Blastomeres Of Embryos Research Articles

Cleavage-Stage Embryo Segmentation Using SAM-Based Dual Branch Pipeline: Development and Evaluation with the CleavageEmbryo Dataset.

Embryo selection is one of the critical factors in determining the success of pregnancy in in vitro fertilization (IVF) procedures. Using artificial intelligence to aid in embryo selection could effectively address the current time-consuming, expensive, subjectively influenced process of embryo assessment by trained embryologists. However, current deep learning-based methods often focus on blastocyst segmentation, grading, or predicting cell development via time-lapse videos, often overlooking morphokinetic parameters or lacking interpretability. Given the significance of both morphokinetic and morphological evaluation in predicting the implantation potential of cleavage-stage embryos, as emphasized by previous research, there is a necessity for an automated method to segment cleavage-stage embryos to improve this process. In this article, we introduce the SAM-based Dual Branch Segmentation Pipeline for automated segmentation of blastomeres in cleavage-stage embryos. Leveraging the powerful segmentation capability of SAM, the instance branch conducts instance segmentation of blastomeres, while the semantic branch performs semantic segmentation of fragments. Due to the lack of publicly available datasets, we construct the CleavageEmbryo dataset, the first dataset of human cleavage-stage embryos with pixel-level annotations containing fragment information. We train and test a series of state-of-the-art segmentation algorithms on CleavageEmbryo. Our experiments demonstrate that our method outperforms existing algorithms in terms of objective metrics (mAP 0.748 on blastomeres, Dice 0.694 on fragments) and visual quality, enabling more accurate segmentation of cleavage-stage embryos. The code and sample data in this study can be found at: Https://github.com/12austincc/Cleavage-StageEmbryoSegmentation. Supplementary data are available at Bioinformatics online.

Proteome asymmetry in mouse and human embryos before fate specification.

Pre-patterning of the embryo, driven by spatially localized factors, is a common feature across several non-mammalian species 1-4 . However, mammals display regulative development and thus it was thought that blastomeres of the embryo do not show such pre-patterning, contributing randomly to the three lineages of the blastocyst: the epiblast, primitive endoderm and trophectoderm that will generate the new organism, the yolk sac and placenta respectively 4-6 . Unexpectedly, early blastomeres of mouse and human embryos have been reported to have distinct developmental fates, potential and heterogeneous abundance of certain transcripts 7-12 . Nevertheless, the extent of the earliest intra-embryo differences remains unclear and controversial. Here, by utilizing multiplexed and label-free single-cell proteomics by mass-spectrometry 13 , we show that 2-cell mouse and human embryos contain an alpha and a beta blastomere as defined by differential abundance of hundreds of proteins exhibiting strong functional enrichment for protein synthesis, transport, and degradation. Such asymmetrically distributed proteins include Gps1 and Nedd8, depletion or overexpression of which in one blastomere of the 2-cell embryo impacts lineage segregation. These protein asymmetries increase at 4-cell stage. Intriguingly, halved mouse zygotes display asymmetric protein abundance that resembles alpha and beta blastomeres, suggesting differential proteome localization already within zygotes. We find that beta blastomeres give rise to a blastocyst with a higher proportion of epiblast cells than alpha blastomeres and that vegetal blastomeres, which are known to have a reduced developmental potential, are more likely to be alpha. Human 2-cell blastomeres also partition into two clusters sharing strong concordance with clusters found in mouse, in terms of differentially abundant proteins and functional enrichment. To our knowledge, this is the first demonstration of intra-zygotic and inter-blastomere proteomic asymmetry in mammals that has a role in lineage segregation.

The pluripotency state of human embryonic stem cells derived from single blastomeres of eight-cell embryos

Human embryonic stem cells (hESCs) derived from blastocyst stage embryos present a primed state of pluripotency, whereas mouse ESCs (mESCs) display naïve pluripotency. Their unique characteristics make naïve hESCs more suitable for particular applications in biomedical research. This work aimed to derive hESCs from single blastomeres and determine their pluripotency state, which is currently unclear. We derived hESC lines from single blastomeres of 8-cell embryos and from whole blastocysts, and analysed several naïve pluripotency indicators, their transcriptomic profile and their trilineage differentiation potential. No significant differences were observed between blastomere-derived hESCs (bm-hESCs) and blastocyst-derived hESCs (bc-hESCs) for most naïve pluripotency indicators, including TFE3 localization, mitochondrial activity, and global DNA methylation and hydroxymethylation, nor for their trilineage differentiation potential. Nevertheless, bm-hESCs showed an increased single-cell clonogenicity and a higher expression of naïve pluripotency markers at early passages than bc-hESCs. Furthermore, RNA-seq revealed that bc-hESCs overexpressed a set of genes related to the post-implantational epiblast. Altogether, these results suggest that bm-hESCs, although displaying primed pluripotency, would be slightly closer to the naïve end of the pluripotency continuum than bc-hESCs.

