Segmentation of mature human oocytes provides interpretable and improved blastocyst outcome predictions by a machine learning model.

Within the medical field of human assisted reproductive technology, a method for interpretable, non-invasive, and objective oocyte evaluation is lacking. To address this clinical gap, a workflow utilizing machine learning techniques has been developed involving automatic multi-class segmentation of two-dimensional images, morphometric analysis, and prediction of developmental outcomes of mature denuded oocytes based on feature extraction and clinical variables. Two separate models have been developed for this purpose-a model to perform multiclass segmentation, and a classifier model to classify oocytes as likely or unlikely to develop into a blastocyst (Day 5-7 embryo). The segmentation model is highly accurate at segmenting the oocyte, ensuring high-quality segmented images (masks) are utilized as inputs for the classifier model (mask model). The mask model displayed an area under the curve (AUC) of 0.63, a sensitivity of 0.51, and a specificity of 0.66 on the test set. The AUC underwent a reduction to 0.57 when features extracted from the ooplasm were removed, suggesting the ooplasm holds the information most pertinent to oocyte developmental competence. The mask model was further compared to a deep learning model, which also utilized the segmented images as inputs. The performance of both models combined in an ensemble model was evaluated, showing an improvement (AUC 0.67) compared to either model alone. The results of this study indicate that direct assessments of the oocyte are warranted, providing the first objective insights into key features for developmental competence, a step above the current standard of care-solely utilizing oocyte age as a proxy for quality.

High histone crotonylation modification in bovine fibroblasts promotes cell proliferation and the developmental efficiency of preimplantation nuclear transfer embryos.

Lysine crotonylation (Kcr) is a recently discovered histone acylation modification that is closely associated with gene expression, cell proliferation, and the maintenance of stem cell pluripotency and indicates the transcriptional activity of genes and the regulation of various biological processes. During cell culture, the introduction of exogenous croconic acid disodium salt (Nacr) has been shown to modulate intracellular Kcr levels. Although research on Kcr has increased, its role in cell growth and proliferation and its potential regulatory mechanisms remain unclear compared to those of histone methylation and acetylation. Our investigation demonstrated that the addition of 5 mM Nacr to cultured bovine fibroblasts increased the expression of genes associated with Kcr modification, ultimately promoting cell growth and stimulating cell proliferation. Somatic cell nuclear transfer of donor cells cultured in 5 mM Nacr resulted in 38.1% blastocyst development, which was significantly greater than that in the control group (25.2%). This research is important for elucidating the crotonylation modification mechanism in fibroblast proliferation to promote the efficacy of somatic cell nuclear transfer.

Development and evaluation of a usable blastocyst predictive model using the biomechanical properties of human oocytes.

PURPOSE: The purposes of this study were to determine whether biomechanical properties of mature oocytes could predict usable blastocyst formation better than morphological information or maternal factors, and to demonstrate the safety of the aspiration measurement procedure used to determine the biomechanical properties of oocytes. METHODS: A prospective split cohort study was conducted with patients from two IVF clinics who underwent in vitro fertilization. Each patient's oocytes were randomly divided into a measurement group and a control group. The aspiration depth into a micropipette was measured, and the biomechanical properties were derived. Oocyte fertilization, day 3 morphology, and blastocyst development were observed and compared between measured and unmeasured cohorts. A predictive classifier was trained to predict usable blastocyst formation and compared to the predictions of four experienced embryologists. RESULTS: 68 patients and their corresponding 1252 oocytes were included in the study. In the safety analyses, there was no significant difference between the cohorts for fertilization, while the day 3 and 5 embryo development were not negatively affected. Four embryologists predicted usable blastocyst development based on oocyte morphology with an average accuracy of 44% while the predictive classifier achieved an accuracy of 71%. Retaining the variables necessary for normal fertilization, only data from successfully fertilized oocytes were used, resulting in a classifier an accuracy of 81%. CONCLUSIONS: To date, there is no standard guideline or technique to aid in the selection of oocytes that have a higher likelihood of developing into usable blastocysts, which are chosen for transfer or vitrification. This study provides a comprehensive workflow of extracting biomechanical properties and building a predictive classifier using these properties to predict mature oocytes' developmental potential. The classifier has greater accuracy in predicting the formation of usable blastocysts than the predictions provided by morphological information or maternal factors. The measurement procedure did not negatively affect embryo culture outcomes. While further analysis is necessary, this study shows the potential of using biomechanical properties of oocytes to predict embryo developmental outcomes.

