Loss of KANSL3 leads to defective inner cell mass and early embryonic lethality.

e-Lysine acetylation is a prominent histone mark found at transcriptionally active loci. Among many lysine acetyl transferases, nonspecific lethal complex (NSL) members are known to mediate the modification of histone H4. In addition to histone modifications, the KAT8 regulatory complex subunit 3 gene (Kansl3), a core member of NSL complex, has been shown to be involved in several other cellular processes such as mitosis and mitochondrial activity. Although functional studies have been performed on NSL complex members, none of the four core proteins, including Kansl3, have been studied during early mouse development. Here we show that homozygous knockout Kansl3 embryos are lethal at peri-implantation stages, failing to hatch out of the zona pellucida. When the zona pellucida is removed in vitro, Kansl3 null embryos form an abnormal outgrowth with significantly disrupted inner cell mass (ICM) morphology. We document lineage-specific defects at the blastocyst stage with significantly reduced ICM cell number but no difference in trophectoderm cell numbers. Both epiblast and primitive endoderm lineages are altered with reduced cell numbers in null mutants. These results show that Kansl3 is indispensable during early mouse embryonic development and with defects in both ICM and trophectoderm lineages.

MEK signalling pathway is required for hypoblast specification and migration in ovine.

In brief: MEK signalling pathway is required for hypoblast differentiation in mouse embryos, but its role in ungulate embryos remains controversial. This paper demonstrates that MEK is required for hypoblast specification in the inner cell mass of the ovine blastocyst and that it plays a role during the hypoblast migration occurring following blastocyst hatching. Abstract: Early embryo development requires the differentiation of three cell lineages in two differentiation events. The second lineage specification differentiates the inner cell mass into epiblast, which will form the proper fetus, and hypoblast, which together with the trophectoderm will form the extraembryonic membranes and the fetal part of the placenta. MEK signalling pathway is required for hypoblast differentiation in mouse embryos, but its role in ungulate embryos remains controversial. The aim of this work was to analyse the role of MEK signalling on hypoblast specification at the blastocyst stage and on hypoblast migration during post-hatching stages in vitro in the ovine species. Using well-characterized and reliable lineage markers, and different MEK inhibitor concentrations, we demonstrate that MEK signalling pathway is required for hypoblast specification in the inner cell mass of the ovine blastocyst, and that it plays a role during the hypoblast migration occurring following blastocyst hatching. These results show that the role of MEK signalling pathway on hypoblast specification is conserved in phylogenetically distant mammals.

Improved prediction of clinical pregnancy using artificial intelligence with enhanced inner cell mass and trophectoderm images.

This study aimed to assess the performance of an artificial intelligence (AI) model for predicting clinical pregnancy using enhanced inner cell mass (ICM) and trophectoderm (TE) images. In this retrospective study, we included static images of 2555 day-5-blastocysts from seven in vitro fertilization centers in South Korea. The main outcome of the study was the predictive capability of the model to detect clinical pregnancies (gestational sac). Compared with the original embryo images, the use of enhanced ICM and TE images improved the average area under the receiver operating characteristic curve for the AI model from 0.716 to 0.741. Additionally, a gradient-weighted class activation mapping analysis demonstrated that the enhanced image-trained AI model was able to extract features from crucial areas of the embryo in 99% (506/512) of the cases. Particularly, it could extract the ICM and TE. In contrast, the AI model trained on the original images focused on the main areas in only 86% (438/512) of the cases. Our results highlight the potential efficacy of using ICM- and TE-enhanced embryo images when training AI models to predict clinical pregnancy.

Differential Gene Regulation of the Human Blastocyst Trophectoderm and Inner Cell Mass by Progesterone.

Knowledge of action of progesterone (P4) on the human preimplantation embryo is lacking. The objective of this study was to determine expression of a mitochondrial P4 receptor (PR-M) in the trophectoderm (TE) and the inner cell mass (ICM) of the human blastocyst and to determine P4-induced gene expression during growth from the cleavage to the blastocyst stage. Previously cryopreserved cleavage stage embryos were treated with P4 (10-6 M) or vehicle until blastocyst development. Cells from the TE and the ICM of dissected euploid embryos underwent RNA-seq analysis, while other embryos were used for analysis of nuclear PR (nPR) and PR-M expression.PR-M expression was confirmed in the TE, the ICM, and a human embryonic stem cell line (HESC). Conversely, nPR expression was absent in the TE and the ICM with low expression in the HESC line. RNA-seq analysis revealed P4 effects greater in the TE with 183 significant pathway changes compared to 27 in the ICM. The TE response included significant upregulation of genes associated with DNA replication, cell cycle phase transition and others, exemplified by a 7.6-fold increase in the cell proliferation gene, F-Box Associated Domain Containing. The majority of ICM pathways were downregulated including chromosome separation, centromere complex assembly and chromatin remodeling at centromere. This study confirms that human blastocysts express PR-M in both the TE and the ICM, but lack expression of nPR. P4-induced gene regulation differs greatly in the two cell fractions with the predominant effect of cell proliferation in the TE and not the ICM.

