Cell Stage Research Articles

P-112 Magnetic resonance spectroscopy at the micro scale (Micro-MRS) for non-invasively assessing individual mammalian embryos’ metabolism

Abstract Study question Can Micro-Magnetic Resonance Spectroscopy (micro-MRS) reveal the metabolic fingerprint of mammalian pre-implantation embryo? Summary answer Our research demonstrates that rapid, non-invasive analysis of embryos using Micro-Magnetic Resonance Spectroscopy (micro-MRS) can effectively uncover the significance of embryonic metabolites during development. What is known already Non-invasive selection of optimal embryos for transfer is a crucial challenge in assisted reproduction for both humans and animals. Magnetic Resonance Spectroscopy (MRS) emerges as a promising solution due to its chemical sensitivity and non-invasive nature. Despite its effectiveness in analysing large organisms, MRS faces obstacles in small sample handling and sensitivity, particularly with embryos, limiting its clinical and research applications. Our study overcomes these challenges using innovative microchip-based MRS sensors. This approach enabled successful MRS at the embryo scale, revealing metabolic biomarker linked to developmental potential in pre-implantation bovine embryos and opening new possibilities in assisted reproduction technology. Study design, size, duration Multiple MRS measuring sessions were conducted on two distinct embryo cohorts. The first cohort involved naturally arrested IVP embryos at early (8-cell) and late (morula/blastocyst) stages, establishing metabolic biomarkers in mammalian pre-implantation embryos. The second cohort involved 8-cell embryos which were further cultured over 10 days post-MRS with imaging at specific intervals (3 hours post-thawing, days 5, 8, and 10). This step was crucial to back-correlate biomarkers with in-vitro development based on bright field microscopy. Participants/materials, setting, methods Both studies involved vitrified samples that were subjected to a 50-minute MRS measurement following a 3-hour post-warming culture period. Single bovine embryo analysis was performed using our 1nL micro-MRS probe. Pre-implantation bovine embryos were obtained through IVF (n = 32 samples for cohort one and, n = 61 for cohort two). Post-MRS, the embryos were cultured in equilibrated IVC medium in a humidified incubator with 5% CO2 and 6% O2 at 38 °C. Statistical significance was set at p < 0.05. Main results and the role of chance Micro-Magnetic Resonance Spectroscopy (Micro-MRS) was utilized to identify metabolic markers in single bovine embryos, providing insights into their pre-implantation developmental stages. The spectroscopy data revealed distinct peak regions in the spectra of both cohorts, indicative of essential life-supporting fatty acids. Notably, up to six lipid markers were identified in the first cohort, five of which displayed statistically significant differences (p < 0.05). However, the majority of these biomarkers in the first cohort did not show significant expression differences in the groups of embryos assigned to the second cohort. An exception was noted in the case of the Principal Lipid Component (PLC) marker, which was significantly less prevalent in the arrested embryos than in those progressing to the blastocyst stage (p = 0.0016). Further data analysis based on AI-classification algorithms revealed up to 80% precision in predicting the development to the blastocyst stage solely based on their metabolic profiles at the 8-cell stage. Limitations, reasons for caution Magnetic Resonance Spectroscopy (MRS) shows promise for clinical and research use, requiring reliable signal detection from cellular components. While suitable for research, clinical deployment demands further safety assessments and protocol refinement. Additionally, larger sample sizes and data on post-MRS implantation and pregnancy rates are needed to validate AI-classification algorithms. Wider implications of the findings Micro-Magnetic Resonance Spectroscopy (Micro-MRS) holds potential as a reliable and non-invasive method for embryo screening prior to transfer. This innovative tool can shed light on the role of embryonic metabolites during development making Micro-MRS a key instrument in advancing both embryology research and human assisted reproduction techniques. Trial registration number not applicable

P-798 A feasible clinical model to generate genotyped oocytes for infertile women

