2-cell Embryos Research Articles

MicroRNA-148b secreted by bovine oviductal extracellular vesicles enhance embryo quality through BPM/TGF-beta pathway

BackgroundExtracellular vesicles (EVs) and their cargoes, including MicroRNAs (miRNAs) play a crucial role in cell-to-cell communication. We previously demonstrated the upregulation of bta-mir-148b in EVs from oviductal fluid of cyclic cows. This miRNA is linked to the TGF-β pathway in the cell proliferation. Our aim was to verify whether miR-148b is taken up by embryos through gymnosis, validate its target genes, and investigate the effect of miR-148b supplementation on early embryo development and quality.MethodsZygotes were cultured in SOF + 0.3% BSA (Control) or supplemented with: 1 µM miR-148b mimics during: D1-D7 (miR148b) or D1-D4 (miR148b-OV: representing miRNA effect in the oviduct) or D4-D7 (miR148b-UT: representing miRNA effect in the uterus) or 1 µM control mimics was used during: D1-D7 (CMimic). Embryos at ≥ 16-cells and D7 blastocysts (BD7) were collected to examine the mRNA abundance of transcripts linked to the TGF-β pathway (TGFBR2, SMAD1, SMAD2, SMAD3, SMAD5, BMPR2, RPS6KB1, POU5F1, NANOG), total cell number (TC), trophectoderm (TE), and inner cell mass (ICM) were also evaluated. One-way ANOVA was used for all analyses.ResultsWe demonstrated that miR-148b can be taken up in both 16-cell embryos and BD7 by gymnosis, and we observed a decrease in SMAD5 mRNA, suggesting it’s a potential target of miR-148b. Cleavage and blastocysts rates were not affected in any groups; however, supplementation of miR-148b mimics had a positive effect on TC, TE and ICM, with values of 136.4 ± 1.6, 92.5 ± 0.9, 43.9 ± 1.3 for miR148b and 135.3 ± 1.5, 92.6 ± 1.2, 42.7 ± 0.8, for miR148b-OV group. Furthermore, mRNA transcripts of SMAD1 and SMAD5 were decreased (P ≤ 0.001) in 16-cell embryos and BD7 from miR148b and miR148b-OV groups, while POU5F1 and NANOG were upregulated (P ≤ 0.001) in BD7 and TGFBR2 was only downregulated in 16-cell embryos. pSMAD1/5 levels were higher in the miR148b and miR148b-OV groups.ConclusionsOur findings suggest that supplementation of bta-miR-148b mimics during the entire culture period (D1 - D7) or from D1 - D4 improves embryo quality and influences the TGF-β signaling pathway by altering the transcription of genes associated with cellular differentiation and proliferation. This highlights the importance of miR-148b on embryo quality and development.

Dynamics of Mitochondrial DNA Copy Number and Membrane Potential in Mouse Pre-Implantation Embryos: Responses to Diverse Types of Oxidative Stress.

Mitochondria undergo a myriad of changes during pre-implantation embryo development, including shifts in activity levels and mitochondrial DNA (mtDNA) replication. However, how these distinct aspects of mitochondrial function are linked and their responsiveness to diverse stressors is not well understood. Here, we show that mtDNA content increased between 8-cell embryos and the blastocyst stage, with similar copy numbers per cell in the inner cell mass (ICM) and trophectoderm (TE). In contrast, mitochondrial membrane potential (MMP) was higher in TE than ICM. Culture in ambient oxygen (20% O2) altered both aspects of mitochondrial function: the mtDNA copy number was upregulated in ICM, while MMP was diminished in TE. Embryos cultured in 20% O2 also exhibited delayed development kinetics, impaired implantation, and reduced mtDNA levels in E18 fetal liver. A model of oocyte mitochondrial stress using rotenone showed only a modest effect on on-time development and did not alter the mtDNA copy number in ICM; however, following embryo transfer, mtDNA was higher in the fetal heart. Lastly, endogenous mitochondrial dysfunction, induced by maternal age and obesity, altered the blastocyst mtDNA copy number, but not within the ICM. These results demonstrate that mitochondrial activity and mtDNA content exhibit cell-specific changes and are differentially responsive to diverse types of oxidative stress during pre-implantation embryogenesis.