Efficient induction and sustenance of pluripotent stem cells from bovine somatic cells.

Although derivation of naïve bovine embryonic stem cells is unachieved, the possibility for generation of bovine induced pluripotent stem cells (biPSCs) has been generally reported. However, attempts to sustain biPSCs by promoting self-renewal have not been successful. Methods established for maintaining murine and human induced pluripotent stem cells (iPSCs) do not support self-renewal of iPSCs for any bovid species. In this study, we examined methods to enhance complete reprogramming and concurrently investigated signaling relevant to pluripotency of the bovine blastocyst inner cell mass (ICM). First, we identified that forced expression of SV40 large T antigen together with the reprogramming genes (OCT4, SOX2, KLF4 and MYC) substantially enhanced the reprogramming efficacy of bovine fibroblasts to biPSCs. Second, we uncovered that TGFß signaling is actively perturbed in the ICM. Inhibition of ALK4/5/7 to block TGFß/activin/nodal signaling together with GSK3ß and MEK1/2 supported robust in vitro self-renewal of naïve biPSCs with unvarying colony morphology, steady expansion, expected pluripotency gene expression and committed differentiation plasticity. Core similarities between biPSCs and stem cells of the 16-cell-stage bovine embryo indicated a stable ground state of pluripotency; this allowed us to reliably gain predictive understanding of signaling in bovine pluripotency using systems biology approaches. Beyond defining a high-fidelity platform for advancing biPSC-based biotechnologies that have not been previously practicable, these findings also represent a significant step towards understanding corollaries and divergent aspects of bovine pluripotency. This article has an associated First Person interview with the joint first authors of the paper.

Klf5 establishes bi-potential cell fate by dual regulation of ICM and TE specification genes.

Early blastomeres of mouse preimplantation embryos exhibit bi-potential cell fate, capable of generating both embryonic and extra-embryonic lineages in blastocysts. Here we identify three major two-cell-stage (2C)-specific endogenous retroviruses (ERVs) as the molecular hallmark of this bi-potential plasticity. Using the long terminal repeats (LTRs) of all three 2C-specific ERVs, we identify Krüppel-like factor 5 (Klf5) as their major upstream regulator. Klf5 is essential for bi-potential cell fate; a single Klf5-overexpressing embryonic stem cell (ESC) generates terminally differentiated embryonic and extra-embryonic lineages in chimeric embryos, and Klf5 directly induces inner cell mass (ICM) and trophectoderm (TE) specification genes. Intriguingly, Klf5 and Klf4 act redundantly during ICM specification, whereas Klf5 deficiency alone impairs TE specification. Klf5 is regulated by multiple 2C-specific transcription factors, particularly Dux, and the Dux/Klf5 axis is evolutionarily conserved. The 2C-specific transcription program converges on Klf5 to establish bi-potential cell fate, enabling a cell state with dual activation of ICM and TE genes.

Mitochondrial maturation in the trophectoderm and inner cell mass regions of bovine blastocysts.

Cellular differentiation induces various morphological changes, including elongation, in mitochondria. Preimplantation embryos have round-shaped mitochondria, characteristic of undifferentiated cells. However, there is controversy regarding the precise mitochondrial morphology in blastocyst embryos, which are generated from two cell lineages: undifferentiated inner cell mass (ICM) and differentiated trophectoderm (TE). This study attempted to precisely determine mitochondrial morphology in these two blastocyst regions. Transmission electron microscopy analyses were conducted using more than 1000 mitochondria from blastocyst embryos. No significant differences were observed in the configuration of mitochondrial cristae and frequencies of hooded mitochondria, which are specific to embryos of livestock animals, between the ICM and TE. To accurately compare mitochondrial roundness between the ICM and TE, oblateness was calculated based on both the major and minor axes. Average oblateness was significantly greater in the TE than in the ICM (P < 0.01). These results indicate tissue-specific mitochondrial maturation with complete elongation in the TE at the blastocyst stage. Since mitochondrial elongation is closely associated with cellular metabolism and differentiation, the present study provides new insights for better understanding of early embryonic development in cattle.