Abstract Study question Is it possible to generate genotyped oocytes by utilizing accessible somatic cells in a somatic cell nuclear transfer model? Summary answer Genotyped oocytes generated through haploidization of endometrial somatic cells exhibited reliably completed embryo development and yielded healthy offspring. What is known already The generation of genotyped oocytes through neogametogensis or somatic cell nuclear transfer (SCNT) would be the ultimate solution to treat female infertility due to primary ovarian insufficiency or agonadism. Among the various approaches, somatic cell haploidization appears more feasible (Lee, et al 2022). Unfortunately, this approach has only been successful in a murine model using cumulus cells. However, this donor cell type is not ideal when translated into clinical setting, due to the absence of cumulus cells in patients who are void of oocytes, which arguably is the patient population that would potentially benefit the most from this technique. Study design, size, duration In a mouse SCNT model, endometrial stromal cells (EMSCs) were injected into enucleated oocytes. Cumulus cells (CCs) were used as another source of nuclear donor. Intact MII oocytes were used as control. Post-SCNT oocytes and controls were inseminated by spermatozoa from B6-EGFP mice to monitor paternal genome presence through embryo development and offspring. Embryos were cultured in a time-lapse microscope. To prove gamete competence, blastocysts were transferred into pseudo-pregnant recipients and offspring health was assessed. Participants/materials, setting, methods Endometrial stromal cells were cultured for 4 days and exposed to aphidicolin overnight to synchronize at G1 phase of cell cycle. Cumulus cells were collected after denudation. Oocytes from B6D2F1 mice were enucleated under polarized light microscope. SCNT was performed with CCs or EMSCs by lipofectamine-mediate fusion. Oocytes were inseminated with spermatozoa from GFP-positive transgenic mice. Embryos morphokinesis were assessed by a time-lapse system. Blastocysts were transferred into 2.5-dpc surrogate CD1 mice. Main results and the role of chance Control oocytes(n = 75) fertilized at 93.3%(70/75) and progressed to 2-cell stage at 93.3%(70/75), 8-cell at 93.3%(70/75) and blastocyst at 89.3%(67/75). The control embryos started syngamy at 14.4±1h, reaching the 4-cell stage at 37.3±3h and blastulation at 80.6±8h. A total of 484 oocytes were enucleated, 470(97.1%) survived and underwent SCNT using CCs(n = 85) or EMSCs(n = 385). Fusion rate was 95.5% for CC with a haploidization rate of 70.9%(56/79) that led to a fertilization rate of 64.3%(36/56). The CC cohort developed to 2-cell, 8-cell, and morula at 47.4%, 35.4%, and 22.8%, with a delay in development at syngamy at 19.0±8h(P<0.0001). In the EMSC cohort, fusion rate was 96.0%(361/376). Haploidization rate was 65.4%(236/361), comparable to the CC cohort(P = 0.35). Fertilization was lower in the EMSC cohort at 47.0%(P<0.01) compared to the CC cohort. Subsequently, EMSC cohort yielded similar cleavage rates to 2-cell stage at 30.7%, 8-cell stage at 29.4%, and morula stage at 28.5%. The blastocyst rate was higher in the EMSC cohort at 28.0%(P<0.05). The EMSC cohort yielded similar developmental timing comparing to controls from pronuclear appearance to 4-cell cleavage but a delay manifested afterward at 37.3±3h(P<0.0001). In the EMSC cohort, 42 blastocysts were transferred, yielding 3 healthy pups(2 male,1 female). Limitations, reasons for caution While we generated consistent haploidization and satisfactory fertilization, full pre-implantation development remained low. Even more limited was the live birth rate, which probably related to subtle factors such as an imprinting issue due to insufficient reprogramming of the somatic cell donor. Wider implications of the findings The SCNT model described to generate genotyped oocytes, although still with limited efficiency, represents a feasible reproductive option for women with agonadism and compromised ovarian reserve. This model is still a better approach to complete neogametogenesis. Trial registration number not applicable

