Human Preimplantation Development Research Articles

X-Chromosome Inactivation during Preimplantation Development and in Pluripotent Stem Cells

X dosage compensation between XX female and XY male mammalian cells is achieved by a process known as X-chromosome inactivation (XCI). XCI initiates early during preimplantation development in female cells, and it is subsequently stably maintained in somatic cells. However, XCI is a reversible process that occurs in vivo in the inner cell mass of the blastocyst, in primordial germ cells or in spermatids during reprogramming. Erasure of transcriptional gene silencing can occur though a mechanism named X-chromosome reactivation (XCR). XCI and XCR have been substantially deciphered in the mouse, whereas they still remain debated in the human. In this review, we summarized the recent advances in the knowledge of X-linked gene dosage compensation during mouse and human preimplantation development and in pluripotent stem cells.

Machine Learning of Single-Cell Transcriptome Highly Identifies mRNA Signature by Comparing F-Score Selection with DGE Analysis

Human preimplantation development is a complex process involving dramatic changes in transcriptional architecture. For a better understanding of their time-spatial development, it is indispensable to identify key genes. Although the single-cell RNA sequencing (RNA-seq) techniques could provide detailed clustering signatures, the identification of decisive factors remains difficult. Additionally, it requires high experimental cost and a long experimental period. Thus, it is highly desired to develop computational methods for identifying effective genes of development signature. In this study, we first developed a predictor called EmPredictor to identify developmental stages of human preimplantation embryogenesis. First, we compared the F-score of feature selection algorithms with differential gene expression (DGE) analysis to find specific signatures of the development stage. In addition, by training the support vector machine (SVM), four types of signature subsets were comprehensively discussed. The prediction results showed that a feature subset with 1,881 genes from the F-score algorithm obtained the best predictive performance, which achieved the highest accuracy of 93.3% on the cross-validation set. Further function enrichment demonstrated that the gene set selected by the feature selection method was involved in more development-related pathways and cell fate determination biomarkers. This indicates that the F-score algorithm should be preferentially proposed for detecting key genes of multi-period data in mammalian early development.

C-kit signaling promotes human pre-implantation 3PN embryonic development and blastocyst formation

BackgroundAlthough in vitro culture system has been optimized in the past few decades, the problem of few or no high quality embryos has been still not completely solved. Accordingly, fully understanding the regulatory mechanism of pre-implantation embryonic development would be beneficial to further optimize the in vitro embryo culture system. Recent studies have found the expression of c-kit in mouse embryo and its promotion effects on mouse embryonic development. However, it is unclear the expression, the role and the related molecular regulatory mechanism of c-kit in human pre-implantation embryo development. Therefore, the present study is to determine whether c-kit is expressed in human pre-implantation embryos, and to investigate the possible regulatory mechanism of c-kit signaling in the process of embryonic development.MethodsThe present study includes human immature oocytes and three pronucleus (3PN) embryos collected from 768 women (28–32 ages) undergoing IVF, and normal 2PN embryos collected from ICR mice. Samples were distributed randomly into three different experimental groups: SCF group: G-1™ (medium for culture of embryos from the pro-nucleate stage to day 3) or G-2™ (medium for culture of embryos from day3 to blastocyst stage) + HSA (Human serum album) solution + rhSCF; SCF + imanitib (c-kit inhibitor) group: G-1™ or G-2™ + HSA solution + rhSCF + imanitib; SCF + U0126 (MEK/ERK inhibitor) group: G-1™ or G-2™ + HSA solution + rhSCF + U0126; Control group: G-1™ or G-2™ + HSA solution + PBS; The rate of good quality embryos at day 3, blastulation at day 6 and good quality blastulation at day 6 were analysis. RT-PCR, western blot and immunofluorescence staining were applied to detect the target genes and proteins in samples collected from human or mice, respectively.Resultsc-kit was expressed ubiquitously in all human immature oocytes, 3PN embryos and 3PN blastocysts. In the experiment of human 3PN embryos, compared with other groups, SCF group showed obviously higher rate of good quality at day 3, better rate of blastocyst formation at day 6 and higher rate of good quality blastocyst formation at day 6. Furthermore, we observed a higher ETV5 expression in SCF group than that in other groups. Similar results were also found in animal experiment. Interestingly, we also found a higher phosphorylation level of MEK/ERK signal molecule in mice embryos from SCF group than those from other groups. Moreover, inhibition of MEK/ERK signaling would remarkably impeded the mice embryonic development, which might be due to the reduced ETV5 expression.ConclusionsThe present study firstly revealed that c-kit signaling might promote the human pre-implantation embryonic development and blastocyst formation by up-regulating the expression of ETV5 via MEK/ERK pathway. Our findings provide a new idea for optimizing the in vitro embryo culture condition during ART program, which is beneficial to obtain high quality embryos for infertile patients.