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation.

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair templates and CRISPR-Cas reagents is the most common delivery method into embryos. However, this type of micromanipulation necessitates access to specialized equipment, is laborious, and requires a certain level of technical skill. Moreover, microinjection techniques often result in lower embryo survival due to the mechanical stress on the embryo. In this protocol, we developed an optimized method to deliver large DNA repair templates to work in conjunction with CRISPR-Cas9 genome editing without the need for microinjection. This protocol combines AAV-mediated DNA delivery of single-stranded DNA donor templates along with the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) by electroporation to modify 2-cell embryos. Using this novel strategy, we have successfully produced targeted knock-in rat models carrying insertion of DNA sequences from 1.2 to 3.0 kb in size with efficiencies between 42% and 90%.

Early onset of APC/C activity renders SAC inefficient in mouse embryos.

Control mechanisms of spindle assembly and chromosome segregation are vital for preventing aneuploidy during cell division. The mammalian germ cells and embryos are prone to chromosome segregation errors, and the resulting aneuploidy is a major cause of termination of development or severe developmental disorders. Here we focused on early mouse embryos, and using combination of methods involving microinjection, immunodetection and confocal live cell imaging, we concentrated on the Spindle Assembly Checkpoint (SAC) and Anaphase Promoting Complex/Cyclosome (APC/C). These are two important mechanisms cooperating during mitosis to ensure accurate chromosome segregation, and assessed their activity during the first two mitoses after fertilization. Our results showed, that in zygotes and 2-cell embryos, the SAC core protein Mad1 shows very low levels on kinetochores in comparison to oocytes and its interaction with chromosomes is restricted to a short time interval after nuclear membrane disassembly (NEBD). Exposure of 2-cell embryos to low levels of spindle poison does not prevent anaphase, despite the spindle damage induced by the drug. Lastly, the APC/C is activated coincidentally with NEBD before the spindle assembly completion. This early onset of APC/C activity, together with precocious relocalization of Mad1 from chromosomes, prevents proper surveillance of spindle assembly by SAC. The results contribute to the understanding of the origin of aneuploidy in early embryos.

The impact of clinical and laboratory parameters on clinical pregnancy and live birth rates in fresh cycles: a retrospective study of 9608 high-quality cleavage-stage embryos

BackgroundEvidence from the Istanbul consensus workshop suggests correlations between morphological parameters and embryo developments. 8-cell embryos are the best blastomere stage on day 3. No good quality evidence exists to support high-quality embryonic selection following blastulation and clinical outcomes. This study aimed to investigate the factors that affect blastocyst formation, blastocyst quality, and clinical outcomes of high-quality cleavage-stage embryos in fresh cycles.MethodsThis study was a retrospective analysis of 9608 high-quality cleavage-stage embryos from 2987 couples between January 2017 to June 2021, namely 1520 embryos categorized as “812” (8-cell, grade 2, mild fragmentation), 2961 as “821” (8-cell, grade 2, mild asymmetry), 896 as “711” (7-cell, grade 1), and 517 as “911” (9-cell, grade 1) compared with 3714 embryos categorized as “811” (8-cell, grade 1). The primary outcomes were clinical pregnancy rate (CPR) and live birth rate (LBR). Blastulation rate (BR), available late blastocyst rate (ABR) and high-quality late blastocyst rate (HBR) were secondary outcome measures.ResultsBR, ABR, and HBR had significant differences among the five groups (P < 0.001), while CPR and LBR were also significantly different in cleavage-stage fresh transfer (P < 0.01). The multivariable multilevel logistic regression analysis revealed a significant association between cell number, cell size, blastocyst development and clinical outcomes. For 7 to 9-cell highest-quality embryo, mild fragmentation and more blastomeres were more conducive to blastocyst formation and clinical outcomes. While cleavage-stage embryos developed into blastocysts, the negative impact of their initial morphology on clinical outcomes would be erased.ConclusionsOur study firstly evaluated blastocyst development and clinical outcomes of high-quality cleavage-stage embryos in fresh cycles, with rankings of 811, 812, 911, 821, and 711. We found the initial morphological characteristics of the high-quality cleavage-stage embryos did not adversely impact clinical outcomes, even as they progressed to the blastocyst stage.