P-805 Sperm and embryo genome editing by CRISPR-Cas9 in a mouse model

Abstract Study question We wonder whether it is possible to edit a specific gene in individual mammalian spermatozoa as well as embryo by using CRISPR-Cas9? Summary answer We were able to successfully edit the coat pigment gene on individual spermatozoa as well as embryos by injecting CRISPR-Cas9 during Piezo-ICSI. What is known already Earlier investigations into embryo genome editing with CRISPR-Cas9 were conducted during the S-phase or zygote stage, encountering obstacles such as mosaicism and loss of heterozygosity. To overcome these challenges, performing genome editing at the gamete level is considered more favorable. While it may be achievable in oocytes, sperm genome editing can be ambitious due to the tightly supercoiled DNA around the protamine cores. In initial attempts, simple permeabilization of the sperm membrane allowed CRISPR-Cas9 penetration into the cell; however, the attempts at genomic editing proved to be unsuccessful. Study design, size, duration In the past several months, oocytes were divided into two groups: half of them were used to perform embryo genome editing, whilst the other half was used to edit exclusively the sperm genome through oocyte-mediated sperm decondensation (OMSD), where a single spermatozoon was injected into an enucleated oocyte and used the ooplasmic machinery for DNA decondensation and editing. In both models, CRISPR-Cas9 was used to knock out Tyr gene to create an albino phenotype. Participants/materials, setting, methods B6D2F1 mice were used to retrieve oocytes and spermatozoa. A cohort of oocytes used for the OSMD approach were enucleated, whilst the others were maintained with an intact nucleus. All oocytes, enucleated or not, were injected with a single spermatozoon with Tyr-sgRNA and Cas9 protein. Embryos were cultured until the 8-cell stage and processed for T7E1 cleavage analysis. Genome editing efficiency was calculated following the manufacturer protocol. Main results and the role of chance Of the 40 oocytes used for the study, 20 intact oocytes were used to perform embryo genome editing in the standard fashion, while 20 were enucleated for OMSD experiments. After undergoing Piezo-ICSI with CRISPR-Cas9 solution, 90.0% (18/20) of intact oocytes fertilized. After 48 hours in culture, diploid embryos reached the 8-cell stage at 88.9% (16/18). The 584-bp region of the target site of extracted DNA was amplified and gene modification of the edited diploid embryo cohort was confirmed in 87.5% (14/16). Compared to the OMSD cohort, enucleated oocytes fertilized at 85.0% (17/20) while cleavage rate was lower at 64.7% (11/17) and sperm genome modification at 81.8% (9/11). Both diploid embryos and haploid androgenetic embryos evidence an editing efficiency of 36.8%. Therefore, we verified that genome editing on embryos and sperm achieved the same editing yield. Limitations, reasons for caution While the experimental observations are still limited, it is important to note that these results in the embryo model may be underestimated by the T7E1 cleavage assay that only recognize heterozygous modification. The technique needs further refinement to optimize targeting and editing efficiency. Wider implications of the findings Sperm genome decondensation through an OMSD approach allows CRISPR-Cas9 to edit the sperm genome in order to correct eventual pathogenic mutations present in the gamete. Moreover, these would be the only method to absolutely ensure uniform generation of resulting embryos. Trial registration number not applicable

P-175 Morphokinetic growth variance score as a tool to identify euploid embryos with the highest live birth potential