Blastocyst morphology is associated with the incidence of monozygotic twinning in assisted reproductive technology.

BACKGROUND: An increased incidence of monozygotic twinning after a blastocyst transfer has been previously reported in assisted reproductive technology treatment. It is uncertain whether this phenomenon is due to the extended culture time, culture medium, or inherent blastocyst parameters. OBJECTIVE: This study aimed to investigate the association between blastocyst parameters (in vitro culture time, blastocyst stage, and inner cell mass and trophectoderm grading) and the incidence of monozygotic twinning after assisted reproductive technology. STUDY DESIGN: This was a retrospective cohort study employing data from a multicenter, large, electronic database from 4 academic hospitals. All clinical pregnancies after a single blastocyst transfer between January 2014 and February 2020 were included. Blastocyst morphology was evaluated based on the Gardner grading system, considering the blastocyst stage, and inner cell mass and trophectoderm grading (grades A, B, and C). Monozygotic twinning was defined as ≥2 fetal heartbeats in a single gestational sac or 2 gestational sacs with sex concordance at birth. The multivariable predicted marginal proportions from logistic regression models were used to compute adjusted relative risks for the association between blastocyst parameters and the incidence of monozygotic twinning. RESULTS: The overall monozygotic twinning rate was 1.53% (402 of 26,254 cases). The monozygotic twinning was not associated with the culture time in vitro (day 5 vs day 6) or blastocyst stage (early, blastocyst, expanded, hatching, and hatched). Alternatively, monozygotic twinning was associated with lower inner cell mass grading (B vs A: adjusted relative risk, 1.67 [95 % confidence interval, 1.28-2.25]; C vs A: adjusted relative risk, 1.98 [95% confidence interval, 1.18-3.11]) and higher trophectoderm grading (B vs C: adjusted relative risk, 1.38 [95% confidence interval, 1.03-1.92]; A vs C: adjusted relative risk, 2.14 [95% confidence interval, 1.45-3.20]). The incidence of monozygotic twinning was the lowest in blastocysts with grade A inner cell mass and grade B or C trophectoderm (0.82%, as the reference) and the highest in blastocysts with grade B or C inner cell mass and grade A trophectoderm (2.40%; adjusted relative risk, 2.62; 95% confidence interval, 1.60-4.43). The incidence of monozygotic twinning in blastocysts with consistent inner cell mass or trophectoderm grading was somewhere in between (both A: 1.58%; adjusted relative risk, 1.86 [95% confidence interval, 1.23-3.04]; both B or C: 1.59%; adjusted relative risk, 1.84 [95% confidence interval, 1.29-2.90]). CONCLUSION: Higher risk of monozygotic twinning was associated with blastocyst morphology specific to those blastocysts with loosely arranged inner cell mass cells combined with tightly packed trophectoderm cells.

Lysophosphatidic Acid Accelerates Bovine In Vitro-Produced Blastocyst Formation through the Hippo/YAP Pathway.

The segregation of trophectoderm (TE) and inner cell mass in early embryos is driven primarily by the transcription factor CDX2. The signals that trigger CDX2 activation are, however, less clear. In mouse embryos, the Hippo-YAP signaling pathway is important for the activation of CDX2 expression; it is less clear whether this relationship is conserved in other mammals. Lysophosphatidic acid (LPA) has been reported to increase YAP levels by inhibiting its degradation. In this study, we cultured bovine embryos in the presence of LPA and examined changes in gene and protein expression. LPA was found to accelerate the onset of blastocyst formation on days 5 and 6, without changing the TE/inner cell mass ratio. We further observed that the expression of TAZ and TEAD4 was up-regulated, and YAP was overexpressed, in LPA-treated day 6 embryos. However, LPA-induced up-regulation of CDX2 expression was only evident in day 8 embryos. Overall, our data suggest that the Hippo signaling pathway is involved in the initiation of bovine blastocyst formation, but does not affect the cell lineage constitution of blastocysts.

Retinoic acid signaling is critical during the totipotency window in early mammalian development.

Totipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such '2-cell-like-cells' (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.

Roles of cell differentiation factors in preimplantation development of domestic animals.

