Embryo Arrest Research Articles

Comparison of Preimplantation Mouse Embryos with Different Genetic Backgrounds as Models for Evaluating Human Embryo Culture Media Composition.

Can the best culture medium be determined by mouse embryo testing? Zygotes/embryos of four different mouse strains: inbred Balb/c (n = 79) and C57Bl/6 (n = 95), F1 hybrid DBA/2*C57Bl/6 (n = 133), and outbred ICR (n = 69) were incubated in vitro in four culture media: 1-Step Culture Medium (VitaVitro), CSC medium (Irvine Scientific), Sage 1-Step medium (Origio), G-TL medium (Vitrolife). The embryos were cultured under standard conditions at atmospheric oxygen: 37℃; 6% CO2, 100% humidity. Standard MEA-test parameters (number of expanded blastocysts on E4.5), as well as additional characteristics (embryo development on E2.5-E4.5, number of hatching and hatched blastocysts on E4.5, embryo arrest and degeneration) were assessed for each of the strain-medium combinations. The results were compared across strains as well as across media. Embryo development depended on both the media and the mouse strain. Embryos from inbred and outbred mice were more sensitive to suboptimal culture conditions compared to the hybrid strain; this was reflected in reduced blastocyst and hatching rates, as well as an increased percentage of arrested and degraded embryos. The choice of optimal media depended strongly on the strain: CSC was better for Balb/c, Sage 1-step for C57Bl/6, while both were preferred for hybrids, and G-TL for outbreds. VitaVitro was quite good for hybrids and performed worse for other strains. Overall, the embryos of each strain behaved differently in different media, and it was not possible to make a real preference for any of them over human embryos based on the results of any single mouse strain. Only a pooled sample of different mouse strains can be used for comprehensive media MEA testing. MEA testing with any strain of mice is doomed to error due to their specific culture requirements. Hybrid mice are too reproductively efficient and cannot be used to distinguish media based on their subtle differences. Outbred and inbred mice are too sensitive and may be delayed or degraded in culture media that is well suited to the requirements of human embryos. Combined analysis of multiple mouse strains should be used to test media more fully and may serve as a model for the heterogeneity of human embryos in IVF clinics.

Loss of Cell-Cell Contact Inhibits Cellular Differentiation of α-Catenin Knock Out P19 Embryonal Carcinoma Cells and Their Colonization into the Developing Mouse Embryos.

Cadherin-catenin cell-cell adhesion complexes, composed of cadherin, β-catenin or plakoglobin, and α-catenin (α-cat) molecules, are crucial for maintaining cell-cell contact and are commonly referred to as "adherens junctions (AJs)." Inactivating this system leads to loss of cell-cell contact and developmental arrest in early embryos. However, it remains unclear whether the loss of cell-cell contact affects the differentiation of embryonic cells. In this study, we explored the use of a murine embryonal carcinoma cell line, P19, as an in vitro model for early embryogenesis. P19 cells easily form embryoid bodies (EBs) and are susceptible to cellular differentiation in response to retinoic acid (RA) and teratoma formation. Using CRISPR/Cas9 technology to disrupt the endogenous α-cat gene in P19 cells, we generated α-cat knockout (KO) cells that exhibited a loss of cell-cell contact. When cultivated on non-coated dishes, these α-cat KO cells formed EBs, but their structures were labile. In the RA-containing medium, the α-cat KO EBs failed to produce differentiated cells on their outer layer and continued to express SSEA-1, an antigen specific to pluripotent cells. Teratoma formation assays revealed an absence of overt differentiated cells in tumors derived from α-cat KO P19 cells. Aggregation assays revealed the inability of the KO cells to colonize into the zona pellucida-denuded 8-cell embryos. These findings suggest that the AJs are essential for promoting the early stages of cellular differentiation and for the colonization of early-developing embryos.