Chromosome Division in Early Embryos-Is Everything under Control? And Is the Cell Size Important?

Chromosome segregation in female germ cells and early embryonic blastomeres is known to be highly prone to errors. The resulting aneuploidy is therefore the most frequent cause of termination of early development and embryo loss in mammals. And in specific cases, when the aneuploidy is actually compatible with embryonic and fetal development, it leads to severe developmental disorders. The main surveillance mechanism, which is essential for the fidelity of chromosome segregation, is the Spindle Assembly Checkpoint (SAC). And although all eukaryotic cells carry genes required for SAC, it is not clear whether this pathway is active in all cell types, including blastomeres of early embryos. In this review, we will summarize and discuss the recent progress in our understanding of the mechanisms controlling chromosome segregation and how they might work in embryos and mammalian embryos in particular. Our conclusion from the current literature is that the early mammalian embryos show limited capabilities to react to chromosome segregation defects, which might, at least partially, explain the widespread problem of aneuploidy during the early development in mammals.

Chimeric PRMT6 protein produced by an endogenous retrovirus promoter regulates cell fate decision in mouse preimplantation embryos†.

Murine endogenous retrovirus with leucine tRNA primer, also known as MERVL, is expressed during zygotic genome activation in mammalian embryos. Here we show that protein arginine N-methyltransferase 6 (Prmt6) forms a chimeric transcript with MT2B2, one of the long terminal repeat sequences of murine endogenous retrovirus with leucine tRNA primer, and is translated into an elongated chimeric protein (PRMT6MT2B2) whose function differs from that of the canonical PRMT6 protein (PRMT6CAN) in mouse preimplantation embryos. Overexpression of PRMT6CAN in fibroblast cells increased asymmetric dimethylation of the third arginine residue of both histone H2A (H2AR3me2a) and histone H4 (H4R3me2a), while overexpression of PRMT6MT2B2 increased only H2AR3me2a. In addition, overexpression of PRMT6MT2B2 in one blastomere of mouse two-cell embryos promoted cell proliferation and differentiation of the blastomere into epiblast cells at the blastocyst stage, while overexpression of PRMT6CAN repressed cell proliferation. This is the first report of the translation of a chimeric protein (PRMT6MT2B2) in mouse preimplantation embryos. Our results suggest that analyzing chimeric transcripts with murine endogenous retrovirus with leucine tRNA primer will provide insight into the relationship between zygotic genome activation and subsequent intra- and extra-cellular lineage determination.

Blastomere size in the human 2-cell embryo predicts the division order that leads to imbalanced lineage contribution to the future body.

Retrospective tracing of somatic mutations predicted that most cells in the human body could be traced back to a single cell of the 2-cell stage embryo. Accordingly, a recent prospective study of the developmental trajectory of blastomeres in human embryos confirmed that progeny of the first 2-cell stage blastomere to divide generates more epiblast cells (future body). How the 2-cell blastomeres differ is unknown. Here, we show that 2-cell stage blastomeres in human embryos are asymmetric; they differ in size and the bigger blastomere divides first to 4-cell stage. We propose that this asymmetry might originate differences in cell fate.

Resveratrol intake by males increased the mitochondrial DNA copy number and telomere length of blastocysts derived from aged mice.

The present study examined whether male resveratrol intake affected mitochondrial DNA copy number (mt-cn) and telomere length (TL) in blastocysts fathered by young and aged male mice. C57BL/6N male mice supplied with water or water containing 0.1 mM resveratrol were used for embryo production at 14-23 and 48-58 weeks of age. Two-cell-stage embryos were collected from the oviducts of superovulated female mice (8-15 weeks old) and cultured for 3 days until the blastocyst stage. Mt-cn and TL levels were measured by real-time polymerase chain reaction. Resveratrol intake did not affect body weight or water consumption. Resveratrol intake increased the expression levels of SIRT1 in the liver, the antioxidative ability of serum, and extended TL in the heart, whereas there was no significant difference in mt-cn in the heart or TL in sperm. The rate of blastocyst development was significantly lower in aged male mice than in younger mice, and resveratrol intake increased the total number of blastocysts derived from both young and aged males. Resveratrol intake did not affect mt-cn or TL in blastomeres of blastocyst-stage embryos derived from young mice, but significantly increased both mt-cn and TL in blastomeres of blastocysts derived from aged fathers. In conclusion, resveratrol intake increased mt-cn and TL levels in blastocysts derived from aged male mice.