Determining the potency of primordial germ cells by injection into early mouse embryos

Primordial germ cells (PGCs) are the earliest precursors of the gametes. During normal development, PGCs only give rise to oocytes or spermatozoa. However, PGCs can acquire pluripotency invitro by forming embryonic germ (EG) cells and invivo during teratocarcinogenesis. Classic embryological experiments directly assessed the potency of PGCs by injection into the pre-implantation embryo. As no contribution to embryos or adult mice was observed, PGCs have been described as unipotent. Here, we demonstrate that PGCs injected into 8-cell embryos can initially survive, divide, and contribute to the developing inner cell mass. Apoptosis-deficient PGCs exhibit improved survival in isolated epiblasts and can form naive pluripotent embryonic stem cell lines. However, contribution to the post-implantation embryo is limited, with no functional incorporation observed. In contrast, PGC-like cells show an extensive contribution to mid-gestation chimeras. We thus propose that PGC formation invivo establishes a latent form of pluripotency that restricts chimera contribution.

Maternal KLF17 controls zygotic genome activation by acting as a messenger for RNA Pol II recruitment in mouse embryos

Initiation of timely and sufficient zygotic genome activation (ZGA) is crucial for the beginning of life, yet our knowledge of transcription factors (TFs) contributing to ZGA remains limited. Here, we screened the proteome of early mouse embryos after cycloheximide (CHX) treatment and identified maternally derived KLF17 as a potential TF for ZGA genes. Using a conditional knockout (cKO) mouse model, we further investigated the role of maternal KLF17 and found that it promotes embryonic development and full fertility. Mechanistically, KLF17 preferentially binds to promoters and recruits RNA polymerase II (RNA Pol II) in early 2-cell embryos, facilitating the expression of major ZGA genes. Maternal Klf17 knockout resulted in a downregulation of 9% of ZGA genes and aberrant RNA Pol II pre-configuration, which could be partially rescued by introducing exogenous KLF17. Overall, our study provides a strategy for screening essential ZGA factors and identifies KLF17 as a crucial TF in this process.

Propyl gallate exposure affects the mouse 2-cell stage embryonic development through inducing oxidative stress and autophagy

Propyl gallate (PG), owing to its exceptional antioxidant properties, is extensively used in industries such as food processing. The potential harmful impacts of PG have sparked concern among people. It has been reported that exposure of PG has certain reproductive toxicity, which can affect the maturation of mouse oocytes and induce testicular dysfunction. However, its impact on early embryonic development is still unclear. In this study, we explored the toxic effects and potential mechanisms of PG on mouse 2-cell stage embryonic development. The results showed that exposure of PG can decrease the development of 2-cell stage embryos and repress the development of 4-cell stage embryos. Further study found that PG could induce intracellular oxidative stress and the accumulation of DNA damage in 2-cell stage embryos. Moreover, exposure of PG impaired the function of mitochondria and lysosomes in 2-cell stage embryos, thereby triggering the occurrence of autophagy. In addition, exposure of PG altered the epigenetic modification of 2-cell stage embryos, displaying a decreased level of DNA methylation and an increased level of H3K4me3. In summary, our results indicated that exposure of PG can damage the development of mouse 2-cell stage embryos by inducing oxidative stress, DNA damage, and autophagy, and altering epigenetic modification.

Zygotic Splicing Activation of the Transcriptome is a Crucial Aspect of Maternal-to-Zygotic Transition and Required for the Conversion from Totipotency to Pluripotency.

During maternal-to-zygotic transition (MZT) in the embryo, mRNA undergoes complex post-transcriptional regulatory processes. However, it is unclear whether and how alternative splicing plays a functional role in MZT. By analyzing transcriptome changes in mouse and human early embryos, dynamic changes in alternative splicing during MZT are observed and a previously unnoticed process of zygotic splicing activation (ZSA) following embryonic transcriptional activation is described. As the underlying mechanism of RNA splicing, splicing factors undergo dramatic maternal-to-zygotic conversion. This conversion relies on the key maternal factors BTG4 and PABPN1L and is zygotic-transcription-dependent. CDK11-dependent phosphorylation of the key splicing factor, SF3B1, and its aggregation with SRSF2 in the subnuclear domains of 2-cell embryos are prerequisites for ZSA. Isoforms generated by erroneous splicing, such as full-length Dppa4, hinder normal embryonic development. Moreover, alternative splicing regulates the conversion of early embryonic blastomeres from totipotency to pluripotency, thereby affecting embryonic lineage differentiation. ZSA is an essential post-transcriptional process of MZT and has physiological significance in generating new life. In addition to transcriptional activation, appropriate expression of transcript isoforms is also necessary for preimplantation embryonic development.

