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One week after fertilization, human embryos implant into the uterus. This event requires the embryo to form a blastocyst consisting of a sphere encircling a cavity lodging the embryo proper. Stem cells can form a blastocyst model that we called a blastoid 1 . Here we show that naive human pluripotent stem cells cultured in PXGL medium 2 and triply inhibited for the Hippo, TGF-β and ERK pathways efficiently (with more than 70% efficiency) form blastoids generating blastocyst-stage analogues of the three founding lineages (more than 97% trophectoderm, epiblast and primitive endoderm) according to the sequence and timing of blastocyst development. Blastoids spontaneously form the first axis, and we observe that the epiblast induces the local maturation of the polar trophectoderm, thereby endowing blastoids with the capacity to directionally attach to hormonally stimulated endometrial cells, as during implantation. Thus, we propose that such a human blastoid is a faithful, scalable and ethical model for investigating human implantation and development 3 , 4 . Blastoids derived from naive PXGL-cultured human pluripotent stem cells in which Hippo, TGF-β and ERK pathways are inhibited closely recapitulate aspects of blastocyst development, form cells resembling blastocyst-stage cells and thus provide a model system for implantation and development studies.

PurposeThis paper aims to investigate the efficacy of IVF with preimplantation genetic testing for aneuploidy (PGT-A), using only best-scoring blastocysts from young (≤ 35 years) infertile patients undergoing single blastocyst frozen embryo transfers (FET).MethodIn this randomized controlled trial (RCT) registered 29 March 2017, 302 infertile patient-couples eligible to participate underwent autologous ICSI blastocyst freeze-all cycles. Two-hundred and twenty patient-couples satisfied the inclusion criteria (i.e., female age ≤ 35 years, two-day 5 ≥ 2BB blastocysts) and were randomized to either the PGT-A (PGT-A group, n = 109) selection arm or morphology score (morphology group, n = 111) selection arm. In both arms, the highest ranking (by morphological score) blastocysts were selected for FET.ResultsOf the 109 best-scoring blastocysts that underwent PGT-A, 80 were predicted to be euploid (73.4%) and were transferred in FET (euploid subgroup). There was no statistical difference in LB rate between the euploid subgroup and morphology group (56.3% vs 58.6%, odds ratio 0.91 (95% CI 0.51–1.63), p = 0.750). In a multiple logistic regression, the transfer of euploid blastocysts was not found to be a significant predictor of LB when adjusting for female age, infertility duration, antral follicle count, and blastocyst quality, with the independent odds expressed as 0.91 (95% CI 0.50–1.66, p = 0.760).ConclusionIn young (≤ 35 years) infertile patients with at least two ≥ 2BB blastocysts, PGT-A blastocyst selection does not result in an enhanced LB rate, with the evidence suggesting that the effectivity of PGT-A may be limited by the effectivity of TE biopsy.Trial registration: ClinicalTrials.gov ID: NCT03095053.

Purpose A subset of preimplantation stage embryos may possess mosaicism of chromosomal constitution, representing a possible limitation to the clinical predictive value of comprehensive chromosome screening (CCS) from a single biopsy. However, contemporary methods of CCS may be capable of predicting mosaicism in the blastocyst by detecting intermediate levels of aneuploidy within a trophectoderm biopsy. This study evaluates the sensitivity and specificity of aneuploidy detection by two CCS platforms using a cell line mixture model of a mosaic trophectoderm biopsy. Methods Four cell lines with known karyotypes were obtained and mixed together at specific ratios of six total cells (0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0). A female euploid and a male trisomy 18 cell line were used for one set, and a male trisomy 13 and a male trisomy 15 cell line were used for another. Replicates of each mixture were prepared, randomized, and blinded for analysis by one of two CCS platforms (quantitative polymerase chain reaction (qPCR) or VeriSeq next-generation sequencing (NGS)). Sensitivity and specificity of aneuploidy detection at each level of mosaicism was determined and compared between platforms. Results With the default settings for each platform, the sensitivity of qPCR and NGS were not statistically different, and 100 % specificity was observed (no false positives) at all levels of mosaicism. However, the use of previously published custom criteria for NGS increased sensitivity but also significantly decreased specificity (33 % false-positive prediction of aneuploidy). Conclusions By demonstrating increased false-positive diagnoses when reducing the stringency of predicting an abnormality, these data illustrate the importance of preclinical evaluation of new testing paradigms before clinical implementation.