Abstract Study question Is the longitudinal growth pattern of euploid embryos associated with live birth rate? Summary answer Embryos with lower growth variance score (GVS), i.e., pointing towards a steadier growth during time-lapse monitoring (TLM), have higher live birth rates. What is known already Dynamic TLM imaging has been increasingly adopted by fertility clinics as an attempt to improve the ability of selecting embryos with the highest potential for implantation. Many authors have identified and incorporated markers of embryonic morphokinetics into decision algorithms for embryo (de)selection. However, longitudinal changes during this temporal process, and the impact of such changes on embryonic competence remains unknown. Aiming to model the reference ranges of euploid embryonic growth as a single growth trajectory might provide an additive value to blastocyst morphological grade to identify highly competent euploid embryos. Study design, size, duration This observational cohort study was performed in a tertiary referral IVF centre between October 2017 and June 2021, including a total of 340 single euploid autologous frozen embryo transfers. Pre-implantation genetic diagnosis for aneuploidy (PGT-A) performed by next generation sequency (NGS). Pregnancy outcomes were assessed as live birth, pregnancy loss or not pregnant. Participants/materials, setting, methods Standard morphokinetic parameters (tPB2 to tEB) of all euploid embryos cultured in TLM system were used to develop the reference ranges for the growth variance score (GVS) model. Nuclear errors at two- and four-cell stages, and blastocyst grade before trophectoderm (TE)-biopsy were also annotated. GVS of euploid embryos was evaluated for its association with pregnancy outcomes, and to its predictive ability to live birth potential over TE and inner cell mass (ICM) grade morphology. Main results and the role of chance A total of 189 (55.6%) euploid transfers resulted in a live birth, 48 (14.1%) in pregnancy loss and 103 (30. 3%) did not result in pregnancy. The median time for euploid embryo blastulation was 109.9 hours (95% CI:98.8-121.0 hours). Lower quality ICM (OR: 0.13, 95% CI: 0.05-0.33, P < 0.001) and TE (OR: 0.46, 95% CI:0.22-0.94, P = 0.034), multinucleation at 4-cell stage (OR: 0.042, 95% CI:0.21-0.84, P = 0.016) and higher GVS (OR: 0.59, 95% CI: 0.45-0.77, P < 0.001), significantly decreased the odds of live birth. In the multivariable analysis, only GVS (OR: 0.62, 95% CI: 0.46-0.84, P = 0.002) and lower ICM quality (OR: 0.21, 95% CI: 0.06-0.65, P = 0.009) had a significant effect on live birth. There was a significant trend towards higher live birth rates with lower GVS among embryos of similar ICM grades: 85% vs 65% live births in low vs. high GVS for embryos with ICM type A, 76% vs. 48% for ICM type B, 67% vs. 21% for ICM type C, respectively (P for trend <0.001). Addition of the GVS to embryo morphology model (ICM, TE, biopsy day) significantly improved the model’s ability to predict live birth (AUC: 0.67 vs. 0.62, P = 0.015). Limitations, reasons for caution The exclusion of IVF cases limits the utility of the model to ICSI-derived embryos. The utility of GVS should be replicated further in external cohorts to confirm its reproducibility in other clinical and laboratory settings. Wider implications of the findings By capturing the dynamic growth pattern of embryos, GVS may serve as a valuable tool in prioritizing PGT-A tested embryos for transfer. Its impact was even more prominent for selecting suboptimal blastocysts, which is a more challenging subset of embryos to choose due to their likelihood of reduced viability. Trial registration number Not Applicable

P-191 Pronuclear transfer overcomes embryonic developmental arrest in Padi6-deficient mice