Locational and functional characterization of PI4KB in the mouse embryo.

Phosphatidylinositol 4-kinase beta (PI4KB) is a member of the PI4K family, which is mainly enriched and functions in the Golgi apparatus. The kinase domain of PI4KB catalyzes the phosphorylation of phosphatidylinositol to form phosphatidylinositol 4-phosphate, a process that regulates various sub-cellular events, such as non-vesicular cholesterol and ceramide transport, protein glycosylation, and vesicle transport, as well as cytoplasmic division. In this study, a strain of PI4KB knockout mouse, immunofluorescence, reverse transcription polymerase chain reaction and microinjection were used to characterize the cytological location and biological function of PI4KB in the mouse embryos. we found that knocking down Pi4kb in mouse embryos resulted in embryonic lethality at around embryonic day (E) 7.5. Additionally, we observed dramatic fluctuations in PI4KB expression during the development of preimplantation embryos, with high expression in the 4-cell and morula stages. PI4KB colocalized with the Golgi marker protein TGN46 in the perinuclear and cytoplasmic regions in early blastomeres. Postimplantation, PI4KB was highly expressed in the epiblast of E7.5 embryos. Treatment of embryos with PI4KB inhibitors was found to inhibit the development of the morula into a blastocyst and the normal progression of cytoplasmic division during the formation of a 4-cell embryo. These findings suggest that PI4KB plays an important role in mouse embryogenesis by regulating various intracellular vital functions of embryonic cells.

Overexpression of BRG1 improves early development of porcine somatic cell nuclear transfer embryos

The epigenetic modification levels of donor cells directly affect the developmental potential of somatic cell nuclear transfer (SCNT) embryos. BRG1, as an epigenetic modifying enzyme, has not yet been studied in donor cells and SCNT embryos. In this study, BRG1 was overexpressed in porcine fetal fibroblasts (PFFs), its effect on chromatin openness and gene transcription was examined, subsequently, the development potential of porcine SCNT embryos was investigated. The results showed that compared with the control group, the percentage of G1 phase cells was significantly increased (32.3 % ± 0.87 vs 25.7 % ± 0.81, P < 0.05) in the experimental group. The qRT-PCR results showed that the expression of H3K9me3-related genes was significantly decreased (P < 0.05), HAT1 was significantly increased (P < 0.05). Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) results revealed that SMARCA4、NANOG、SOX2、MAP2K6 and HIF1A loci had more open chromatin peaks in the experimental group. The RNA-seq results showed that the upregulated genes were mainly enriched in PI3K/AKT and WNT signaling pathways, and the downregulated genes were largely focused on disease development. Interestingly, the developmental rate of porcine SCNT embryos was improved (27.33 % ± 1.40 vs 17.83 % ± 2.02, P < 0.05), the expression of zygotic gene activation-related genes in 4-cell embryos, and embryonic development-related genes in blastocysts was significantly upregulated in the experimental group (P < 0.05). These results suggest that overexpression of BRG1 in donor cells is benefit for the developmental potential of porcine SCNT embryos.

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.

Chromatin structure in totipotent mouse early preimplantation embryos.

Totipotency refers to the ability of a single cell to give rise to all the different cell types in the body. Terminally differentiated germ cells (sperm and oocytes) undergo reprogramming, which results in the acquisition of totipotency in zygotes. Since the 1990s, numerous studies have focused on the mechanisms of totipotency. With the emergence of the concept of epigenetic reprogramming, which is important for the undifferentiated and differentiated states of cells, the epigenomes of germ cells and fertilized oocytes have been thoroughly analyzed. However, in early immunostaining studies, detailed epigenomic information was difficult to obtain. In recent years, the explosive development of next-generation sequencing has made it possible to acquire genome-wide information and the rise of genome editing has facilitated the analysis of knockout mice, which was previously difficult. In addition, live imaging can effectively analyze zygotes and 2-cell embryos, for which the number of samples is limited, and provides biological insights that cannot be obtained by other methods. In this review, the progress of our research using these advanced techniques is traced back from the present to its earliest years.

