Post-implantation Development Research Articles

A comprehensive review: synergizing stem cell and embryonic development knowledge in mouse and human integrated stem cell-based embryo models.

Mammalian stem cell-based embryo models have emerged as innovative tools for investigating early embryogenesis in both mice and primates. They not only reduce the need for sacrificing mice but also overcome ethical limitations associated with human embryo research. Furthermore, they provide a platform to address scientific questions that are otherwise challenging to explore in vivo. The usefulness of a stem cell-based embryo model depends on its fidelity in replicating development, efficiency and reproducibility; all essential for addressing biological queries in a quantitative manner, enabling statistical analysis. Achieving such fidelity and efficiency requires robust systems that demand extensive optimization efforts. A profound understanding of pre- and post-implantation development, cellular plasticity, lineage specification, and existing models is imperative for making informed decisions in constructing these models. This review aims to highlight essential differences in embryo development and stem cell biology between mice and humans, assess how these variances influence the formation of partially and fully integrated stem cell models, and identify critical challenges in the field.

Oct4 controls basement membrane development during human embryogenesis

Basement membranes (BMs) are sheet-like structures of extracellular matrix (ECM) that provide structural support for many tissues and play a central role in signaling. They are key regulators of cell behavior and tissue functions, and defects in their assembly or composition are involved in numerous human diseases. Due to the differences between human and animal embryogenesis, ethical concerns, legal constraints, the scarcity of human tissue material, and the inaccessibility of the invivo condition, BM regulation during human embryo development has remained elusive. Using the post-implantation amniotic sac embryoid (PASE), we delineate BM assembly upon post-implantation development and BM disassembly during primitive streak (PS) cell dissemination. Further, we show that the transcription factor Oct4 regulates the expression of BM structural components and receptors and controls BM development by regulating Akt signaling and the small GTPase Rac1. These results represent a relevant step toward a more comprehensive understanding of early human development.

Influence of the storage conditions of embryo culture media on mouse development.

The culture of preimplantation embryos in vitro is an important method for human and mouse reproductive technology. This study aims to investigate the influence of different conditions of culture media on the preimplantation stage of mouse embryos cultured in vitro, and monitor the post-implantation development of new mice after embryo transfer to surrogate females. We demonstrated here that mouse embryos cultured in vitro in fresh M16, KSOM, Global, and HTF embryo culture media from one cell to the blastocyst stage and the subsequent embryo transfer to surrogate females are able to proceed through post-implantation development and, after birth, develop into healthy mice. However, culture of embryos in differently aged media shows various (often unpredictable) results. To find the optimal storage conditions of culture media, we suggest that the freezing and long-term storage of these media at - 80°C will not influence the quality of the media. To test this hypothesis, we grew embryos from one cell to blastocysts in vitro in the selected media after thawing and subsequently transferring them to surrogate females. Embryo culture in these four media after thawing does not affect preimplantation and postnatal mouse development. Thus, we have shown that storage of embryo culture media at low temperature (- 80°C) does not impact the quality of the media, and subsequently, it can be used for the culture of embryos for the full preimplantation period, the same as in fresh media.

The emergence of human gastrulation upon in vitro attachment

While studied extensively in model systems, human gastrulation remains obscure. The scarcity of fetal biological material as well as ethical considerations limit our understanding of this process. Invitro attachment of natural blastocysts shed light on aspects of the second week of human development in the absence of the morphological manifestation of gastrulation. Stem cell-derived blastocyst models, blastoids, provide the opportunity to reconstitute pre- to post-implantation development invitro. Here we show that upon invitro attachment, human blastoids self-organize a BRA+ population and undergo gastrulation. Single-cell RNA sequencing of these models replicates the transcriptomic signature of the human gastrula. Analysis of developmental timing reveals that in both blastoid models and natural human embryos, the onset of gastrulation as defined by molecular markers, can be traced to timescales equivalent to 12days post fertilization. In all, natural human embryos and blastoid models self-organize primitive streak and mesoderm derivatives upon invitro attachment.

