Individual Blastomeres Research Articles

Genome-wide equine preimplantation genetic testing enabled by simultaneous haplotyping and copy number detection

In different species, embryonic aneuploidies and genome-wide errors are a major cause of developmental failure. The increasing number of equine embryos being produced worldwide provides the opportunity to characterize and rank or select embryos based on their genetic profile prior to transfer. Here, we explored the possibility of generic, genome-wide preimplantation genetic testing concurrently for aneuploidies (PGT-A) and monogenic (PGT-M) traits and diseases in the horse, meanwhile assessing the incidence and spectrum of chromosomal and genome-wide errors in in vitro-produced equine embryos. To this end, over 70,000 single nucleotide polymorphism (SNP) positions were genotyped in 14 trophectoderm biopsies and corresponding biopsied blastocysts, and in 26 individual blastomeres from six arrested cleavage-stage embryos. Subsequently, concurrent genome-wide copy number detection and haplotyping by haplarithmisis was performed and the presence of aneuploidies and genome-wide errors and the inherited parental haplotypes for four common disease-associated genes with high carrier frequency in different horse breeds (GBE1, PLOD1, B3GALNT2, MUTYH), and for one color coat-associated gene (STX17) were compared in biopsy-blastocyst combinations. The euploid (n = 12) or fully aneuploid (n = 2) state and the inherited parental haplotypes for 42/45 loci of interest of the biopsied blastocysts were predicted by the biopsy samples in all successfully analyzed biopsy-blastocyst combinations (n = 9). Two biopsies showed a loss of maternal chromosome 28 and 31, respectively, which were confirmed in the corresponding blastocysts. In one of those biopsies, additional complex aneuploidies not present in the blastocyst were found. Five out of six arrested embryos contained chromosomal and/or genome-wide errors in most of their blastomeres, demonstrating their contribution to equine embryonic arrest in vitro. The application of the described PGT strategy would allow to select equine embryos devoid of genetic errors and pathogenetic variants, and with the variants of interest, which will improve foaling rate and horse quality. We believe this approach will be a gamechanger in horse breeding.

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

O-223 Sperm Genome Editing by CRISPR-Cas9 through an Ooplasm-Mediated Approach

Abstract Study question Can we edit the sperm genome by CRISPR-Cas9 through an ooplasmic-mediated approach? Summary answer The ooplasm-mediated technique induced the decondensation of the male gamete nucleus and allowed CRISPR-Cas9 to access the sperm DNA to carry out successful edits. What is known already Previous studies on genome editing using CRISPR-Cas9 have been performed at the S-phase or zygote stage, but challenges were represented by mosaicism and possible off-target edits. To address these issues, gene editing at the gamete level would be ideal. While this may be feasible in oocytes, it appears ambitious in the spermatozoon due to the DNA hypercoiling around the protamine core and the chromatin compaction. In preliminary experiments, permeabilization of sperm membrane allowed penetration of CRISPR-Cas9 to enter the cell, however, genomic editing was proved unsuccessful. Study design, size, duration In the past 5 months, a total of 128 oocytes were divided into 2 groups. To edit exclusively the male genome through oocyte-mediated sperm decondensation (OMSD), a single spermatozoon was injected into an enucleated oocyte to produce a haploid androgenetic embryo. The control cohort consisted of embryos generated through standard ICSI with the established heritable genome editing (HGE) approach. Both groups were treated with CRISPR-Cas9 aiming to knockout Tyr gene to create an albino phenotype. Participants/materials, setting, methods B6D2F1 mice were used to retrieve oocytes and spermatozoa. A cohort of oocytes used for OSMD approach were enucleated. While the intact oocytes were used for HGE control. All oocytes were injected with a single spermatozoon together with CRISPR-Cas9 solution, containing Tyr gRNA. All embryos were cultured up to the 8-cell stage. Individual blastomeres were isolated and sequenced to proof editing. DNA was extracted and amplified for T7E1 analysis to validate genome editing efficiency. Main results and the role of chance Of the 128 oocytes used for the study, 51 were enucleated for the OMSD experiments, while 77 were used for HGE control. After undergoing ICSI with CRISPR-Cas9 solution, 84.3% (43/51) of the OMSD cohort displayed a single male pronucleus and 79.2% (61/77) control HGE fertilized. In a time-lapse microscopy, 95.3% (41/43) of the OMSD experimental group developed to the 2-cell stage, comparable to the HGE control development at 96.7% (59/61). After 48 hours in culture, the HGE control reached 8-cell development at 88.5% (54/61), while the OMSD cohort cleaved at the lower rate at 60.4% (26/43, P<0.001). A 423-bp region around the CRISPR target site was amplified on DNA extracted by isolated blastomere. Gene modification at the target site was confirmed in 33.7% (70/208) of OMSD cohort and 29.7% (88/296) of the HGE control embryos. Limitations, reasons for caution While the experimental observations are still limited, it is important to note that these results may be underestimated since embryos with completely uniform modifications may go undetected by T7E1 assay. The technique needs further refinement to optimize targeting efficiency and support embryo development. Wider implications of the findings Once the genetic editing method is optimized, individual pseudo-blastomeres generated through this OMSD approach can be utilized as male gametes to fertilize oocyte. The generation of live offspring with the corrected haplotype will serve to prove the safety of genome editing through this technique. Trial registration number N/A

RNA exosome ribonuclease DIS3 degrades Pou6f1 to promote mouse pre-implantation cell differentiation.