Live birth derived from a markedly large polar body oocyte: a rare case report.

Oocytes with excessively large first polar bodies (PB1) often occur in assisted reproductive procedures. Many times these oocytes are discarded without insemination and, as a result, the application of this portion of oocytes has scarcely been reported to date. Few studies have examined large PB1 oocytes in infertile women and have virtually entirely studied genetic variations for large PB1 oocyte abnormalities. Here, we describe an unusual case of a live birth from a remarkably large PB1 oocyte in a frozen embryo transfer (FET) cycle. This is the first instance of a successful live birth resulting from a PB1 oocyte with an extremely large polar body measuring 80 µM × 40 µM in size. The large PB1 oocyte was performed by an early rescue intracytoplasmic sperm injection (r-ICSI) and was formed into a blastocyst on day 5. Following FET, a healthy boy baby weighing 3100 g was finally delivered by caesarean section at 37 weeks and 5 days after conception. Additionally, there were no complications throughout the antenatal period or the perinatal phase of this following full-term delivery. In this study, it is revealed for the first time that a huge PB1 oocyte can be fertilized, resulting in the growth of a blastocyst, a subsequent pregnancy, and a live birth. This new information prompts us to reconsider the use of large PB1 oocytes. More insightful talks should be given attention to prevent the waste of embryos because not all oocytes with aberrant morphology are unavailable.

Functional sensory circuits built from neurons of two species.

A central question for regenerative neuroscience is whether synthetic neural circuits, such as those built from two species, can function in an intact brain. Here, we apply blastocyst complementation to selectively build and test interspecies neural circuits. Despite approximately 10-20 million years of evolution, and prominent species differences in brain size, rat pluripotent stem cells injected into mouse blastocysts develop and persist throughout the mouse brain. Unexpectedly, the mouse niche reprograms the birth dates of rat neurons in the cortex and hippocampus, supporting rat-mouse synaptic activity. When mouse olfactory neurons are genetically silenced or killed, rat neurons restore information flow to odor processing circuits. Moreover, they rescue the primal behavior of food seeking, although less well than mouse neurons. By revealing that a mouse can sense the world using neurons from another species, we establish neural blastocyst complementation as a powerful tool to identify conserved mechanisms of brain development, plasticity, and repair.

A geometrical model of cell fate specification in the mouse blastocyst.

The lineage decision that generates the epiblast and primitive endoderm from the inner cell mass (ICM) is a paradigm for cell fate specification. Recent mathematics has formalized Waddington's landscape metaphor and proven that lineage decisions in detailed gene network models must conform to a small list of low-dimensional stereotypic changes called bifurcations. The most plausible bifurcation for the ICM is the so-called heteroclinic flip that we define and elaborate here. Our re-analysis of recent data suggests that there is sufficient cell movement in the ICM so the FGF signal, which drives the lineage decision, can be treated as spatially uniform. We thus extend the bifurcation model for a single cell to the entire ICM by means of a self-consistently defined time-dependent FGF signal. This model is consistent with available data and we propose additional dynamic experiments to test it further. This demonstrates that simplified, quantitative and intuitively transparent descriptions are possible when attention is shifted from specific genes to lineages. The flip bifurcation is a very plausible model for any situation where the embryo needs control over the relative proportions of two fates by a morphogen feedback.

Fresh or frozen day 6 blastocyst transfer: is there still a question?