MTOR hypoactivity leads to trophectoderm cell failure by enhancing lysosomal activation and disrupting the cytoskeleton in preimplantation embryo

BackgroundMetabolic homeostasis is closely related to early impairment of cell fate determination and embryo development. The protein kinase mechanistic target of rapamycin (mTOR) is a key regulator of cellular metabolism in the body. Inhibition of mTOR signaling in early embryo causes postimplantation development failure, yet the mechanisms are still poorly understood.MethodsPregnancy mice and preimplantation mouse embryo were treated with mTOR inhibitor in vivo and in vitro respectively, and subsequently examined the blastocyst formation, implantation, and post-implantation development. We used immunofluorescence staining, RNA-Seq smart2, and genome-wide bisulfite sequencing technologies to investigate the impact of mTOR inhibitors on the quality, cell fate determination, and molecular alterations in developing embryos.ResultsWe showed mTOR suppression during preimplantation decreases the rate of blastocyst formation and the competency of implantation, impairs the post implantation embryonic development. We discovered that blocking mTOR signaling negatively affected the transformation of 8-cell embryos into blastocysts and caused various deficiencies in blastocyst quality. These included problems with compromised trophectoderm cell differentiation, as well as disruptions in cell fate specification. mTOR suppression significantly affected the transcription and DNA methylation of embryos. Treatment with mTOR inhibitors increase lysosomal activation and disrupts the organization and dynamics of the actin cytoskeleton in blastocysts.ConclusionsThese results demonstrate that mTOR plays a crucial role in 8-cell to blastocyst transition and safeguards embryo quality during early embryo development.

Complete human day 14 post-implantation embryo models from naive ES cells

The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation1. Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking1,2. Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs)3. Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions)4. Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13–14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior–posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development.

In Vitro Embryogenesis and Gastrulation Using Stem Cells in Mice and Humans.

During early mammalian embryonic development, fertilized one-cell embryos develop into pre-implantation blastocysts and subsequently establish three germ layers through gastrulation during post-implantation development. In recent years, stem cells have emerged as a powerful tool to study embryogenesis and gastrulation without the need for eggs, allowing for the generation of embryo-like structures known as synthetic embryos or embryoids. These in vitro models closely resemble early embryos in terms of morphology and gene expression and provide a faithful recapitulation of early pre- and post-implantation embryonic development. Synthetic embryos can be generated through a combinatorial culture of three blastocyst-derived stem cell types, such as embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm cells, or totipotent-like stem cells alone. This review provides an overview of the progress and various approaches in studying in vitro embryogenesis and gastrulation in mice and humans using stem cells. Furthermore, recent findings and breakthroughs in synthetic embryos and gastruloids are outlined. Despite ethical considerations, synthetic embryo models hold promise for understanding mammalian (including humans) embryonic development and have potential implications for regenerative medicine and developmental research.

3D-cultured blastoids model human embryogenesis from pre-implantation to early gastrulation stages.

Naive human pluripotent stem cells have the remarkable ability to self-organize into blastocyst-like structures ("blastoids") that model lineage segregation in the pre-implantation embryo. However, the extent to which blastoids can recapitulate the defining features of human post-implantation development remains unexplored. Here, we report that blastoids cultured on thick three-dimensional (3D) extracellular matrices capture hallmarks of early post-implantation development, including epiblast lumenogenesis, rapid expansion and diversification of trophoblast lineages, and robust invasion of extravillous trophoblast cells by day 14. Extended blastoid culture results in the localized activation of primitive streak marker TBXT and the emergence of embryonic germ layers by day 21. We also show that the modulation of WNT signaling alters the balance between epiblast and trophoblast fates in post-implantation blastoids. This work demonstrates that 3D-cultured blastoids offer a continuous and integrated invitro model system of human embryonic and extraembryonic development from pre-implantation to early gastrulation stages.

3D-cultured human blastoids model trophoblast expansion, diversification, and invasion during early post-implantation development

3D-cultured human blastoids model trophoblast expansion, diversification, and invasion during early post-implantation development

Mitochondrial DNA mutations can influence the post-implantation development of human mosaic embryos.