Morphology of 16-cell embryo in bovine: Inside cells, compaction, fragmentation and effects of X-sorted semen.

In mouse embryos, inside cells are allocated in 16-cell embryos through a well-orchestrated sequence of events involving compaction and polarization. The emergence of inside cells is of great importance as itl later gives rise to the inner cell mass and epiblast. In this study, we report the sequence of critical events in embryology (compaction, inside cells allocation and fragmentation) in bovine 72 h.p.i. 9-16 cell embryos, while also investigating the effects of X-sorted semen on these events. We found a wide distribution of total cell numbers among embryos, attributed to an asynchronous cleavage pattern and blastomere death. Additionally, 13% of embryos displayed irregular shapes. The establishment of the inside cell compartment increased (p < 0.01) in embryos with more cells. However, only 53.8% of 16-cell embryos presented inside cells. Compaction was present in 32.4% embryos and was positively correlated (p = 0.03, OR 3.02) with the establishment of inside cells, occurring independently of cell number. Fragmentation was present in 36% embryos, being more frequent (p = 0.01) in embryos with lower cell numbers. A possible association between irregular shape and fragmentation was considered (p = 0.06). The use of X-sorted semen had no effect on most evaluated parameters. However, it did have a marked effect on cleavage rate (p < 0.01) and the arrest of 2- and 4- cell embryos. In conclusion, bovine embryos exhibit an asynchronous cleavage pattern, high levels of fragmentation, and demonstrate compaction and inside cell allocation later in development compared to mouse embryos. Semen X-sorting has major effects on cleavage and embryo arrest. Further studies are needed to elucidate the association between irregularly shaped embryos and fragmentation, as well as the effects of sex on inside cell allocation.

Microarray evidence that 8-cell human embryos express some hormone family members including oxytocin.

This study is to discover hormone pathways active in early cleaving human embryos. A list of 152 hormones and receptors were compiled to query the microarray database of mRNAs in 8-cell human embryos, two lines of human embryonic stem cells plus human fibroblasts before and after induced pluripotency. Over half of the 152 hormones and receptors were silent on the arrays of all cell types, and more were detected at high or moderate levels on the 8-cell arrays than on the pluripotent cell or fibroblast arrays. Eight hormone family genes were uniquely detected at least 22-fold higher on the 8-cell arrays than the stem cell arrays: AVPI1, CCK, CORT, FSTL4, GIP, GPHA2, OXT, and PPY suggesting novel roles for these proteins in early development. Oxytocin was detected by pilot immunoassay in culture media collected from Day 3 embryos. Robust detection of CRHR1 and EPOR suggests the 8-cell embryo may be responsive to maternal CRH and EPO. The over-expression of POMC and GHITM suggests POMP peptide products may have undiscovered roles in early development and GHITM may contribute to mitochondrial remodeling. Under-detected on the 8-cell arrays at least tenfold were two key enzymes in steroid biosynthesis, DHCR24 and FDPS. The 8-cell human embryo may be secreting oxytocin, which could stimulate its own progress down the fallopian tube as well as play a role in early neural precursor development. The 8-cell embryo does not synthesize reproductive steroid hormones. As previously reported for growth factor families, the early embryo over-expresses more hormones than hormone receptors.