Mammalian development is precisely controlled by cell differentiation. Identifying new regulators and investigating their interactions provide insight into genetic networks defining pre-implantation development. We established a knockout mouse model of Dis3, an exosome associated ribonuclease. Homozygous Dis3 null embryos arrest at the morula stage of development. Using single-embryo RNA sequencing (RNA-seq), we observed persistence of Pou6f1 mRNA in homozygous null Dis3 embryos and that the cognate protein represses transcription of Nanog and Cdx2. The resultant defects in cell differentiation disrupt the morula-to-blastocyst transition and are embryonic lethal. Microinjection of Dis3 mRNA into zygotes rescues the phenotype. Point mutations of Dis3 ribonuclease in individual blastomeres prevents their incorporation into embryos. To overcome the paucity of embryos, we derived homozygous Dis3 null mouse embryonic stem cells to identify additional gene targets of POU6F1. Our findings delineate a regulatory pathway of DIS3-POU6F1 in pre-implantation mammalian embryogenesis.

Whole genome sequencing of CCR5 CRISPR-Cas9-edited Mauritian cynomolgus macaque blastomeres reveals large-scale deletions and off-target edits.

Introduction: Genome editing by CRISPR-Cas9 approaches offers promise for introducing or correcting disease-associated mutations for research and clinical applications. Nonhuman primates are physiologically closer to humans than other laboratory animal models, providing ideal candidates for introducing human disease-associated mutations to develop models of human disease. The incidence of large chromosomal anomalies in CRISPR-Cas9-edited human embryos and cells warrants comprehensive genotypic investigation of editing outcomes in primate embryos. Our objective was to evaluate on- and off-target editing outcomes in CCR5 CRISPR-Cas9-targeted Mauritian cynomolgus macaque embryos. Methods: DNA isolated from individual blastomeres of two embryos, along with paternal and maternal DNA, was subjected to whole genome sequencing (WGS) analysis. Results: Large deletions were identified in macaque blastomeres at the on-target site that were not previously detected using PCR-based methods. De novo mutations were also identified at predicted CRISPR-Cas9 off-target sites. Discussion: This is the first report of WGS analysis of CRISPR-Cas9-targeted nonhuman primate embryonic cells, in which a high editing efficiency was coupled with the incidence of editing errors in cells from two embryos. These data demonstrate that comprehensive sequencing-based methods are warranted for evaluating editing outcomes in primate embryos, as well as any resultant offspring to ensure that the observed phenotype is due to the targeted edit and not due to unidentified off-target mutations.

P-190 The first cleavage plane in human embryos is dictated by the topology of pronuclei, not by the polar body position

Abstract Study question Is the first cleavage plane pre-determined in human embryos? Summary answer Unlike in mice, the human zygotes tend to divide at the right angle to the longitudinal pronuclear axis, irrespective of polar body position. What is known already In lower species, the orientation of the first cleavage plane defines the development of individual blastomeres and the three-dimensional (3D) body plan. The existence of embryonic pre-patterning in mammals remains controversial. Mouse studies suggested that the juxtaposed pronuclei (PN) are aligned with the polar body (PB) and cleavage furrow bisects the zygote along the polar axis thus ensuring symmetrical distribution of maternal resources to arising blastomeres. However, whether the same spatial control applies to humans is unclear. Study design, size, duration Retrospective observational analysis of high-resolution footage from 300 human embryos that gave rise to live births after a single embryo transfer. Participants/materials, setting, methods Embryo monitoring has been performed by the GERI time-lapse incubator. Image acquisition of individual embryo development lasted 6 days, starting within 10 minutes after intracytoplasmic sperm injection (ICSI). Transmitted light images have been automatically captured in 5-minute intervals and 11 focal planes covering the whole embryo volume. Analysis of 3D image data was performed at the time frame corresponding to the onset of the first mitosis. Main results and the role of chance The systematic analysis of the zygotic division pattern showed that the PN axis (defined by apposed pronuclei) was not stereotypically aligned with the animal-vegetal (AV) axis (with animal pole defined by the PB). Instead, PN/AV arrangement categories were equally represented in our cohort (PN/AV angle 0-30° - 34.7% vs. 30-60° - 36.0% and 60-90° - 29.3%; chi-square test p=.91351). Remarkably, we observed that the direction of the first cleavage was preferentially perpendicular to the PN axis (65.3%), while only 2.7% of embryos divided along the longitudinal PN axis. The remaining 32% embryos showed intermediated phenotype with cleavage plane/PN axis angle 30-60o. The results of this human embryo analysis contrast with the previous reports from mice. We hypothesize that this discrepancy might be attributed to the difference in centrosome inheritance and configuration of the zygotic spindle apparatus. Limitations, reasons for caution All analyzed embryos were produced by ICSI. Deposition of spermatozoa might affect the pronuclei positioning. Nevertheless, preliminary data from conventional IVF cycles are in agreement with the ICSI group results. Wider implications of the findings Our observations challenge the textbook view of the first division’s geometry, which has been derived from experiments in mice. The analysis of clinical time-lapse records improves our understanding of symmetry breaking during normal human development and creates scientific ground for evidence-based (de)selection of embryos for infertility treatment. Trial registration number not applicable