Introduction: Several healthy euploid births have been reported following the transfer of mosaic embryos, including both euploid and aneuploid blastomeres. This has been attributed to a reduced number of aneuploid cells, as previously reported in mice, but remains poorly explored in humans. We hypothesized that mitochondrial function, one of the most critical factors for embryonic development, can influence human post-implantation embryonic development, including a decrease of aneuploid cells in mosaic embryos. Methods: To clarify the role of mitochondrial function, we biopsied multiple parts of each human embryo and observed the remaining embryos under in vitro culture as a model of post-implantation development (n = 27 embryos). Karyotyping, whole mitochondrial DNA (mtDNA) sequencing, and mtDNA copy number assays were performed on all pre- and post-culture samples. Results: The ratio of euploid embryos was significantly enhanced during in vitro culture, whereas the ratio of mosaic embryos was significantly reduced. Furthermore, post-culture euploid and culturable embryos had significantly few mtDNA mutations, although mtDNA copy numbers did not differ. Discussion: Our results indicate that aneuploid cells decrease in human embryos post-implantation, and mtDNA mutations might induce low mitochondrial function and influence the development of post-implantation embryos with not only aneuploidy but also euploidy. Analyzing the whole mtDNA mutation number may be a novel method for selecting a better mosaic embryo for transfer.

Pluripotent stem cell-derived model of the post-implantation human embryo

The human embryo undergoes morphogenetic transformations following implantation into the uterus, but our knowledge of this crucial stage is limited by the inability to observe the embryo in vivo. Models of the embryo derived from stem cells are important tools for interrogating developmental events and tissue–tissue crosstalk during these stages1. Here we establish a model of the human post-implantation embryo, a human embryoid, comprising embryonic and extraembryonic tissues. We combine two types of extraembryonic-like cell generated by overexpression of transcription factors with wild-type embryonic stem cells and promote their self-organization into structures that mimic several aspects of the post-implantation human embryo. These self-organized aggregates contain a pluripotent epiblast-like domain surrounded by extraembryonic-like tissues. Our functional studies demonstrate that the epiblast-like domain robustly differentiates into amnion, extraembryonic mesenchyme and primordial germ cell-like cells in response to bone morphogenetic protein cues. In addition, we identify an inhibitory role for SOX17 in the specification of anterior hypoblast-like cells2. Modulation of the subpopulations in the hypoblast-like compartment demonstrates that extraembryonic-like cells influence epiblast-like domain differentiation, highlighting functional tissue–tissue crosstalk. In conclusion, we present a modular, tractable, integrated3 model of the human embryo that will enable us to probe key questions of human post-implantation development, a critical window during which substantial numbers of pregnancies fail.

O-179 Fertilization topologies that occur seldom or not at all in nature give rise to distinct functional classes of mouse embryos