PurposeLaevo (l)-carnitine plays important roles in reducing the cytotoxic effects of free fatty acids by forming acyl-carnitine and promoting beta-oxidation, leading to alleviation of cell damage. Recently, the mitochondrial functions in morula has been shown to decrease with the maternal age. Here, we assessed the effect of l-carnitine on mitochondrial function in human embryos and embryo development.MethodsTo examine the effect of l-carnitine on mitochondrial function in morulae, 38 vitrified–thawed embryos at the 6–11-cell stage on day 3 after ICSI were donated from 19 couples. Each couple donated two embryos. Two siblings from each couple were divided randomly into two groups and were cultured in medium with or without 1 mM l-carnitine. The oxygen consumption rates (OCRs) were measured at morula stage. The development of 1029 zygotes cultured in medium with or without l-carnitine was prospectively analyzed.ResultsAddition of l-carnitine to the culture medium significantly increased the OCRs of morulae and improved the morphologically-good blastocyst formation rate per zygote compared with sibling embryos. Twenty healthy babies were born from embryos cultured in l-carnitine-supplemented medium after single embryo transfers.Conclusion(s)l-carnitine is a promising culture medium supplement that might be able to counteract the decreased mitochondrial function in human morula stage embryos.

In the preimplantation mouse embryo, TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP sequencing to define genomewide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation toward the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification.

During mammalian blastocyst development, inner cell mass (ICM) cells differentiate into epiblast (Epi) or primitive endoderm (PrE). These two fates are characterized by the expression of the transcription factors NANOG and GATA6, respectively. Here, we investigate the spatio-temporal distribution of NANOG and GATA6 expressing cells in the ICM of the mouse blastocysts with quantitative three-dimensional single cell-based neighbourhood analyses. We define the cell neighbourhood by local features, which include the expression levels of both fate markers expressed in each cell and its neighbours, and the number of neighbouring cells. We further include the position of a cell relative to the centre of the ICM as a global positional feature. Our analyses reveal a local three-dimensional pattern that is already present in early blastocysts: 1) Cells expressing the highest NANOG levels are surrounded by approximately nine neighbours, while 2) cells expressing GATA6 cluster according to their GATA6 levels. This local pattern evolves into a global pattern in the ICM that starts to emerge in mid blastocysts. We show that FGF/MAPK signalling is involved in the three-dimensional distribution of the cells and, using a mutant background, we further show that the GATA6 neighbourhood is regulated by NANOG. Our quantitative study suggests that the three-dimensional cell neighbourhood plays a role in Epi and PrE precursor specification. Our results highlight the importance of analysing the three-dimensional cell neighbourhood while investigating cell fate decisions during early mouse embryonic development.

Tight regulation of Nanog dose at the chromosome level is important for the acquisition of pluripotency during development. Nanog under tight rein in stem cells The homeobox protein Nanog is a key factor in reprogramming and stem-cell pluripotency. This study shows that tight regulation of Nanog dose at the chromosome level is important for acquisition of naive pluripotency. The initial expression of Nanog , but not Oct4 , is monoallelic in early pre-implantation embryos before formation of the definitive pluripotent compartment, the epiblast. Nanog then undergoes a progressive switch to biallelic expression during the transition towards the naive pluripotency in the epiblast of late blastocysts. The findings reveal a previously unknown level of regulation of reprogramming. Pluripotency is established through genome-wide reprogramming during mammalian pre-implantation development, resulting in the formation of the naive epiblast. Reprogramming involves both the resetting of epigenetic marks and the activation of pluripotent-cell-specific genes such as Nanog and Oct4 (also known as Pou5f1 ) 1 , 2 , 3 , 4 . The tight regulation of these genes is crucial for reprogramming, but the mechanisms that regulate their expression in vivo have not been uncovered. Here we show that Nanog —but not Oct4 —is monoallelically expressed in early pre-implantation embryos. Nanog then undergoes a progressive switch to biallelic expression during the transition towards ground-state pluripotency in the naive epiblast of the late blastocyst. Embryonic stem (ES) cells grown in leukaemia inhibitory factor (LIF) and serum express Nanog mainly monoallelically and show asynchronous replication of the Nanog locus, a feature of monoallelically expressed genes 5 , but ES cells activate both alleles when cultured under 2i conditions, which mimic the pluripotent ground state in vitro . Live-cell imaging with reporter ES cells confirmed the allelic expression of Nanog and revealed allelic switching. The allelic expression of Nanog is regulated through the fibroblast growth factor–extracellular signal-regulated kinase signalling pathway, and it is accompanied by chromatin changes at the proximal promoter but occurs independently of DNA methylation. Nanog -heterozygous blastocysts have fewer inner-cell-mass derivatives and delayed primitive endoderm formation, indicating a role for the biallelic expression of Nanog in the timely maturation of the inner cell mass into a fully reprogrammed pluripotent epiblast. We suggest that the tight regulation of Nanog dose at the chromosome level is necessary for the acquisition of ground-state pluripotency during development. Our data highlight an unexpected role for allelic expression in controlling the dose of pluripotency factors in vivo , adding an extra level to the regulation of reprogramming.