External bending of cryodevice during vitrification leads to cryoprotectant cracks and damage to embryo blastomeres

Research questionEmbryo blastomeres and the zona pellucida are occasionally damaged during vitrification; is this a result of crack-induced mechanical damage in the glass state, caused by external bending of the device? DesignA stereomicroscope was used to observe external bending-induced cracks in a cryoprotectant. Thereafter, 309 human cleavage-stage embryos derived from abnormally fertilized eggs were used to assess embryo damage under two external bending conditions: forward bending and backward bending, with three bending degrees applied. Three distinct embryo positions were used to examine the correlation between bending and embryo damage. Damage was assessed by looking at blastomere lysis rates, and overall rates of damaged and surviving embryos. ResultsA series of parallel cracks were identified in the cryoprotectant used for external bending, which led to damage to the embryo blastomeres. Compared with forward bending and control, the embryos were found to be more easily damaged by backward bending, indicated by significantly higher blastomere lysis and embryo damage rates, and lower embryo survival rate of backward bending than forward bending (P < 0.001). The degree of embryo damage also increased as the degree of external forces increased. Embryo position correlated with degree of embryo damage. ConclusionsCryoprotectant crack-induced damage was identified as the cause of embryo damage. Mechanical damage to the glass state occurs because of improper external bending of the cryodevice strip in liquid nitrogen during vitrification. To prevent damage, bending of the strip should be avoided and the embryos should be placed near the tip of the strip.

#23 : Correlation of Multinucleated Embryo and Pregnancy Outcome

Background and Aims: Single embryo transfer is increasingly done in practice to avoid multiple pregnancy. Therefore, it is important to ensure the embryo with the best pregnancy outcome is selected. Although new criteria involving genomic, transcriptomic, and proteomic have been developed to predict embryo outcome, morphology of the embryo remains as the main indicator because it is easy to implement and less controversial. To study the correlation of multinucleated embryos and pregnancy outcomes Method: This is a retrospective review of a total of 411 embryos from 225 women undergoing their first IVF or ICSI cycle in University Malaya Medical Centre, Kuala Lumpur between January 2019 and October 2021. Multinucleation was defined as the presence of more than one nucleus within at least one of the blastomeres of a 2-cell embryo and a time-lapse imaging system was used to observe embryo development (Embryoscope; Unisense Fertilitech). Group allocation was based on the presence or absence of multinucleation on day 2 after egg retrieval. Embryos with evidence of one or more blastomere having multinucleation were included in the studied group (n = 67 embryos). Embryos without any blastomere exhibiting multinucleation were included in the control group (n = 344 embryos). Results: The data were based on 225 transfers of 411 embryos consisting of 16.3% (n = 67) from studied group and 83.7% (n = 344) from the control group. The biochemical pregnancy rate and implantation rate is lower in the studied group compared to the control group although not statistically significant (Table 1). Conclusion: Multinucleated embryos reduce the implantation rate and clinical pregnancy rate although not statistically significant.

In situ quantification of osmotic pressure within living embryonic tissues

Mechanics is known to play a fundamental role in many cellular and developmental processes. Beyond active forces and material properties, osmotic pressure is believed to control essential cell and tissue characteristics. However, it remains very challenging to perform in situ and in vivo measurements of osmotic pressure. Here we introduce double emulsion droplet sensors that enable local measurements of osmotic pressure intra- and extra-cellularly within 3D multicellular systems, including living tissues. After generating and calibrating the sensors, we measure the osmotic pressure in blastomeres of early zebrafish embryos as well as in the interstitial fluid between the cells of the blastula by monitoring the size of droplets previously inserted in the embryo. Our results show a balance between intracellular and interstitial osmotic pressures, with values of approximately 0.7 MPa, but a large pressure imbalance between the inside and outside of the embryo. The ability to measure osmotic pressure in 3D multicellular systems, including developing embryos and organoids, will help improve our understanding of its role in fundamental biological processes.

