Human Preimplantation Development Research Articles

An atlas of small non-coding RNAs in human preimplantation development

Understanding the molecular circuitries that govern early embryogenesis is important, yet our knowledge of these in human preimplantation development remains limited. Small non-coding RNAs (sncRNAs) can regulate gene expression and thus impact blastocyst formation, however, the expression of specific biotypes and their dynamics during preimplantation development remains unknown. Here we identify the abundance of and kinetics of piRNA, rRNA, snoRNA, tRNA, and miRNA from embryonic day (E)3-7 and isolate specific miRNAs and snoRNAs of particular importance in blastocyst formation and pluripotency. These sncRNAs correspond to specific genomic hotspots: an enrichment of the chromosome 19 miRNA cluster (C19MC) in the trophectoderm (TE), and the chromosome 14 miRNA cluster (C14MC) and MEG8-related snoRNAs in the inner cell mass (ICM), which may serve as ‘master regulators’ of potency and lineage. Additionally, we observe a developmental transition with 21 isomiRs and in tRNA fragment (tRF) codon usage and identify two novel miRNAs. Our analysis provides a comprehensive measure of sncRNA biotypes and their corresponding dynamics throughout human preimplantation development, providing an extensive resource. Better understanding the sncRNA regulatory programmes in human embryogenesis will inform strategies to improve embryo development and outcomes of assisted reproductive technologies. We anticipate broad usage of our data as a resource for studies aimed at understanding embryogenesis, optimising stem cell-based models, assisted reproductive technology, and stem cell biology.

Distinct dynamics of parental 5-hydroxymethylcytosine during human preimplantation development regulate early lineage gene expression

The conversion of DNA 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by TET enzymes represents a significant epigenetic modification, yet its role in early human embryos remains largely unknown. Here we showed that the early human embryo inherited a significant amount of 5hmCs from an oocyte, which unexpectedly underwent de novo hydroxymethylation during its growth. Furthermore, the generation of 5hmC in the paternal genome after fertilization roughly followed the maternal pattern, which was linked to DNA methylation dynamics and regions of sustained methylation. The 5hmCs persisted until the eight-cell stage and exhibited high enrichment at OTX2 binding sites, whereas knockdown of OTX2 in human embryos compromised the expression of early lineage genes. Specifically, the depletion of 5hmC affected the activation of embryonic genes, which was further evaluated by ectopically expressing mouse Tet3 in human early embryos. These findings revealed distinct dynamics of 5hmC and unravelled its multifaceted functions in early human embryonic development.

O-030 The impact of aneuploidy on human peri-implantation development: findings from the extended in vitro culture of 191 human embryos