Abstract Study question Would new embryo properties emerge if fertilization was imposed on oocytes in regions, such as the animal pole, where it does not take place naturally? Summary answer Oocyte fertilization at the animal pole, vegetal pole or equator results in 2-cell embryos with distinguishable transcriptomes and functional peculiarities. What is known already There is fertilization bias in embryos used in basic and clinical research, because: 1) sperm–oocyte fusion hardly occurs at the surface above the meiotic spindle – the animal pole; 2) the region opposite the spindle - vegetal pole - is poorly accessible to sperm due to tiny perivitelline space. These regions are also avoided during intracytoplasmic sperm injection (ICSI) for fear of damaging the spindle or losing the sperm nucleus into the 2nd polar body. Pole materials are held non-essential for mouse development, but this conclusion relies on the 2nd polar body, which is an unreliable topological marker. Study design, size, duration Oocytes were rotated using a micromanipulator fitted with Nomarski optics, using the meiotic spindle as a landmark. Between 9:30 and 10:30 am single sperm heads were microinjected (ICSI) at the animal or vegetal pole (treatments) vs. the naturally prevalent equatorial region (control). This way two fertilization topologies and possibly also two classes of zygotes were created that are otherwise seldom or not at all represented in natural fertilization, in vitro insemination or conventional ICSI. Participants/materials, setting, methods Metaphase II oocytes were collected from 8-week-old B6C3F1 mice stimulated with 10 I.U. eCG+hCG. Sperm heads from a single batch of cryopreserved CD1 semen were deposited via ICSI in the cortex at the animal pole, vegetal pole or half-way between poles i.e. equatorially. Zygotes were cultured in KSOM(aa) and analyzed (triplicate or more) for: cleavage rates, transcriptomes at the 2-cell stage (RNAseq), blastocyst germ layers (immunostaining for trophectoderm, primitive endoderm, epiblast), and postimplantation development. Main results and the role of chance Although full development was supported irrespective of ICSI site, embryos clustered by site, as revealed by single-cell RNAseq of 21, 21 and 13 two-cell embryos whose oocytes were fertilized at the animal pole, vegetal pole or equator, respectively. When examining the sister blastomeres together, 462 genes of the shared transcriptome were differently expressed between ICSI sites, with the equatorial class contributing most to the difference (adj.p<0.05, Wilcoxon test). This was true also when examining the sister blastomeres separately: interblastomere differences of the equatorial class exceeded those of the pole classes (72% vs. 14% of differently expressed genes). Ontology analysis of the differently expressed genes using Enrichr pointed at the endomembrane system – an acquaintance of oocyte polarity studies (PMID 10545249; PMID 29746690). Follow-up of 2-cell embryos to blastocysts revealed that sister blastomere contribution to each germ layer was less balanced in the equatorial class, as measured by linear correlation of cell numbers (e.g. equatorial R2=0.00 vs. polar R2>0.23 for the epiblast). Summing up, it is difficult to reconcile these data with a mainstream view that fertilization at the animal pole is harmful. Rather they support that the topology of fertilization defines functional classes of 2-cell embryos with distinguishable transcriptomes. Limitations, reasons for caution This is an animal study. Mouse ICSI uses mercury-loaded piezo-driven needles, human ICSI does not. The higher consistency of using the one and same batch of cryopreserved spermatozoa was traded off against lower developmental rates. Single-cell resolution posed a limit on RNAseq depth. Results need confirmation in other mouse strains. Wider implications of the findings The higher blastomere similarity observed after ICSI at the vegetal pole compared to ICSI at the equator is not consistent with the prevalent model of first zygotic cleavage that is driven by the topology of the two apposing pronuclei. Trial registration number not applicable

P-282 The role of activin A in the development of embryo: a preliminary insight using a mouse in vitro model

Abstract Study question Is activin A involved in the preimplantation development of mouse embryo? Do maternal-derived and zygotic protein have a different impact on embryogenesis? Summary answer Deletion of zygotic activin A does not impair the preimplantation development of mouse embryos. However, reduced level of maternal activin A affects female fertility. What is known already Activin A is a protein secreted by the reproductive system of females, both in mouse and human. Disruption of its signalling pathway is correlated with the occurrence of ectopic pregnancies and miscarriages. Activin A is also a known regulator of the hormone FSH, affecting the growth of ovarian follicles and, consequently, the formation of functional gametes. Furthermore, expression of this protein is detected in embryos themselves and is imperative during postimplantation development. Although activin has been reported to be present from the preimplantation stages, due to the multiple sources of secretion, its role during this period of embryogenesis remains elusive. Study design, size, duration In the first part of our project, we compared the phenotype of zygotic knockout mouse embryos (Inhba−/−, n = 43) with stage-matched heterozygotes (Inhba+/−, n = 113) and wild-type (Inhba+/+, n = 42) embryos. In addition, the blastocyst outgrowth assay has been used as an in vitro implantation model. The second part of our studies focuses on the protein provided by the mother. Using hypomorphic mutants, we investigated the impact of reduced level of activin A on mouse female fertility. Participants/materials, setting, methods We analysed the morphokinetic parameters of Inhba−/−, Inhba+/− and Inhba+/+ embryos by recording their development using time-lapse microscopy. Then blastocysts were immunostained and the percentage of each cell lineage was quantified using Imaris software. To assess the fertility of hypomorphic mice, we mated them with wild-type individuals. We evaluated the time needed to receive the offspring and the size of the litter. Furthermore, we examined the functionality of oocytes of hypomorphs by in vitro fertilisation. Main results and the role of chance We revealed that the deletion of zygotic activin A does not affect the rate and course of development of embryos from the zygote to the blastocyst stage. Inhba−/− embryos have similar timing of cleavage divisions, compaction, cavitation and synchrony of the cleavage rounds to the stage-matched Inhba+/− and Inhba+/+ embryos. Moreover, Inhba−/− embryos do not significantly differ in the number and percentage of cells contributing to the cell lineages of a blastocyst. Despite the reports on the possible involvement of activin A in the embryo implantation process, the obtained Inhba−/− outgrowths have normal morphology and a similar surface area to the corresponding control embryos. Therefore, our results indicate that zygotic activin A is indispensable for preimplantation development and embryo implantation. To study the effect of activin A secreted by the maternal reproductive system on embryogenesis, we used mice with a hypomorphic mutation of the activin A gene. We revealed that homozygotic mice have a characteristic phenotype and significantly reduced fertility. Crossing hypomorphic homozygous females with wild-type males resulted in the pregnancy of only one of the seven females. These results, combined with successful oocyte in vitro fertilisation experiments, point to the role of maternal-derived activin A in mouse reproduction. Limitations, reasons for caution Literature data indicate that oocytes have a pool of protein that is a product of the maternal genome. This maternal activin A can partially compensate for the lack of zygotic protein. Therefore, our further research will address the consequences of maternal and maternal-zygotic activin A knockout on embryo development. Wider implications of the findings Separate examination of zygotic and maternal-derived activin A allowed a comprehensive investigation of the contribution of this protein to mouse reproduction and preimplantation embryo development. Despite using a mouse as a model organism, the obtained results may become an argument for discussion of the impact of activin on human pregnancy. Trial registration number not applicable