BACKGROUND: The Live Birth Rate (LBR) after day 5 (D5) blastocyst transfer is significantly higher than that with D6 embryos in both fresh and frozen-vitrified embryo transfer cycles, according to the most recently published meta-analyses. Therefore, for women obtaining only D6 blastocysts, the chances of pregnancy may be lower but nonetheless sufficient to warrant transferring such embryos. The best strategy for transfer (i.e., in fresh versus frozen cycles) remains unclear and there is a paucity of data on this subject. METHODS: A total of 896 couples with D6 single blastocyst transfers were retrospectively analyzed: patients receiving a fresh D6 embryo transfer (Fresh D6 transfer group, n = 109) versus those receiving a frozen-thawed D6 embryo transfer (Frozen D6 transfer group, n = 787). A subgroup comprising a freeze-all cycle without any previous fresh or frozen D5 embryo transfers (Elective frozen D6, n = 77) was considered and also compared with the Fresh D6 transfer group. We compared LBR between these two groups. Correlation between D6 blastocyst morphology according to Gardner's classification and live birth occurrence was also evaluated. Statistical analysis was carried out using univariate and multivariate logistic regression models. RESULTS: The LBR was significantly lower after a fresh D6 blastocyst transfer compared to the LBR with a frozen-thawed D6 blastocyst transfer [5.5% (6/109) vs. 12.5% (98/787), p = 0.034]. Comparison between LBR after Elective frozen D6 group to the Fresh D6 blastocyst transfers confirmed the superiority of frozen D6 blastocyst transfers. Statistical analysis of the blastocyst morphology parameters showed that both trophectoderm (TE) and inner cell mass (ICM) grades were significantly associated with the LBR after D6 embryo transfer (p < 0.001, p = 0.037). Multiple logistic regression revealed that frozen D6 thawed transfer was independently associated with a higher LBR compared with fresh D6 transfer (OR = 2.54; 95% CI: [1.05-6.17]; p = 0.038). Our results also show that transferring a good or top-quality D6 blastocyst increased the chances of a live birth by more than threefold. CONCLUSIONS: Our results indicate that transferring D6 blastocysts in frozen cycles improves the LBR, making it the best embryo transfer strategy for these slow-growing embryos. CLINICAL TRIAL NUMBER: Not applicable.

[Analysis of related factors influencing the detection rate of mosaic embryo and the pregnancy outcomes with mosaic embryo transfers].