Abstract Study question How does aneuploidy affect lineage specification and the developmental potential of human embryos cultured up to 12 days post fertilization (D12)? Summary answer While distinct types of aberrations carry their own risk of embryonic arrest, prevailing aneuploidies appear to compromise lineage specification beyond the blastocyst stage of development. What is known already Chromosomal instability is a hallmark of human preimplantation development, with up to 50% of embryos diagnosed as aneuploid following preimplantation genetic testing (PGT). Aneuploidies are a major cause of implantation failure, pregnancy loss and human congenital defects. Yet despite their clinical relevance, the impact of distinct aberrations on early human development remains poorly understood. Innovative platforms for culturing human embryos up to D12 provide an important opportunity for investigating chromosomal instability. Understanding the effects of specific aneuploidies on human peri-implantation development will be key for advancing human embryo research and improving the interpretation of findings from in vitro embryo models. Study design, size, duration A total of 191 PGT blastocysts obtained from 103 patients were included in the study. Blastocysts were selected based on their original diagnosis, including embryos with single whole-chromosome aneuploidies (single trisomies, n = 37 and single monosomies, n = 71), single segmental aberrations (n = 22), embryos with complex chromosomal constitutions (two or more abnormalities, n = 44), as well as control euploid embryos (n = 17). Participants/materials, setting, methods Vitrified blastocysts were warmed and cultured up to D12 using an optimized protocol to generate embryo outgrowths. We correlated the original PGT diagnoses to developmental outcomes, including outgrowth viability, morphology and outgrowth size. We evaluated blastocyst quality using the Gardner grading system. Following culture, we immunostained 38 viable outgrowths for lineage-specific markers, including OCT4 (epiblast), GATA6 (hypoblast) and CK-7 (trophoblast). Fisher’s exact test was used for statistical analysis (p < 0.05 was considered significant). Main results and the role of chance Following extended culture, 43.5% of embryos remained viable and attached, while 56.5% degenerated and detached. No significant correlation was found between embryo grade and attachment rate. Euploid embryos exhibited the highest attachment rate, with 88.2% (15/17) forming viable outgrowths. Euploid embryos also generated significantly larger outgrowths than trisomic and monosomic embryos (p = 0.01). Embryos diagnosed with trisomies and segmental aberrations exhibited attachment rates comparable to euploid embryos, at 70.3% (p = 0.18) and 68.2% (p = 0.25), respectively. However, embryos with monosomies and complex chromosomal constitutions arrested more readily (28.2% and 15.9% viable, <0.00001, respectively). All euploid embryos (6/6) retained a discernible OCT4+ epiblast-like structure. This was similar to embryos with segmental aberrations and trisomies, with 75% (3/4) and 53.8% (7/13) of outgrowths containing OCT4+ cells, respectively. Nonetheless, distinct trisomies exerted varying effects on peri-implantation development in vitro. For instance, viable trisomies (13, 18, 21) developed similarly to euploid embryos, while outgrowths carrying a trisomy 22 had fewer OCT4+ cells (p = 0.02) and a higher number of GATA6+ cells (p < 0.01), suggesting a potential lineage proliferation defect. Monosomies had the most profound effects on development. Compared to euploid embryos, monosomic embryos formed significantly fewer OCT4+ outgrowths (28.6%, p = 0.02) and contained fewer epiblast (p < 0.0001) and hypoblast-like (p = 0.003) cells. Limitations, reasons for caution The limited number of embryos in certain groups warrants careful interpretation. The central drawback of the models is that embryo outgrowths are predominately flattened, which confounds identification of three-dimensional structures formed during normal embryogenesis. Future studies should encompass additional lineage specification markers and potential culture refinements to enhance our interpretation. Wider implications of the findings Our findings underscore the critical importance of reporting the chromosomal composition of human embryos utilized for research. This practice will enable a more nuanced interpretation of results from in vitro embryo models and ultimately advance our capabilities in human embryo research. Trial registration number not applicable

Capturing totipotency in human cells through spliceosomal repression

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures invitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.

A maternal ketogenic diet alters oviduct fluid nutrients and embryo histone acetylation in mice.

A ketogenic diet (KD) elevates blood β-hydroxybutyrate to concentrations that are known to perturb the development, metabolism, histone acetylation and viability of preimplantation mouse embryos in culture. This study shows that a maternal KD changes available nutrient levels in the oviduct, leading to altered embryo development and epigenetic state in vivo. A ketogenic diet elevates blood β-hydroxybutyrate to concentrations that perturb the development, metabolism, histone acetylation (H3K27ac) and viability of preimplantation mouse embryos in vitro. However, whether a ketogenic diet alters β-hydroxybutyrate concentrations within female reproductive fluid is unknown. This study aimed to quantify glucose and β-hydroxybutyrate within mouse blood and oviduct fluid following standard diet and ketogenic diet consumption and to assess whether a maternal periconceptional ketogenic diet impacts in vivo embryo development and blastocyst H3K27ac. Female C57BL/6 × CBA mice were fed a standard or ketogenic diet (n = 24 each) for 24-27 days. Glucose and β-hydroxybutyrate were quantified in blood via an electronic monitoring system and in oviduct fluid via ultramicrofluorescence. The developmental grade of flushed blastocysts was recorded, and blastocyst cell number and H3K27ac were assessed via immunofluorescence. A maternal ketogenic diet elevated β-hydroxybutyrate in day 24 blood (P < 0.001) and oviduct fluid (P < 0.05) compared with a standard diet, whereas glucose was unchanged. A periconceptional ketogenic diet did not impact blastocyst cell number; however, it significantly delayed blastocyst development (P < 0.05) and reduced trophectoderm-specific H3K27ac (P < 0.05) compared with standard diet-derived embryos. Maternal ketogenic diet consumption is, therefore, associated with reproductive tract nutrient changes and altered embryonic development and epigenetics in vivo. Future studies to assess whether periconceptional/gestational ketogenic diet consumption impacts human preimplantation, fetal, and long-term offspring development and health are warranted.