Non-invasive, label-free optical analysis to detect aneuploidy within the inner cell mass of the preimplantation embryo.

Can label-free, non-invasive optical imaging by hyperspectral autofluorescence microscopy discern between euploid and aneuploid cells within the inner cell mass (ICM) of the mouse preimplantation embryo? Hyperspectral autofluorescence microscopy enables discrimination between euploid and aneuploid ICM in mouse embryos. Euploid/aneuploid mosaicism affects up to 17.3% of human blastocyst embryos with trophectoderm biopsy or spent media currently utilized to diagnose aneuploidy and mosaicism in clinical in vitro fertilization. Based on their design, these approaches will fail to diagnose the presence or proportion of aneuploid cells within the foetal lineage ICM of some blastocyst embryos. The impact of aneuploidy on cellular autofluorescence and metabolism of primary human fibroblast cells and mouse embryos was assessed using a fluorescence microscope adapted for imaging with multiple spectral channels (hyperspectral imaging). Primary human fibroblast cells with known ploidy were subjected to hyperspectral imaging to record native cell fluorescence (4-6 independent replicates, euploid n = 467; aneuploid n = 969). For mouse embryos, blastomeres from the eight-cell stage (five independent replicates: control n = 39; reversine n = 44) and chimeric blastocysts (eight independent replicates: control n = 34; reversine n = 34; 1:1 (control:reversine) n = 30 and 1:3 (control:reversine) n = 37) were utilized for hyperspectral imaging. The ICM from control and reversine-treated embryos were mechanically dissected and their karyotype confirmed by whole genome sequencing (n = 13 euploid and n = 9 aneuploid). Two models were employed: (i) primary human fibroblasts with known karyotype and (ii) a mouse model of embryo aneuploidy where mouse embryos were treated with reversine, a reversible spindle assembly checkpoint inhibitor, during the four- to eight-cell division. Individual blastomeres were dissociated from control and reversine-treated eight-cell embryos and either imaged directly or used to generate chimeric blastocysts with differing ratios of control:reversine-treated cells. Individual blastomeres and embryos were interrogated by hyperspectral imaging. Changes in cellular metabolism were determined by quantification of metabolic co-factors (inferred from their autofluorescence signature): NAD(P)H and flavins with the subsequent calculation of the optical redox ratio (ORR: flavins/[NAD(P)H + flavins]). Autofluorescence signals obtained from hyperspectral imaging were examined mathematically to extract features from each cell/blastomere/ICM. This was used to discriminate between different cell populations. An increase in the relative abundance of NAD(P)H and decrease in flavins led to a significant reduction in the ORR for aneuploid cells in primary human fibroblasts and reversine-treated mouse blastomeres (P < 0.05). Mathematical analysis of endogenous cell autofluorescence achieved separation between (i) euploid and aneuploid primary human fibroblast cells, (ii) control and reversine-treated mouse blastomeres cells, (iii) control and reversine-treated chimeric blastocysts, (iv) 1:1 and 1:3 chimeric blastocysts and (v) confirmed euploid and aneuploid ICM from mouse blastocysts. The accuracy of these separations was supported by receiver operating characteristic curves with areas under the curve of 0.97, 0.99, 0.87, 0.88 and 0.93, respectively. We believe that the role of chance is low as mathematical features separated euploid from aneuploid in both human fibroblasts and ICM of mouse blastocysts. N/A. Although we were able to discriminate between euploid and aneuploid ICM in mouse blastocysts, confirmation of this approach in human embryos is required. While we show this approach is safe in mouse, further validation is required in large animal species prior to implementation in a clinical setting. We have developed an original, accurate and non-invasive optical approach to assess aneuploidy within the ICM of mouse embryos in the absence of fluorescent tags. Hyperspectral autofluorescence imaging was able to discriminate between euploid and aneuploid human fibroblast and mouse blastocysts (ICM). This approach may potentially lead to a new diagnostic for embryo analysis. K.R.D. is supported by a Mid-Career Fellowship from the Hospital Research Foundation (C-MCF-58-2019). This study was funded by the Australian Research Council Centre of Excellence for Nanoscale Biophotonics (CE140100003) and the National Health and Medical Research Council (APP2003786). The authors declare that there is no conflict of interest.