P-264 Correlations between the artificial intelligence scoring system (iDAScore v1.0) and live birth outcomes in preimplantation genetic testing for aneuploidy cycles

Abstract Study question Is the iDAScore v1.0 score correlated with pregnancy outcomes in single embryo transfer (SET) cycles following preimplantation genetic testing for aneuploidy (PGT-A)? Summary answer We demonstrated that elevated iDAScore v1.0 scores are positively correlated with the probabilities of pregnancy and live birth (LB) in SETs following PGT-A. What is known already To select the blastocysts with high development potential in advance, several groups have proposed the implementation of artificial intelligence (AI)-based software is capable of predicting implantation in women undergoing in vitro fertilization (IVF). It has also been known that using high resolution next generation sequencing preimplantation genetic screening (hr-NGS) for PGT-A can enable the exclusion of aneuploid embryos before embryo transfers and thus improve pregnancy outcomes of SETs. However, the capacity of AI-based assessments has remained unclear in IVF cycles with PGT-A. Study design, size, duration This retrospective study, approved by the Institutional Review Board of Chung Sun Medical University, was performed to assess the dataset of 317 SET cycles after PGT-A from January 2017 to September 2019. A single euploid or mosaic blastocyst with an AI score calculated using the iDAScore software (v1.0) was selected for frozen embryo transfer according to the given priority of blastocyst morphology. The iDAScore groups were categorized by quartiles of AI scores. Participants/materials, setting, methods Embryos were cultured in a time-lapse incubator and qualified blastocysts underwent next generation sequencing (NGS)-based PGT-A. The confounding factors associated with LB were evaluated using logistic regression analysis in generalized estimating equations (GEEs). The differences between iDAScore groups were assessed using the Mann–Whitney U test or Fisher’s exact test, as applicable. The receiver operating characteristic (ROC) curve analysis was used to estimate the predictive powers. A P value < 0.05 was considered statistically significant. Main results and the role of chance The results revealed the patient age, anti-Müllerian hormone, body mass index, oocyte sources (autologous or donor), PGT-A results (euploidy, low-level mosaicism, or high-level mosaicism), aberrant chromosome types (none, whole chromosome, segmental chromosome, or whole with segmental chromosome), and aberrant chromosome sites (0, 1, 2, or > 2) were not correlated with LB. However, embryo biopsy days (day 5 vs. day 6, OR = 2.271, 95% CI = 1.534–14.824), blastocyst morphology scores (OR = 1.185, 95% CI = 1.034–1.359), and AI scores (OR = 1.640, 95% CI = 1.231–2.183) were significantly correlated with LB. The AI scores were then divided into quartiles (group 1: 5.0–7.9; group 2: 8.0–8.5; group 3: 8.6–8.9; and group 4: 9.0–9.5). The ongoing pregnancy (29.1% vs. 54.2%–57.5%) and live birth (27.8% vs. 52.8%–56.3%) rates of group 1 were significantly lower than other groups. The blastocyst morphology score (5.3 ± 1.2), and KIDScore D5 scores (v1 = 3.0 ± 1.7; v3 = 4.6 ± 1.5) were also lowest in group 1. The ROC curve analysis confirmed a significant but limited LB prediction capability for iDAScore v1.0 (AUC = 0.6), which was similar to the KIDScore D5 (AUC = 0.61–0.62). Limitations, reasons for caution Because of the retrospective nature, the major limitation was the lack of randomization, which may present a risk of selection bias. According to the criteria of blastocyst selection for SETs, the embryos with low (< 5.0) or high (> 9.5) AI scores were not present in this dataset. Wider implications of the findings Although prediction capability of iDAScore remains perfectible, the AI score is still significantly associated with LB probability. Euploid or mosaic blastocysts with low AI scores (<8.0) processed a decreased LB rate, indicating the potential of annotation-free iDAScore system as a decision support tool for deselecting embryos with poor post-implantation development. Trial registration number not applicable

