Erratum: Pluripotent state transitions coordinate morphogenesis in mouse and human embryos.

This corrects the article DOI: 10.1038/nature24675.

Pluripotent state transitions coordinate morphogenesis in mouse and human embryos.

The foundations of mammalian development lie in a cluster of embryonic epiblast stem cells. In response to extracellular matrix signalling, these cells undergo epithelialization and create an apical surface in contact with a cavity, a fundamental event for all subsequent development. Concomitantly, epiblast cells transit through distinct pluripotent states, before lineage commitment at gastrulation. These pluripotent states have been characterized at the molecular level, but their biological importance remains unclear. Here we show that exit from an unrestricted naive pluripotent state is required for epiblast epithelialization and generation of the pro-amniotic cavity in mouse embryos. Embryonic stem cells locked in the naive state are able to initiate polarization but fail to undergo lumenogenesis. Mechanistically, exit from naive pluripotency activates an Oct4-governed transcriptional program that results in expression of glycosylated sialomucin proteins and the vesicle tethering and fusion events of lumenogenesis. Similarly, exit of epiblasts from naive pluripotency in cultured human post-implantation embryos triggers amniotic cavity formation and developmental progression. Our results add tissue-level architecture as a new criterion for the characterization of different pluripotent states, and show the relevance of transitions between these states during development of the mammalian embryo.

Effects of thyroid hormone on mitochondria and metabolism of human preimplantation embryos.

Thyroid hormones are regarded as the major controllers of metabolic rate and oxygen consumption in mammals. Although it has been demonstrated that thyroid hormone supplementation improves bovine embryo development in vitro, the cellular mechanisms underlying these effects are so far unknown. In this study, we investigated the role of thyroid hormone in development of human preimplantation embryos. Embryos were cultured in the presence or absence of 10-7 M triiodothyronine (T3) till blastocyst stage. Inner cell mass (ICM) and trophectoderm (TE) were separated mechanically and subjected to RNAseq or quantification of mitochondrial DNA copy number. Analyses were performed using DESeq (v1.16.0 on R v3.1.3), MeV4.9 and MitoMiner 4.0v2018 JUN platforms. We found that the exposure of human preimplantation embryos to T3 had a profound impact on nuclear gene transcription only in the cells of ICM (1178 regulated genes-10.5% of 11 196 expressed genes) and almost no effect on cells of TE (38 regulated genes-0.3% of expressed genes). The analyses suggest that T3 induces in ICM a shift in ribosome and oxidative phosphorylation activity, as the upregulated genes are contributing to the composition and organization of the respiratory chain and associated cofactors involved in mitoribosome assembly and stability. Furthermore, a number of genes affecting the citric acid cycle energy production have reduced expression. Our findings might explain why thyroid disorders in women have been associated with reduced fertility and adverse pregnancy outcome. Our data also raise a possibility that supplementation of culture media with T3 may improve outcomes for women undergoing in vitro fertilization.

Definition and validation of a custom protocol to detect miRNAs in the spent media after blastocyst culture: searching for biomarkers of implantation.