Analysis of two layered peristaltic-ciliary transport of Jeffrey fluid and in vitro preimplantation embryo development

The analysis of peristaltic-ciliary transport in the human female fallopian tube, specifically in relation to the growing embryo, is a matter of considerable physiological importance. This paper proposes a biomechanical model that incorporates a finite permeable tube consisting of two layers, where the Jeffrey fluid model characterizes the viscoelastic properties of the growing embryo and continuously secreting fluid. Jeffrey fluid entering with some negative pressure gradient forms the core fluid layer while continuously secreting Jeffrey fluid forms the peripheral fluid layer. The resulting partial differential equations are solved for closed-form solutions after employing the assumption of long wavelength. The analysis delineated that increasing the constant secretion velocity, Darcy number, and Reynolds number leads to a decrease in the appropriate residue time of the core fluid layer and a reduction in the size of the secreting fluid bolus in the peripheral fluid layer. Eventually, the boluses completely disappear when the constant secretion velocity exceeds 3.0 Progesterone (P4\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$P_4$$\\end{document}) and estradiol (E2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$E_2$$\\end{document}) directly regulate the transportation of the growing embryo, while luteinizing hormone (LH) and follicle-stimulating hormone (FSH), prolactin, anti-mullerian hormone (AMH), and thyroid-stimulating hormone (TSH) have an indirect effects. Based on the number and size of blastomeres, the percentage of fragmentation, and the presence of multinucleated blastomeres two groups were formed in an in vitro experiment. Out of 50 patients, 26 (76.5%) were pregnant in a group of the good quality embryos, and only 8 (23.5%) were in a group of the bad quality embryos. The transport of growing embryo in the human fallopian tube and preimplantation development of human embryos in in vitro are constraint by baseline hormones FSH, LH, prolactin, E2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$E_{2}$$\\end{document}, AMH, and TSH.

A temporal extracellular transcriptome atlas of human pre-implantation development

Non-invasively evaluating gene expression products in human pre-implantation embryos remains a significant challenge. Here, we develop a non-invasive method for comprehensive characterization of the extracellular RNAs (exRNAs) in a single droplet of spent media that was used to culture human invitro fertilization embryos. We generate the temporal extracellular transcriptome atlas (TETA) of human pre-implantation development. TETA consists of 245 exRNA sequencing datasets for five developmental stages. These data reveal approximately 4,000 exRNAs at each stage. The exRNAs of the developmentally arrested embryos are enriched with the genes involved in negative regulation of the cell cycle, revealing an exRNA signature of developmental arrest. Furthermore, a machine-learning model can approximate the morphology-based rating of embryo quality based on the exRNA levels. These data reveal the widespread presence of coding gene-derived exRNAs at every stage of human pre-implantation development, and these exRNAs provide rich information on the physiology of the embryo.

Evidence for Functional Roles of MicroRNAs in Lineage Specification During Mouse and Human Preimplantation Development.

Proper formation of the blastocyst, including the specification of the first embryonic cellular lineages, is required to ensure healthy embryo development and can significantly impact the success of assisted reproductive technologies (ARTs). However, the regulatory role of microRNAs in early development, particularly in the context of preimplantation lineage specification, remains largely unknown. Taking a cross-species approach, this review aims to summarize the expression dynamics and functional significance of microRNAs in the differentiation and maintenance of lineage identity in both the mouse and the human. Findings are consolidated from studies conducted using in vitro embryonic stem cell models representing the epiblast, trophectoderm, and primitive endoderm lineages (modeled by naïve embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm stem cells, respectively) to provide insight on what may be occurring in the embryo. Additionally, studies directly conducted in both mouse and human embryos are discussed, emphasizing similarities to the stem cell models and the gaps in our understanding, which will hopefully lead to further investigation of these areas. By unraveling the intricate mechanisms by which microRNAs regulate the specification and maintenance of cellular lineages in the blastocyst, we can leverage this knowledge to further optimize stem cell-based models such as the blastoids, enhance embryo competence, and develop methods of non-invasive embryo selection, which can potentially increase the success rates of assisted reproductive technologies and improve the experiences of those receiving fertility treatments.