P-152 Velocity of cytoplasmic movement in trophectodermal cells depends on the keratin cytoskeleton

Abstract Study question Can dysfunction of the keratin cytoskeleton, known to severely hinder postimplantation development of mammalian embryos, be detected by analysis of cytoplasmic movement in trophectodermal cells? Summary answer Depletion of keratins increases cytoplasmic movement in trophectodermal cells in extent depending on the developmental stage and on the location of the trophectodermal cells (mural/polar). What is known already Recent literature data indicate that keratins, intermediate filament proteins, are key regulators of trophectoderm function in mouse and human embryos. Keratin knockouts display trophoblast fragility, placental bleeding, and lethality after implantation. However, depletion of keratin 8 (Krt8) and keratin 18 (Krt18), which are most abundant in preimplantation embryos, does not lead to severe phenotype up to the blastocyst stage at least in mice and cattle, so can easily remain unnoticed. However, it affects the biomechanical properties of the trophectoderm. Analysis of cytoplasmic movement allows for biomechanical assessment of the trophectoderm in a non-invasive way. Study design, size, duration We analyzed 188 mouse embryos (66 control, 60 with Krt8 depletion, and 62 with Krt18 depletion) to quantitatively assess changes in the velocity of cytoplasmic movement caused by a knockdown of Krt8 and Krt18. Additionally, we correlated the velocity of cytoplasmic movement in trophectodermal cells (n = 70 embryos) with the ability to form outgrowths. Appropriate statistical tests were used to determine the statistical significance of the obtained results. A value of p < 0.05 was considered statistically significant. Participants/materials, setting, methods Zygotes were injected with Krt8 and Krt18 siRNAs or with scrambled siRNAs used as a negative control. The efficiency of the knockdown was tested on mRNA and protein levels. The embryos were cultured for 3 or 4 days and the velocity of cytoplasmic movement in polar and mural trophectoderm cells was measured. Additionally, we correlated the cytoplasmic movement velocity in trophectodermal cells in E3.5 embryos with their ability to implant in vitro (i.e., form outgrowths). Main results and the role of chance Our research showed that the velocity of cytoplasmic movement of trophectodermal cells varies depending on the embryo's developmental stage. Namely, the velocity of cytoplasmic movement was higher in E3.5 blastocysts than in E4.5 blastocysts. In addition, the velocity of cytoplasmic movement was higher in polar (8.45 ± 2.64 nm/s in E3.5 and 6.79 ± 1.96 nm/s in E4.5 control embryos) than in mural (6.30 ± 2.30 nm/s in E3.5 and 4.98 ± 1.89 nm/s in E4.5 control embryos) trophectodermal cells. Noteworthy, the depletion of Krt8 and Krt18 increased the velocity of cytoplasmic movement both in the mural and polar trophectodermal cells. However, it appears that changes caused by the depletion of these keratins depend on the embryo's developmental stage. For example, the depletion of Krt18 increased the velocity of cytoplasmic movement in mural trophectodermal cells in E3.5 embryos (9.61 ± 3.80 nm/s), while the depletion of Krt8 - in E4.5 embryos (6.65 ± 2.35 nm/s). In addition, data from our preliminary experiments with in vitro implantation suggested that embryos characterized by the higher cytoplasmic movement velocity were unable to form an outgrowth. The above data suggest that the velocity of cytoplasmic movement depends on the keratin cytoskeleton and probably can be used to predict an embryo’s ability to implant. Limitations, reasons for caution We used mouse embryos, a well-established model of early mammalian embryo development. However, analogous experiments should be repeated on other mammalian species, including humans, if we wish to pursue the possibility that cytoplasmic velocity in trophectodermal cells is an embryo quality marker. Wider implications of the findings Our data indicate that cytoplasmic velocity in trophectodermal cells may be a valuable tool in embryo quality assessment, as it reflects the functionality of the keratin cytoskeleton in embryos as well as correlates with the embryos’ ability to implant. Trial registration number not applicable