STUDY QUESTION: Can miRNAs be reliably detected in the spent blastocyst media (SBM) after IVF as putative biomarkers of the implantation potential of euploid embryos? SUMMARY ANSWER: Adjustment of the data for blastocyst quality and the day of full-expansion hinders the predictive power of a fast, inexpensive, reproducible and user-friendly protocol based on the detection of 10 selected miRNAs from SBM. WHAT IS KNOWN ALREADY: Euploidy represents so far the strongest predictor of blastocyst competence. Nevertheless, ~50% of the euploid blastocysts fail to implant. Several studies across the years have suggested that a dialogue exists between the embryo and the endometrium aimed at the establishment of a pregnancy. MicroRNAs have been proposed as mediators of such a dialogue and investigated in this respect. Several expensive, time-consuming and complex protocols have been adopted and promising results have been produced, but conclusive evidence from large clinical studies is missing. STUDY DESIGN, SIZE, DURATION: This study was conducted in two phases from September 2015 to December 2017. In Phase 1, the human blastocyst miRNome profile was defined from the inner cell mass (ICM) and the corresponding whole-trophectoderm (TE) of six donated blastocysts. Two different protocols were adopted to this end. In parallel, 6 pools of 10 SBM each were run (3 from only implanted euploid blastocysts, IEBs; and 3 from only not-implanted euploid blastocysts, not-IEBs). A fast, inexpensive and user-friendly custom protocol for miRNA SBM profiling was designed. In Phase 2, 239 SBM from IEB and not-IEB were collected at three IVF centres. After 18 SBM from poor-quality blastocysts were excluded from the analysis, data from 107 SBM from not-IEB and 114 from IEB were produced through the previously developed custom protocol and compared. The data were corrected through logistic regressions. PARTICIPANT/MATERIALS, SETTINGS, METHODS: Donated blastocysts underwent ICM and whole-TE isolation. SBM were collected during IVF cycles characterized by ICSI, blastocyst culture in a continuous media, TE biopsy without zona pellucida opening in Day 3, quantitative PCR (qPCR)-based aneuploidy testing and vitrified-warmed single euploid embryo transfer. Not-IEB and IEB were clustered following a negative pregnancy test and a live birth, respectively. The Taqman Low Density Array (TLDA) cards and the Exiqon microRNA human panel I+II qPCR analysis protocols were adopted to analyse the ICM and whole-TE. The latter was used also for SBM pools. A custom protocol and plate was then designed based on the Exiqon workflow, validated and finally adopted for SBM analysis in study Phase 2. This custom protocol allows the analysis of 10 miRNAs from 10 SBM in 3 hours from sample collection to data inspection. MAIN RESULTS AND ROLE OF THE CHANCE: The TLDA cards protocol involved a higher rate of false positive results (5.6% versus 2.8% with Exiqon). There were 44 miRNAs detected in the ICM and TE from both the protocols. One and 24 miRNAs were instead detected solely in the ICM and the TE, respectively. Overall, 29 miRNAs were detected in the pooled SBM: 8 only from not-IEB, 8 only from IEB and 13 from both. Most of them (N = 24/29, 82.7%) were also detected previously in both the ICM and TE with the Exiqon protocol; two miRNAs (N = 2/29, 6.9%) were previously detected only in the TE, and three (N = 3/29, 10.3%) were never detected previously. In study Phase 2, significant differences were shown between not-IEB and IEB in terms of both miRNA detection and relative quantitation. However, when the data were corrected for embryo morphology and day of full development (i.e. SBM collection), no significant association was confirmed. LIMITATIONS, REASONS FOR CAUTION: This study did not evaluate specifically exosomal miRNAs, thereby reducing the chance of identifying the functional miRNAs. Ex-vivo experiments are required to confirm the role of miRNAs in mediating the dialogue with endometrial cells, and higher throughput technologies need to be further evaluated for miRNA profiling from clinical SBM samples. WIDER IMPLICATIONS OF THE FINDINGS: Although no clinical predictive power was reported in this study, the absence of invasiveness related with SBM analysis and the evidence that embryonic genetic material can be reliably detected and analysed from SBM make this waste product of IVF an important source for further investigations aimed at improving embryo selection. STUDY FUNDING/COMPETING INTEREST(S): This project has been financially supported by Merck KgaA (Darmstadt, Germany) with a Grant for Fertility Innovation (GFI) 2015. The authors have no conflict of interest to declare related with this study. TRIAL REGISTRATION NUMBER: None.

A thyroid hormone receptor/KLF9 axis in human hepatocytes and pluripotent stem cells.