Debate 4: Morphological Assessment of Embryos is Outdated

Motion: For The Outdated Significance of Morphological Assessment in Embryo Selection and the Rise of Advanced Technologies in Reproductive Medicine This symposium lecture presents a compelling debate, shedding light on the diminishing relevance of traditional static morphological assessment as a primary criterion for embryo. The speaker aims to challenge the long-standing reliance on morphological evaluation and advocate for the integration of cutting-edge technologies as a more reliable and objective means of embryo quality assessment. During the debate, the speaker will examine the limitations of morphological assessment, acknowledging its subjective nature, potential bias, and lack of comprehensive insight into embryonic development. The traditional morphological assessment based solely on visual appearance may not adequately predict an embryo’s true potential for successful implantation and healthy pregnancy. A variety of available new technologies will be highlighted during the lecture, showcasing alternatives to morphological embryo assessment. Attendees will gain insight into how these innovative methods can offer more objective and precise measures of embryo quality, enhancing the success rates of assisted reproductive procedures. The symposium will foster an engaging environment, encouraging active participation and thoughtful discussions among attendees from the reproductive medicine community. Participants will have the opportunity to challenge prevailing notions and explore the implications of adopting new technologies in clinical practice. In summary, this debate aims to catalyze a paradigm shift in embryo evaluation, emphasizing the need to move beyond traditional morphological assessment towards embracing advanced technologies, to optimize embryo selection and lead to improved outcomes in IVF. Motion: Against Morphological Assessment of Embryos is Not Outdated In the era of new technologies and artificial intelligence, looking good has never been more important! On-time progression through specific events is requisite for human preimplantation development and provides important visual insights into the health and physiology of the embryo. The formation of 2 distinct pronuclei, the times and patterns of cleavage, and how the cells compact are all key markers of development. Following compaction, the formation of the blastocyst is not only a temporally regulated event, but also an energetically demanding process, one that reflects the metabolic activity (a biomarker of viability) of the embryo. Further, at the blastocyst stage it is possible to visualise and quantitate the formation of both the inner cell mass (ICM) and trophectoderm, with all parameters of blastocyst development linked to transfer outcomes. The development of time-lapse incubation, which can be described as embryo morphology over time, has led to the incorporation of several key morphological features to support embryo selection, further reflecting the significance of embryo morphology. Interestingly, AI algorithms used for embryo selection reflect predominantly those morphological parameters that are known to affect transfer outcomes, such as blastocyst quality. While only specific molecular tests can accurately determine the precise chromosomal composition of an embryo, key morphological features and events can also be used to tag those embryos at greatest risk of being genetically abnormal. In conclusion, key morphological developmental events indirectly inform us of the physiology of the embryo (degree of blastocyst expansion reflects metabolic activity for example), and hence this is why morphological grading systems (such as the Gardner Grade for blastocysts) have been shown to be effective in embryo selection for over the past 25 years, and further have proven to be as useful as AI or biomarker analysis for the majority of patients.

Isoform-resolved transcriptome of the human preimplantation embryo

Human preimplantation development involves extensive remodeling of RNA expression and splicing. However, its transcriptome has been compiled using short-read sequencing data, which fails to capture most full-length mRNAs. Here, we generate an isoform-resolved transcriptome of early human development by performing long- and short-read RNA sequencing on 73 embryos spanning the zygote to blastocyst stages. We identify 110,212 unannotated isoforms transcribed from known genes, including highly conserved protein-coding loci and key developmental regulators. We further identify 17,964 isoforms from 5,239 unannotated genes, which are largely non-coding, primate-specific, and highly associated with transposable elements. These isoforms are widely supported by the integration of published multi-omics datasets, including single-cell 8CLC and blastoid studies. Alternative splicing and gene co-expression network analyses further reveal that embryonic genome activation is associated with splicing disruption and transient upregulation of gene modules. Together, these findings show that the human embryo transcriptome is far more complex than currently known, and will act as a valuable resource to empower future studies exploring development.

Human Embryo Live Imaging Reveals Nuclear DNA Shedding During Blastocyst Expansion and Biopsy

