Understanding how the first cell lineages in human development are specified and maintained has fundamental importance and clinical implications for regenerative medicine, infertility and pregnancy loss. Although mouse models have provided valuable insights into transcription factors regulating early development, translating these findings to human embryos has been limited by ethical, technical and biological constraints. Functional studies of transcription factors in human embryos have been hindered by nuclease-based genome editing approaches that induce genotoxicity1-3. Here, to overcome this, we applied ABE8e adenine base editing4,5 to precisely target an exon splice donor site, resulting in a splicing defect and functional knockout of the developmental regulator NANOG in human embryos. This approach did not trigger genotoxicity and showed limited off-target editing. Loss of NANOG disrupts pluripotent epiblast specification and instead cells differentiate towards a primitive endoderm (yolk sac) or trophectoderm (placental) transcriptional programme. Retention of primitive endoderm differentiation in NANOG-edited human embryos reveals a functional compensation that is distinct from mouse, underscoring the importance of directly investigating human development. Our findings demonstrate an essential role for NANOG in human pluripotency and epiblast specification and highlight the utility of base editing for functional interrogation of human development.

Aneuploidy in human eggs, which rises sharply with age, is a leading cause of infertility, IVF failure, and miscarriage. This age-related aneuploidy is primarily driven by premature sister chromatid separation (PSSC), resulting from loss of the cohesin complex that holds chromatids together. How cohesin is destabilized in the long-lived mammalian oocyte is poorly understood. Here, we show that in mouse oocytes, pericentromeric transcription is essential for maintaining the cohesion protector Shugoshin 1 (SGO1) and PP2A at centromeres, which together safeguard the cohesin subunit REC8. With age, mouse oocytes lose pericentromeric transcription, SGO1, and PP2A, leading to destabilized cohesion and increased PSSC. Supplementing aged mouse oocytes with Sgo1 restores centromeric protection, and reduces PSSC to youthful levels. Aged human oocytes also show reduced SGO1, and SGO1 supplementation reduces the fraction of human eggs with PSSC by approximately half. These findings establish SGO1 supplementation as a potential strategy to preserve chromatid cohesion in aging oocytes. ### Competing Interest Statement D.S. and M.S. are listed as inventors on a patent application filed by the Max Planck Society for the Advancement of Science (application no. PCT/EP2025/063405) based on the data presented in this study. M.S. and A.P.Z. are co-founders of Ovo Labs and hold equity in the company. A.P.Z. is Co-Chief Executive Officer of Ovo Labs. M.S. serves as a scientific advisor to Ovo Labs. S.M. is an employee of Ovo Labs and holds equity in the company. The remaining authors declare no competing interests. German Heart Research Foundation, https://ror.org/02yjb3a91, EXC 2067/1-390729940 DFG Leibniz Prize, SCHU 3047/1-1

The placenta supports the foetus by mediating nutrient and gas exchange, hormone production, and immune protection. Yet, human placental biology remains poorly understood due to limited in vitro models. The foetal part of the placenta, the chorion, consists of a trophoblast-derived layer and a mesenchymal zone containing extraembryonic mesoderm cells. Here, we show that human blastocysts can give rise to both trophoblast organoids and extraembryonic mesoderm cells under the same culture conditions. Trophoblast organoids originate from both outer trophoblast cells and inner cells of the blastocyst, while extraembryonic mesoderm cells derive exclusively from inner cells. These organoids recapitulate the cellular composition, morphology, and function of the in vivo trophoblast. Moreover, given the high rate of aneuploidy at the blastocyst stage, aneuploid trophoblast organoids can be readily generated. These models reflect the trophoblast’s unique ability to tolerate aneuploidy and offer a valuable platform to study human placental development and pathophysiology. ### Competing Interest Statement The authors have declared no competing interest. Gates Cambridge Trust Funai Foundation for Information Technology UK Research and Innovation, https://ror.org/001aqnf71, EP/Y009924/1, MC\_UP\_1201/24, EP/X023044/1 Wellcome Trust, https://ror.org/029chgv08, 220540/Z/20/A Natural Sciences and Engineering Research Council of Canada Discovery, RGPIN-2020-05378 Canadian Institutes of Health Research, 202109PAV-468535-CA2 European Molecular Biology Organization, https://ror.org/04wfr2810