Comparative analysis of short versus long co-incubation of gametes on post-insemination outcomes and embryo morphokinetics: a sibling oocyte randomized study

Research questionDoes a short co-incubation of gametes in conventional IVF impact post-insemination outcomes and embryo morphokinetics? DesignSibling oocyte randomized pilot study performed between December 2020 and March 2023. Eligible couples (n=55) were female aged 18-43 years with body mass index (BMI) ≤35 and male normal semen parameters. Cumulus oocyte complexes (COCs) (min 6 - max 12) were randomized in a 1:1:2 proportion in long (16-18 hrs), or short (2 hrs) co-incubation IVF exposure, and ICSI, respectively. All oocytes inseminated via IVF were monitored by time-lapse (TL). Blastocysts of ≥BL3CC were subjected to trophectoderm (TE) biopsy on day (D)5, D6 or D7 by next generation sequencing (NGS). ResultsA total of 954 COCs were distributed to long co-incubation (n=235, 24.6%), short co-incubation (n=235, 24.6%), and ICSI (n=484, 50.3%). In total, 202 were MII after long co-incubation, 209 after short co-incubation and 394 at ICSI. Regression analyses showed both short co-incubation groups (OR: 0.72, 95% CI: 0.46–1.11, P=0.139) and ICSI (OR: 1.43, 05% CI: 0.95–2.15, P=0.090) did not significantly impact normal fertilization rates (2PN) when compared to long co-incubation. Usable blastocysts per MII, per 2PN, and euploid blastocysts per MII, per 2PN and per tested blastocysts were similar among the groups. The rate of embryo arrest (HR: HR: 0.79, 95% CI: 0.57-1.11, P=0.183) and timings to reach morphokinetic milestones among embryos reaching blastulation were similar between IVF groups (P>0.05, for all). ConclusionA short 2-hour co-incubation of gametes in IVF yields comparable fertilization, blastocyst, and euploidy rates without adverse embryo morphokinetic events, compared to standard overnight co-incubation.

New Immunological Indexes for the Effect of Systemic Inflammation on Oocyte and Embryo Development in Women With Unexplained Infertility: Systemic Immune Response Index and Pan-Immune-Inflammation Value.

Predicting the impact of systemic inflammation on oocyte and embryonic development in unexplained infertile women using the new immunological indexes. This retrospective cohort study was conducted using the records of the In Vitro Fertilization Department of Ankara Gülhane Training and Research Hospital. After reviewing the records of patients who had undergone in vitro fertilization (IVF) for unexplained infertility (UI) and excluding all known factors that could cause systemic immune inflammation, the systemic immune response index (SIRI), and pan-immune score were calculated from the pre-treatment hemogram parameters between the embryo arrest (EA) group and the embryo transfer group. It was investigated whether there was a statistical difference between the two groups and whether an SIRI value affecting embryo quality was found. A receiver operating characteristic (ROC) curve analysis was performed to determine the optimal cut-off values for inflammatory markers to predict EA. The 108 EA group (embryos that were arrested during their development and could not be transferred) and the 140 embryo transfer group showed statistically significant differences in the parameters of systemic inflammatory index (SII), SIRI, pan-immune inflammation value (PIV), and neutrophil/lymphocyte ratio (NLR) (p < 0.05). These inflammatory parameters, which were examined before ovulation induction, also correlated positively with the required total dose of gonadotropin and negatively with the ovarian sensitivity index (OSI). SII, SIRI, PIV, and NLR have specific cut-off values with ROC analysis and determine the effect of the inflammatory status of the environment in which the oocyte develops on EA (p < 0.005). In women with UI, high levels of systemic immune inflammation have a negative impact on oocyte and embryo development, and treatments to suppress inflammation may improve IVF success.