Abstract Study question Can pronuclear transfer (PNT) overcome embryonic developmental arrest (EDA) observed in the created Padi6-/- mouse model? Summary answer Applying PNT significantly improved the blastocyst rate of Padi6-/- zygotes, indicating its potential in overcoming female-related infertility. What is known already Various components of the subcortical maternal complex (SCMC), including peptidyl arginine deiminase 6 (PADI6), play a crucial role in early embryonic development and female fertility. Recent studies have revealed that mutations in PADI6 can result in EDA and thus female infertility, which is hard to overcome by current available treatment methods. The PNT technique, designed to replace compromised cytoplasm with a healthy counterpart, has been proposed to overcome specific female-related infertility indications. The Padi6-/- mouse displaying EDA, offers a valuable model to investigate the efficacy of PNT to overcome female-related infertility. Study design, size, duration From April 2022 to December 2023, an experimental study was conducted with CRISPR/Cas9 created Padi6 knockout mice. For fertility evaluation, Padi6-/- and Padi6+/- females were paired with wildtype males. Next, 30 wildtype and 25 Padi6-/- oocytes were collected for parthenogenetic activation. A total of 81, 54 and 145 zygotes were collected from wildtype, Padi6+/- and Padi6-/- females, respectively, for evaluating embryonic development and the effectiveness of PNT in overcoming EDA. Participants/materials, setting, methods The C57BL/6J Padi6-/- mouse model was established by introducing Padi6 c.1577_1578insT with CRISPR/Cas9. Female mice aged 6-12 weeks underwent ovarian hyperstimulation and mated with wildtype males. Oocytes were parthenogenetically activated with SrCl2 in presence of Cytochalasin D, and checked for 2-cell and blastocyst rates. PNT treatment was applied by transferring the 2PN from Padi6-/- to wildtype enucleated zygotes using electrofusion, and the efficiency was evaluated in terms of embryonic developmental potential. Main results and the role of chance After mating Padi6-/- female mice with wild-type males, no offspring could be obtained. In contrast, Padi6+/- females exhibited normal fertility, yielding an average of 7.3 offspring per litter. In addition, Padi6-/- males are also fertile. Twenty-eight wildtype oocytes (n = 30) survived parthenogenetic activation, with 85.71% (24/28) reaching the 2-cell stage, and 75% (18/24) subsequently forming blastocysts. In contrast, all Padi6-/- oocytes (n = 25) survived after activation, but only 20% (5/25) reached the 2-cell stage (P < 0.0001), with none progressing to blastocyst (P = 0.0039). Regarding zygote development, 82.14% (46/56) of wildtype zygotes and 98.14% (53/54) of Padi6+/-embryos reached the 2-cell stage, showing blastocyst rates of 95.65% (44/46) and 100% (53/53) respectively. For zygotes obtained from Padi6-/- females, 72.50% (87/120) of them progressed to the 2-cell stage (P = 0.1908, vs wildtype), but only 4.60% (4/87) reached blastocysts (P< 0.0001, vs wildtype), exhibiting the EDA phenotype. Following PNT to wildtype cytoplasm (n = 25), 23 reconstructed PNT zygotes were obtained. Of these, 91.30% (21/23) reached the 2-cell stage (P = 0.4924, vs wildtype), and 85,71% (18/21) developed to the blastocyst stage (P = 0.3151, vs wildtype), which is equivalent to the wildtype zygotes. Limitations, reasons for caution This study is limited by the small sample size of the PNT, which will still be extended. Next to the blastocyst rate, additional assessment methods such as multi-omics analyses will be employed to assess the efficiency and safety of PNT treatment. Wider implications of the findings This study provides further insight into the potential of the nuclear transfer technology for female-related infertility issues due to cytoplasmic incompetence. For couples experiencing recurrent EDA after ICSI due to inferior oocyte quality, PNT still offers them the possibility of having genetically related children. Trial registration number Not applicable

P-526 Identification of mouse cumulus cell-specific miRNAs and their role in the acquisition of the oocyte developmental competence

Abstract Study question Do cumulus cells (CCs), during mouse germinal vesicle (GV)-to-metaphase II (MII) transition, produce miRNAs with a role in the acquisition of the oocyte developmental competence? Summary answer We identified a group of 74 miRNAs with a known role in key processes of oocytes maturation and developmental competence. What is known already We recently developed a co-culture platform whereby CC-free, denuded, GV oocytes (DOs) were matured to MII upon a feeder layer (FL) of CCs derived either from developmentally competent (FL-SN-CCs) or incompetent (FL-NSN-CCs) oocytes. When cultured upon FL-SN-CCs, DOs displayed a developmental competence equal to that showed by control cumulus-enclosed oocytes (COCs). Instead, when cultured upon FL-NSN-CCs, all DOs arrested at the 2-cell stage. We found that CCs released into the medium large amounts of extracellular vesicles (EVs), whose content analysis by NGS led to the identification of candidate miRNAs. Study design, size, duration Five-eight-week-old CD1 mouse females (Charles River) were injected with 10 I.U. Pregnant Mare Serum Gonadotropin (PMSG). After 48 hr, the ovaries were isolated and their surface punctured with a thin sterile needle to collect fully-grown antral oocytes which were separated from the surrounding CCs. Eight-ten DOs were cultured upon FL-SN-CCs or FL-NSN-CCs for 15 hr in 50 µl exosome-free alpha-MEM Glutamax medium for EVs production. Experiments were performed in triplicates. Participants/materials, setting, methods EVs were collected and processed for EVs characterisation by Amnis-Imaging-flow-cytometer and transmission electron microscopy (TEM), or for RNA extraction (miRNeasy Micro Kit, Qiagen), libraries production (Small RNA-Seq Library Prep kit, Lexogen) and NGS (Illumina Genome Analyzer and the NextSeq 500/550 High Output v2.5). To identify miRNA’s target genes, we used “mirPath v.3” server of the DIANA tools website (https://dianalab.e-ce.uth.gr/html/mirpathv3/index.php?r=mirpath). Then, STRING and KEGG were used to highlight the pathways in which these genes are involved. Main results and the role of chance Imaging flow cytometry recorded an amount of released EVs in both FL-SN-CCs or FL-NSN-CCs in the mean range of 5-5.9 x 10(8) particles/ml (p > 0.05), with >96% positive to CD9 exosome surface marker. TEM analysis showed an EVs size in diameter comprised between 18.5-54.9 nm. By comparing FL-SN-CCs vs. FL-NSN-CCs, NGS of EVs’ miRNAs content displayed 74 differentially expressed miRNAs, 43 up- and 31 down-regulated. Of them, 7 resulted involved in the regulation of 71 target genes with a known function in meiosis resumption (N = 24), follicle growth (N = 23), fertilisation (N = 1) and in the acquisition of the oocyte’s developmental competence (N = 23). Limitations, reasons for caution We used an in vitro platform to mimic the cumulus-oocyte-complex environment and, thus, the artificial set-up may differ from that of the natural condition. Also, our hypothesis on the role of these miRNAs, needs to be further confirmed by functional assays (e.g., miRNA inactivation). Wider implications of the findings Overall, our results indicate CC-derived miRNAs as candidates of the CC-to-oocyte bidirectional communication and suggest a group of miRNAs as potential regulative molecules in the acquisition of the oocyte developmental competence. Trial registration number NOT APPLICABLE