To evaluate the effect of melatonin supplementation in maturation medium for human 'rescue IVM' and investigate differences in transcriptomic profile of blastocysts developed from oocytes matured in vitro with/without melatonin treatment and in vivo, a total of 314 GV oocytes and 320 MI oocytes were collected from 200 patients younger than 35 years old undergoing ICSI cycle. The oocytes were randomly distributed in the control group (no melatonin) and four other groups of varying melatonin concentrations (10−11, 10−9, 10−7, 10−5 mol/l). Gene profiling was performed on blastocysts developed from in vivo maturation oocytes (in vivo group), and in vitro maturation (IVM) oocytes with an optimal concentration of melatonin treatment (IVM-anti group) or without melatonin (IVM group). The ratio of high quality blastocysts was significantly higher in the groups treated with 10−5 mol/l melatonin compared with others groups. The large-scale analysis of the transcriptome revealed significant differences in mRNA expression levels. In each group, nine blastocysts were selected for gene expression profiling. The differentially expressed genes were involved in cysteine and methionine metabolism, regulation of apoptotic process, mineral absorption, steroid hormone biosynthesis, Wnt signaling, p53 signaling pathway and other functions. The findings indicated that the IVM procedure may potentially affect DNA methylation and the canonical Wnt signaling pathway. Exogenous melatonin positively influenced quality of blastocysts, which may be mediated via upregulation of p53 signaling and correcting DNA methylation changes caused by 'rescue IVM'. However, this study reflected what was generally referred to as 'rescue IVM' and was not a true reflection of clinical IVM techniques. Therefore, melatonin required further investigation as a promising supplement for use in IVM.

Limited access to embryos has hampered the study of human embryogenesis and disorders that occur during early pregnancy. Human pluripotent stem cells provide an alternative means to study human development in a dish 1 – 7 . Recent advances in partial embryo models derived from human pluripotent stem cells have enabled human development to be examined at early post-implantation stages 8 – 14 . However, models of the pre-implantation human blastocyst are lacking. Starting from naive human pluripotent stem cells, here we developed an effective three-dimensional culture strategy with successive lineage differentiation and self-organization to generate blastocyst-like structures in vitro. These structures—which we term ‘human blastoids’—resemble human blastocysts in terms of their morphology, size, cell number, and composition and allocation of different cell lineages. Single-cell RNA-sequencing analyses also reveal the transcriptomic similarity of blastoids to blastocysts. Human blastoids are amenable to embryonic and extra-embryonic stem cell derivation and can further develop into peri-implantation embryo-like structures in vitro. Using chemical perturbations, we show that specific isozymes of protein kinase C have a critical function in the formation of the blastoid cavity. Human blastoids provide a readily accessible, scalable, versatile and perturbable alternative to blastocysts for studying early human development, understanding early pregnancy loss and gaining insights into early developmental defects. An in vitro culture strategy enables the generation of blastocyst-like structures termed human blastoids from naive human pluripotent stem cells, providing a model for studying human embryogenesis.

Human pluripotent and trophoblast stem cells have been essential alternatives to blastocysts for understanding early human development 1 – 4 . However, these simple culture systems lack the complexity to adequately model the spatiotemporal cellular and molecular dynamics that occur during early embryonic development. Here we describe the reprogramming of fibroblasts into in vitro three-dimensional models of the human blastocyst, termed iBlastoids. Characterization of iBlastoids shows that they model the overall architecture of blastocysts, presenting an inner cell mass-like structure, with epiblast- and primitive endoderm-like cells, a blastocoel-like cavity and a trophectoderm-like outer layer of cells. Single-cell transcriptomics further confirmed the presence of epiblast-, primitive endoderm-, and trophectoderm-like cells. Moreover, iBlastoids can give rise to pluripotent and trophoblast stem cells and are capable of modelling, in vitro, several aspects of the early stage of implantation. In summary, we have developed a scalable and tractable system to model human blastocyst biology; we envision that this will facilitate the study of early human development and the effects of gene mutations and toxins during early embryogenesis, as well as aiding in the development of new therapies associated with in vitro fertilization. Human fibroblasts are reprogrammed to generate blastocyst-like structures called iBlastoids, which recapitulate aspects of embryo implantation.