P–166 Machine learning for automated cell segmentation in embryos

Abstract Study question Is it possible to automate the process of detecting individual blastomeres within a 4-cell embryo? Summary answer Deep learning models are capable of identifying individual cells in single focal plane images of 4-cell embryos. What is known already As individual blastomeres within a 4-cell embryo maintain totipotency, their intercellular junctions are critical in maintaining and directing communication. These junctions are determined by the zygote’s cleavage patterns, and can affect the overall embryo ‘shape’, which can be described as either ‘tetrahedral’ or ‘planar’. Planar embryos carry significantly worse outcomes both in the short and long term, such as more compromised blastulation, clinical pregnancy and live birth rates. Therefore, more accurate identification of cell borders at the 4-cell stage may contribute to improved classification of cell shape and embryo visualisation. Study design, size, duration This was a retrospective cohort analysis of 222 single focal plane images from 3 clinics. Each image captured an embryo at the 4-cell stage and was taken using the Embryoscope™ time-lapse incubator at the central focal plane. Images from two of the clinics were split into training (n = 161) and validation (n = 17) sets. Images from the third clinic formed a blind testing set (n = 44). Participants/materials, setting, methods Ground truth masks were manually created by two human operators using the VGG Image Annotator software. A MaskRCNN neural network model with a pre-trained ResNet–50 backbone was trained to segment individual blastomeres from training images. Data augmentation (flips, rotations, Gaussian noise, cropping, brightness changes and optical distortion) were used during the training process. The model’s performance was evaluated using the IoU metric (a measure of overlap between model-predicted and human-annotated masks). Main results and the role of chance The model was evaluated on a blind test set of 44 images. The model had a mean IoU of 0.92 for individual cells (Standard Deviation (SD) = 0.05) with precision and sensitivity of 0.95 and 0.97 respectively. The mean IoU for the entire embryo (in relation to all 4 blastomeres combined) was 0.92 (SD 0.02). Furthermore, the model was able to count the number of cells in the images with 70% accuracy, and deviating by no more than 1 cell in each error. The nature of these errors can be broken down into the detection of fragmentation as a cell (2 cases); the detection of two cells as one (1 case); the cell being directly under another cell (4 cases); and duplicate detection of the same cell (6 cases). This last issue could be resolved by rejecting detections with significant overlap. Our results demonstrate that our model can be used across different clinics. Limitations, reasons for caution Inaccuracies in segmentation and cell counting sometimes occurred when a cell’s borders were unclear or obscured (e.g. in a different focal plane). The inclusion of multiple focal planes will be key for improving performance. Moreover, as only one focal plane was used, ambiguous cases were annotated with a ‘best guess’. Wider implications of the findings: The creation of a model capable of detecting individual cells would be highly beneficial in the IVF industry. Aside from automating laborious processes for embryologists, it may also prove a useful tool for future research such as in identifying intercellular contact points or rendering three-dimensional embryo visualisations. Trial registration number N/A

O-090 Correcting a PLCζ mutation in the human germ line to overcome hereditary infertility