The human blastocyst contains the pluripotent epiblast from which human embryonic stem cells (hESCs) can be derived. ACTIVIN/NODAL signaling maintains expression of the transcription factor NANOG and in vitro propagation of hESCs. It is unknown whether this reflects a functional requirement for epiblast development in human embryos. Here, we characterized NODAL signaling activity during pre-implantation human development. We showed that NANOG is an early molecular marker restricted to the nascent human pluripotent epiblast and was initiated prior to the onset of NODAL signaling. We further demonstrated that expression of pluripotency-associated transcription factors NANOG, SOX2, OCT4, and KLF17 were maintained in the epiblast in the absence of NODAL signaling activity. Genome-wide transcriptional analysis showed that NODAL signaling inhibition did not decrease NANOG transcription or impact the wider pluripotency-associated gene regulatory network. These data suggest differences in the signaling requirements regulating pluripotency in the pre-implantation human epiblast compared with existing hESC culture.

A couple presenting with more than 3 years’ history of infertility and three miscarriages was tested for serum homocysteine levels and for the two principal MTHFR SNPs: 677C < T and 1298A < C, as per our general policy for patients with infertility of long duration. The woman was found to be wild type for both MTHFR SNPs with a serum homocysteine 10.5 µM, slightly higher than our accepted normal value of 8.5 µM. The man was found to be a carrier of a triple mutation for 677C < T and 1298 A < C, with an elevated homocysteine level of 19 µM. Since high doses of folic acid (FA) exacerbate perturbations in the DNA methylome, the husband was treated with 5-methyl tetrahydrofolate together with nutritional support of the one-carbon cycle (OCC). After 3 months of treatment, the couple conceived, and the woman delivered a healthy female baby. Three years later, she again conceived and delivered a second healthy female baby. Our treatment can restore fertility in males affected by a triple SNP mutation in the MTHFR gene; this confirms that Hcy and MTHFR SNP testing should not be overlooked in patients affected by long duration of infertility/repeat miscarriages.

Studies in the mouse demonstrate the importance of fibroblast growth factor (FGF) and extra-cellular receptor tyrosine kinase (ERK) in specification of embryo-fated epiblast and yolk-sac-fated hypoblast cells from uncommitted inner cell mass (ICM) cells prior to implantation. Molecular mechanisms regulating specification of early lineages in human development are comparatively unclear. Here we show that exogenous FGF stimulation leads to expanded hypoblast molecular marker expression, at the expense of the epiblast. Conversely, we show that specifically inhibiting ERK activity leads to expansion of epiblast cells functionally capable of giving rise to naïve human pluripotent stem cells. Single-cell transcriptomic analysis indicates that these epiblast cells downregulate FGF signalling and maintain molecular markers of the epiblast. Our functional study demonstrates the molecular mechanisms governing ICM specification in human development, whereby segregation of the epiblast and hypoblast lineages occurs during maturation of the mammalian embryo in an ERK signal-dependent manner.

Existing methods to image chromosome segregation errors are not suitable for studying human embryos at advanced preimplantation stages. As chromosomal errors are a leading cause of miscarriage and infertility, it remains unclear whether missegregation arises postfertilization. Here we optimize nuclear DNA labeling via messenger RNA electroporation and apply light-sheet live imaging to reveal chromosome segregation errors immediately before implantation. We show that embryos at advanced preimplantation stages display missegregation, including multipolar spindle formation, lagging chromosomes, misalignment and mitotic slippage. Most lagging chromosomes are passively inherited rather than reincorporated. To trace individual nuclei, we developed an open-source, semi-automated segmentation method using a customized deep learning model optimized for variability in embryo size, shape and signal. With this approach, we find most labeled cells remain externally positioned, consistent with placental rather than inner cell mass fate. Our findings raise questions about clinical uses of preimplantation genetic testing for aneuploidy, while providing broadly applicable imaging and segmentation methods for studying diverse cellular structures in human embryos. A method to label cultured human embryos finds chromosome segregation errors in placenta-fated cells.