Transcriptome analysis reveals that defects in cell cycle regulation contribute to preimplantation embryo arrest

Patients with preimplantation embryo arrest (PREMBA) often experience assisted reproductive failure primarily due to the lack of transferable embryos, and the molecular mechanisms underlying PREMBA remain unclear. In our study, the embryos from five women with recurrent preimplantation embryo arrest and three women with tubal factor infertility were used for single-embryo transcriptome sequencing. Meanwhile, the transcriptomes of normal human preimplantation embryos obtained from GSE36552 were utilized to perform a comparative analysis with the transcriptomes of PREMBA embryos. Our results showed dysregulation of the cell cycle phase transition might be a potential pathogenic factor contributing to PREMBA. Through integrated analysis of the differentially expressed genes (DEGs) and weighted gene co-expression network analysis (WGCNA), we identified a number of hub genes using the protein-protein interaction network. The top 5 hub genes were as follows: CCNB2, BUB1B, CDC25A, CCNB3, and PLK3. The expression of hub genes was validated in PREMBA embryos and donated embryos using RT-qPCR. The knockdown of Ccnb2 in mouse zygotes led to an increase in embryo fragmentation, a rise in apoptosis, and a reduction in blastocyst formation. Furthermore, silencing the expression of CDC25A in HEK293T cells resulted in a decrease in cell proliferation and an increase in apoptosis, providing further support for our findings. Our findings could predict the development outcomes of preimplantation embryos and be used as potential therapeutic targets to prevent recurrent failures of IVF/ICSI attempts.

Biallelic variants in α-tubulin isotypes cause female infertility characterised as recurrent preimplantation embryo arrest

BackgroundRecurrent preimplantation embryo developmental arrest (RPEA) is the most common phenotype in assisted reproductive technology treatment failure associated with identified genetic abnormalities. Currently known maternal genetic variants explain only a...

P-086 Consequences of elevated levels of DNA fragmentation and its influence on pregnancy – A multicentric cohort analysis

Abstract Study question Does an abnormal DNA Fragmentation Index impact the clinical outcomes of patients undergoing IVF treatment? Summary answer Statistical differences were seen in biochemical, implantation, clinical, and ongoing pregnancy rate in sperms with high and low DFI scores with autologous and donated oocytes. What is known already Sperm chromatin integrity is associated with embryo development and clinical outcomes of an IVF cycle. DNA Fragmentation Index (DFI) is a diagnostic tool for the detection of spermatozoa with damaged chromosomes in the ejaculate. DNA fragmentation is a significant cause of day 3 embryo arrest and the development of poor-quality embryos. The interventions associated with elevated levels of DFI are ICSI, lifestyle changes and antioxidant therapies. However, the published data regarding the potential of embryo development with high DFI is limited to the sample size thus, encouraging a need for more research in this area. Study design, size, duration The study is population-based and included 768 male patients who underwent DFI score detection across 44 centers at Indira IVF Hospital Private Limited from January 2023 to September 2023. The abstinence period for all the patients was recorded during the DFI detection. After the DFI assessment patients were classified into normal DFI score ( &amp;lt; =15%) (n = 203) and abnormal DFI score (&amp;gt;15%) (n = 565). Participants/materials, setting, methods The retrospective cohort analysis was performed on our databank on the male patients regardless of their age (21-50), ethnicity, and medical history. DFI was calculated using a Sperm Chromatin Dispersion assay. Patients were categorized into two groups: Normal DFI ( &amp;lt; =15%) and Abnormal DFI (&amp;gt;15%). The characteristics of population along with clinical outcomes were evaluated after transferring embryos fertilized with normal DFI sperm and abnormal DFI sperm. The data was analyzed and estimated by SPSS software. Main results and the role of chance Our analyses discovered that embryos fertilized with sperms of normal DFI score have significantly enhanced the likelihood of implantation compared with embryos fertilized with sperms of abnormal DFI (50.27% v/s 44.40%; p = 0.047). Simultaneously, early pregnancy was evaluated by determining the values of beta-HCG in blood, also known as biochemical pregnancy rate (BPR). The embryos fertilized with sperms of normal DFI score significantly enhanced the BPR compared with embryos fertilised with sperms of abnormal DFI (67% v/s 59.1%; p = 0.048). Clinical Pregnancy Rate (CPR) defined as presence of a heartbeat of gestational sac observed during the 6-week scan. Furthermore, it was evaluated that the CPR showed a significant enhancement in embryos fertilized with sperms of normal DFI score in comparison with embryos fertilized with sperms of abnormal DFI (65.5% v/s 57.9%; p = 0.034). However, it was noticed that male DFI score had no impact on the miscarriage rate of embryos inseminated with normal DFI score and abnormal DFI score (7.52% v/s 7.34%; p = 0.687). Moreover, the pregnancy was tracked, and it was revealed that embryos inseminated with sperms of normal DFI score significantly enhanced the ongoing pregnancy till 12 weeks compared with embryos inseminated with sperms of abnormal DFI score (60.6% v/s 53.6%; p = 0.039). Limitations, reasons for caution Embryos are known to have a self-correct mechanism to correct the presence of mosaicism in the embryos. Hence, the oocyte quality also affects the outcomes of the IVF cycle. Therefore, using a high grade of the oocyte for the patients with abnormal DFI score can be useful. Wider implications of the findings Our data suggest that abnormal DFI have an impact on the clinical outcomes of an IVF cycle. Therefore, examining the levels of DNA Fragmentation is utmost imperative for a successful pregnancy. Trial registration number not applicable