Biological processes require close cooperation of multiple transcription factors that integrate different signals. Thyroid hormone receptors (TRs) induce Krüppel-like factor 9 (KLF9) to regulate neurogenesis. Here, we show that triiodothyronine (T3) also works through TR to induce KLF9 in HepG2 liver cells, mouse liver, and mouse and human primary hepatocytes and sought to understand TR/KLF9 network function in the hepatocyte lineage and stem cells. Knockdown experiments reveal that KLF9 regulates hundreds of HepG2 target genes and modulates T3 response. Together, T3 and KLF9 target genes influence pathways implicated in stem cell self-renewal and differentiation, including Notch signaling, and we verify that T3 and KLF9 cooperate to regulate key Notch pathway genes and work independently to regulate others. T3 also induces KLF9 in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC) and this effect persists during differentiation to definitive endoderm and hiPSC-derived hepatocytes. Microarray analysis reveals that T3 regulates hundreds of hESC and hiPSC target genes that cluster into many of the same pathways implicated in TR and KLF9 regulation in HepG2 cells. KLF9 knockdown confirms that TR and KLF9 cooperate to regulate Notch pathway genes in hESC and hiPSC, albeit in a partly cell-specific manner. Broader analysis of T3 responsive hESC/hiPSC genes suggests that TRs regulate multiple early steps in ESC differentiation. We propose that TRs cooperate with KLF9 to regulate hepatocyte proliferation and differentiation and early stages of organogenesis and that TRs exert widespread and important influences on ESC biology.

Developmental clock compromises human twin model created by embryo splitting.

STUDY QUESTION: Is the quality of the human embryos generated by twinning in vitro comparable to the quality of the embryos created by fertilization? SUMMARY ANSWER: Our data suggest that the human twin embryos created in vitro are unsuitable not only for clinical use but also for research purposes. WHAT IS KNOWN ALREADY: Pregnancy from in vitro generated monozygotic twins by embryo splitting or twinning leads to live birth of healthy animals. Similar strategies, however, have been less successful in primates. Recent reports suggest that the splitting of human embryos might result in viable, morphologically adequate blastocysts, although the qualitative analyses of the embryos created in such a way have been very limited. STUDY DESIGN, SIZE, DURATION: This study was a comparative analysis of embryos generated by twinning in vitro and the embryos created by in vitro fertilization. PARTICIPANTS/MATERIALS, SETTING, METHODS: We analysed morphokinetics and developmental competence of 176 twin embryos created by splitting of 88 human embryos from either early (2-5 blastomeres, n = 43) or late (6-10 blastomeres, n = 45) cleavage stages. We compared the data with morphometrics of embryos created by in vitro fertilization and resulting in pregnancy and live birth upon single blastocyst transfer (n = 42). MAIN RESULTS AND THE ROLE OF CHANCE: The morphokinetic data suggested that the human preimplantation development is subjected to a strict temporal control. Due to a 'developmental clock', the size of twin embryos was proportionate to the number of cells used for their creation. Furthermore, the first fate decision was somewhat delayed; the inner cell mass (ICM) became distinguishable later in the twin than in the normal blastocysts obtained through fertilization. If an ICM was present at all, it was small and of poor quality. The majority of the cells in the twin embryos expressed ICM and trophectoderm markers simultaneously. LIMITATIONS, REASONS FOR CAUTION: We created monozygotic twins by blastomere separation from cleavage stage embryos. Embryo twinning by blastocyst bisection may circumvent limitations set by the developmental clock. WIDER IMPLICATIONS OF THE FINDINGS: Taken together, our data suggest that the human twin embryos created in vitro are unsuitable not only for clinical use but also for research purposes.

Segregation of mitochondrial DNA heteroplasmy through a developmental genetic bottleneck in human embryos.

Mitochondrial DNA (mtDNA) mutations cause inherited diseases and are implicated in the pathogenesis of common late-onset disorders, but how they arise is not clear1,2. Here we show that mtDNA mutations are present in primordial germ cells (PGCs) within healthy female human embryos. Isolated PGCs have a profound reduction in mtDNA content, with discrete mitochondria containing ~5 mtDNA molecules. Single-cell deep mtDNA sequencing of in vivo human female PGCs showed rare variants reaching higher heteroplasmy levels in late PGCs, consistent with the observed genetic bottleneck. We also saw the signature of selection against non-synonymous protein-coding, tRNA gene and D-loop variants, concomitant with a progressive upregulation of genes involving mtDNA replication and transcription, and linked to a transition from glycolytic to oxidative metabolism. The associated metabolic shift would expose deleterious mutations to selection during early germ cell development, preventing the relentless accumulation of mtDNA mutations in the human population predicted by Muller's ratchet. Mutations escaping this mechanism will show shifts in heteroplasmy levels within one human generation, explaining the extreme phenotypic variation seen in human pedigrees with inherited mtDNA disorders.