Spatio-Temporal Analysis of Human Preimplantation Development Reveals Dynamics of Epiblast and Trophectoderm Specification

Recent technological advances such as single-cell (sc) RNAseq and CRISPR-CAS9-mediated knock-out have allowed an unprecedented access to processes orchestrating human preimplantation development. However, the sequence of events which occur during human preimplantation development are still unknown. In particular, the timing of the first human lineage specification, the process by which the morula cells acquire a specific fate, remains elusive. Here, we present a mouse and human preimplantation development model based on transcriptomic pseudotime modelling of scRNAseq biologically validated by spatial information and precise time-lapse staging. We show that in human, trophectoderm (TE) and inner cell mass (ICM) specification in not complete until the B3 blastocyst stage. This reveals a delay with the mouse first specification. We identified novel markers enabling precise staging of human preimplantation embryos: IFI16, associated to the developed epiblast (EPI) and NR2F2, associated with TE development. Strikingly, NR2F2 positive TE cells arise from the polar side, shortly after specification, supporting a model of faster polar TE development leading to polar implantation in the endometrium. Altogether, our study unravels preimplantation lineage specification in human embryos and provides a browsable resource for mapping spatio-temporal events orchestrating human preimplantation development.

Distribution and dynamics of mitochondrial DNA methylation in oocytes, embryos and granulosa cells

Comparison of mitochondrial DNA (mtDNA) methylation patterns in oocytes, blastocysts and ovarian granulosa cells indicates hitherto unsuspected dynamics. Oocytes and blastocysts recovered from cows subjected to ovarian stimulation and from non-stimulated abattoir ovaries were analyzed using bisulphite transformation of DNA followed by whole genome sequencing. The cow is a recognized as a good model for human oocyte and pre-implantation development. The number of mtDNA copies is high in oocytes (200,000–400,000) and early embryos, resulting in very high coverage (>3000x) and very low p values for each of 716 cytosine-based nucleosides. Methylation ratio was lowest in oocytes, following by blastocysts then granulosa cells and was not restricted to CG sites but was found also at CHG and CHH sites. The initial methylation pattern is conserved during the first week of life but not in somatic cells. RNA analysis of mitochondria encoded genes showed a significant inverse correlation between methylation and expression for almost all sequences. Methylation was more extensive in somatic tissues from mature animals than in immature pre-pubertal animals. Our findings suggest that mtDNA methylation might play a programming role during gametogenesis and would be subject to epigenetic regulation according to environment and/or maternal maturity.

An integrated chromatin accessibility and transcriptome landscape of human pre-implantation embryos

Human pre-implantation embryonic development involves extensive changes in chromatin structure and transcriptional activity. Here, we report on LiCAT-seq, a technique that enables simultaneous profiling of chromatin accessibility and gene expression with ultra-low input of cells, and map the chromatin accessibility and transcriptome landscapes for human pre-implantation embryos. We observed global difference in chromatin accessibility between sperm and all stages of embryos, finding that the accessible regions in sperm tend to occur in gene-poor genomic regions. Integrative analyses between the two datasets reveals strong association between the establishment of accessible chromatin and embryonic genome activation (EGA), and uncovers transcription factors and endogenous retrovirus (ERVs) specific to EGA. In particular, a large proportion of the early activated genes and ERVs are bound by DUX4 and become accessible as early as the 2- to 4-cell stages. Our results thus offer mechanistic insights into the molecular events inherent to human pre-implantation development.

BMP4 plays a role in apoptosis during human preimplantation development.