P-730 A feasibility study of the impact of the level of exposure to ambient air pollutants on outcomes of assisted reproductive technologies

Abstract Study question What is the impact of air pollution exposure on outcomes for couples having IVF treatment? Summary answer Environmental exposure to high levels of air pollutants may negatively influence IVF outcomes with potential implications for fertility treatment. What is known already Air pollutants are harmful substances in the atmosphere that have adverse effects on human health. Increased levels of air pollutants have been associated with respiratory disorders, cardiovascular disease, and increased morbidity in humans across all age groups. Existing evidence suggests that increased exposure to air pollutants may adversely affect fertility. The relationship between air pollutants and IVF outcomes remains poorly understood. Study design, size, duration This was an ethically approved prospective observational feasibility study conducted over a two-year period. Ten couples due to undergo an IVF cycle were recruited from a fertility centre in the UK. Environmental exposure to ambient air pollutant for particulate matter (PM) 2.5 and 10 micron size was measured using portable AirBeam monitors over the course of two weeks from commencement of the ovarian stimulation regime. Participants/materials, setting, methods Eligible participants were those aged 18-35 years old, undergoing fresh IVF, with a good ovarian reserve and ability to provide informed consent. Data of interest included: BMI, age, parity, ethnicity, smoking status. IVF data included: number of oocytes retrieved, embryo quality and cycle outcomes. AirBeam device data was extracted, analysed and correlated with IVF outcomes. Participants feedback was sought for qualitative input for future studies. Main results and the role of chance Total of 28,269 data points of ambient air pollutant data for PM 2.5 and 10 micron size were recorded amongst the ten participants. One participant was exposed to significantly high air pollutant levels during ovarian stimulation on certain days (PM2.5 - 81.06 µg/m³ and PM10 - 154.48 µg/m³) compared to standard annual air pollution level for the UK of 25 µg/m³ for PM2.5 and 40 µg/m³ for PM10. Two women did not have embryo transfer due to embryo arrest. Of those who had transfer, 50% (n = 4) had positive pregnancy tests however three experienced early pregnancy losses. At the time of writing there was one ongoing single viable pregnancy. On correlation of air quality data with clinical outcomes, the one participant who exhibited fluctuant very high exposure levels to the measured air pollutants, had unexpected low fertilization rate (20%) with IVF and no transfer due to embryo arrest. Of the nine participants returning the questionnaire feedback, all but one rated it acceptable to most acceptable to carry the device around. The main barrier was the size of device and battery life. All respondents were willing to recommend others to participate in a future similar study confirming feasibility to recruit. Limitations, reasons for caution As a feasibility study, with a limited sample size, our findings should be interpreted cautiously. The small cohort limits the generalisability of our results. Further research with larger sample sizes is required to validate our findings. Wider implications of the findings Understanding the impact of air pollutants on IVF outcomes is important for developing evidence-based guidance and interventions to optimise fertility treatment efficacy. Our study demonstrates the feasibility of personal air quality monitoring and emphasises the necessity for larger scale research and the practicality of pollutant exposure measurements in fertility treatments. Trial registration number REC reference: 21/NE/0233 IRAS project ID: 294240. Theramex BIRTH Grant

Research progress on the effect of sperm chromatin integrity on function and its detection methods.