Telomere Length in Human Spermatogenic Cells as a New Potential Predictor of Clinical Outcomes in ART Treatment with Intracytoplasmic Injection of Testicular Spermatozoa.

Predicting the clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles that use the testicular spermatozoa of azoospermic patients presents a challenge. Thus, the development of additional approaches to assessing the competence of a testicular-sperm-derived embryo without causing damage to gametes or the embryo is necessary. One of the key parameters in determining such developmental competence is telomere length (TL). We aimed to analyze TLs in spermatogenic cells from the testicular biopsy samples of azoospermic patients and determine how this parameter influences embryo competence for pre- and post-implantation development. Using Q-FISH, we studied the TL of the chromosomes in spermatogonia and spermatocytes I from the TESE biopsy samples of 30 azoospermic patients. An increase in TL was detected during the differentiation from spermatogonia to spermatocytes I. The patients' testicular spermatozoa were used in 37 ICSI cycles that resulted in 22 embryo transfers. Nine pregnancies resulted, of which, one was ectopic and eight ended in birth. The analysis of embryological outcomes revealed a dependence between embryo competence for development to the blastocyst stage and the TL in spermatogenic cells. The TLs in spermatogonia and spermatocytes I in the testicular biopsy samples were found to be higher in patients whose testicular sperm ICSI cycles resulted in a birth. Therefore, the length of telomeres in spermatogenic cells can be considered as a potential prognostic criterion in assessing the competence of testicular-sperm-derived embryos for pre- and post-implantation development. The results of this study provide the basis for the development of a laboratory test for the prediction of testicular sperm ICSI cycle outcomes.

Evaluation of the post-implantation development of mouse embryos derived from round spermatid injection

Round spermatid injection (ROSI), one of the assistant reproductive technologies, was used to treat partial infertility patients suffering from non-obstructive azoospermia. However, the development efficiency and birth rate of ROSI embryos are extremely low, and it is urgent to investigate the underlying mechanisms of low efficiency to improve the clinical application of ROSI technology. Here, we analyzed and compared the genome stability of the mouse blastocyst and the post-implantation development between ROSI and ICSI embryos. We first sequenced the genome of blastocysts from mouse ROSI embryos that can correctly form male and female pronuclei (2 PN) and found that the genomes of 7 blastocysts were normal. Furthermore, the implantation rate of ROSI 2 PN embryos on embryonic day 7.5 is similar to that of ICSI embryos, and at this time, 37.50% (9/24) of deciduas have no normal gestational sac. The proportion of embryos that survived to embryonic day 11.5 in the ROSI 2 PN group, ROSI non-2 PN group, parthenogenesis group, and ICSI 2 PN group was 51.61%, 7.14%, 0.00%, and 55.00%, respectively. And two smaller fetuses were found in the ROSI 2 PN group, which is not found in the other three groups. In addition, the physiological indexes, including fetus and placenta weight, sex ratio, growth rate, and the natural breeding ability for the offspring obtained from mouse ROSI, were evaluated; ROSI mice exhibited no obvious defect or abnormality and implied that the progeny were safe. Our results provided new evidence to promote the clinical application of ROSI technology.