Objective: To explore the related factors influencing the detection rate of mosaic embryo and the pregnancy outcomes of mosaic embryo transfer in preimplantation genetic testing for aneuploidy (PGT-A) based on next generation sequencing (NGS) technology. Methods: A retrospective study was performed to analyze the clinical data of patients in 745 PGT-A cycles from January 2019 to May 2023 at Chongqing Health Center for Women and Children, including 2 850 blastocysts. The biopsy cells were tested using NGS technology, and the embryos were divided into three groups based on the test results, namely euploid embryos, aneuploid embryos and mosaic embryos. The influence of population characteristics and laboratory-related parameters on the detection rate of mosaic embryo were analyzed, and the pregnancy outcomes of 98 mosaic embryo transfer cycles and 486 euploid embryo transfer cycles were compared during the same period, including clinical pregnancy rate and live birth rate. Results: Among the embryos tested (n=2 850), the number and proportion of euploid embryos, aneuploid embryos and mosaic embryos were 1 489 (52.2%, 1 489/2 850), 917 (32.2%, 917/2 850) and 444 (15.6%, 444/2 850), respectively. Among mosaic embryos, 245 (55.2%, 245/444) were segmental mosaic embryos, 118 (26.6%, 118/444) were whole-chromosome mosaic embryos, and 81 (18.2%, 81/444) were complex mosaic embryos. NGS technology was performed in 4 genetic testing institutions and the detection rate of mosaic embryo fluctuated from 13.5% to 27.0%. The distributions of female age, level of anti-Müllerian hormone, PGT-A indications, ovulation-inducing treatments, gonadotropin (Gn) dosage, Gn days, inner cell mass grade, trophectoderm cell grade, genetic testing institutions and developmental stage of blastocyst were significantly different among the three groups (all P<0.05). Multi-factor analysis showed that the trophectoderm cell grade and genetic testing institutions were significantly related to the detection rate of mosaic embryo; compared with the trophectoderm cell graded as A, the detection rate of mosaic embryo was significantly increased in the trophectoderm cell graded as B-(OR=1.59, 95%CI: 1.04-2.44, P=0.033); compared with genetic testing institution a, the detection rate of mosaic embryo was significantly higher (OR=2.89, 95%CI: 2.10-3.98, P<0.001) in the testing institution c. The clinical pregnancy rate and live birth rate with mosaic embryos transfer were significantly lower than those of euploid embryos transfer (clinical pregnancy rate: 51.0% vs 65.2%, P=0.008; live birth rate: 39.4% vs 53.2%, P=0.017). After adjustment for age, PGT-A indications, trophectoderm cell grade and days of embryo culture in vitro, the clinical pregnancy rate and live birth rate with mosaic embryos transfer were significantly lower than those of euploid embryos transfer (clinical pregnancy rate: OR=0.52, 95%CI: 0.32-0.83, P=0.007; live birth rate: OR=0.50, 95%CI: 0.31-0.83, P=0.007). Conclusions: The trophectoderm cell grade and genetic testing institutions are related to the detection rate of mosaic embryo. Compared with euploid embryos transfer, the clinical pregnancy rate and live birth rate with mosaic embryos transfer are significantly reduced. For infertile couple without euploid embryos, transplantable mosaic embryos could be recommended according to the mosaic ratio and mosaic type in genetic counseling to obtain the optimal pregnancy outcome.

Sex ratio shift after frozen single blastocyst transfer in relation to blastocyst morphology parameters.

The sex ratio shift was observed in peoples who underwent ART treatment. Moreover, there is limited evidence on differences in sex ratio between single frozen-thawed blastocyst morphology, insemination type and transfer days. So further research is needed in this area with regard to factors possibly affecting the sex ratio. Retrospective study based on multicenter including two large assisted reproduction centers in Shanghai and Wuhan in China. A total of 6361 singleton delivery offspring after frozen-thawed blastocyst transfer. Propensity score weighting and logistic regression models were used to estimate the associations between blastocyst morphology grading and child sex ratio. The main outcome measures is singleton sex ratio. In our study, the primary outcome measure was sex ratio which was calculated as the proportion of male newborns among all live births. Higher quality blastocysts resulted in a higher sex ratio than single poor-quality frozen-thawed blastocyst transfer. Among the three blastocyst morphological parameters of trophectoderm (TE), Grade A and B were significantly associated with a higher sex ratio than Grade C. The similar trend was observed in both IVF and ICSI treated subgroups. As compared with expansion (4 + 3), expansion degree 6 achieved a higher sex ratio in overall populations and IVF treated subgroup. Transferring blastocysts of day 6 had the highest sex ratio both in IVF group and ICSI group. A 6.95% higher sex ratio in transferring blastocysts of day 5 in IVF group than those in ICSI group. No significant association between inner cell mass degree and sex ratio was observed. However, as compared with IVF treatment, all morphology parameters achieved the similar or the biased sex ratio favoring female in ICSI treated subgroup. Quality of blastocysts was positively associated with sex ratio. TE score and expansion degree rather than ICM were significantly associated with sex ratio at birth. ICSI treatment promotes the biased sex ratio favoring female.

WISE: whole-scenario embryo identification using self-supervised learning encoder in IVF.