Sperm chromatin not only carries genetic information such as paternal DNA, but also carries structural proteins, epigenetic information, and higher-order chromatin structures (such as matrix attachment regions and telomeres), etc. These information play an important role in embryonic development. This article mainly reviews the effects of these different information carried by sperm chromatin on sperm function and embryonic development and the research progress of related detection methods, in order to provide a theoretical basis and scientific diagnosis and treatment strategies for the etiology screening of clinical infertility, embryo arrest and recurrent miscarriage, so as to improve the pregnancy outcomes of natural conception and assisted reproduction. Keywords: sperm chromatin; epigenetics; sperm DNA damage; sperm function; higher-order chromatin structures.

Inhibition of neddylation disturbs zygotic genome activation through histone modification change and leads to early development arrest in mouse embryos

Post-translational modification and fine-tuned protein turnover are of great importance in mammalian early embryo development. Apart from the classic protein degradation promoting ubiquitination, new forms of ubiquitination-like modification are yet to be fully understood. Here, we demonstrate the function and potential mechanisms of one ubiquitination-like modification, neddylation, in mouse preimplantation embryo development. Treated with specific inhibitors, zygotes showed a dramatically decreased cleavage rate and almost all failed to enter the 4-cell stage. Transcriptional profiling showed genes were differentially expressed in pathways involving cell fate determination and cell differentiation, including several down-regulated zygotic genome activation (ZGA) marker genes. A decreased level of phosphorylated RNA polymerase II was detected, indicating impaired gene transcription inside the embryo cell nucleus. Proteomic data showed that differentially expressed proteins were enriched in histone modifications. We confirmed the lowered in methyltransferase (KMT2D) expression and a decrease in histone H3K4me3. At the same time, acetyltransferase (CBP/p300) reduced, while deacetylase (HDAC6) increased, resulting in an attenuation in histone H3K27ac. Additionally, we observed the up-regulation in YAP1 and RPL13 activities, indicating potential abnormalities in the downstream response of Hippo signaling pathway. In summary, we found that inhibition of neddylation induced epigenetic changes in early embryos and led to abnormalities in related downstream signaling pathways. This study sheds light upon new forms of ubiquitination regulating mammalian embryonic development and may contribute to further investigation of female infertility pathology.

LINE-1 transcription activates long-range gene expression.

Long interspersed nuclear element-1 (LINE-1 or L1) is a retrotransposon group that constitutes 17% of the human genome and shows variable expression across cell types. However, the control of L1 expression and its function in gene regulation are incompletely understood. Here we show that L1 transcription activates long-range gene expression. Genome-wide CRISPR-Cas9 screening using a reporter driven by the L1 5' UTR in human cells identifies functionally diverse genes affecting L1 expression. Unexpectedly, altering L1 expression by knockout of regulatory genes impacts distant gene expression. L1s can physically contact their distal target genes, with these interactions becoming stronger upon L1 activation and weaker when L1 is silenced. Remarkably, L1s contact and activate genes essential for zygotic genome activation (ZGA), and L1 knockdown impairs ZGA, leading to developmental arrest in mouse embryos. These results characterize the regulation and function of L1 in long-range gene activation and reveal its importance in mammalian ZGA.

What importance do donors and recipients attribute to the nuclear DNA-related genetic heritage of oocyte donation?