Abstract Study question Can clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing result in the correction of a single base pair substitution that causes male infertility? Summary answer CRISPR/Cas9 administration during intracytoplasmic sperm injection (ICSI) leads to correction attempts of mutant phospholipase C zeta (PLCζ), howeverc loss-of-heterozygosity (LOH). What is known already Failed fertilization after ICSI can be caused by mutations in the sperm-related oocyte factor PLCζ which can be overcome by assisted oocyte activation (AOA). In this way, children may inherit the infertility-causing mutation. Mutation transmission can be overcome through CRISPR/Cas9 delivery during ICSI. In previous studies using CRISPR/Cas9 in the human germline for mutation correction, loss-of-heterozygosity (LOH, loss of the allele of one of the parents) was observed. Two different explanations were given, namely partial or complete paternal chromosomal loss or the correction of the mutation by using the maternal wild-type allele instead of the exogeneous supplied repair template. Study design, size, duration We injected a gRNA-Cas9 protein complex to target the PLCζ mutant allele, a repair template harboring the desired nucleotide substitution and an additional synonymous variant to track template usage, together with patient’s sperm. To overcome fertilization failure, AOA was applied during ICSI. After a culture period of maximal 6 days the embryos were collected. At day 3, some embryos were dissociated in individual blastomeres. The extracted DNA was analyzed through different genetic sequencing techniques. Participants/materials, setting, methods Donated sperm of a patient experiencing complete fertilization failure after routine ICSI, harboring a heterozygous base pair substitution in PLCZ1 (c.136-1G&amp;gt;C), was utilized. Sperm was injected in donated in vitro matured oocytes or in vivo matured oocytes containing clusters of smooth endoplasmic reticulum. Next-generation sequencing was used to assess correction potential. Short tandem repeat (STR) and single nucleotide polymorphism (SNP) assays were used to determine whether the sperm contained the mutation and to evaluate LOH. Main results and the role of chance CRISPR/Cas9 injections had no significant impact (p &amp;gt; 0.05) on embryonic development. Due to the heterozygous nature of the mutation, 47% (27/58) of the embryos originated from mutated sperm injection. The CRISPR components showed a high specificity with absence of insertions/deletions in 97% of the embryos originating from wild-type sperm (n = 31). Embryos originating from mutant sperm (n = 27) fall into three categories:(1) 22% showed the untargeted mutant allele, (2) 52% showed additional mutagenesis and (3) 26% showed the wild-type allele, which could be explained by correction. Mosaicism, defined as various editing events, was present in 17% (1), 21% (2) and 71% (3) of the embryos. The low occurrence of the synonymous variant, incorporated in the repair template, suggests that the template is not used during correction attempts. In only 29% (2/7) and 14% (1/7) of the ‘corrected embryos’, respectively long (&amp;gt;18Mb) or medium width LOH (4Mb) was observed through STR analysis. SNP analysis in closer proximity showed in 71% (5/7) of the embryos LOH, even in the absence of LOH through STR, suggesting also the occurrence of short width LOH. These results will be studied in more detail before definitive conclusions can be made. Chromosomal LOH will be studied by ddRADseq. Limitations, reasons for caution The occurrence of mosaicism and LOH might complicate the use of traditional CRISPR/Cas9 in human embryos and should be studied in detail to draw definite conclusions on its potential future use. To this end, genomic data have been produced from both individual blastomeres and whole-embryos which will be further analyzed. Wider implications of the findings Our findings demonstrate caution to use CRISPR/Cas9 to correct mutations in the germ line. They seem to contradict other reports that show predominant lack of mosaicism and presence of long width LOH. A deeper evaluation will be undertaken to define the length and type of LOH in this study. Trial registration number Not Applicable

O-083 Non-invasive, label-free optical analysis to detect aneuploidy within the inner cell mass of the preimplantation embryo

Abstract Study question Can we separate between control and reversine-treated cells within the inner cell mass (ICM) of the mouse preimplantation embryo by using label-free and non-invasive hyperspectral microscopy? Summary answer Hyperspectral microscopy is able to discern between control and reversine-treated cells using cellular autofluorescence in the complete absence of fluorescence tags. What is known already Embryo mosaicism (containing cells that are euploid (46 chromosomes) and aneuploid (deviation from the expected number of chromosomes)) affects up to 17.3% of human blastocyst embryos. Current diagnosis of aneuploidy in the IVF clinic involves a biopsy of trophectoderm (TE) cells or spent media followed by sequencing. In some blastocyst embryos these approaches will fail to diagnose of the proportion of aneuploid cells within the fetal lineage (ICM). Study design, size, duration The impact of aneuploidy on cellular metabolism was assessed by using cellular autofluoresence and hyperspectral microscopy (broad spectral profile). Two models were employed: (i) Primary human fibroblast cells with known karyotypes (4-6 independent replicates, euploid n = 467; aneuploid n = 969) and reversine induced aneuploidy in mouse embryos (5-8 independent replicates, 30-44 cells per group). Both models were subjected to hyperspectral imaging to quantify native cell fluorescence. Participants/materials, setting, methods The human model is comprised of euploid (male and female) and aneuploid (triploid and trisomies: 13, 18, 21, XXX, and XXY) primary human fibroblast cells. For the mouse model, we treated embryos with reversine, a reversible spindle assembly checkpoint inhibitor, during the 4- to 8-cell division. Individual blastomeres were dissociated from control and reversine treated 8-cell embryos. Blastomeres were either imaged directly or used to generate chimeric blastocysts with differing ratios of control:reversine-treated cells. Main results and the role of chance Following unsupervised linear unmixing, the relative abundance of metabolic cofactors was quantified: reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavins with the subsequent calculation of the optical redox ratio (ORR: Flavins/[NAD(P)H + Flavins]). Primary human fibroblast cells displayed an increase in the relative abundance of NAD(P)H with a decrease in flavins, leading to a significant reduction in the ORR for aneuploid cells (P &amp;lt; 0.05). The mouse embryos displayed an identical trend as the human model between control and reversine-treated embryos. Mathematical algorithms were applied and able to distinguish between (i) euploid and aneuploid primary human fibroblast cells, (ii) control and reversine-treated mouse blastomeres and (iii) chimeric blastocysts with differing ratios of control and reversine-treated cells. The accuracy of these separations was supported by receiver operating characteristic curves with areas under the curve. We also showed that hyperspectral imaging of the preimplantation embryo does not impact on embryo developmental competence, pregnancy outcome and offspring health in a mouse model. We believe the role of chance is low as both human somatic cells and mouse embryos showed a consistent shift in cellular metabolism in response to human fibroblast cells that are aneuploid and reversine treated mouse embryos. Limitations, reasons for caution Further validation of our approach could include sequencing of the ICM of individual blastocysts to determine the proportion of aneuploid cells in ICM and correlate this with the metabolic profile obtained through hyperspectral imaging. Wider implications of the findings With hyperspectral imaging able to discriminate between (i) euploid and aneuploid human fibroblast cells and (ii) control and reversine-treated mouse embryos, this could be an accurate, non-invasive and label-free optical imaging approach to assess mosaicism within the ICM of mouse embryos, potentially leading to a new diagnostic tool for embryos. Trial registration number Not applicable

