Apico-basal polarization of cells within the embryo is critical for the segregation of distinct lineages during mammalian development. Polarized cells become the trophectoderm (TE), which forms the placenta, and apolar cells become the inner cell mass (ICM), the founding population of the fetus. The cellular and molecular mechanisms leading to polarization of the human embryo and its timing during embryogenesis have remained unknown. Here, we show that human embryo polarization occurs in two steps: it begins with the apical enrichment of F-actin and is followed by the apical accumulation of the PAR complex. This two-step polarization process leads to the formation of an apical domain at the 8-16 cell stage. Using RNA interference, we show that apical domain formation requires Phospholipase C (PLC) signaling, specifically the enzymes PLCB1 and PLCE1, from the 8-cell stage onwards. Finally, we show that although expression of the critical TE differentiation marker GATA3 can be initiated independently of embryo polarization, downregulation of PLCB1 and PLCE1 decreases GATA3 expression through a reduction in the number of polarized cells. Therefore, apical domain formation reinforces a TE fate. The results we present here demonstrate how polarization is triggered to regulate the first lineage segregation in human embryos.
The formation of differential cell lineages in the mammalian blastocyst from the totipotent zygote is crucial for implantation and the success of the whole pregnancy. The first lineage segregation generates the polarised trophectoderm (TE) tissue, which forms the placenta, and the apolar inner cell mass (ICM), which mainly gives rise to all foetal tissues and also the yolk sac1-3. The mechanism underlying this cell fate segregation has been extensively studied in the mouse embryo4,5. However, when and how it takes place in the human embryo remains unclear. Here, using time-lapse imaging and 325 surplus human embryos, we provide a detailed characterisation of morphological events and transcription factor expression and localisation to understand how they lead to the first lineage segregation in human embryogenesis. We show that the first lineage segregation of the human embryo is triggered by cell polarisation that occurs at the 8-cell stage in two sequential steps. In the first step, F-actin becomes apically polarised concomitantly with embryo compaction. In the second step, the Par complex becomes polarised to form the apical cellular domain. Mechanistically, we show that activation of Phospholipase C (PLC) triggers actin polarisation and is therefore essential for apical domain formation, as is the case in mouse embryos6. Finally, we show that, in contrast to the mouse embryo, the key extra-embryonic determinant GATA37,8 is expressed not only in extra-embryonic lineage precursors upon blastocyst formation. However, the cell polarity machinery enhances the expression and nuclear accumulation of GATA3. In summary, our results demonstrate for the first time that cell polarisation reinforces the first lineage segregation in the human embryo.
Aneuploidy in preimplantation embryos is a major cause of human reproductive failure. Unlike uniformly aneuploid embryos, embryos diagnosed as diploid-aneuploid mosaics after preimplantation genetic testing for aneuploidy (PGT-A) can develop into healthy infants. However, the reason why these embryos achieve full reproductive competence needs further research. Current RNA sequencing techniques allow for the investigation of the human preimplantation transcriptome, providing new insights into the molecular mechanisms of embryo development. In this prospective study, using euploid embryo gene expression as a control, we compared the transcriptome profiles of inner cell mass and trophectoderm samples from blastocysts with different levels of chromosomal mosaicism. A total of 25 samples were analyzed from 14 blastocysts with previous PGT-A diagnosis, including five low-level mosaic embryos and four high-level mosaic embryos. Global gene expression profiles visualized in cluster heatmaps were correlated with the original PGT-A diagnosis. In addition, gene expression distance based on the number of differentially expressed genes increased with the mosaic level, compared to euploid controls. Pathways involving apoptosis, mitosis, protein degradation, metabolism, and mitochondrial energy production were among the most deregulated within mosaic embryos. Retrospective analysis of the duration of blastomere cell cycles in mosaic embryos revealed several mitotic delays compared to euploid controls, providing additional evidence of the mosaic status. Overall, these findings suggest that embryos with mosaic results are not simply a misdiagnosis by-product, but may also have a genuine molecular identity that is compatible with their reproductive potential.
Study question Does transcriptome of remaining trophectoderm (TE) reflect the developmental potential of mosaic blastocysts after preimplantation genetic testing for aneuploidy (PGT-A)? Summary answer: TE from low-degree mosaic (Low-mos) and high-degree mosaic (High-mos) blastocysts are transcriptionally equivalent, standing between euploid and aneuploid categories and displaying key deregulated developmental processes. What is known already Blastocysts classified as mosaic by PGT-A are associated with lower implantation and higher miscarriage rates than those classified as euploid, yet they still lead to healthy babies. Unveiling the true developmental identity of these embryos faces a dilemma: understanding to which extent they represent technical artefacts or whether they hold own potential to implant and give rise to normal pregnancies. Current RNA sequencing (RNA-seq) techniques allow for the determination of whole transcriptomic profiles even from single cells, which paves the way for the identification of new molecular keys of embryonic competence. Study design, size, duration Prospective study comparing RNA-seq data of remaining TE from blastocysts classified as euploid (n = 4), Low-mos (n = 5), High-mos (n = 4) and aneuploid (n = 6) by PGT-A. Participants were recruited between October 2018 and November 2019 at IVI-RMA Valencia. Participants/materials, setting, methods Chromosomal mosaicism was defined in the range 30%-<50% (Low-mos) and 50%-<70% (High-mos) using a next-generation sequencing (NGS) validated algorithm. Whole TE fractions were separately collected and processed for RNA-seq. Differentially expressed genes (DEGs) were calculated with DESeq2 package [Benjamini-Hochberg (BH)-adjusted p < 0.01 & abs(log2FoldChange)>2 significant]. Fgsea algorithm was used for enrichment analysis on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms (BH-adjusted p < 0.01 significant). Main results and the role of chance For comparisons, TE from euploid blastocysts were used as control. At the gene level, 15 DEGs were found in Low-mos, 20 DEGs in High-mos, and 64 DEGs in aneuploid blastocysts. To address the functional implications of these differences, pathways significantly deregulated according to KEGG and GO categories were identified. TE from aneuploid blastocysts displayed significant downregulation in up to 115 KEGG and GO processes directly involved in processing and integrity maintenance of nuclear and mitochondrial genomes, a reflection of their aberrant chromosomal identity. In addition, TE from High-mos and Low-mos were transcriptionally equivalent (0 DEGs between both groups), with 23 overlapping KEGG and GO processes significantly downregulated compared with control. Importantly, main significantly-affected processes included mitotic sister chromatid segregation, NIK NF-kB activity, regulation of apoptosis, and pathways related to the biosynthesis and metabolism of proteins, fatty acids, carbohydrates and steroid hormones. These findings indicate that mosaic embryos comprise a unique developmental entity, which swims between the euploid and aneuploid waterfronts and may regulate survival by diverse mechanisms, including cell proliferation and apoptosis. Limitations, reasons for caution This is a descriptive, single-center study with limited sample size. TE fractions were obtained by micromanipulation, which may have led to potential cross-contamination with the inner cell mass. Wider implications of the findings: Transcriptomic equivalence between Low-mos and High-mos TE fractions questions the biological significance of inferring mosaicism degrees from single biopsies. Deregulated processes in these embryos support their reduced developmental and live birth potential, pointing to mechanisms that may mediate survival in the presence of aneuploid cells, as shown in the mouse. Trial registration number Not applicable
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