Comprehensive understanding of lineage differentiation and apoptosis processes is important to increase our knowledge of human preimplantation development in vitro. We know that BMP signaling is important for different processes during mammalian development. In mouse preimplantation embryos, BMP signaling has been shown to play a role in the differentiation into extra-embryonic trophectoderm (TE) and primitive endoderm (PE). In this study, we aimed to investigate the effect of bone morphogenetic protein 4 (BMP4) supplementation on human preimplantation embryos cultured in vitro. The BMP4 treatment impaired human blastocyst formation. No differences in the expression of the early lineage markers NANOG, CDX2, GATA3, and GATA6 were found between BMP4-treated embryos and controls. Instead, BMP4 supplementation triggered apoptosis in the human blastocyst. We focused on P53, which is known to play a major role in the apoptosis. In BMP4-treated embryos, the P53 responsive gene expression was not altered; however, the P53 deacetylase SIRT1 was downregulated and acetylated P53 was increased in mitochondria. Altogether, our findings suggest that BMP4 plays a role in the apoptosis during human preimplantation development.

Expression of endogenous retroviruses in pre‐implantation stages of bovine embryo

Endogenous retroviruses (ERVs) are involved in cellular proliferation, pluripotency, tissue-specific remodelling and regulation of developmental processes. These elements are transcriptionally active in mouse and human pre-implantation embryos. Empirical evidence indicates that regulatory networks involved with ERV transcripts are responsible for pluripotency and totipotency at certain stages of mouse and human pre-implantation development. Yet, the expression in pre-implantation bovine embryo remains unidentified. To determine whether two members of bovine endogenous retroviruses, BERV-K1 and BERV-K2, are expressed in the pre-implantation bovine embryo, each embryonic stage developed in vitro and was subjected to RNA release, reverse transcription and quantitative PCR. We found that BERV-K1 and BERV-K2 are expressed throughout different stages of pre-implantation development. The higher level of expression was detected in embryonic blastomeres with totipotent/pluripotent status (two-cell to 16-cell stages), while the more differentiated blastocyst stage showed significantly lower levels of ERVs expression. These findings suggest a possible role for endogenous retroviruses in the establishment of totipotent and pluripotent states in pre-implantation bovine embryo, similar to functions which have been suggested for these elements in human and mouse embryos.

Single-cell multi-omics sequencing of human early embryos.

DNA methylation, chromatin states and their interrelationships represent critical epigenetic information, but these are largely unknown in human early embryos. Here, we apply single-cell chromatin overall omic-scale landscape sequencing (scCOOL-seq) to generate a genome-wide map of DNA methylation and chromatin accessibility at single-cell resolution during human preimplantation development. Unlike in mice, the chromatin of the paternal genome is already more open than that of the maternal genome at the mid-zygote stage in humans, and this state is maintained until the 4-cell stage. After fertilization, genes with high variations in DNA methylation, and those with high variations in chromatin accessibility, tend to be two different sets. Furthermore, 1,797 out of 5,155 (35%) widely open chromatin regions in promoters closed when transcription activity was inhibited, indicating a feedback mechanism between transcription and open chromatin maintenance. Our work paves the way for dissecting the complex, yet highly coordinated, epigenetic reprogramming during human preimplantation development.

The Role of Xist in X-Chromosome Dosage Compensation.

In each somatic cell of a female mammal one X chromosome is transcriptionally silenced via X-chromosome inactivation (XCI), initiating early in development. Although XCI events are conserved in mouse and human postimplantation development, regulation of X-chromosome dosage in preimplantation development occurs differently. In preimplantation development, mouse embryos undergo imprinted form of XCI, yet humans lack imprinted XCI and instead regulate gene expression of both X chromosomes by dampening transcription. The long non-coding RNA Xist/XIST is expressed in mouse and human preimplantation and postimplantation development to orchestrate XCI, but its role in dampening is unclear. In this review, we discuss recent advances in our understanding of the role of Xist in X chromosome dosage compensation in mouse and human.

DNA methylomes of bovine gametes and in vivo produced preimplantation embryos.