How do oocyte donors and recipients perceive the genetic link related to the transfer of nuclear DNA between donors and offspring? Whether they are donors or recipients, individuals attach great importance to the transmission of their genetic heritage, since 94.5% would opt for the pronuclear transfer method to preserve this genetic link in the context of oocyte donation. Since 1983, the use of oocyte donation has increased worldwide. Performed in France since the late 1980s and initially offered to women with premature ovarian insufficiency, its indications have progressively expanded and now it is proposed in many indications to prevent the transmission of genetically inherited diseases. This has resulted in an increase in the waiting time for access to oocyte donation due to the difficulty in recruiting oocyte donors in French ART centres. Several articles have discussed how to fairly distribute donor oocytes to couples, but few have interviewed women in the general population to record their feelings about oocyte donation, as either the donor or recipient and the importance given to the genetic link between the oocyte donors and the children born. Mitochondrial replacement therapy (MRT) is a technique originally developed for women at risk of transmitting a mitochondrial DNA mutation. Recently, MRT has been considered for embryo arrest and oocyte rejuvenation as it could help females to reproduce with their own genetic material through the transfer of their oocyte nucleus into a healthy donor oocyte cytoplasm. We conducted an opinion survey from January 2021 to December 2021, during which 1956 women completed the questionnaire. Thirteen participants were excluded from the analysis due to incomplete responses to all the questions. Consequently, 1943 women were included in the study. We specifically developed a questionnaire for this study, which was created and distributed using the Drag'n Survey® software. The questionnaire consisted of 21 items presented alongside a video created with whiteboard animation software. The aim was to analyse whether certain factors, such as age, education level, marital status, number of children, use of ART for pregnancy, video viewing, and knowledge about oocyte donation, were associated with feelings towards oocyte donation, by using a univariate conditional logistic regression model. This statistical method was also used to assess whether women would be more inclined to consider oocyte donation with the pronuclear transfer technique rather than the whole oocyte donation. All parameters found to be statistically significant in the univariate analysis were subsequently tested in a multivariate model using logistic regression. Most women were concerned about the biological genetic contribution of the donated oocyte (94.8%). The most common reason for a women's reluctance to donate their oocytes was their unwillingness to pass on their genetic material (33.3%). Nearly 70% of women who were initially hesitant to donate their oocytes indicated that they would reconsider their decision if the oocyte donation was conducted using donated cytoplasm and the pronuclear transfer technique. Concomitantly, >75% of the respondents mentioned that it would be easier to receive a cytoplasm donation. The largest proportion of the population surveyed (94.5%) expressed their support for its legalization. In this study, a substantial portion of the responses came from individuals with medical or paramedical backgrounds, potentially introducing a recruitment bias among potential donors. The rate of missing responses to the question regarding the desire to become an oocyte donor was 13.6%, while the question about becoming an oocyte cytoplasm donor had a missing response rate of 23%. These missing responses may introduce a bias in the interpretation of the data. This study was the first to demonstrate that, for the French population studied, the combination of oocyte cytoplasm donation with pronuclear transfer could offer a promising approach to enhance the acceptance of oocyte donation for both the donor and the recipient. No external funding was used for this study. The authors have no conflicts of interest. N/A.

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.

A maternal-effect Padi6 variant causes nuclear and cytoplasmic abnormalities in oocytes, as well as failure of epigenetic reprogramming and zygotic genome activation in embryos.

Maternal inactivation of genes encoding components of the subcortical maternal complex (SCMC) and its associated member, PADI6, generally results in early embryo lethality. In humans, SCMC gene variants were found in the healthy mothers of children affected by multilocus imprinting disturbances (MLID). However, how the SCMC controls the DNA methylation required to regulate imprinting remains poorly defined. We generated a mouse line carrying a Padi6 missense variant that was identified in a family with Beckwith-Wiedemann syndrome and MLID. If homozygous in female mice, this variant resulted in interruption of embryo development at the two-cell stage. Single-cell multiomic analyses demonstrated defective maturation of Padi6 mutant oocytes and incomplete DNA demethylation, down-regulation of zygotic genome activation (ZGA) genes, up-regulation of maternal decay genes, and developmental delay in two-cell embryos developing from Padi6 mutant oocytes but little effect on genomic imprinting. Western blotting and immunofluorescence analyses showed reduced levels of UHRF1 in oocytes and abnormal localization of DNMT1 and UHRF1 in both oocytes and zygotes. Treatment with 5-azacytidine reverted DNA hypermethylation but did not rescue the developmental arrest of mutant embryos. Taken together, this study demonstrates that PADI6 controls both nuclear and cytoplasmic oocyte processes that are necessary for preimplantation epigenetic reprogramming and ZGA.