In mammalian embryos, the first visible differentiation event is the segregation of the inner cell mass (ICM) and trophectoderm (TE) during the transition from the morula to the blastocyst stage. The ICM, which is attached to the inside of the TE, develop into the fetus and extraembryonic tissues, while the TE, which is a single layer surrounding the fluid-filled cavity called the blastocoel, will provide extraembryonic structures such as the placenta. ICM/TE differentiation is regulated by the interaction between various transcriptional factors. However, little information is available on the segregation of the ICM and TE lineages in preimplantation embryos of domestic animals, such as cattle and pigs. This review focuses on the roles of cell differentiation factors that regulate the ICM/TE segregation of preimplantation bovine and porcine embryos. Understanding the mechanism of cell differentiation in early embryos is necessary to improve the in vitro production systems for bovine and porcine embryos.

Rescuing monopronucleated-derived human blastocysts: a model to study chromosomal topography and fingerprinting.

OBJECTIVE: To quantify the percentage of monopronuclear-derived blastocysts (MNBs) that are potentially useful for reproductive purposes using classic and state-of-the-art chromosome analysis approaches, and to study chromosomal distribution in the inner cell mass (ICM) and trophectoderm (TE) for intertissue/intratissue concordance comparison. DESIGN: Prospective experimental study. SETTING: Single-center in vitro fertilization clinic and reproductive genetics laboratory. PATIENT(S): A total of 1,128 monopronuclear zygotes were obtained between June 2016 and December 2018. INTERVENTION(S): MNBs were whole-fixed or biopsied to obtain a portion of ICM and 2 TE portions (TE1 and TE2) and were subsequently analyzed by fluorescence in situ hybridization, new whole-genome sequencing, and fingerprinting by single-nucleotide polymorphism array-based techniques (a-SNP). MAIN OUTCOME MEASURE(S): We assessed MNB rate, ploidy rate, and chromosomal constitution by new whole-genome sequencing, and parental composition by comparative a-SNP, performed in a "trio"-format (embryo/parents). The 24-chromosome distribution was compared between the TE and the ICM and within the TE. RESULT(S): A total of 18.4% of monopronuclear zygotes progressed to blastocysts; 77.6% of MNBs were diploid; 20% of MNBs were male and euploid, which might be reproductively useful. Seventy-five percent of MNBs were biparental and half of them were euploid, indicating that 40% might be reproductively useful. Intratissue concordance (TE1/TE2) was established for 93.3% and 73.3% for chromosome matching. Intertissue concordance (TE/ICM) was established for 78.8%, but 57.6% for chromosome matching. When segmental aneuploidy was not considered, intratissue concordance and chromosome matching increased to 100% and 80%, respectively, and intertissue concordance and chromosome matching increased to 84.8% and 75.8%, respectively. CONCLUSION(S): The a-SNP-trio strategy provides information about ploidy, euploidy, and parental origin in a single biopsy. This approach enabled us to identify 40% of MNBs with reproductive potential, which can have a significant effect in the clinical setting. Additionally, segmental aneuploidy is relevant for mismatched preimplantation genetic testing of aneuploidies, both within and between MNB tissues. Repeat biopsy might clarify whether segmental aneuploidy is a prone genetic character.

Inherent mosaicism and extensive mutation of human placentas.

Placentas can exhibit chromosomal aberrations that are absent from the fetus1. The basis of this genetic segregation, which is known as confined placental mosaicism, remains unknown. Here we investigated the phylogeny of human placental cells as reconstructed from somatic mutations, using whole-genome sequencing of 86 bulk placental samples (with a median weight of 28 mg) and of 106 microdissections of placental tissue. We found that every bulk placental sample represents a clonal expansion that is genetically distinct, and exhibits a genomic landscape akin to that of childhood cancer in terms of mutation burden and mutational imprints. To our knowledge, unlike any other healthy human tissue studied so far, the placental genomes often contained changes in copy number. We reconstructed phylogenetic relationships between tissues from the same pregnancy, which revealed that developmental bottlenecks genetically isolate placental tissues by separating trophectodermal lineages from lineages derived from the inner cell mass. Notably, there were some cases with full segregation-within a few cell divisions of the zygote-of placental lineages and lineages derived from the inner cell mass. Such early embryonic bottlenecks may enable the normalization of zygotic aneuploidy. We observed direct evidence for this in a case of mosaic trisomic rescue. Our findings reveal extensive mutagenesis in placental tissues and suggest that mosaicism is a typical feature of placental development.