DNA methylation is an important epigenetic modification that undergoes dynamic changes in mammalian embryogenesis, during which both parental genomes are reprogrammed. Despite the many immunostaining studies that have assessed global methylation, the gene-specific DNA methylation patterns in bovine preimplantation embryos are unknown. Using reduced representation bisulfite sequencing, we determined genome-scale DNA methylation of bovine sperm and individual in vivo developed oocytes and preimplantation embryos. We show that (1) the major wave of genome-wide demethylation was completed by the 8-cell stage; (2) promoter methylation was significantly and inversely correlated with gene expression at the 8-cell and blastocyst stages; (3) sperm and oocytes have numerous differentially methylated regions (DMRs)-DMRs specific for sperm were strongly enriched in long terminal repeats and rapidly lost methylation in embryos; while the oocyte-specific DMRs were more frequently localized in exons and CpG islands (CGIs) and demethylated gradually across cleavage stages; (4) DMRs were also found between in vivo and in vitro matured oocytes; and (5) differential methylation between bovine gametes was confirmed in some but not all known imprinted genes. Our data provide insights into the complex epigenetic reprogramming of bovine early embryos, which serve as an important model for human preimplantation development.

Chromatin analysis in human early development reveals epigenetic transition during ZGA.

Upon fertilization, drastic chromatin reorganization occurs during preimplantation development 1 . However, the global chromatin landscape and its molecular dynamics in this period remain largely unexplored in humans. Here we investigate chromatin states in human preimplantation development using an improved assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) 2 . We find widespread accessible chromatin regions in early human embryos that overlap extensively with putative cis-regulatory sequences and transposable elements. Integrative analyses show both conservation and divergence in regulatory circuitry between human and mouse early development, and between human pluripotency in vivo and human embryonic stem cells. In addition, we find widespread open chromatin regions before zygotic genome activation (ZGA). The accessible chromatin loci are readily found at CpG-rich promoters. Unexpectedly, many others reside in distal regions that overlap with DNA hypomethylated domains in human oocytes andare enriched for transcription factor-binding sites. A large portion of these regions then become inaccessible after ZGA in a transcription-dependent manner. Notably, such extensive chromatin reorganization during ZGA is conserved in mice and correlates with the reprogramming of the non-canonical histone mark H3K4me3, which is uniquely linked to genome silencing3-5. Taken together, these data not only reveal a conserved principle that underlies the chromatin transition during mammalian ZGA, but also help to advance our understanding of epigenetic reprogramming during human early development and in vitro fertilization.

Investigation of LAMTOR1 gene and protein expressions in germinal vesicle and metaphase II oocytes and embryos from 1-cell to blastocyst stage in a mouse model

Improving the success of in vitro fertilization (IVF) and infertility treatment depend on understanding basic cellular and molecular mechanisms of human preimplantation development. Pre-implantation mouse embryo model is an ideal empiric system to understand these mechanisms. This study was aimed to investigate the gene and protein expressions of LAMTOR1 in mouse oocytes and pre-implantation embryos at different developmental stages. The findings demonstrate that LAMTOR1 was detected in the oocytes and in subsequent all stages of embryo development. The expression was increased progressively from MII-stage oocyte to morula stage embryo (p < 0.05), highest expression was identified in morula stage (p < 0.05), and decreased in blastocyst stage (p < 0.05). Immunofluorescence analysis showed outer and inner nuclear membranes and cytoplasmic subcellular localizations of LAMTOR1 in oocytes and pre-implantation embryos. The LAMTOR1 immunoexpression was gradually increased from MII oocyte and the highest level was detected at the morula stage of embryo development (p < 0.05). The lowest LAMTOR1 immunoexpression was detected at GV-stage oocyte (p < 0.05) and no clear difference in M2 oocyte, I-cell, 2-cell, and blastocyst stage embryos. In conclusion, both the mRNA and protein levels of LAMTOR1 increase progressively in cleavage-stage mouse embryos. LAMTOR1 has a significant higher embryonic expression at 2-cell to morula stage. LAMTOR1 may play a role in the oogenesis process and probably required for further developmental stages and it may play a possible role in the process of compaction and cavitation in mice. Therefore, further studies are needed to explore the LAMTOR1 expression especially in the different stages of embryonal development.

Human Pre-gastrulation Development.