Endogenous retroviruses shape pluripotency specification in mouse embryos.

The smooth and precise transition from totipotency to pluripotency is a key process in embryonic development, generating pluripotent stem cells capable of forming all cell types. While endogenous retroviruses (ERVs) are essential for early development, their precise roles in this transition remains mysterious. Using cutting-edge genetic and biochemical techniques in mice, we identify MERVL-gag, a retroviral protein, as a crucial modulator of pluripotent factors OCT4 and SOX2 during lineage specification. MERVL-gag tightly operates with URI, a prefoldin protein that concurs with pluripotency bias in mouse blastomeres, and which is indeed required for totipotency-to-pluripotency transition. Accordingly, URI loss promotes a stable totipotent-like state and embryo arrest at 2C stage. Mechanistically, URI binds and shields OCT4 and SOX2 from proteasome degradation, while MERVL-gag displaces URI from pluripotent factor interaction, causing their degradation. Our findings reveal the symbiotic coevolution of ERVs with their host cells to ensure the smooth and timely progression of early embryo development.

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.

Engineering TALE-linked deaminases to facilitate precision adenine base editing in mitochondrial DNA

DddA-derived cytosine base editors (DdCBEs) and transcription activator-like effector (TALE)-linked deaminases (TALEDs) catalyze targeted base editing of mitochondrial DNA (mtDNA) in eukaryotic cells, a method useful for modeling of mitochondrial genetic disorders and developing novel therapeutic modalities. Here, we report that A-to-G-editing TALEDs but not C-to-T-editing DdCBEs induce tens of thousands of transcriptome-wide off-target edits in human cells. To avoid these unwanted RNA edits, we engineered the substrate-binding site in TadA8e, the deoxy-adenine deaminase in TALEDs, and created TALED variants with fine-tuned deaminase activity. Our engineered TALED variants not only reduced RNA off-target edits by>99% but also minimized off-target mtDNA mutations and bystander edits at a target site. Unlike wild-type versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse embryos. As a result, we obtained mice with pathogenic mtDNA mutations, associated with Leigh syndrome, which showed reduced heart rates.

#38 : Maternal Age Affects Morula Compaction and Cell Polarity of Human Embryos

Background and Aims: With the exception of chromosome segregation errors, the molecular and biochemical mechanisms responsible for advanced maternal age (AMA)-related reduced embryo developmental competence remain unclear. In women of AMA, the blastocyst formation rate has been reported to decline due to an increase in embryo arrest at the morula stage. Therefore, we aimed to reveal the impact of AMA on cellular events during compaction, as assessed using morphokinetic analysis. We also assessed the possible influence of AMA on the expression of cell polarity markers, yes-associated protein (YAP) and protein kinase C-[Formula: see text] (PKC[Formula: see text]), during blastomere compaction. Method: We retrospectively analysed 2,058 fertilized oocytes, which were stratified by maternal age according to the Society for Assisted Reproductive Technology classification (&lt; 35, 35-37, 38–40, 41–42, and &gt; 42 years). The effects of AMA were assessed in relation to embryo morphokinetics and morphological alterations. Furthermore, 20 voluntarily donated human embryos were used for immunofluorescence staining of YAP and PKC-[Formula: see text]. Results: Delayed embryonic compaction and an increased number of extruded blastomeres post compaction were observed in the &gt; 42-year group. The time interval from the eight-cell stage to compaction initiation was prolonged with increasing age. The number of YAP-positive cells in the outer cells of the morulae was significantly higher in the young age group than in the AMA groups. The PKC[Formula: see text] fluorescence intensity in the cortical region of the morulae was significantly lower in the AMA groups than in the young age groups. Conclusion: Delayed compaction and blastomere extrusion occur more frequently in embryos from women of AMA due to insufficient regulation of cell polarity, which may lead to morula arrest. Further research is required to reveal the impacts of AMA on the mechanism underlying the expression of cell polarity factors and develop methods to avoid AMA-associated morula arrest.

