosis which might explain why a chromosomally mosaic embryo can lead to a healthy live-birth. Also, aneuploidy impaired PrE development, which might explain why aneuploid embryo fail to implant.

Overall, we revealed specifiers of the first and second lineage segregation event and several cellular consequences of aneuploidy during the first week of human pre-implantation development. These results contribute to our understanding of fundamental processes during early human development. Since we highlighted profound differences with the mouse embryo, we consider research on human embryos to be highly relevant.