Author Correction: Segregation of mitochondrial DNA heteroplasmy through a developmental genetic bottleneck in human embryos.

In the version of this Letter originally published, an author error led to the affiliations for Brendan Payne, Jonathan Coxhead and Gavin Hudson being incorrect. The correct affiliations are: Brendan Payne: 3Wellcome Trust Centre for Mitochondrial Research, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK. 6Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UK; this is a new affiliation 6 and subsequent existing affiliations have been renumbered. Jonathan Coxhead: 11Genomic Core Facility, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK; this is a new affiliation 11 and subsequent existing affiliations have been renumbered. Gavin Hudson: 3Wellcome Trust Centre for Mitochondrial Research, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK. In addition, in Fig. 2d, the numbers on the x-axis of the left plot were incorrectly labelled as negative; they should have been positive. These errors have now been corrected in all online versions of the Letter.

Potential of human twin embryos generated by embryo splitting in assisted reproduction and research.

BACKGROUND: Embryo splitting or twinning has been widely used in veterinary medicine over 20 years to generate monozygotic twins with desirable genetic characteristics. The first human embryo splitting, reported in 1993, triggered fierce ethical debate on human embryo cloning. Since Dolly the sheep was born in 1997, the international community has acknowledged the complexity of the moral arguments related to this research and has expressed concerns about the potential for reproductive cloning in humans. A number of countries have formulated bans either through laws, decrees or official statements. However, in general, these laws specifically define cloning as an embryo that is generated via nuclear transfer (NT) and do not mention embryo splitting. Only the UK includes under cloning both embryo splitting and NT in the same legislation. On the contrary, the Ethics Committee of the American Society for Reproductive Medicine does not have a major ethical objection to transferring two or more artificially created embryos with the same genome with the aim of producing a single pregnancy, stating that 'since embryo splitting has the potential to improve the efficacy of IVF treatments for infertility, research to investigate the technique is ethically acceptable'. OBJECTIVE AND RATIONALE: Embryo splitting has been introduced successfully to the veterinary medicine several decades ago and today is a part of standard practice. We present here an overview of embryo splitting experiments in humans and non-human primates and discuss the potential of this technology in assisted reproduction and research. SEARCH METHODS: A comprehensive literature search was carried out using PUBMED and Google Scholar databases to identify studies on embryo splitting in humans and non-human primates. 'Embryo splitting' and 'embryo twinning' were used as the keywords, alone or in combination with other search phrases relevant to the topics of biology of preimplantation embryos. OUTCOMES: A very limited number of studies have been conducted in humans and non-human primates. The published material, especially the studies with human embryos, is controversial. Some reports suggest that twinning technology will find clinical use in reproductive medicine in the future, whereas others conclude the opposite that human twin embryos created in vitro are unsuitable not only for clinical, but also for research, purposes. WIDER IMPLICATIONS: The blastomere biopsy technique of embryo splitting seems to be unsuitable for either clinical or research purposes; however, embryo bisection, a preferable method of cloning in veterinary medicine, has not yet been tested on human embryos.

Human embryos from induced pluripotent stem cell-derived gametes: ethical and quality considerations.

Protocols for successful differentiation of male and female gametes from induced pluripotent stem cells have been published. Although culture of precursor cells in a natural microenvironment remains necessary to achieve terminal differentiation, the creation of human preimplantation embryos from induced pluripotent stem cell-derived gametes is technically feasible. Such embryos could provide a solution to the scarcity of human cleavage-stage embryos donated for research. Here, we discuss current technology, major research-related ethical concerns and propose the norms that would assure the quality and reliability of such embryos.