Many mammals can temporally uncouple conception from parturition by pacing down their development around the blastocyst stage. In mice, this dormant state is achieved by decreasing the activity of the growth-regulating mTOR signaling pathway. It is unknown whether this ability is conserved in mammals in general and in humans in particular. Here, we show that decreasing the activity of the mTOR signaling pathway induces human pluripotent stem cells (hPSCs) and blastoids to enter a dormant state with limited proliferation, developmental progression, and capacity to attach to endometrial cells. These in vitro assays show that, similar to other species, the ability to enter dormancy is active in human cells around the blastocyst stage and is reversible at both functional and molecular levels. The pacing of human blastocyst development has potential implications for reproductive therapies.

Meiotic and mitotic chromosome segregation errors in human development have been studied mostly prior to and at the time of fertilisation. Despite chromosomal errors being a leading cause of miscarriage and infertility, chromosome missegregation has not been extensively studied at later stages of human development. Here we optimised labelling, light-sheet live imaging and semi-quantitative analysis of human embryos and reveal chromosome segregation errors just prior to implantation. We found that human embryos exhibited a number of chromosome missegregation events including multipolar spindle formation, lagging chromosomes, misalignment and chromosome slippage. We found that the majority of lagging chromosomes were passively inherited by one of the daughter cells, instead of reincorporating into the nuclei, suggesting a distinct pattern of micronuclei inheritance. By semi-automated segmentation, we tracked the position of labelled cells in human embryos and observed that while most labelled cells remained segregated to the outside, and therefore restricted to a placental-progenitor fate, there was evidence of a rare cell migration to cells positioned on the inside, which suggests that there may be plasticity. Altogether, we found that mitotic chromosome segregation errors arise just prior to implantation, which has implications for our understanding of biological events that contribute to aneuploidy mosaicism. ### Competing Interest Statement The authors have declared no competing interest.

Studies in the mouse demonstrate the importance of fibroblast growth factor (FGF) and extra-cellular receptor tyrosine kinase (ERK) in specification of embryo-fated epiblast and yolk-sac-fated hypoblast cells from uncommitted inner cell mass (ICM) cells prior to implantation. Molecular mechanisms regulating specification of early lineages in human development are comparatively unclear. Here we show that exogenous FGF stimulation leads to expanded hypoblast molecular marker expression, at the expense of the epiblast. Conversely, we show that specifically inhibiting ERK activity leads to expansion of epiblast cells functionally capable of giving rise to naïve human pluripotent stem cells. Single-cell transcriptomic analysis indicates that these epiblast cells downregulate FGF signalling and upregulate molecular markers associated with naïve pluripotency. Our functional study demonstrates for the first time the molecular mechanisms governing ICM specification in human development, whereby segregation of the epiblast and hypoblast lineages occurs during maturation of the mammalian embryo in an ERK signal-dependent manner.### Competing Interest StatementThe authors have declared no competing interest.