151 Multipolar zygotic divisions result in multinuclear and anuclear blastomeres in cattle

The mammalian zygotic cleavage is expected to result in two mononuclear blastomeres. However, zygotes undergoing multipolar divisions resulting in direct cleavage into three or four cells are frequently observed in bovine and human embryonic development and have been associated with decreased euploidy rates of resulting blastocysts and a lower pregnancy rate (Somfai et al. 2010 J. Reprod. Dev. 56, 200-207; https://doi.org/10.1262/jrd.09-097a; Zhan et al. 2016 PLoS ONE 11, 1-19; https://doi.org/10.1371/journal.pone.0166398; Sugimura et al. 2017 J. Reprod. Dev. 63, 353-357; https://doi.org/10.1262/jrd.2017-041). Therefore, multipolar zygotic divisions may underly genetic abnormalities by aberrant segregation of the chromosomal material resulting in multinucleated or anuclear blastomeres. These abnormal blastomeres have been observed in human cleavage-stage embryos (Nogueira et al. 2000 Fertil. Steril. 74, 295-298; https://doi.org/10.1016/s0015-0282(00)00642-7; Chatzimeletiou et al. 2006 Hum. Reprod. 20, 672–682; https://doi.org/10.1093/humrep/deh652), but the prevalence in bovine embryos and the direct association with the multipolar division in both bovine and human embryos remains unknown. We hypothesised that anuclear and multinuclear blastomeres also occur in bovine embryos, and we aimed to unravel the link between multipolar zygotic divisions and genome segregation errors by determining the nuclear blastomere content in a bovine model. Therefore, oocytes from 5 cows were matured and fertilized in vitro by the same bull according to our standard in vitro production procedure (Wydooghe et al. 2014 Reproduction 148, 519-529). The first cleavage was monitored the by time-lapse imaging. Forty-three blastomeres from 22 bipolar zygotic divisions, and 65 blastomeres from 20 multipolar zygotic divisions were collected immediately after the first cleavage, using pronase to isolate the individual blastomeres. The area of each blastomere was measured and the number of nuclei was determined after fixation and staining with Hoechst 33342. Generalized mixed effect models were built to identify the effect of the type of cleavage (bipolar vs. multipolar) on the number of nuclei (mononuclear vs. anuclear or multinuclear) in the blastomeres. Linear mixed models were built to determine the effect of the type of cleavage and the nuclear content on the size of the blastomeres. Embryos presented a greater number of blastomeres with a normal nuclear content (92.6 ± 0.4%) after a bipolar cleavage compared with multipolar division (73.2 ± 0.7%; P=0.03). Moreover, blastomeres presented a 28% larger blastomere area (P&amp;lt;0.001) after bipolar division compared with multipolar division. Notably, anuclear blastomeres tended to be smaller than multi- and mononuclear blastomeres (P=0.09 for both), while no difference was found between mono and multinucleated blastomeres (P=0.84). In conclusion, this is one of the first reports on the association between nuclear blastomere content in bovine embryos and the dynamics of the first zygotic division. Even though sample size was limited, these results confirm the hypothesised link between multipolar division and abnormal genome segregation as determined by multinuclear and anuclear blastomeres in the resulting blastomeres. Therefore, multipolar cell divisions at the zygotic division may underly at least some of the genetic abnormalities observed in embryos at early development.

Development of PCR based approach to detect potential mosaicism in porcine embryos

Direct injection of genome editing tools such as CRISPR/Cas9 system into developing embryos has been widely used to generate genetically engineered pigs. The approach allows us to produce pigs carrying targeted modifications at high efficiency without having to apply somatic cell nuclear transfer. However, the targeted modifications during embryogenesis often result in mosaicism, which causes issues in phenotyping founder animals and establishing a group of pigs carrying intended modifications. This study was aimed to establish a genomic PCR and sequencing system of a single blastomere in the four-cell embryos to detect potential mosaicism. We performed genomic PCR in four individual blastomeres from four-cell embryos. We successfully amplified target genomic region from single blastomeres of 4-cell stage embryo by PCR. Sanger sequencing of the PCR amplicons obtained from the blastomeres suggested that PCR-based genotyping of single blastomere was a feasible method to determine mutation type generated by genome editing technology such as CRISPR/Cas9 in early stage embryos. In conclusion, we successfully genotyped single blastomeres in a single 4-cell stage embryo to detect potential mosaicism in porcine embryos. Our approach offers a simple platform that can be used to screen the prevalence of mosaicism from designed CRISPR/Cas9 systems.