PURPOSE: To study the effectiveness of whole-scenario embryo identification using a self-supervised learning encoder (WISE) in in vitro fertilization (IVF) on time-lapse, cross-device, and cryo-thawed scenarios. METHODS: WISE was based on the vision transformer (ViT) architecture and masked autoencoders (MAE), a self-supervised learning (SSL) method. To train WISE, we prepared three datasets including the SSL pre-training dataset, the time-lapse identification dataset, and the cross-device identification dataset. To identify whether pairs of images were from the same embryos in different scenarios in the downstream identification tasks, embryo images including time-lapse and microscope images were first pre-processed through object detection, cropping, padding, and resizing, and then fed into WISE to get predictions. RESULTS: WISE could accurately identify embryos in the three scenarios. The accuracy was 99.89% on the time-lapse identification dataset, and 83.55% on the cross-device identification dataset. Besides, we subdivided a cryo-thawed evaluation set from the cross-device test set to have a better estimation of how WISE performs in the real-world, and it reached an accuracy of 82.22%. There were approximately 10% improvements in cross-device and cryo-thawed identification tasks after the SSL method was applied. Besides, WISE demonstrated improvements in the accuracy of 9.5%, 12%, and 18% over embryologists in the three scenarios. CONCLUSION: SSL methods can improve embryo identification accuracy even when dealing with cross-device and cryo-thawed paired images. The study is the first to apply SSL in embryo identification, and the results show the promise of WISE for future application in embryo witnessing.

In silico-designed vitrification protocols: an approach to improve survival of in vitro produced-bovine embryos.

In brief: In silico predictions validated in this study demonstrate the potential for designing shorter equilibration protocols that improve post-warming re-expansion and hatching rates of D7 and D8 in vitro-produced bovine embryos. Our results benefit the livestock industry by providing a refined and reproducible approach to cryopreserving bovine embryos, which, in addition, could be useful for other mammalian species. Abstract: The cryopreservation of in vitro-produced (IVP) embryos is vital in the cattle industry for genetic selection and crossbreeding programs. Despite its importance, there is no standardized protocol yielding pregnancy rates comparable to fresh embryos. Current approaches often neglect the osmotic tolerance responses to cryoprotectants based on temperature and time. Hereby, we propose improved vitrification methods using shorter dehydration-based protocols. Blastocysts cultured for 7 (D7) or 8 days (D8) were exposed to standard equilibration solution (ES) at 25ºC and 38.5ºC. Optimized exposure times for each temperature and their impact on post-warming re-expansion, hatching rates, cell counts, and apoptosis rate were determined. In silico predictions aligned with in vitro observations, showing original volume recovery within 8 min 30 s at 25ºC or 3 min 40 s at 38.5ºC (D7 blastocysts) and 4 min 25 s at 25ºC and 3 min 15 s at 38.5ºC (D8 blastocysts) after exposure to ES. Vitrification at 38.5ºC resulted in D7 blastocysts re-expansion and hatching rates (93.1% and 38.1%, respectively) comparable to fresh embryos (100.0% and 32.4%, respectively), outperforming the 25ºC protocol (86.2% and 24.4%, respectively; P < 0.05). No differences were observed between D7 and D8 blastocysts using the 38.5ºC protocol. Total cell number was maintained for D7 and D8 blastocysts vitrified at 38.5ºC but decreased at 25ºC (P < 0.05). Apoptosis rates increased post-warming (P < 0.05), except for D8 blastocysts vitrified at 38.5ºC, resembling fresh controls. In conclusion, based on biophysical permeability data, new ES incubation times of 3 min 40 s for D7 blastocysts and 3 min 15 s for D8 blastocysts at 38.5ºC were validated for optimizing vitrification/warming methods for bovine IVP blastocysts.

MEK signalling pathway is required for hypoblast specification and migration in ovine.