In our practice, testing hypo-fertile patients for circulating homocysteine (Hcy) and the two principal MTHFR SNPs (677C > T and 1298A > C) has been routine for the past 7 years. Couples carrying a genetic background known to be associated with the disease were proposed treatment regimens consisting of 5-methyl tetrahydrofolate (5-MTHF) together with nutritional support of the one-carbon cycle (1-CC). Some patients preferred to continue with folic acid (FA) as prescribed by their referring gynecologist/obstetrician: this gave us the opportunity to compare outcomes between the two groups of patients. After successful live birth deliveries, we compared health characteristics and circulating Hcy in the offspring from ages 2 to 6 years, i.e., after cessation of breastfeeding and before puberty. Follow-up included children of 21 couples who were treated with FA vs 36 couples treated with 5-MTHF. In the FA-treated group, we found two children with autism spectrum disorder (ASD) syndrome, one child with significantly elevated circulating Hcy (19 µM at the age of 2 years), and one child affected by oculo-auriculo-vertebral spectrum (OAVS), a syndrome known to be linked to DNA methylation. No pathology of any kind was detected in children of the 5-MTHF treatment group. Treatment with 5-MTHF is safe and effective for both males and females. It should be implemented in order to avoid disruption of methylation linked to folate metabolism during oocyte maturation and pregnancy, and subsequently in the offspring. This type of treatment should be considered to avoid metabolic diseases linked to elevated homocysteine.

Development requires coordinated interactions between the epiblast, which generates the embryo proper; the trophectoderm, which generates the placenta; and the hypoblast, which forms both the anterior signalling centre and the yolk sac. These interactions remain poorly understood in human embryogenesis because mechanistic studies have only recently become possible. Here we examine signalling interactions post-implantation using human embryos and stem cell models of the epiblast and hypoblast. We find anterior hypoblast specification is NODAL dependent, as in the mouse. However, while BMP inhibits anterior signalling centre specification in the mouse, it is essential for its maintenance in human. We also find contrasting requirements for BMP in the naive pre-implantation epiblast of mouse and human embryos. Finally, we show that NOTCH signalling is important for human epiblast survival. Our findings of conserved and species-specific factors that drive these early stages of embryonic development highlight the strengths of comparative species studies.

ABSTRACT During the first week of development, human embryos form a blastocyst composed of an inner cell mass and trophectoderm (TE) cells, the latter of which are progenitors of placental trophoblast. Here, we investigated the expression of transcripts in the human TE from early to late blastocyst stages. We identified enrichment of the transcription factors GATA2, GATA3, TFAP2C and KLF5 and characterised their protein expression dynamics across TE development. By inducible overexpression and mRNA transfection, we determined that these factors, together with MYC, are sufficient to establish induced trophoblast stem cells (iTSCs) from primed human embryonic stem cells. These iTSCs self-renew and recapitulate morphological characteristics, gene expression profiles, and directed differentiation potential, similar to existing human TSCs. Systematic omission of each, or combinations of factors, revealed the crucial importance of GATA2 and GATA3 for iTSC transdifferentiation. Altogether, these findings provide insights into the transcription factor network that may be operational in the human TE and broaden the methods for establishing cellular models of early human placental progenitor cells, which may be useful in the future to model placental-associated diseases.

20 21 Recent advances in single cell omics have been transformative in the characterisation of 22 challenging to study biological contexts, including when the source material is precious, such 23 as the early human embryo. Single cell datasets are technically challenging to infer 24 transcription factor-gene regulatory interactions, due to low read-depth leading to zero inflated 25 data. Here we have systematically assessed the application of four different machine learning 26 linear or non-linear gene regulatory network prediction strategies to single cell simulated and 27 human embryo transcriptome datasets. We have also compared how the method of gene 28 expression normalisation impacts on regulatory network predictions. Integrating chromatin 29 accessibility together with transcript expression datasets improved the reproducibility of the 30 predicted gene regulatory networks. We found that the application of a non-linear network 31 prediction method based on mutual information (MI) to single cell transcriptome datasets 32 refined with chromatin accessibility