ABSTRACT The proportion of babies born from in vitro fertilization (IVF) is rising at an exponential rate, highlighting the importance that we have a comprehensive understanding of human preimplantation development and determinants of embryo quality. The early divisions of the embryo after fertilization but before implantation and resulting mitotic errors have been studied primarily in the mouse embryo; however, thus far we have lacked the technology to characterize these critical steps in humans. Establishing an approach that can bypass genetic manipulation and microinjections of DNA or mRNA into human embryos would allow us to better uncover the processes patterning preimplantation human development. This study aimed to evaluate human blastocyst preimplantation development with noninvasive imaging using membrane permeable fluorescent dyes. First, the dyes SPY650-DNA, which labels genomic DNA, and SPY555-actin, which labels F-actin, were validated in the mouse embryo where they produced high contrast labelling with similar results to microinjection of fluorescent mRNAs and produced offspring at a similar rate to nondyed control embryos. Live imaging data using these dyes enabled 3-dimensional scans of the embryo at 5- to 10-minute intervals of the following central events during preimplantation development: the main phases of mitosis, visualizing the major changes in cell shape that characterize embryo compaction at the 8-cell stage, detecting cell polarization at the 8-cell stage, establishing apical F-actin rings at the 16-cell stage, tracking the expansion and zippering of F-actin rings, detecting the first internalized cells within the 16-cell embryo, and visualizing blastocyst expansion and hatching from the zona pellucida. Cleavage-stage human IVF embryos were then studied using the fluorescent dyes to track early cellular and morphogenetic processes. Early cell-cycle dynamics and mitotic stages revealed that the duration of human mitosis is similar to that of mice, but interphase is 27% ± 4% longer in humans (16.1 ± 0.9 vs 12.7 ± 0.4 hours; P &lt; 0.01). Using live imaging, compaction dynamics such as increased cell-cell contact and angle between apical membranes with a decrease in cell sphericity were observed beginning at the 12-cell stage and differed significantly from development in mice. In comparison to mice, human compaction was found to be more asynchronous and did not have clear links to apical polarization or inner-outer segregation. Next, chromosomal segregation was analyzed to determine if this approach allowed detection of segregation errors in the human embryo leading to aneuploidy. Lagging chromosomes detected in human embryos using SPY-DNA appeared morphologically similar to those found in mouse embryos and had similar segregation dynamics. During blastocyst expansion, a subset of trophectoderm cell nuclei forms protruding bud-like that is shed into cytoplasm producing cytoplasmic DNA structures (cytDNA), suggesting an additional process of DNA loss different from chromosome segregation errors during mitosis. A perinuclear keratin network developing at the early cavitation stage appears to protect from formation of cytDNA structures, and the mechanical stress of blastocyst expansion appeared to disrupt the keratin network in certain cells, increasing DNA shedding. Next, trophectoderm biopsy was conducted in mouse, and human embryos to determine if the mechanical stress would increase DNA shedding. Biopsy of human blastocysts resulted in a significant increase in nuclear budding (6.0% vs 0.70%; P = 0.0022), suggesting a link between mechanical stress and DNA shedding. This study demonstrates the use of fluorescent dyes and live imaging in characterization of key cellular and morphogenetic processes patterning the preimplantation human embryo and describes a process of DNA shedding in response to mechanical stressors that warrants future research.

Transcriptomic differences between human 8-cell-like cells reprogrammed with different methods.

Embryonic genome activation (EGA) is a critical step in embryonic development. However, while EGA has been studied in mice using mouse 2-cell-like cells, human EGA remains incompletely elucidated due to the lack of an invitro cell model recapitulating the early blastomere stage in humans. Recently, five groups independently reported human 8-cell-like cells (8CLCs, also called induced blastomere-like cells) developed from pluripotent stem cells and used single-cell RNA sequencing (scRNA-seq) to specify their cellular identities. Here we summarize the methods developed to produce the 8CLCs and compare their transcriptomic profiles by integrating them with the scRNA-seq datasets of human embryos. These observations will allow comparison and validation of the models, stimulate further in-depth research to characterize the genes involved in human EGA and pre-implantation development, and facilitate studies on human embryogenesis.

Assessing the influence of distinct culture media on human pre-implantation development using single-embryo transcriptomics.

The use of assisted reproductive technologies is consistently rising across the world. However, making an informed choice on which embryo culture medium should be preferred to ensure satisfactory pregnancy rates and the health of future children critically lacks scientific background. In particular, embryos within their first days of development are highly sensitive to their micro-environment, and it is unknown how their transcriptome adapts to different embryo culture compositions. Here, we determined the impact of culture media composition on gene expression in human pre-implantation embryos. By employing single-embryo RNA-sequencing after 2 or 5days of the post-fertilization culture in different commercially available media (Ferticult, Global, and SSM), we revealed medium-specific differences in gene expression changes. Embryos cultured pre-compaction until day 2 in Ferticult or Global media notably displayed 266 differentially expressed genes, which were related to essential developmental pathways. Herein, 19 of them could have a key role in early development, based on their previously described dynamic expression changes across development. When embryos were cultured after day 2 in the same media considered more suitable because of its amino acid enrichment, 18 differentially expressed genes thought to be involved in the transition from early to later embryonic stages were identified. Overall, the differences were reduced at the blastocyst stage, highlighting the ability of embryos conceived in a suboptimal in vitro culture medium to mitigate the transcriptomic profile acquired under different pre-compaction environments.