Blastomeres of 8-cell mouse embryos differ in their ability to generate embryonic stem cells and produce lines with different transcriptional signatures.

Embryonic stem cell (ESC) derivation from single blastomeres of 8-cell mouse embryos results in lower derivation rates than that from whole blastocysts, raising a biological question about the developmental potential of sister blastomeres. We aimed to assess the ability of 8-cell blastomeres to produce epiblast cells and ESC lines after isolation, and the properties of the resulting lines. Our results revealed unequal competence among sister blastomeres to produce ESC lines. At least half of the blastomeres possess a lower potential to generate ESCs, although culture conditions and blastomeres plasticity can redirect their non-pluripotent fate towards the epiblast lineage, allowing us to generate up to seven lines from the same embryo. Lines originated from the same embryo segregated into two groups according to their transcriptional signatures. While the expression of genes related to pluripotency and development was higher in one group, no differences were found in their trilineage differentiation ability. These results may help to improve our understanding of the ESC derivation process from single blastomeres and cell fate determination in the preimplantation mouse embryos.

Single-cell RNA sequencing reveals blastomere heterogeneity of 2-cell embryos in pigs.

In mammals, single blastomeres from as early as 2-cell embryos demonstrate heterogeneous developmental capacity and fate decision into different cell lineages. However, mechanisms underlying blastomere heterogeneity of 2-cell embryos remain largely unresolved. Here, we analysed the molecular heterogeneity of full-length mRNAs and their 3'UTR regions, based on the single-cell RNA-seq data of pig 2-cell embryos generated from in vivo fertilization (in vivo), in vitro fertilization (in vitro) and parthenogenetic activation (PA), respectively. First, unsupervised clustering helped discover two different groups of blastomeres for 2-cell pig embryos. Between these two groups of blastomeres in pig 2-cell embryos, 35, 301 and 428 full-length mRNAs respectively in in vivo, in vitro and PA embryo types were identified to be differentially expressed (padj ≤ .05 and |log2 [fold change]| ≥1) (DE mRNAs), while 92, 89 and 42 mRNAs were shown to be with significantly different 3'UTR lengths (3'UTR DE) (padj ≤ .05). Gene enrichment for both DE mRNAs and 3'UTR DE mRNAs found multiple signalling pathways, including cell cycle, RNA processing. Few numbers of common DE mRNAs and 3'UTR DE mRNAs existed between in vitro and in vivo blastomeres derived from 2-cell embryos, indicating the larger differences between in vitro and in vivo fertilized embryos. Integrative genomics viewer analysis further identified that 3'UTRs of HSDL2 and SGTA (in vivo), FAM204A and phosphoserine phosphatase (in vitro), PRPF40A and RPIA (PA) had >100 nt average length changes. Moreover, numbers and locations of regulatory elements (polyadenylation site, cytoplasmic polyadenylation element and microRNA binding sites) within 3'UTRs of these DE mRNAs were predicted. These results indicate that molecular heterogeneity existed among blastomeres from different types of pig 2-cell embryos, providing useful information and novel insights into future functional investigation on its relationship with the subsequent embryo development and differentiation.

Spatial positioning of preimplantation mouse embryo cells is regulated by mTORC1 and m7G-cap-dependent translation at the 8- to 16-cell transition.

Preimplantation mouse embryo development involves temporal-spatial specification and segregation of three blastocyst cell lineages: trophectoderm, primitive endoderm and epiblast. Spatial separation of the outer-trophectoderm lineage from the two other inner-cell-mass (ICM) lineages starts with the 8- to 16-cell transition and concludes at the 32-cell stages. Accordingly, the ICM is derived from primary and secondary contributed cells; with debated relative EPI versus PrE potencies. We report generation of primary but not secondary ICM populations is highly dependent on temporal activation of mammalian target of Rapamycin (mTOR) during 8-cell stage M-phase entry, mediated via regulation of the 7-methylguanosine-cap (m7G-cap)-binding initiation complex (EIF4F) and linked to translation of mRNAs containing 5' UTR terminal oligopyrimidine (TOP-) sequence motifs, as knockdown of identified TOP-like motif transcripts impairs generation of primary ICM founders. However, mTOR inhibition-induced ICM cell number deficits in early blastocysts can be compensated by the late blastocyst stage, after inhibitor withdrawal; compensation likely initiated at the 32-cell stage when supernumerary outer cells exhibit molecular characteristics of inner cells. These data identify a novel mechanism specifically governing initial spatial segregation of mouse embryo blastomeres, that is distinct from those directing subsequent inner cell formation, contributing to germane segregation of late blastocyst lineages.