EpigenomicsVol. 15, No. 21 CommentaryHomocysteine testing is a significant predictor of health for couples trying to conceive and their future childrenYves Ménézo, Kay Elder, Arthur Clement, Pasquale Patrizio, Michel Brack & Patrice ClementYves Ménézo *Author for correspondence: E-mail Address: yves.menezo@gmail.comhttps://orcid.org/0000-0001-6861-8905Laboratoire CLEMENT, Genetics and Assisted reproduction, 17 avenue d'Eylau, 75016, Paris France, Kay Elder https://orcid.org/0000-0003-3510-8268Bourn Hall Clinic, High St, Bourn, Cambridge, CB23 2TN, UK, Arthur Clement https://orcid.org/0000-0003-2425-3953Laboratoire CLEMENT, Genetics and Assisted reproduction, 17 avenue d'Eylau, 75016, Paris France, Pasquale Patrizio https://orcid.org/0000-0003-4796-7078Reproductive Endocrinology & Infertility, 1400 Northwest, 12th Avenue, Miami, FL 33136, USA, Michel BrackThe oxidative stress College, 92250 La garenne Colombes, France & Patrice Clement https://orcid.org/0000-0001-8449-1035Laboratoire CLEMENT, Genetics and Assisted reproduction, 17 avenue d'Eylau, 75016, Paris FrancePublished Online:13 Nov 2023https://doi.org/10.2217/epi-2023-0333AboutSectionsView ArticleView Full TextSupplemental MaterialPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail View articleKeywords: gametogenesishealthhomocysteinemethylationobstetricspregnancyReferences1. 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Influence of early identification and therapy on long-term outcomes in early-onset MTHFR deficiency. J. Inherit. Metab. Dis. 45(4), 848–861 (2022).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetails Vol. 15, No. 21 STAY CONNECTED Supplemental Materials Metrics Downloaded 17 times History Received 18 September 2023 Accepted 25 October 2023 Published online 13 November 2023 Published in print November 2023 Information© 2023 Future Medicine LtdKeywordsgametogenesishealthhomocysteinemethylationobstetricspregnancySupplementary dataTo view the supplementary data that accompany this paper please visit the journal website at: www.futuremedicine.com/doi/suppl/10.2217/epi-2023-0333Financial disclosureThe authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.Competing interests disclosureThe authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.Writing disclosureNo writing assistance was utilized in the production of this manuscript.PDF download

Accurate chromosome segregation during meiosis is crucial for reproduction. Human and porcine oocytes transiently cluster their chromosomes before the onset of spindle assembly and subsequent chromosome segregation. The mechanism and function of chromosome clustering are unknown. Here we show that chromosome clustering is required to prevent chromosome losses in the long gap phase between nuclear envelope breakdown and the onset of spindle assembly, and to promote the rapid capture of all chromosomes by the acentrosomal spindle. The initial phase of chromosome clustering is driven by a dynamic network of Formin-2- and Spire-nucleated actin cables. The actin cables form in the disassembling nucleus and migrate towards the nuclear centre, moving the chromosomes centripetally by interacting with their arms and kinetochores as they migrate. A cage of stable microtubule loops drives the late stages of chromosome clustering. Together, our data establish a crucial role for chromosome clustering in accurate progression through meiosis.

Our understanding of the molecular events driving cell specification in early mammalian development relies mainly on mouse studies, and it remains unclear whether these mechanisms are conserved across mammals, including humans. We have shown that the establishment of cell polarity via aPKC is a conserved event in the initiation of the trophectoderm (TE) placental programme in mouse, cow and human embryos. However, the mechanisms transducing cell polarity into cell fate in cow and human embryos are unknown. Here, we have examined the evolutionary conservation of Hippo signalling, which is thought to function downstream of aPKC activity, in four different mammalian species: mouse, rat, cow and human. In all four species, inhibition of the Hippo pathway by targeting LATS kinases is sufficient to drive ectopic TE initiation and downregulation of SOX2. However, the timing and localisation of molecular markers differ across species, with rat embryos more closely recapitulating human and cow developmental dynamics, compared with the mouse. Our comparative embryology approach uncovered intriguing differences as well as similarities in a fundamental developmental process among mammals, reinforcing the importance of cross-species investigations.