P-399 Oocyte vitrification does not impair clinical outcomes in women of advanced maternal age. An analysis of 1268 cycles using fresh and vitrified oocytes

Abstract Study question Does oocyte vitrification impair clinical outcomes in women of advanced maternal age (AMA, &amp;gt;35 years old)? Summary answer Up to the age of 39 years, clinical outcomes were comparable amongst cycles using fresh and vitrified oocytes. What is known already Primarily recommended for young women, oocyte vitrification is the most efficient method for elective fertility preservation. Nevertheless, various social and economic circumstances often compel women to undergo fertility preservation when already at an AMA. Recent studies in mouse suggest that AMA increases the vulnerability of oocytes to molecular and subcellular damage during vitrification, ultimately compromising embryo viability and implantation potential. However, the effects of oocyte vitrification on human preimplantation development and clinical outcomes in women of AMA currently remain unknown. Study design, size, duration This is a retrospective cohort single-center study. We compared outcomes of 1268 patients undergoing their first fresh ICSI cycle using autologous fresh (n = 1087) or vitrified oocytes (n = 181), performed between January 2019 and October 2022. All vitrified oocytes were obtained following elective fertility preservation for age-related fertility decline. Outcomes were stratified according to maternal age at oocyte pick-up: control group (≤35) and AMA groups (36-37, 38-39 and ≥40 years). Participants/materials, setting, methods We compared outcomes between fresh and vitrified oocytes across matching maternal age groups. These included fertilization rates, proportion of viable embryos on day 3, clinical pregnancy and live birth rates. Univariate and multivariate analyses (2-contrast linear and logistic regression) were used to establish correlations. Analyses were adjusting for sperm origin (partner or donor), day of transfer (3 or 5), number of embryos transferred and endometrial age. All p values &amp;lt;0.05 were considered significant. Main results and the role of chance Sperm donation and homologous cycles were equally distributed among the study groups (45% sperm donor and 55% male partner across the entire cohort). In our cohort, blastocyst transfer was performed less frequently in older women, 23.8% for women ≥40 years compared to 45.6% for women ≤35 years (p &amp;lt; 0.0001). When comparing oocyte status across matching maternal age groups, fertilization rates were ∼10% lower when vitrified oocytes were used (p &amp;lt; 0.05). Our adjusted analysis confirmed this negative impact of oocyte vitrification on fertilization rates. Oocyte vitrification also affected ongoing cleavage, as the proportion of viable embryos on day 3 was significantly lower across all groups when vitrified oocytes were used (p &amp;lt; 0.05). Nevertheless, this effect did not translate to clinical pregnancy rates, which were comparable across all maternal age groups regardless of oocyte status. As expected, live birth rate decreased steadily with advancing maternal age (from 38.5% in women ≤35 to 15.0% in women ≥40). While our adjusted analysis showed a negative effect of oocyte vitrification on live birth rates for patients ≥40 (OR: 0.60, 95%CI: 0.18-2.06), we observed no such effect in AMA patients ≤39 years. Limitations, reasons for caution The main limitation of this study is its retrospective design. The low number of women ≥40 undergoing fertility presentation (n = 39) warrants careful interpretation. Our results cannot be generalized to all patient populations and indications for fertility preservation. Wider implications of the findings Clinical outcomes were comparable between fresh and vitrified oocytes up to the age of 39 years. While oocyte vitrification before 35 years old remains the most established and effective method for preventing age-related fertility decline, our findings offer a valuable clinical resource for counselling AMA patients considering elective fertility preservation. Trial registration number Not applicable

P-802 TFAP2C is required for proper segregation of developmental markers regulating trophectoderm commitment during mouse preimplantation development