Understanding the progression of early human embryonic development prior to implantation is of fundamental biological importance. Greater insights into early developmental events may lead to clinical improvements, not only via the establishment of novel stem cell models with increased potential or more physiological relevance, but also by uncovering some underlying causes of infertility, miscarriages, and developmental disorders. The majority of human embryos available for study are those donated to research once they are surplus to family building following in vitro fertilization, though in some countries it is also possible to create embryos using donated gametes. As human embryo development is surprisingly inefficient, with only 40% reaching the blastocyst stage in vitro (French, Sabanegh, Goldfarb, & Desai, 2010; Gardner, Lane, Stevens, Schlenker, & Schoolcraft, 2000), many embryos may not develop to a stage suitable for study. Where legally permitted, the oversight of human embryo research is subject to either ethics approval from a local institutional review board (i.e., China and the United States) or both a national regulator as well as a regional research ethics committee (i.e., the United Kingdom). The study of human development has historically been by necessity comparative, relying on model organisms and stem cell lines to inform analyses. Preimplantation mouse and human embryos in particular exhibit remarkably similar gross morphologies at these early stages of development, although key differences have been identified in gene expression patterns and developmental timing. While recent advances in high-resolution transcriptomic analyses at the single cell level have improved our capability to interrogate expression patterns directly in the human embryo, we still lack an understanding of basic molecular events in the human embryo, including how the first cell lineages become specified. Here, we present a current overview of the major developmental events during human preimplantation development, from fertilization to delineation of the embryonic and extraembryonic lineages prior to implantation. Comparisons to both the mouse and alternative models are included where these have formed the basis for similar investigations in a human context.

Time-Lapse Imaging for the Detection of Chromosomal Abnormalities in Primate Preimplantation Embryos.

The use of time-lapse microscopic imaging has proven to be a powerful tool for the study of mitotic divisions and other cellular processes across diverse species and cell types. Although time-lapse monitoring (TLM) of human preimplantation development was first introduced to the in vitro fertilization (IVF) community several decades ago, it was not until relatively recently that TLM systems were commercialized for clinical embryology purposes. Traditionally, human IVF embryos are assessed by successful progression and morphology under a stereomicroscope at distinct time points prior to selection for transfer. Due to the high frequency of aneuploidy, embryos may also be biopsied at the cleavage or blastocyst stage for preimplantation genetic screening (PGS) of whole and/or partial chromosomal abnormalities. However, embryo biopsy is invasive and can hinder subsequent development, and there are additional concerns over chromosomal mosaicism and resolution with PGS. Moreover, embryos are typically outside of the incubator in suboptimal culture conditions for extended periods of time during these procedures. With TLM systems, embryos remain in the stable microenvironment of an incubator and are simultaneously imaged for noninvasive embryo evaluation using a fraction of the light exposure as compared to a stereomicroscope. Each image is then compiled into a time-lapse movie, the information from which can be extrapolated to correlate morphological, spatial, and temporal parameters with embryo quality and copy number status. Here, we describe the various TLM systems available for clinical and/or research use in detail and provide step-by-step instructions on how the measurement of specific timing intervals and certain morphological criteria can be implemented into IVF protocols to enhance embryo assessment and avoid the selection of aneuploid embryos. We also discuss the biological significance of processes unique to mitotically dividing embryos and the likelihood that complex chromosomal events such as chromothripsis occur during preimplantation development in humans and other mammals, particularly nonhuman primates.

ROCK and RHO Playlist for Preimplantation Development: Streaming to HIPPO Pathway and Apicobasal Polarity in the First Cell Differentiation.