The Morphology of Inner Cell Mass Is the Strongest Predictor of Live Birth After a Frozen-Thawed Single Embryo Transfer.

Background: The scoring system for human blastocysts is traditionally based on morphology; however, there are controversies on the effect of morphology parameters on pregnancy outcomes. The aim of this study is to evaluate the predicting value of each morphology parameter on pregnancy outcomes in a setting of single embryo transfer. Methods: This is a retrospective cohort study on patients undergoing frozen-thawed single blastocyst transfer at our center, between Jan. 2009 and Dec. 2018. A total of 10,482 cycles were analyzed. The blastocysts were scored according to the expansion and hatching status, morphology of inner cell mass (ICM), and cells of trophectoderm (TE). The primary outcome measure was live birth rate. One-way analysis of variance, chi-square test, and multiple logistic regression were used for statistical analysis. Results: The clinical pregnancy rate was lower in the blastocysts of stage 3 (48.15%), compared with those of stage 4 (56.15%), stage 5 (54.91%), and stage 6 (53.37%). The live birth rate was lower in the blastocysts of stage 3 (37.07%), compared with those of stage 4 (44.21%) and stage 5 (41.67%). The rates of clinical pregnancy (A: 66.60%, B: 53.25%, C: 39.33%) and live birth (A: 54.62%, B: 41.29%, C: 28.45%) were both decreased with decreasing grade of ICM morphology, and these differences were pairwise significant. The miscarriage rate of blastocysts with ICM grade A was lower, compared with ICM grade C (17.53 vs. 27.66%). Blastocysts with TE morphology of C had lower rates of clinical pregnancy (43.53%) and live birth (32.57%), compared with those with TE morphology of A and B (clinical pregnancy rate: 64.26% for A, 58.11% for B; live birth rate: 52.74% for A, 45.64% for B). There were no significant differences in rates of clinical pregnancy, live birth, and miscarriage between the blastocysts with TE grade A and B. Conclusions: The blastocyst expansion stage, ICM grade, and TE grade are all associated with pregnancy outcomes. ICM grade is the strongest predictor of live birth. A blastocyst with stage 4-5, ICM grade A, and TE grade A/B should be given priority for single embryo transfer.

A workflow for simultaneous DNA copy number and methylome analysis of inner cell mass and trophectoderm cells from human blastocysts.

OBJECTIVE: To establish a workflow for isolating single trophectoderm (TE) and inner cell mass (ICM) cells and to simultaneously evaluate these cells for copy number variation (CNV) as well as methylome development. DESIGN: Experimental. SETTING: Academic medical center. PATIENT(S): Donated genetically abnormal blastocysts. INTERVENTION(S): Single cells were isolated, followed by bisulfite conversion and sequencing to identify CNV and methylome profiles. MAIN OUTCOME MEASURE(S): CNV and methylation profiling. RESULT(S): Two embryos were dissociated, isolating 46 single cells, with 17 ICM and 12 TE cells selected for further downstream analysis. Chromosome ploidies and embryo sex were concordant with the results from conventional aneuploidy testing. In 3 of the 29 cells, additional aneuploidies were discovered, indicating possible mosaicism undetected by routine preimplantation genetic testing for aneuploidy. CpG methylation frequency was higher in ICM cells compared with TE cells (44.3% vs. 32.4%), respectively, while non-CpG methylation frequency was similar among both cell types. CpG methylation levels accurately distinguished ICM from TE cells epigenetically. CONCLUSION(S): We describe an effective workflow for isolating and sequencing single ICM and TE cells from human blastocysts. The use of methylation profiling can help distinguish these two cell populations better then morphologic identification alone. TE cells had significantly lower levels of DNA methylation, which may be explained in part by the fact that these cells have begun the process of differentiation and are transcriptionally more active than ICM. This approach may be used to explore the genetic complexities within human embryos, specifically among the two primary cell types seen at this stage of development.

OCT4 induces embryonic pluripotency via STAT3 signaling and metabolic mechanisms.

OCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 establishes and protects the pluripotent lineage in the embryo, we used comparative single-cell transcriptomics and quantitative immunofluorescence on control and OCT4 null blastocyst inner cell masses at two developmental stages. Surprisingly, activation of most pluripotency-associated transcription factors in the early mouse embryo occurs independently of OCT4, with the exception of the JAK/STAT signaling machinery. Concurrently, OCT4 null inner cell masses ectopically activate a subset of trophectoderm-associated genes. Inspection of metabolic pathways implicates the regulation of rate-limiting glycolytic enzymes by OCT4, consistent with a role in sustaining glycolysis. Furthermore, up-regulation of the lysosomal pathway was specifically detected in OCT4 null embryos. This finding implicates a requirement for OCT4 in the production of normal trophectoderm. Collectively, our findings uncover regulation of cellular metabolism and biophysical properties as mechanisms by which OCT4 instructs pluripotency.

Sex-specific epigenetic profile of inner cell mass of mice conceived in vivo or by IVF.

The preimplantation stage of development is exquisitely sensitive to environmental stresses, and changes occurring during this developmental phase may have long-term health effects. Animal studies indicate that IVF offspring display metabolic alterations, including hypertension, glucose intolerance and cardiac hypertrophy, often in a sexual dimorphic fashion. The detailed nature of epigenetic changes following in-vitro culture is, however, unknown. This study was performed to evaluate the epigenetic (using whole-genome bisulfite sequencing (WGBS) and assay for transposase-accessible chromatin using sequencing (ATAC-seq)) and transcriptomic changes (using RNA-seq) occurring in the inner cell mass (ICM) of male or female mouse embryos generated in vivo or by IVF. We found that the ICM of IVF embryos, compared to the in-vivo ICM, differed in 3% of differentially methylated regions (DMRs), of which 0.1% were located on CpG islands. ATAC-seq revealed that 293 regions were more accessible and 101 were less accessible in IVF embryos, while RNA-seq revealed that 21 genes were differentially regulated in IVF embryos. Functional enrichment analysis revealed that stress signalling (STAT and NF-kB signalling), developmental processes and cardiac hypertrophy signalling showed consistent changes in WGBS and ATAC-seq platforms. In contrast, male and female embryos showed minimal changes. Male ICM had an increased number of significantly hyper-methylated DMRs, while only 27 regions showed different chromatin accessibility and only one gene was differentially expressed. In summary, this study provides the first comprehensive analysis of DNA methylation, chromatin accessibility and RNA expression changes induced by IVF in male and female ICMs. This dataset can be of value to all researchers interested in the developmental origin of health and disease (DOHaD) hypothesis and might lead to a better understanding of how early embryonic manipulation may affect adult health.

The inclusion of blastomeres into the inner cell mass in early-stage human embryos depends on the sequence of cell cleavages during the fourth division.

The fate of the ICM in humans is still unknown, due to the ethical difficulties surrounding experimentation in this field. In this study we have explored the existing time-lapse recording data of embryos in the early stages of development, taking advantage of the large refractile bodies (RBs) within blastomeres as cellular markers. Our study found that the cellular composition of the ICM in humans is largely determined at the time of the fourth division and blastomeres which cleave first to fourth, during the fourth division from 8 cells to 16 cells, have the potential to be incorporated in the ICM.

Initiation of a conserved trophectoderm program in human, cow and mouse embryos.

Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac1. Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human2-5 and cow6. Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical-basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos.

Analysis of accessible chromatin landscape in the inner cell mass and trophectoderm of human blastocysts.

Early embryonic development is characterized by drastic changes in chromatin structure that affects the accessibility of the chromatin. In human, the chromosome reorganization and its involvement in the first linage segregation are poorly characterized due to the difficulties in obtaining human embryonic material and limitation on low input technologies. In this study, we aimed to explore the chromatin remodeling pattern in human preimplantation embryos and gain insight into the epigenetic regulation of inner cell mass (ICM) and trophectoderm (TE) differentiation. We optimized ATAC-seq (an assay for transposase-accessible chromatin using sequencing) to analyze the chromatin accessibility landscape for low DNA input. Sixteen preimplantation human blastocysts frozen on Day 6 were used. Our data showed that ATAC peak distributions of the promoter regions (<1 kb) and distal regions versus other regions were significantly different between ICM versus TE samples (P < 0.01). We detected that a higher percentage of accessible binding loci were located within 1 kb of the transcription start site in ICM compared to TE (P < 0.01). However, a higher percentage of accessible regions was detected in the distal region of TE compared to ICM (P < 0.01). In addition, eight differential peaks with a false discovery rate <0.05 between ICM and TE were detected. This is the first study to compare the landscape of the accessible chromatin between ICM and TE of human preimplantation embryos, which unveiled chromatin-level epigenetic regulation of cell lineage specification in early embryo development.