A genome-wide screen reveals new regulators of the 2-cell-like cell state.

In mammals, only the zygote and blastomeres of the early embryo are totipotent. This totipotency is mirrored in vitro by mouse '2-cell-like cells' (2CLCs), which appear at low frequency in cultures of embryonic stem cells (ESCs). Because totipotency is not completely understood, we carried out a genome-wide CRISPR knockout screen in mouse ESCs, searching for mutants that reactivate the expression of Dazl, a gene expressed in 2CLCs. Here we report the identification of four mutants that reactivate Dazl and a broader 2-cell-like signature: the E3 ubiquitin ligase adaptor SPOP, the Zinc-Finger transcription factor ZBTB14, MCM3AP, a component of the RNA processing complex TREX-2, and the lysine demethylase KDM5C. All four factors function upstream of DPPA2 and DUX, but not via p53. In addition, SPOP binds DPPA2, and KDM5C interacts with ncPRC1.6 and inhibits 2CLC gene expression in a catalytic-independent manner. These results extend our knowledge of totipotency, a key phase of organismal life.

Proteomics reveals the underlying mechanism by which the first uneven division affects embryonic development in pig

One of the most typical abnormal cleavage patterns during early embryonic development is uneven division, but the first uneven division of pig zygote is common. Uneven division results in different daughter cell sizes and an uneven distribution of organelles such as lipid droplet, mitochondria, but the developmental capacity of daughter cells and proteomic changes of daughter cells are still unclear. Therefore, the developmental ability and proteomic quantification were investigated on blastomeres from even division (ED) or uneven division (UD) embryos at 2-cell stage in the present study. Firstly, the developmental ability was affected by the blastomeric size, when compared with medium blastomeres (MBs), the large blastomeres (LBs) with the higher cleavage rate but the small blastomeres (SBs) with the lower rate was observed. Subsequently, proteomic analysis was performed on blastomeres of LBs, MBs and SBs, a total of 109 DEPs were detected, which were involved in protein metabolism and processing, energy metabolism and ribosome. In particular, DEPs in LBs vs. SBs were focused on RNA binding and actin cytoskeletal tissue. Two protein-dense networks associated with RNA binding and cytoskeleton were revealed by further protein-protein interaction (PPI) analysis of DEPs in LBs vs. SBs, that DDX1 related to RNA binding and ACTB related to cytoskeleton were confirmed in UD embryos. Therefore, a briefly information of DEPs in blastomeres of 2-cell stage pig embryos was described in the present study, and it further confirmed that the formation of uneven division of the first cell cycle of pig embryos might be controlled by the cytoskeleton; the developmental capacity of daughter cells might be affected by the energy metabolism, RNA binding and ribosome, and further account for the developmental potential of the whole embryo.

Multi-level Fgf4- and apoptosis-dependent regulatory mechanism ensures the plasticity of ESC-chimaeric mouse embryo.

The preimplantation mammalian (including mouse and human) embryo holds remarkable regulatory abilities, which have found their application for example in the preimplantation genetic diagnosis of human embryos. Another manifestation of this developmental plasticity is the possibility of obtaining chimaeras by combining either two embryos or embryos and pluripotent stem cells, which enables the verification of the cell pluripotency and generation of genetically modified animals used to elucidate gene function. Using mouse chimaeric embryos (constructed by injection of embryonic stem cells into the 8-cell embryos) as a tool, we aimed to explore the mechanisms underlying the regulatory nature of the preimplantation mouse embryo. We comprehensively demonstrated the functioning of a multi-level regulatory mechanism involving the FGF4/MAPK signalling as a leading player in the communication between both components of the chimaera. This pathway, coupled with the apoptosis, cleavage division pattern, and cell cycle duration controlling the size of ESC component and giving it a competitive advantage over host embryo blastomeres, provides a cellular and molecular basis for regulative development, ensuring the generation of the embryo characterised by proper cellular composition.