Abstract Study question What is the effect of CRISPR/Cas9-mediated knock-out of trophectoderm markers TFAP2A/C on polarization and lineage commitment in mouse? Summary answer CRISPR/Cas9-mediated KO revealed discrepancies in TFAP2A/C function, wherein TFAP2C is dispensable for blastocyst formation but controls the segregation of transcription factors in the mouse embryo. What is known already TFAP2C is an important transcription factor that is expressed solely in trophectoderm (TE) in mouse, and both TE and epiblast in human. Recently, genetic ablation of both TFAP2C and TEAD4 (player of the HIPPO-pathway) completely abolished polarization in mouse, which indicates that TFAP2C might already act before the first lineage segregation takes place. In addition, TFAP2C RNA depletion experiments in mouse embryos revealed a compensatory upregulation of TFAP2A, which indicates functional redundancy. Nonetheless, the question remains how exactly TFAP2C regulates polarization on a molecular level in mouse and whether there is also a functional implication of its isoform TFAP2A. Study design, size, duration Guide RNAs were designed targeting exon 5 of Tfap2c and exon 2 of the Tfap2a gene. CRISPR/Cas9 ribonucleoprotein complexes were delivered into mouse zygotes via electroporation. Additionally, appropriate non-targeted and scramble (inactive crRNA) control groups were included. Morphological analysis, immunofluorescence and next-generation sequencing (NGS) were applied to check for gene editing efficiency and the impact of KO on embryonic development and lineage segregation. Participants/materials, setting, methods Targeted mouse embryos and controls were cultured for a maximum of 4.5 days in vitro. They were stained for different developmental markers, including CDX2 (TE), SOX2 (early ICM), NANOG (epibast, EPI) and SOX17 (hypoblast, PrE) at different stages of development, such as polarization (E2.5), first (E3.5) and second (E4.5) lineage segregation. Immunostaining was used to determine cell number, TE/ICM fraction, marker localization and fluorescence intensity. Embryos were subjected to genetic analysis to determine on-target efficiency. Main results and the role of chance CRISPR/Cas9-mediated electroporation of mouse zygotes generated efficiently complete knock-out embryos. Of the 39 mouse zygotes targeted for TFAP2C, 38 (97%) of them were edited. From the 38 embryos edited for TFAP2C, 24 (62%) embryos displayed 100% frameshift mutations, and were considered KO. TFAP2C KO mouse embryos cultured until E4.5 were still able to form blastocysts (13/15, 87%), but were associated with inferior blastocyst quality compared to the control groups. Furthermore, TFAP2c-null blastocysts could hatch but herniated at multiple places, in contrast to wild-type blastocysts, which typically herniate at one place. In full frameshift TFAP2C KO mouse embryos, we observed delayed CDX2 expression at E2.5 (n = 7). At E3.5 (n = 5), however, CDX2 expression could still be pertained in KO embryos, whereas nuclear localization of SOX2 was disturbed. At E4.5 (n = 6), some primitive endoderm cells were observed in the presumed ICM (SOX17-positive), whereas no NANOG-positive (EPI) cells could be detected. However, some cells of presumed TE of E4.5 TFAP2C-KO embryos displayed erroneous SOX17 or NANOG expression, indicating that they adopted another cell fate irrespective of cell localization. Secondly, we generated TFAP2A KO mouse embryos (3/5, 60%) which exhibited complete morula arrest (100%; n = 3) at E4.5. Limitations, reasons for caution CRISPR/Cas9 is limited by the occurrence of mosaicism (more than one genotype present in an embryo) and potential off-target editing, which we will assess at in silico predicted off-target sites via NGS in mouse embryonic stem cells. The observations of the study will be consolidated by increasing the sample size. Wider implications of the findings Gene editing studies enable us to unravel the molecular interactions that are required for human preimplantation development. Obtaining novel insights into the molecular networks of the TFAP2-transcription factor family could improve our fundamental understanding on the spatial segregation of key transcription factors, which is crucial for successful implantation. Trial registration number Not applicable

Lineage segregation in human pre-implantation embryos is specified by YAP1 and TEAD1.

Lineage segregation in human pre-implantation embryos is specified by YAP1 and TEAD1.

Updates on preimplantation embryo research

Preimplantation development is the only stage of human development that can be studied outside the body in real time, as human embryos can be produced by invitro fertilization and cultured in the laboratory as self-contained structures until the blastocyst stage. Here, we focus some of the key cellular and morphogenetic processes by which the 1-cell embryo is transformed gradually into a blastocyst ready for implantation. Although most of our knowledge about the dynamic series of events patterning preimplantation human development derives from work in mouse embryos, we discuss key differences that could exist with humans. Furthermore, we highlight how new approaches may enable to reveal many of the unknown processes driving human preimplantation development, particularly using noninvasive imaging and genetic technologies.