P-200 Vitrification induces a deviation in cleavage morphokinetic patterns and transcriptomic signatures along mouse embryo development

Abstract Study question Are there cellular, molecular and/or epigenetic risks associated with cryopreservation that may affect embryo development and compromise the health of future newborns? Summary answer Cryopreservation significantly impacted mice pre-implantation embryo morphokinetics, altered transcriptomic profiles, and cell lineage distribution. However, global DNA methylation remained unaffected by cryopreservation. What is known already Cryopreservation is currently widely used in assisted reproduction. However, epidemiological studies suggest a negative impact in the long-term health of children born after frozen-embryo transfer (FET). Recent population-based register studies have reported: 1) an increased risk of infant mortality from birth up to 1 year of life in singletons conceived using cryopreserved embryos vs natural conceptions; 2) high birth weights for gestational age associated with FET; 3) an increased risk of obstetrical complications in singletons conceived using cryopreserved embryos; 4) a higher incidence of childhood cancer and imprinting disorders associated with FET. Study design, size, duration F1 hybrid (B6/CBA) mice zygotes were collected following uterus dissection (n = 200 embryos/collection), and half of those were vitrified for comparison. Each embryo’s morphokinetics was assessed through time-lapse imaging using GERI, at 37 °C, 6% CO2, 5% O2, up to 105 hours. Extended culture beyond blastocyst stage was also performed. Fresh and cryopreserved blastocysts at E5.0 were cultured up to E9.0-E10 in static conditions (IVC1 and IVC2 medium). Participants/materials, setting, methods Fresh and cryopreserved embryos at several developmental stages were used for: 1) immunofluorescence staining, 2) gene expression analysis through RT-qPCR and 3) single-cell RNAseq. Global methylation activity, cell lineage distribution and single-cell transcriptome were assessed using one of the previously described methodologies. Main results and the role of chance The morphokinetics of preimplantation embryos were examined by tracking the time required to reach 4-cell, 8-cell, compactation, morula, full blastocyst, and expanded blastocyst stage. A comprehensive analysis revealed notable delays in the developmental rhythm of cryopreserved embryos compared to fresh embryos (p < 0.01). Applying single-cell RNA sequencing (scRNAseq), transcriptomic profiles were generated for fresh and cryopreserved embryos, covering the zygote to blastocyst stages. Notably, distinct transcriptomic signatures for fresh and frozen embryos emerged within separate clusters at different developmental stages. These findings suggest a potential functional impact of cryopreservation on embryo development. To complement this, gene expression analysis of DNA methyltransferases and MAT enzymes was conducted through RT-qPCR at 8-cell and blastocyst stages. No significant differences were detected between fresh and cryopreserved conditions. Furthermore, immunofluorescence staining of nuclear 5mC and DNMT1 in 8-cell embryos and blastocysts demonstrated similar levels between fresh and cryopreserved embryos. Additionally, differential staining of embryonic and extraembryonic cells (OCT4, GATA6 and GATA3) in extended embryos (E9 and E10) revealed distinct cell distribution patterns between fresh and cryopreserved conditions. Limitations, reasons for caution Our research, conducted using a mouse experimental model, has limited applicability to humans due to considerable species differences in embryonic development. Additional research is needed to validate our findings and establish their relevance to human embryos, facilitating translational approaches. Wider implications of the findings Our study highlights a noticeable functional impact of vitrification on mouse embryos. Given the global incorporation of this method in fertility clinics and its potential association with increased disease risks, our findings emphasize the need for increased research to reduce uncertainties regarding the health of children born from cryopreservation methods. Trial registration number Not applicable