In brief: MEK signalling pathway is required for hypoblast differentiation in mouse embryos, but its role in ungulate embryos remains controversial. This paper demonstrates that MEK is required for hypoblast specification in the inner cell mass of the ovine blastocyst and that it plays a role during the hypoblast migration occurring following blastocyst hatching. Abstract: Early embryo development requires the differentiation of three cell lineages in two differentiation events. The second lineage specification differentiates the inner cell mass into epiblast, which will form the proper fetus, and hypoblast, which together with the trophectoderm will form the extraembryonic membranes and the fetal part of the placenta. MEK signalling pathway is required for hypoblast differentiation in mouse embryos, but its role in ungulate embryos remains controversial. The aim of this work was to analyse the role of MEK signalling on hypoblast specification at the blastocyst stage and on hypoblast migration during post-hatching stages in vitro in the ovine species. Using well-characterized and reliable lineage markers, and different MEK inhibitor concentrations, we demonstrate that MEK signalling pathway is required for hypoblast specification in the inner cell mass of the ovine blastocyst, and that it plays a role during the hypoblast migration occurring following blastocyst hatching. These results show that the role of MEK signalling pathway on hypoblast specification is conserved in phylogenetically distant mammals.

Towards a more sustainable balance between optimal live birth rate and supernumerary embryos in ART treatments.

PURPOSE: To assess the relation between number of inseminated oocytes and cumulative live birth rate (CLBR) in order to provide guidance for limiting the number of surplus blastocysts. METHODS: The study was a retrospective, single-center cohort analysis of 1223 ART complete cycles. Cycles were stratified according to female age (≤ 34, 35-38, and 39-42 years) and number of inseminated oocytes (1-5, 6-10, and > 10). Inclusion criteria were indication for IVF/ICSI with own spermatozoa and blastocyst culture up to day 6 of all embryos. RESULTS: In patients younger than 35 years, insemination of more than ten oocytes produced an increase in overall blastocyst number, CLBR (40.3%, 54.3%, and 75.8%, respectively, for each oocyte group) and surplus embryo rate (12.9%, 27.8%, and 49.7% of cases for each group). Instead, in the middle age group, the use of more than ten oocytes was solely associated with an increase in the rate of surplus embryos (1.25%, 21.33%, and 28.68% of cases after stratification for oocyte number). In older patients, neither CLBR (9.1%, 23.9%, and 24.7%, respectively) nor rate of surplus embryos (2.0%, 7.1%, and 13.4% of cases for each group) were higher in cycles with more than ten inseminated oocytes. CONCLUSION: In women up to 38 years, sustainable CLBR are achieved while limiting the number of inseminated oocytes and the resulting blastocysts remaining unused. Based on this notion, novel treatment strategies could pursue high outcome rates, while alleviating the problems derived from surplus stored embryos.

Detrimental effects of electromagnetic radiation emitted from cell phone on embryo morphokinetics and blastocyst viability in mice.

Electromagnetic radiation (EMR) has deleterious effects on sperm motility and viability, as well as oocyte membrane and organelle structure. The aim was to assess the effects of cell phone radiation on preimplantation embryo morphokinetics and blastocyst viability in mice. For superovulation, 20 female mice were treated with intraperitoneal (IP) injections of 10 IU pregnant mare's serum gonadotropin (Folligon® PMSG), followed by 10 IU of human chorionic gonadotropin (hCG) after 48 h. The zygotes (n = 150) from the control group were incubated for 4 days. The experimental zygotes (n = 150) were exposed to a cell phone emitting EMR with a frequency range 900-1800 MHz for 30 min on day 1. Then, all embryos were cultured in the time-lapse system and annotated based on time points from the 2-cell stage (t2) to hatched blastocyst (tHDyz), as well as abnormal cleavage patterns. Blastocyst viability was assessed using Hoechst and propidium iodide staining. Significant increases (P < 0.05) were observed in the cleavage division time points of t2, t8, t10, and t12 of the experimental group compared with the controls. In terms of blastocyst formation parameters, a delay in embryo development was observed in the experimental group compared with the controls. Data analysis of the time intervals between the two groups showed a significant difference in the s3 time interval (P < 0.05). Also, the rates of fragmentation, reverse cleavage, vacuole formation, and embryo arrest were significantly higher in the experimental group (P < 0.05). Furthermore, the cell survival rate in the experimental group was lower than the control group (P < 0.05). Exposure to EMR has detrimental consequences for preimplantation embryo development in mice. These effects can manifest as defects in the cleavage stage and impaired blastocyst formation, leading to lower cell viability.