Efficient generation of ETX embryoids that recapitulate the entire window of murine egg cylinder development.

The murine embryonic-trophoblast-extra-embryonic endoderm (ETX) model is an integrated stem cell-based model to study early postimplantation development. It is based on the self-assembly potential of embryonic, trophoblast, and hypoblast/primitive/visceral endoderm-type stem cell lines (ESC, TSC, and XEN, respectively) to arrange into postimplantation egg cylinder-like embryoids. Here, we provide an optimized method for reliable and efficient generation of ETX embryoids that develop into late gastrulation in static culture conditions. It is based on transgenic Gata6-overproducing ESCs and modified assembly and culture conditions. Using this method, up to 43% of assembled ETX embryoids exhibited a correct spatial distribution of the three stem cell derivatives at day 4 of culture. Of those, 40% progressed into ETX embryoids that both transcriptionally and morphologically faithfully mimicked in vivo postimplantation mouse development between E5.5 and E7.5. The ETX model system offers the opportunity to study the murine postimplantation egg cylinder stages and could serve as a source of various cell lineage precursors.

A New Approach to Generate Gastruloids to Develop Anterior Neural Tissues.

Embryonic development is a complex process integrating cell fate decisions and morphogenesis in a spatiotemporally controlled manner. Previous studies with model organisms laid the foundation of our knowledge on post-implantation development; however, studying mammalian embryos at this stage is a difficult and laborious process. Early attempts to recapitulate mammalian development in vitro begun with embryoid bodies (EBs), in which aggregates of mouse embryonic stem cells (mESCs) were shown to differentiate into spatially arranged germ layers. A more revised version of EBs, gastruloids, improved the germ layer differentiation efficiency and demonstrated cell fate patterning on multiple axes. However, gastruloids lack anterior neural progenitors that give rise to brain tissues in the embryo. Here, we report a novel culture protocol to coax mESCs into post-implantation epiblast-like (EPI) aggregates in high throughput on bioengineered microwell arrays. We show that upon inhibition of the Wnt signaling pathway, EPI aggregates establish an extended axial patterning, leading to co-derivation of anterior neural progenitors and posterior tissues. Our approach is amenable to large-scale studies aimed at identifying novel regulators of gastrulation and anterior neural development that is currently out of reach with existing embryoid models. This work should contribute to the advancement of the nascent field of synthetic embryology, opening up exciting perspectives for various applications of pluripotent stem cells in disease modeling and tissue engineering. Key features A new gastruloid culture system to model post-implantation mouse embryonic development in vitro High-throughput formation of epiblast-like aggregates on hydrogel microwells Builds upon conventional gastruloid cultures and provides insight into the role of Wnt signaling for the formation of anterior neural tissues Graphical overview.

Epiblast-like stem cells established by Wnt/β-catenin signaling manifest distinct features of formative pluripotency and germline competence

Different formative pluripotent stem cells harboring similar functional properties have been recently established to be lineage neutral and germline competent yet have distinct molecular identities. Here, we show that WNT/β-catenin signaling activation sustains transient mouse epiblast-like cells as epiblast-like stem cells (EpiLSCs). EpiLSCs display metastable formative pluripotency with bivalent cellular energy metabolism and unique transcriptomic features and chromatin accessibility. We develop single-cell stage label transfer (scSTALT) to study the formative pluripotency continuum and reveal that EpiLSCs recapitulate a unique developmental period invivo, filling the gap of the formative pluripotency continuum between other published formative stem cells. WNT/β-catenin signaling activation counteracts differentiation effects of activin A and bFGF by preventing complete dissolution of naive pluripotency regulatory network. Moreover, EpiLSCs have direct competence toward germline specification, which is further matured by an FGF receptor inhibitor. Our EpiLSCs can serve as an invitro model for mimicking and studying early post-implantation development and pluripotency transition.