A shared pattern of altered gene expression in human embryos affected by mitochondrial diseases.

Does mitochondrial deficiency affect human embryonic preimplantation development? The presence of a pathogenic mitochondrial variant triggers changes in the gene expression of preimplantation human embryos, compromising their development, cell differentiation, and survival. Quantitative and qualitative anomalies of mitochondrial DNA (mtDNA) are reportedly associated with impaired human embryonic development, but the underlying mechanisms remain unexplained. Taking advantage of the preimplantation genetic testing for mitochondrial disorders in at-risk couples, we have compared gene expression of 9 human embryos carrying pathogenic variants in either mtDNA genes or nuclear genes encoding mitochondrial protein to 33 age-matched control embryos. Single-embryo transcriptomic analysis was performed on whole human blastocyst embryos donated to research. Specific pathogenic mitochondrial variants downregulate gene expression in preimplantation human embryos [566 genes in oxidative phosphorylation (OXPHOS)-deficient embryos], impacting transcriptional regulators, differentiation factors, and nuclear genes encoding mitochondrial proteins. These changes in gene expression primarily alter OXPHOS and cell survival pathways. The number of OXPHOS-deficient embryos available for the study was limited owing to the rarity of this material. However, the molecular signature shared by all these embryos supports the relevance of the findings. While identification of reliable markers of normal embryonic development is urgently needed in ART, our study prompts us to consider under-expression of the targeted genes reported here, as predictive biomarkers of mitochondrial dysfunction during preimplantation development. This work was supported by the 'Association Française contre les Myopathies (AFM-Téléthon)' and the 'La Fondation Maladies Rares'. No competing interests to declare. N/A.

Histone remodeling reflects conserved mechanismsof bovine and human preimplantationdevelopment.

How histone modifications regulate changes in gene expression during preimplantation development in any species remains poorly understood. Using CUT&Tag to overcome limiting amounts of biological material, we profiled two activating (H3K4me3 and H3K27ac) and two repressive (H3K9me3 and H3K27me3) marks in bovine oocytes, 2-, 4-, and 8-cell embryos, morula, blastocysts, inner cell mass, and trophectoderm. In oocytes, broad bivalent domains mark developmental genes, and prior to embryonic genome activation (EGA), H3K9me3 and H3K27me3 co-occupy gene bodies, suggesting a global mechanism for transcription repression. During EGA, chromatin accessibility is established before canonical H3K4me3 and H3K27ac signatures. Embryonic transcription is required for this remodeling, indicating that maternally provided products alone are insufficient for reprogramming. Last, H3K27me3 plays a major role in restriction of cellular potency, as blastocyst lineages are defined by differential polycomb repression and transcription factor activity. Notably, inferred regulators of EGA and blastocyst formation strongly resemble those described in humans, as opposed to mice. These similarities suggest that cattle are a better model than rodents to investigate the molecular basis of human preimplantation development.

Cross-species comparison of mouse and human preimplantation development with an emphasis on lineage specification.

Human embryogenesis still remains largely unexplored. This review helps identify some of our current gaps in knowledge pertaining to preimplantation development, which may have implications for understanding fundamental aspects of human development, assisted reproductive technologies, and stem cell biology. Preimplantation development is arguably one of the most critical stages of embryogenesis. Beginning with the formation of the totipotent zygote post-fertilization, a series of cell divisions, and a complex coordination of physical cues, molecular signals and changes in gene expression lead to the formation of the blastocyst, a structure capable of implanting into the uterine wall. The blastocyst is composed of more specified cellular lineages, which will give rise to every tissue of the developing organism as well as the extra-embryonic lineages which support fetal growth. While the mouse has been used as a model to understand the events of preimplantation development for decades, in recent years, an expanding body of work has been conducted using the human embryo. These studies have identified some crucial species differences, particularly in the transcriptional and spatio-temporal expression of lineage markers and responses to cell signaling perturbations. This review compares recent findings on preimplantation development in mouse and human, with a focus on the specification of the first cellular lineages. Highlighting differences and noting mechanisms that require further examination in the human embryo is of critical importance for both the accurate translation of results from the mouse model and our overall understanding of mammalian development. We further highlight the latest advancement in reproductive research, the development of the 3D stem cell-based models known as 'blastoids'. The knowledge discussed in this review has major clinical implications for assisted reproductive technologies such as in vitro fertilization and for applications in stem cell biology.