PFOS and F–53B disrupted inner cell mass development in mouse preimplantation embryo

Perfluorooctane sulfonic acid (PFOS) is a perfluoroalkyl and polyfluoroalkyl substance (PFAS) widely used in daily life. As its toxicity was confirmed, it has been gradually substituted by F–53B (chlorinated polyfluoroalkyl sulfonates, Cl-PFESAs) in China. PFOS exposure during prenatal development may hinder the development of preimplantation embryos, as indicated by recent epidemiological research and in vivo assays. However, the embryotoxicity data for F–53B are scarce. Furthermore, knowledge about the toxicity of F–53B and PFOS exposure to internal follicular fluid concentrations on early preimplantation embryo development remains limited. In this study, internal exposure concentrations of PFOS (10 nM) and F–53B (2 nM) in human follicular fluid were chosen to study the effects of PFAS on early mouse preimplantation embryo development. We found that both PFOS and F–53B treated zygotes exhibited higher ROS activity in 8-cell embryos but not in 2-cell stage embryos. PFOS and F–53B significantly affected the proportion and aggregation of the inner cell mass (ICM) in the blastocyst, but not the total cell number. Mouse embryonic stem cells (mESCs, isolated from the ICM) and embryoid body (EB) assays were employed to assess the toxicity of PFOS and F–53B on the development and differentiation of embryonic pluripotent cells. These results suggested that mESCs exhibited more DNA damage and abnormal germ layer differentiation after brief exposure to PFOS or F–53B. Finally, RNA-sequencing revealed that PFOS and F–53B exposure affected mESCs biosynthetic processes and chromatin-nucleosome assembly. Our results indicate that F–53B has potential risks as an alternative to PFOS, which disrupts ICM development and differentiation.

Effect of lipotoxicity on mitochondrial function and epigenetic programming during bovine in vitro embryo production

Maternal metabolic disorders may cause lipotoxic effects on the developing oocyte. Understanding the timing at which this might disrupt embryo epigenetic programming and how this is linked with mitochondrial dysfunction is crucial for improving assisted reproductive treatments, but has not been investigated before. Therefore, we used a bovine in vitro model to investigate if pathophysiological palmitic acid (PA) concentrations during in vitro oocyte maturation and in vitro embryo culture alter embryo epigenetic patterns (DNA methylation (5mC) and histone acetylation/methylation (H3K9ac/H3K9me2)) compared to control (CONT) and solvent control (SCONT), at the zygote and morula stage. Secondly, we investigated if these epigenetic alterations are associated with mitochondrial dysfunction and changes in ATP production rate, or altered expression of epigenetic regulatory genes. Compared to SCONT, H3K9ac and H3K9me2 levels were increased in PA-derived zygotes. Also, 5mC and H3K9me2 levels were increased in PA-exposed morulae compared to SCONT. This was associated with complete inhibition of glycolytic ATP production in oocytes, increased mitochondrial membrane potential and complete inhibition of glycolytic ATP production in 4-cell embryos and reduced SOD2 expression in PA-exposed zygotes and morulae. For the first time, epigenetic alterations in metabolically compromised zygotes and morulae have been observed in parallel with mitochondrial dysfunction in the same study.

#264 : 3D Time-Lapse Refractive-Index Imaging of Intact Mouse Embryos for Non-Invasive Evaluation of the Embryonic Development

Background and Aims: Time-lapse technology is an emerging assisted reproductive technology (ART) that enables simultaneous culture and monitoring of embryos in vitro during their development. It provides extra information on key events during embryo development, which can assist embryologists in selecting embryos alongside traditional morphology evaluations. Various imaging technologies have been utilised to visualise and evaluate embryos such as brightfield microscopy, darkfield microscopy, and Hoffman modulation contrast microscopy. Although these imaging technologies allow for non-invasive imaging of human embryos in a clinical setting, 3D imaging of a whole embryo with subcellular resolution remains a challenge. Method: We demonstrate 3D time-lapse label-free imaging of live mouse embryos using the 3D refractive index (RI) imaging technique. The proposed method is a quantitative phase imaging technique that measures multiple-intensity images of transmitted light through the embryos via axial scanning to reconstruct the 3D RI map of the embryos. Results: Using the proposed method, live mouse embryos were monitored at different developmental stages, ranging from 2-cell embryos to expanded blastocysts. The measured 3D RI maps of the developing embryos reveal 3D landscape of subcellular features inside the embryos (Fig. 1). In the quantitative assessment of embryonic development, various morphokinematic parameters were extracted from the measured 3D RI maps of the embryos. Conclusion: Based on the results, we envision that our method has broad applications where non-invasive and quantitative assessment of embryonic development is required.

#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.

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

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