Emergence of replication timing during early mammalian development.

DNA replication enables genetic inheritance across the kingdoms of life. Replication occurs with a defined temporal order known as the replication timing (RT) programme, leading to organization of the genome into early- or late-replicating regions. RT is cell-type specific, is tightly linked to the three-dimensional nuclear organization of the genome1,2 and is considered an epigenetic fingerprint3. In spite of its importance in maintaining the epigenome4, the developmental regulation of RT in mammals in vivo has not been explored. Here, using single-cell Repli-seq5, we generated genome-wide RT maps of mouse embryos from the zygote to the blastocyst stage. Our data show that RT is initially not well defined but becomes defined progressively from the 4-cell stage, coinciding with strengthening of the A and B compartments. We show that transcription contributes to the precision of the RT programme and that the difference in RT between the A and B compartments depends on RNA polymerase II at zygotic genome activation. Our data indicate that the establishment of nuclear organization precedes the acquisition of defined RT features and primes the partitioning of the genome into early- and late-replicating domains. Our work sheds light on the establishment of the epigenome at the beginning of mammalian development and reveals the organizing principles of genome organization.

Multifaceted SOX2-chromatin interaction underpins pluripotency progression in early embryos.

Pioneer transcription factors (TFs), such as OCT4 and SOX2, play crucial roles in pluripotency regulation. However, the master TF-governed pluripotency regulatory circuitry was largely inferred from cultured cells. In this work, we investigated SOX2 binding from embryonic day 3.5 (E3.5) to E7.5 in the mouse. In E3.5 inner cell mass (ICM), SOX2 regulates the ICM-trophectoderm program but is dispensable for opening global enhancers. Instead, SOX2 occupies preaccessible enhancers in part opened by early-stage expressing TFs TFAP2C and NR5A2. SOX2 then widely redistributes when cells adopt naive and formative pluripotency by opening enhancers or poising them for rapid future activation. Hence, multifaceted pioneer TF-enhancer interaction underpins pluripotency progression in embryos, including a distinctive state in E3.5 ICM that bridges totipotency and pluripotency.

Derivation of Naïve Human Embryonic Stem Cells Using a CHK1 Inhibitor.

Embryonic development is a continuum in vivo. Transcriptional analysis can separate established human embryonic stem cells (hESC) into at least four distinct developmental pluripotent stages, two naïve and two primed, early and late relative to the intact epiblast. In this study we primarily show that exposure of frozen human blastocysts to an inhibitor of checkpoint kinase 1 (CHK1) upon thaw greatly enhances establishment of karyotypically normal late naïve hESC cultures. These late naïve cells are plastic and can be toggled back to early naïve and forward to early primed pluripotent stages. The early primed cells are transcriptionally equivalent to the post inner cell mass intermediate (PICMI) stage seen one day following transfer of human blastocysts into in vitro culture and are stable at an earlier stage than conventional primed hESC.

LIS1 RNA-binding orchestrates the mechanosensitive properties of embryonic stem cells in AGO2-dependent and independent ways.

Lissencephaly-1 (LIS1) is associated with neurodevelopmental diseases and is known to regulate the molecular motor cytoplasmic dynein activity. Here we show that LIS1 is essential for the viability of mouse embryonic stem cells (mESCs), and it governs the physical properties of these cells. LIS1 dosage substantially affects gene expression, and we uncovered an unexpected interaction of LIS1 with RNA and RNA-binding proteins, most prominently the Argonaute complex. We demonstrate that LIS1 overexpression partially rescued the extracellular matrix (ECM) expression and mechanosensitive genes conferring stiffness to Argonaute null mESCs. Collectively, our data transforms the current perspective on the roles of LIS1 in post-transcriptional regulation underlying development and mechanosensitive processes.