Next-generation sequencing analysis of each blastomere in good-quality embryos: insights into the origins and mechanisms of embryonic aneuploidy in cleavage-stage embryos.

To explore the whole-chromosome status, origins, and mechanisms of chromosomal abnormalities in good-quality cleavage embryos using multiple annealing and looping-based amplification cycle (MALBAC) sequencing. The embryos studied came from7 patients (maternal aged 26-35) who had healthy birth from the same IVF cycles. These 21 frozen day 3 good-quality embryos were thawed and disaggregated into individual blastomere. Each blastomere was collected and analyzed by MALBAC sequencing. Conclusive results were obtained from a high percentage of blastomeres (95.3%). A total of 46.6% of blastomeres were diploid, 53.4% were abnormal, and 28.0% had complex aneuploidy. Out of 21 embryos, 3 (14.3%) were normal and 18 (85.7%) were mosaics, showing the occurrence of mitotic errors; aneuploidy was confirmed in all cells of 4 of the 18 embryos, which showed the coexistence of meiotic errors. Conclusive results were obtained from all blastomeres of 15 embryos (71.4%, 15/21), which enabled us to reconstruct the cell lineage on the basis of the chromosomal content of the blastomeres in each division. There were 9 mitotic errors (8.7%, 9/103): nondisjunction accounted for 88.9% (8/9), and endoreplication accounted for 11.1% (1/9). In good-quality embryos, there was a high rate and diverse array of chromosomal abnormalities. Morphological evaluation does not appear to assist in the reduction in meiotic errors from parental origins. Mitotic errors were common, and nondisjunction was found to be the main mechanism causing malsegregation during the cleavage divisions.

Automatic three-dimensional imaging for blastomere identification in early-stage embryos based on brightfield microscopy

As the first step in manual or automated preimplantation genetic diagnosis (PGD), accurate locating of the blastomere is essential. In order to further improve success rates of the PGD process, it is necessary to obtain three-dimensional information of the blastomeres. In this study, a practical and effective pipeline that allows conventional brightfield microscopy systems to provide 3D imaging feedback for individual blastomeres inside embryos is proposed. The technique consists of two main phases, namely the blastomere identification and the 3D modeling of individual blastomeres. In the first phase, after a series of optical sections are obtained along the z-axis (z-stack), the embryo is located in the z-stack and the blastomere outline is then identified inside the embryo using a coarse-to-fine strategy; In the second phase, a probabilistic model combined with the machine learning approach is designed for the boundary estimation between individual blastomeres, and the 3D coordinate and model of each blastomere are obtained through further relocalization and reconstruction in the z-stack. Experimental results demonstrate that the proposed technique is valid and accurate compared with human perception. Specifically, the proposed technique achieves 90.32% precision rate, and the bias of blastomere position between the proposed method and human segmentation is 1.028 μm.

CARM1 is heterogeneous in mouse four-cell embryo and important to blastocyst development.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a type I arginine methyltransferase that methylates the arginine residues of histone and nonhistone. Carm1 regulates various cellular processes, including transcriptional regulation, mRNA processing, cellular proliferation, and differentiation. Blastomeres with high Carm1 expression levels show cleavage tendency to inner cell mass (ICM) in mouse embryos. However, details about the factors for CARM1 distribution in mouse early embryos and the role of Carm1 in blastocyst development remain unclear. Here, the endonuclear distribution of CARM1 protein was heterogeneous between blastomeres from the late four-cell stage to the blastocyst stage. The heterogeneity of CARM1 distribution in blastomeres at the late four-cell stage was randomly obtained from two-cell stage embryos. From the four-cell stage to morula, CARM1 in individual blastomere remained heterogeneous. In the blastocyst stage, CARM1 protein level in ICM was much higher than that in trophoblast. We found that microRNA (miRNA) miR-181a is an important regulator for Carm1 distribution at the late four-cell stage. The ratio of heterogeneous embryos was reduced in all the embryos when miR-181a was inhibited. CARM1 inhibition reduced the level of symmetrical histone H3 arginine-26 dimethylation and impaired blastocyst development. Silencing Carm1 reduced cell number and increased cell apoptosis at the blastocyst stage. These results show a CARM1 heterogeneous distribution from the four-cell embryos to the blastocysts. miR-181a regulates the control of CARM1 heterogeneous distribution in the four-cell-stage embryos, and CARM1 is an important protein in regulating blastocyst development.

Establishment of a relationship between blastomere geometry and YAP localisation during compaction.