P-228 Reliability of blastomere versus trophectoderm biopsy in preimplantation genetic testing for mitochondrial DNA disorders

Abstract Study question Can a single blastomere or trophectoderm (TE) biopsy accurately reflect the heteroplasmy levels of the whole embryo for mitochondrial DNA (mtDNA) disorders? Summary answer Heteroplasmy levels of single blastomere and TE are comparable to those of the rest of the embryo, including the inner cell mass (ICM). What is known already Oocytes of women carrying mtDNA mutations can display varying mutation loads – percentage of mutated mtDNA copies. Preimplantation genetic testing (PGT) aims at reducing the risk of having affected children by screening for embryos with minimal mtDNA heteroplasmy. The technique involves either a blastomere biopsy on cleavage stage embryos or TE biopsy from blastocysts, followed by genetic analysis to select embryos suitable for transfer. However, it remains unclear whether there is a uniform segregation of heteroplasmic mtDNA during the early stages of embryogenesis, and therefore whether the mutation load from blastomere or TE biopsies are representative of the whole embryo. Study design, size, duration We investigated the suitability of PGT for mtDNA disorders by comparing the mutation load between a single blastomere recovered at the eight-cell stage, TE cells and rest of the same blastocyst (n = 15) in a mouse model heteroplasmic for the m.5024C&amp;gt;T mutation. We also compared the levels of heteroplasmy between TE and ICM of heteroplasmic human blastocysts (n = 5) and between blastomeres of arrested cleavage stage embryos (n = 4) donated by two mtDNA disease patients. Participants/materials, setting, methods To obtain heteroplasmic mouse embryos, we mated female mice carrying a mitochondrial tRNAAla mutation (m.5024C&amp;gt;T) with wild-type males. Whereas in human experiments, we analysed preimplantation embryos from two different carriers of the m.3243A&amp;gt;G mutation, associated with MELAS syndrome, who showed 16% and 32% mutation load in their peripheral blood. After embryo biopsy, NGS was performed in three independent technical replicates and profiles were compared between samples from the same embryo to assess heteroplasmy levels. Main results and the role of chance Analysis of heteroplasmic mouse embryos showed a strong correlation in mtDNA heteroplasmy between blastomere and the corresponding embryo (r2 = 0.76), and between TE and the rest of the blastocyst (r2 = 0.94) (n = 15). Moreover, a strong correlation was observed between the biopsied blastomere and TE from the same embryo (r2 = 0.90). Heteroplasmy levels in the embryos analysed ranged from 55.3% to 88.3%, and the mean overall mutation load was 73.7 ± 7.9%. Development of biopsied embryos was comparable to those of non-manipulated mutant (n = 10) and wild-type embryos (n = 10) (blastocyst rate = 85.7%). For human blastocysts (n = 5), statistical analysis showed a strong correlation in heteroplasmy levels between ICM and TE portions (r2 = 0.98). Moreover, a modest correlation was observed among blastomeres of cleavage stage human embryos (r2 = 0.60) (n = 24 from 4 embryos). The mutation load in the embryos analysed ranged from 10.3% to 57.4%, and the mean overall heteroplasmy was 42.8 ± 12.2% in cleavage stage embryos and 38.6 ± 17.3% in blastocysts. Overall, concordance in heteroplasmy levels between single blastomeres, TE portions and corresponding blastocyst was established in human and mouse embryos. Limitations, reasons for caution These results should be further validated by increasing the sample size and by the inclusion of different mtDNA mutations. Furthermore, current data are suggestive, but not definitive, to guarantee that the mtDNA mutation load in the embryo will remain constant throughout life. Wider implications of the findings Taken together, results suggest that TE biopsy may be better than blastomere biopsy for mtDNA disorders. Moreover, biopsy at the blastocyst stage is likely less harmful for embryo development. Following, we will also investigate how heteroplasmic mtDNA segregates at the peri-implantation stages after the resumption of mtDNA replication. Trial registration number Not applicable

P-295 Application of a Time-Lapse Optical Coherence Tomography (OCT) approach in a pilot study to visualise oocytes and embryos in depth