P-797 Assessment of embryo development following fertilization with de novo male gametes generated in a three-dimensional culture

Abstract Study question Can de novo male gamete from embryonic stem cell contribute to the full pre-implantation development? Summary answer Gametes generated after 29 days of differentiation in our three-dimensional (3D) culture system can support consistent fertilization and normal embryo development to the blastocyst stage. What is known already In regenerative medicine, several 3D culture systems have been proposed and are capable of producing functional tissue implants. In reproductive biology, recent studies have reported preliminary success in generating functional de novo gametes through soft-agar culture and testicular organoids from mouse embryonic stem cells. However, in order to achieve successful fertilization and satisfactory embryo development, an heterologous transplantation in a host seminiferous tubule is required to allow proper spermiogenesis to obtain functional gametes. Here we attempt to perform neogametogensis in a novel 3D niche to generate de novo gamete ready to be used for ICSI. Study design, size, duration Mouse ESCs were first cultured on a gelatin coated 6-well plate with fibroblasts in monolayer and later spherified using sodium alginate. Spheres were submerged in specifically designed conditioned media to encourage differentiation of the mESCs into germ-like cells. Over the course of differentiation, cells were assessed for germ cell differentiation biomarkers. Considering that a normal spermatogenesis occur in 30 days, utilization of the de novo gametes was planned for day-15, 22, 29 and 36. Participants/materials, setting, methods Mouse ESCs were differentiated by submerging the spheres in EpiLC medium containing Activin A, bFGF and KSR for 3 days followed by PGCLC medium containing BMP4, LIF, SCF and EGF for up to 36 days. Differentiation was assessed for markers DAZL (spermatogonium), VASA (spermatocyte), BOULE (post-meiotic stage) and acrosin (spermatid). Differentiated cells were then injected into oocytes and activated by calcium ionophore. Embryo development was monitored in a time-lapse incubator. Main results and the role of chance The evaluation of germline-specific markers through immunofluorescence revealed consistent levels of Vasa expression, a ubiquitous germline marker, with percentages at 15% on day-3, 18% on day-10, 17% on day-15, 19% on day-22, 20% on day-29, and 13% on day-36. Dazl, a specific marker for spermatogonia, exhibited a peak expression of 45% on day-10 in spherified cells. Boule, expressed by secondary spermatocytes, reached its highest level of 10% on day-15 but progressively decreased to 8% on day-22. Acrosin, a marker for spermatids, initially manifested at day-10 at 2% positivity and increased to 5% on day-15, reaching 7% on day-22, and peaked at 20% on day-29. According to spermatogenic marker expression, the neogametes follows a maturational profile similar to in vivo mouse spermatogenesis. To prove neogametes competence, we inject the neogametes into mature oocytes. The control ICSI cohort achieved 89.2% fertilization and 77.8% blastocyst rates. Neogametes generated on day-15, 22, 29 and 36, achieved fertilization rates of 35.0%, 61.1%, 81.8% and 75.0%, respectively, and yielded blastulation rates of 5.0%, 16.7%, 36.4% and 8.3%, respectively. Resulting embryos had morphokinesis comparable to control up to the 8-cell stage, but a delay was observed from compaction to blastocyst hatching. Limitations, reasons for caution In spite of the ability to fertilize normally and support blastulation, efficiency rate remained suboptimal. More importantly, the ability to generate live offspring still has to be proven. Wider implications of the findings This novel 3D differentiation model is capable of generating competent gametes and obviate the need for allo-/xeno-geneic transplantation. Once reproducibility and ability to obtain healthy offspring are confirmed, this method may represent a novel treatment option for azoospermic men Trial registration number N/A