Approaches to Help Older Women in ART

Ancient Chinese emperors sent messengers to the whole country and explored an elixir of life. Determent of aging has been an ultimate hope for humans for a long period. In the modern era, aging become a hot topic to be solved according to longevity. Equality of both sexes in society contributed to the late marriage and start of making children. There happen adverse effects to being conceived in the aged ovaries. The quality and quantity become impaired by the ovarian aging process. In the artificial reproductive treatment, the obtained number of oocytes in ovarian hyperstimulation diminishes in aged women. The aging impairs quality both in the nucleus and cytoplasm. Chromosomal and genetic abnormalities increase lower implantation and higher abortion. In aged women, PGT-A may be effective to effective treatment. On the other hand, aging affects organelles in the cytoplasm of oocytes by protein metabolic dysregulations. Especially it induces mitochondria dysfunction. Mitochondria are called a “powerhouse” and have an essential role in oogenesis and embryogenesis. There exist 200,000-40,000 mitochondria DNA copies in the mature oocytes. Mitochondria dysfunction causes maturation arrest of oocytes, fertilization failure, late cleavage, and fragmented embryos. We have two strategies to solve this problem by enhancement of mitochondrial function. One is the holistic approach. Reactive oxygen species (ROS) or free radicals impair mitochondrial function. Also, mitochondria themselves are known to produce ROS. We measure Diacron-Reactive Oxygen Metabolites (d-Rom) and biological antioxidant potential (BAP) that are predictors of systemic ROS level and antioxidant competence, respectively. Another method is a cell-engineering approach. We have conducted autologous mitochondria transfer into mature oocytes. The quality of the oocytes and embryos were dramatically improved by this method, and we obtained 13 healthy children.

Meiotic and mitotic aneuploidies drive arrest of in vitro fertilized human preimplantation embryos

BackgroundThe high incidence of aneuploidy in early human development, arising either from errors in meiosis or postzygotic mitosis, is the primary cause of pregnancy loss, miscarriage, and stillbirth following natural conception as well as in vitro fertilization (IVF). Preimplantation genetic testing for aneuploidy (PGT-A) has confirmed the prevalence of meiotic and mitotic aneuploidies among blastocyst-stage IVF embryos that are candidates for transfer. However, only about half of normally fertilized embryos develop to the blastocyst stage in vitro, while the others arrest at cleavage to late morula or early blastocyst stages.MethodsTo achieve a more complete view of the impacts of aneuploidy, we applied low-coverage sequencing-based PGT-A to a large series (n = 909) of arrested embryos and trophectoderm biopsies. We then correlated observed aneuploidies with abnormalities of the first two cleavage divisions using time-lapse imaging (n = 843).ResultsThe combined incidence of meiotic and mitotic aneuploidies was strongly associated with blastocyst morphological grading, with the proportion ranging from 20 to 90% for the highest to lowest grades, respectively. In contrast, the incidence of aneuploidy among arrested embryos was exceptionally high (94%), dominated by mitotic aneuploidies affecting multiple chromosomes. In turn, these mitotic aneuploidies were strongly associated with abnormal cleavage divisions, such that 51% of abnormally dividing embryos possessed mitotic aneuploidies compared to only 23% of normally dividing embryos.ConclusionsWe conclude that the combination of meiotic and mitotic aneuploidies drives arrest of human embryos in vitro, as development increasingly relies on embryonic gene expression at the blastocyst stage.