Abstract Study question Can we visualise model oocytes and embryos during embryo development using OCT non-invasively? Summary answer This approach, new to embryology, OCT, allows visualising cells in a 3D perspective, providing more detailed information than standard microscopy from the oocyte or embryo. What is known already Time-lapse is a well-established technique in human IVF, but it is limited in its depth of view due to the limitations of classical microscopy. To overcome this limitation, the approach of Optical Coherence Tomography (OCT), allows non-invasive visualisation through optical cross-sections of the embryo or oocyte to produce 3D images. The obtained information can be extended to 4 or 5 dimensions to track movement and elasticity, using a low-power light source and no staining to ensure the minimum effect on the cell. Here, a pilot study was performed in model (porcine) samples using a newly developed “in incubator” system. Study design, size, duration A pilot basic study was performed where 16 oocytes and 16 cleavage embryos were obtained and prepared to be visualised using OCT. Participants/materials, setting, methods Pig ovaries were obtained from a slaughterhouse, from which follicles were aspirated in order to retrieve oocytes. The best-quality oocytes were cultured for maturation for 44 hours and 16 oocytes were selected for OCT imaging. before fertilisation for 2 hours. Sperm was previously prepared by Percoll gradient. Zygotes were cultured for 6 days until visualisation through OCT. For imaging, samples were prepared in a 16-well Primo Vision dish. Main results and the role of chance Oocytes and embryos were successfully imaged, allowing the identification of distinct cellular features. In oocytes, it was possible to identify the germinal vesicle (nucleus) and polar bodies, individual blastomeres in cleavage stage embryos and trophectoderm, ICM and blastocoel in blastocysts. As images were taken throughout different optical sections, it was possible to correlate the position of the areas within the oocytes/embryos and create a 3D image from a blastocyst, understanding the size of the inner cell mass compared with the embryo's overall size. Interestingly, images were obtained non-invasively and under optimal conditions (in an incubator), demonstrating the future utility of OCT for embryo imaging. Limitations, reasons for caution An experimental OCT system was developed and placed in the incubator, and all images obtained were in a format of a pilot study. Future imaging sessions are planned to obtain more data points and assess the viability of the embryo. Wider implications of the findings Although in the present study, OCT was applied in pig oocytes and embryos, this new approach can be employed in future for human IVF to overcome the current imaging limitations, proving more information on the oocytes and embryos’ quality and assisting Artificial Intelligence analysis. Trial registration number not applicable

Apicobasal RNA asymmetries regulate cell fate in the early mouse embryo

The spatial sorting of RNA transcripts is fundamental for the refinement of gene expression to distinct subcellular regions. Although, in non-mammalian early embryogenesis, differential RNA localisation presages cell fate determination, in mammals it remains unclear. Here, we uncover apical-to-basal RNA asymmetries in outer blastomeres of 16-cell stage mouse preimplantation embryos. Basally directed RNA transport is facilitated in a microtubule- and lysosome-mediated manner. Yet, despite an increased accumulation of RNA transcripts in basal regions, higher translation activity occurs at the more dispersed apical RNA foci, demonstrated by spatial heterogeneities in RNA subtypes, RNA-organelle interactions and translation events. During the transition to the 32-cell stage, the biased inheritance of RNA transcripts, coupled with differential translation capacity, regulates cell fate allocation of trophectoderm and cells destined to form the pluripotent inner cell mass. Our study identifies a paradigm for the spatiotemporal regulation of post-transcriptional gene expression governing mammalian preimplantation embryogenesis and cell fate.

Formation of the brain by stem cell divisions of large neuroblasts in Oikopleura dioica, a simple chordate.

Stem cell division contributes to the generation of various cell types during animal development, especially a diverse pool of neural cells in the nervous system. One example is reiterated unequal stem cell divisions, in which a large stem cell undergoes a series of oriented unequal divisions to produce a chain of small daughter cells that differentiate. We show that reiterated unequal stem cell divisions are involved in the formation of the brain in simple chordate appendicularians (larvaceans). Two large neuroblasts in the anterior and middle of the brain-forming region of hatched larvae were observed. They produced at least 30 neural cells out of 96 total brain cells before completion of brain formation at 10 hours after fertilization by reiterated unequal stem cell divisions. The daughter cells of the anterior neuroblast were postmitotic, and the number was at least 19. The neuroblast produced small daughter neural cells posteriorly every 20 min. The neural cells first moved toward the dorsal side, turned in the anterior direction, aligned in a single line according to their birth order, and showed collective movement to accumulate in the anterior part of the brain. The anterior neuroblast originated from the right-anterior blastomeres of the eight-cell embryos and the right a222 blastomere of the 64-cell embryo. The posterior neuroblast also showed reiterated unequal stem cell divisions, and generated at least 11 neural cells. Sequential unequal stem cell divisions without stem cell growth have been observed in protostomes, such as insects and annelids. The results provide the first examples of this kind of stem cell division during brain formation in non-vertebrate deuterostomes.