In placental mammalian development, the first cell differentiation produces two distinct lineages that emerge according to their position within the embryo: the trophectoderm (TE, placenta precursor) differentiates in the surface, while the inner cell mass (ICM, fetal body precursor) forms inside. Here, we discuss how such position-dependent lineage specifications are regulated by the RHOA subfamily of small GTPases and RHO-associated coiled-coil kinases (ROCK). Recent studies in mouse show that activities of RHO/ROCK are required to promote TE differentiation and to concomitantly suppress ICM formation. RHO/ROCK operate through the HIPPO signaling pathway, whose cell position-specific modulation is central to establishing unique gene expression profiles that confer cell fate. In particular, activities of RHO/ROCK are essential in outside cells to promote nuclear localization of transcriptional co-activators YAP/TAZ, the downstream effectors of HIPPO signaling. Nuclear localization of YAP/TAZ depends on the formation of apicobasal polarity in outside cells, which requires activities of RHO/ROCK. We propose models of how RHO/ROCK regulate lineage specification and lay out challenges for future investigations to deepen our understanding of the roles of RHO/ROCK in preimplantation development. Finally, as RHO/ROCK may be inhibited by certain pharmacological agents, we discuss their potential impact on human preimplantation development in relation to fertility preservation in women.

Focused time-lapse analysis reveals novel aspects of human fertilization and suggests new parameters of embryo viability.

Can focused application of time-lapse microscopy (TLM) lead to a more detailed map of the morphokinetics of human fertilization, revealing novel or neglected aspects of this process? Intensive harnessing of TLM reveals novel or previously poorly characterised phenomena of fertilization, such as a cytoplasmic wave (CW) preceding pronuclear formation and kinetics of pronuclear chromatin polarization, thereby suggesting novel non-invasive biomarkers of embryo quality. In recent years, human preimplantation development has been the object of TLM studies with the intent to develop morphokinetic algorithms able to predict blastocyst formation and implantation. Regardless, our appreciation of the morphokinetics of fertilization remains rather scarce, currently including only times of polar body II (PBII) emission, pronuclear appearance and fading, and first cleavage. This is not consistent with the complexity and importance of this process, calling for further TLM studies aimed at describing previously unrecognized or undetected morphokinetic events and identifying novel developmental biomarkers. The study involved a retrospective observation by TLM of the fertilization process in 500 oocytes utilized in consecutive ICSI cycles carried out in 2016. A maximum of five fertilized oocytes per patients were included in the analysis to reduce possible patient-specific biases. Oocytes of patients with different diagnoses of infertility where included in the analysis, while cases involving cryopreserved gametes or surgically retrieved sperm were excluded. Microinjected oocytes where assessed by a combined TLM-culture system (Embryoscope). Oocytes that were not amenable to TLM assessment, due to excess of residual corona cells or inadequate orientation for the observation of PBII emission, were not analysed. We identified and monitored 28 parameters relevant to meiotic resumption, pronuclear dynamics, chromatin organization, and cytoplasmic/cortical modifications. Times (T) were expressed as mean ± SD hours post-insemination (p.i.) and analysed, where appropriate, by Paired T Student or Fisher's exact tests. PBII emission was occasionally followed (4.3% of cases) by the transient appearance of a protrusion of the cell surface, the fertilization cone (FC), probably resulting from interaction of the male chromatin with the oocyte cortex. Pronuclear formation was always preceded by a radial CW originating from the initial position of the male pronucleus (PN) and extending towards the oocyte periphery. The appearance of the CW followed a precise sequence, occurring always 2-3 h after PBII emission and shortly before PN appearance. Male and female PN appeared virtually simultaneously at approximately 6.2 h p.i. However, while the female PN always formed cortically and near the site of emission of the PBII, the initial position of the male PN was cortical, intermediate, or central (15.2%, 31.2% and 53.6%, respectively). PN juxtaposition involved rapid and straight movement of the female PN towards the male PN. In addition, the initial position of male PN formation was predictive of the position of PN juxtaposition. It was also observed that nucleolar precursor bodies (NPBs) aligned along the juxtaposition area and this happened considerably earlier for the female PN (8.2 ± 2.6 vs.11.2 ± 4.1 h, P = 0.0001). Although it occurred rarely, displacement of juxtaposed PN to the cortex was strongly associated (P < 0.0001) with direct cleavage into three blastomeres at the first cell division. The times of PN breakdown and first cleavage showed a very consistent trend, occurring earlier or progressively later depending on whether initial male PN positioning was central, intermediate or cortical, respectively. Finally, time intervals between discrete fertilization events were strongly associated with embryo quality on Day 3. For example, longer intervals between disappearance of the cytoplasmic halo and PN breakdown were highly predictive of reduced blastomere number and increased fragmentation (P = 0.0001). N/A. Some of the morphokinetic parameters assessed in this study may require better definition to reduce inter-operator annotation variability. To our knowledge, overall, these data represent the most detailed morphokinetic description of human fertilization. Many of the illustrated parameters are novel and may be amenable to further elaboration into algorithms able to predict embryo quality, as suggested by the findings presented in this study. None.