Expression of microRNA let-7 in cleavage embryos modulates cell fate determination and formation of mouse blastocysts†.

After fertilization, the zygote undergoes cell division. Up to the 8-cell stage, the blastomeres of mouse preimplantation embryos are morphologically identical. The first cell differentiation starts in the morula leading to the formation of trophectoderm cells and inner cell mass cells of the blastocyst. The regulation of the differentiation event and the formation of blastocysts are not fully known. Lethal-7 (let-7) is a family of evolutionarily conserved microRNAs. Here, we showed that the expression of let-7a and let-7g decreased drastically from the 1-cell stage to the 2-cell stage, remained low up to the 8-cell stage and slightly increased after the morula stage of mouse embryos. The expression of let-7 in the inner cell mass was higher than that in the trophectoderm. Forced expression of let-7a in embryos at the 1-cell and 4-cell stage inhibited blastocyst formation and downregulated the expression of CDX2 but maintained that of OCT4 in the trophectoderm. Forced expression of other let-7 isoforms exhibited similar inhibitory action on blastulation. On the other hand, inhibition of let-7a at the 4-cell stage and the 8-cell stage enhanced blastocyst formation. Co-injection of green fluorescent protein (GFP) mRNA (lineage tracer) with either precursor of let-7a (pre-let-7a) or scramble control into one blastomere of 2-cell embryos showed that ~75% of the resulting blastocysts possessed GFP+ cells in their inner cell mass only. The biased development towards the inner cell mass with forced expression of let-7 was reproduced in 2-cell chimeric embryos consisting of one wildtype blastomere and one GFP mRNA-injected blastomere from another 2-cell embryo carrying a doxycycline-inducible let-7g gene. Bioinformatics analysis indicated that Tead4 was a potential target of let-7. Let-7 bound to the 3'UTR of Tead4 and let-7 forced expression downregulated the expression of Tead4 in mouse blastocysts. Co-injection of Tead4 mRNA partially nullified the modulatory roles of let-7a in the inner cell mass cell fate. In conclusion, a high level of let-7 at the 2-cell stage favored the formation of the inner cell mass, whereas a low level of let-7 at the 4-cell to 8-cell stage enhanced blastocyst formation. Tead4 mediated the action of let-7 on the inner cell mass cell-fate determination.

YY1 safeguard multidimensional epigenetic landscape associated with extended pluripotency.

Although extended pluripotent stem cells (EPSCs) have the potential to form both embryonic and extraembryonic lineages, how their transcriptional regulatory mechanism differs from that of embryonic stem cells (ESCs) remains unclear. Here, we discovered that YY1 binds to specific open chromatin regions in EPSCs. Yy1 depletion in EPSCs leads to a gene expression pattern more similar to that of ESCs than control EPSCs. Moreover, Yy1 depletion triggers a series of epigenetic crosstalk activities, including changes in DNA methylation, histone modifications and high-order chromatin structures. Yy1 depletion in EPSCs disrupts the enhancer-promoter (EP) interactions of EPSC-specific genes, including Dnmt3l. Yy1 loss results in DNA hypomethylation and dramatically reduces the enrichment of H3K4me3 and H3K27ac on the promoters of EPSC-specific genes by upregulating the expression of Kdm5c and Hdac6 through facilitating the formation of CCCTC-binding factor (CTCF)-mediated EP interactions surrounding their loci. Furthermore, single-cell RNA sequencing (scRNA-seq) experiments revealed that YY1 is required for the derivation of extraembryonic endoderm (XEN)-like cells from EPSCs in vitro. Together, this study reveals that YY1 functions as a key regulator of multidimensional epigenetic crosstalk associated with extended pluripotency.