MicroRNAs in spent blastocyst culture medium are derived from trophectoderm cells and can be explored for human embryo reproductive competence assessment.

OBJECTIVE: To assess whether extracellular microRNAs (miRNAs) can be accurately profiled from spent blastocyst culture media (SBM) and used as embryonic biomarkers. DESIGN: Prospective cohort study. SETTING: Private and academic in vitro fertilization centers. PATIENT(S): Inner cell mass-free trophectoderm (TE) samples and their relative SBM from five good-quality human blastocysts. INTERVENTION(S): Protocol for miRNA purification and analysis based on quantitative polymerase chain reaction set and validated on human embryonic stem cells (hESCs) and on SBM with and without biological variability. MAIN OUTCOMES MEASURE(S): Analysis of miRNAs in culture media in relation with TE cells and comparison of miRNA profiles between implanted and unimplanted euploid blastocysts. RESULT(S): Culture media from embryos in the cleavage, morula, and blastocyst stages were collected to investigate the presence of miRNAs. The SBM were prospectively collected from euploid implanted (n = 25) and unimplanted blastocysts (n = 28) for comparison. We hypothesized that human embryos secrete miRNAs in culture media that can be used as biomarkers. The comparative analysis of TE and SBM samples revealed that 96.6% (57 of 59; 95 CI, 88.3-99.6) of the miRNAs detected in the SBM were expressed from TE cells, suggesting a TE origin. The culture media collected from cleavage and morula stage embryos showed a pattern similar to blanks, suggesting that miRNAs profiling from spent culture media applies only for blastocysts. MicroRNAs analysis of SBM from euploid implanted and unimplanted blastocysts highlighted two miRNAs (miR-20a, miR-30c) that showed increased concentrations in the former and were predicted in silico to be involved in 23 implantation-related pathways. CONCLUSION(S): MicroRNAs secreted from human blastocysts in culture media can be profiled with high reproducibility, and this approach can be further explored for noninvasive embryo selection.

Human Embryos Created by Embryo Splitting Secrete Significantly Lower Levels of miRNA-30c.

Studies reporting term pregnancy and the production of genetically identical offspring from isolated blastomeres of early stage embryos have been carried out in small and large animals. However, very little is known about the effects of embryo splitting on the development and reproductive competency of human embryos. In this study, we investigated the effects of embryo splitting on profile of microRNAs (miRNAs) detected in their spent blastocyst medium (SBM) by comparative analysis of miRNA profiles in SBM of human twin embryos created by blastomere biopsy and SBM of blastocysts that resulted in a healthy pregnancy and live birth following embryo transfer. The profile of miRNA secretion in in vitro culture media consistently distinguishes twin from control embryos. We found that six miRNAs are significantly more abundant in SBM from twin embryos, while nine are significantly more abundant in SBM from euploid implanted blastocysts. These nine include miRNA-30c, a previously reported marker of blastocyst implantation potential. Furthermore, 22.9% of miRNAs secreted by twin embryos were never detected in SBM from normal reproductively competent blastocysts, or from trophectoderm (TE) samples from normal blastocysts donated for the research. The miRNA profile, unique to twin blastocysts, might be a result of differential lineage commitment in these embryos.

Discordant Growth of Monozygotic Twins Starts at the Blastocyst Stage: A Case Study.

Discordant growth is a common complication of monochorionic/diamniotic pregnancies; in approximately 50% of cases, the cause is unknown. The case presented here suggests that discordant growth of monozygotic twins could start during preimplantation development. Two inner cell masses (ICMs) within the same blastocyst may originate in uneven splitting of a single "parental" ICM, or the two ICMs may be formed independently de novo. We studied the transcriptomes of two morphologically distinct ICMs within a single blastocyst using high-resolution RNA sequencing. The data indicated that the two ICM were at different stages of development; one was in the earliest stages of lineage commitment, while the other had already differentiated into epiblast and primitive endoderm. IGF1-mediated signaling is likely to play a key role in ICM growth and to be the major driver behind these differences.