Early X chromosome inactivation during human preimplantation development revealed by single-cell RNA-sequencing

In female mammals, one X chromosome is transcriptionally inactivated (XCI), leading to dosage compensation between sexes, fundamental for embryo viability. A previous study using single-cell RNA-sequencing (scRNA-seq) data proposed that female human preimplantation embryos achieve dosage compensation by downregulating both Xs, a phenomenon named dampening of X expression. Using a novel pipeline on those data, we identified a decrease in the proportion of biallelically expressed X-linked genes during development, consistent with XCI. Moreover, we show that while the expression sum of biallelically expressed X-linked genes decreases with embryonic development, their median expression remains constant, rejecting the hypothesis of X dampening. In addition, analyses of a different dataset of scRNA-seq suggest the appearance of X-linked monoallelic expression by the late blastocyst stage in females, another hallmark of initiation of XCI. Finally, we addressed the issue of dosage compensation between the single active X and autosomes in males and females for the first time during human preimplantation development, showing emergence of X to autosome dosage compensation by the upregulation of the active X chromosome in both male and female embryonic stem cells. Our results show compelling evidence of an early process of X chromosome inactivation during human preimplantation development.

Tripolar mitosis and partitioning of the genome arrests human preimplantation development in vitro

Following in vitro fertilisation (IVF), only about half of normally fertilised human embryos develop beyond cleavage and morula stages to form a blastocyst in vitro. Although many human embryos are aneuploid and genomically imbalanced, often as a result of meiotic errors inherited in the oocyte, these aneuploidies persist at the blastocyst stage and the reasons for the high incidence of developmental arrest remain unknown. Here we use genome-wide SNP genotyping and meiomapping of both polar bodies to identify maternal meiotic errors and karyomapping to fingerprint the parental chromosomes in single cells from disaggregated arrested embryos and excluded cells from blastocysts. Combined with time lapse imaging of development in culture, we demonstrate that tripolar mitoses in early cleavage cause chromosome dispersal to clones of cells with identical or closely related sub-diploid chromosome profiles resulting in intercellular partitioning of the genome. We hypothesise that following zygotic genome activation (ZGA), the combination of genomic imbalance and partial genome loss disrupts the normal pattern of embryonic gene expression blocking development at the morula-blastocyst transition. Failure to coordinate the cell cycle in early cleavage and regulate centrosome duplication is therefore a major cause of human preimplantation developmental arrest in vitro.

Defining the three cell lineages of the human blastocyst by single-cell RNA-seq

Here, we provide fundamental insights into early human development by single-cell RNA-sequencing of human and mouse preimplantation embryos. We elucidate conserved transcriptional programs along with those that are human specific. Importantly, we validate our RNA-sequencing findings at the protein level, which further reveals differences in human and mouse embryo gene expression. For example, we identify several genes exclusively expressed in the human pluripotent epiblast, including the transcription factor KLF17. Key components of the TGF-β signalling pathway, including NODAL, GDF3, TGFBR1/ALK5, LEFTY1, SMAD2, SMAD4 and TDGF1, are also enriched in the human epiblast. Intriguingly, inhibition of TGF-β signalling abrogates NANOG expression in human epiblast cells, consistent with a requirement for this pathway in pluripotency. Although the key trophectoderm factors Id2, Elf5 and Eomes are exclusively localized to this lineage in the mouse, the human orthologues are either absent or expressed in alternative lineages. Importantly, we also identify genes with conserved expression dynamics, including Foxa2/FOXA2, which we show is restricted to the primitive endoderm in both human and mouse embryos. Comparison of the human epiblast to existing embryonic stem cells (hESCs) reveals conservation of pluripotency but also additional pathways more enriched in hESCs. Our analysis highlights significant differences in human preimplantation development compared with mouse and provides a molecular blueprint to understand human embryogenesis and its relationship to stem cells.