ABSTRACTPrecise patterning within the three-dimensional context of tissues, organs and embryos implies that cells can sense their relative position. During preimplantation development, outside and inside cells rely on apicobasal polarity and the Hippo pathway to choose their fate. Despite recent findings suggesting that mechanosensing might be central to this process, the relationship between blastomere geometry (i.e. shape and position) and the Hippo pathway effector YAP remains unknown. We used a highly quantitative approach to analyse information on the geometry and YAP localisation of individual blastomeres of mouse and human embryos. We identified the proportion of exposed cell surface area as most closely correlating with the nuclear localisation of YAP. To test this relationship, we developed several hydrogel-based approaches to alter blastomere geometry in cultured embryos. Unbiased clustering analyses of blastomeres from such embryos revealed that this relationship emerged during compaction. Our results therefore pinpoint the time during early embryogenesis when cells acquire the ability to sense changes in geometry and provide a new framework for how cells might integrate signals from different membrane domains to assess their relative position within the embryo.

Position-Sensing Established During Compaction Dictates Cell Fate in the Mammalian Embryo

Precise patterning within the 3-dimensional context of tissues, organs and embryos implies that cells can sense their relative position. We still understand relatively little about how cells sense their relative position within small groups of cells. During preimplantation development, outside and inside cells rely on apicobasal polarity and the Hippo pathway to choose their fate. Despite recent findings suggesting that mechanosensing may be central to this process, the relationship between blastomere geometry (i.e. shape and position) and the Hippo pathway effector YAP remains unknown. To address this, we used a highly quantitative approach to collect and analyse information on the geometry and YAP localisation of individual blastomeres of mouse and human embryos. We find that the fraction of YAP in the nucleus responds to the proportion of exposed cell surface area rather than blastomere shape. To directly test the influence of blastomere position on YAP localisation we developed an approach using hydrogel based cylindrical cavities to alter blastomere arrangement in cultured embryos. Unbiased clustering analyses of blastomeres from such embryos reveal that the ability to respond to position emerges as early as the 8-cell stage during compaction and that altering the relative position of a blastomere within an embryo can alter its fate. Finally, we demonstrate that this response is lost upon inhibiting PKC signalling, linking it to the cell polarity. Our results pinpoint when during early embryogenesis cell specification decisions are initiated and show how blastomeres can sense their position in a polarity dependent manner to alter YAP localisation.

Taxol Improves Pre-Implantation Development Potential of Mouse Embryos

Aim: The objective of the present study was to investigate the development of mouse embryos and the chromosomal status after the pre-implantation treatment with paclitaxel (Taxol) based on the reports that indicate Taxol improves the developmental potential of vitrified human and mouse oocytes. Methods: Outbred female mice were superovulated and in vitro fertilization (IVF) was carried out using sperms from the same strain. Two-cell stage mouse embryos were cultured in the presence of Taxol for 24 h. After the determination of a non-toxic dose of Taxol, embryo development in control and Taxol-treated groups was compared during 3.5 days post-IVF. The aneuploidy rate of embryos was assessed by fluorescence in situ hybridization for chromosomes 2 and 11. Results: Development to morula and blastocyst stages was considerably enhanced following the addition of Taxol 0.01 µM compared to a similar situation in controls (p < 0.0001). Moreover, the degeneration rate was decreased following treatment with this concentration of Taxol (p < 0.01). The rate of aneuploidy in embryos and individual blastomeres did not vary between groups (p = 0.518 and 0.810 respectively). Conclusion: Pre-implantation treatment with Taxol 0.01 µM had a positive effect on the development to morula/blastocyst stages and decreased the degeneration rate without affecting the aneuploidy rate of chromosomes 2 and 11.

Establishment of porcine and human expanded potential stem cells.

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.

Systems based analysis of human embryos and gene networks involved in cell lineage allocation

BackgroundLittle is understood of the molecular mechanisms involved in the earliest cell fate decision in human development, leading to the establishment of the trophectoderm (TE) and inner cell mass (ICM) stem cell population. Notably, there is a lack of understanding of how transcriptional networks arise during reorganisation of the embryonic genome post-fertilisation.ResultsWe identified a hierarchical structure of preimplantation gene network modules around the time of embryonic genome activation (EGA). Using network models along with eukaryotic initiation factor (EIF) and epigenetic-associated gene expression we defined two sets of blastomeres that exhibited diverging tendencies towards ICM or TE. Analysis of the developmental networks demonstrated stage specific EIF expression and revealed that histone modifications may be an important epigenetic regulatory mechanism in preimplantation human embryos. Comparison to published RNAseq data confirmed that during EGA the individual 8-cell blastomeres are transcriptionally primed for the first lineage decision in development towards ICM or TE.ConclusionsUsing multiple systems biology approaches to compare developmental stages in the early human embryo with single cell transcript data from blastomeres, we have shown that blastomeres considered to be totipotent are not transcriptionally equivalent. Furthermore we have linked the developmental interactome to individual blastomeres and to later cell lineage. This has clinical implications for understanding the impact of fertility treatments and developmental programming of long term health.