P-115 GATAD2B is required for pre-implantation embryo development by delaying zygotic genome activation

Abstract Study question Major zygotic genome activation (ZGA) involving the activation of thousands of genes occurs at the late-2cell stage, but the regulatory mechanisms governing ZGA remain unclear. Summary answer Our results demonstrate that GATAD2B is essential for early embryonic development, in part through facilitating zygotic genome activation (ZGA). What is known already Acetylation modifications of histone tails promote transcriptional activation, and the maternal deletion of H4K16ac leads to failure in ZGA. GATAD2B is one of the core subunits of the Nucleosome remodeling and histone deacetylation complex(NuRD). Study design, size, duration Using mouse embryos as the research model, the study was conducted over a period of approximately two years to ensure a relatively small-scale investigation. Participants/materials, setting, methods We examined the dynamic expression of NURD complex components, including GATAD2B, during pre-implantation embryonic stages. GATAD2B expression was specifically reduced via microinjection of GATAD2B-siRNA during the zygote stage, revealing its impact on early embryonic development in mice. Zygotic genome activation (ZGA), morula compaction, and blastocyst lineage differentiation were investigated using immunofluorescence, western blot, and other techniques. Additionally, we employed RNA-seq, protein immunoprecipitation, and mass spectrometry to uncover underlying molecular mechanisms. Main results and the role of chance Our research has shown that GATAD2B exhibits specific nucleus localization and high protein expression from the late-2-cell to 8-cell. This intriguing phenomenon prompted us to investigate the relationship between GATAD2B and the ZGA. We discovered a distinctive pattern of GATAD2B, starting from the late 2-cell stage with nuclear localization. GATAD2B depletion resulted in defective embryonic development, including increased DNA damage at morula, decreased blastocyst formation rate and abnormal differentiation of ICM/TE lineages. Consistent with the delay during the cleavage stage, the transcriptome analysis of the 2-cell revealed inhibition of the cell cycle G2/M phase transition pathway. Furthermore, the GATAD2B proteomics data provided clear evidence of a certain association between GATAD2B and molecules involved in the cell cycle pathway. As hypothesized, GATAD2B-deficient 2-cell embryos exhibited abnormalities in ZGA during the maternal-to-embryonic transition, with lower expression of the major ZGA mark MERVL. Limitations, reasons for caution Given the limitations of antibody effectiveness and specificity required for the experiments, the exploration of molecular mechanisms is still ongoing. Wider implications of the findings Our findings have elucidated the impact of GATAD2B on early embryonic development through the regulation of zygotic genome activation (ZGA), providing a novel genetic explanation for clinical defects in early embryonic development. Trial registration number not applicable

P-796 A novel method of somatic cell nuclear transfer to generate artificial oocytes

Abstract Study question Can we identify an alternative method to Sendai-Virus fusion for somatic cell nuclear transfer (SCNT) aiming at generating artificial oocyte? Summary answer The utilization lipofectamine proved itself as a valid alternative method to virus-mediated SCNT, allowing successful membrane fusion and supporting proper embryo development. What is known already SCNT involves transferring a diploid cell’s nucleus into an enucleated oocyte. Early attempts of SCNT involves direct injection and electrofusion but both approaches can affect oolemma integrity. To avoid mechanical damage to oolemma, fusogenic agents have been employed for SCNT including polyethylene glycol (PEG) and more efficient Hemagglutinating Virus of Japan Envelope (HVJ-E). However, despite its high efficiency, HVJ-E is difficult to be used in human because it is a virus derivative, and a recent shortage of this compound made it not readily available. On the other hand, cationic phospholipid is fusogenic and is widely utilized in transfection experiments. Study design, size, duration In the past 4 months, SCNT was carried out through PEG-mediated, HVJ-E-mediated, or novel lipofectamine-mediated fusion using cumulus cells as nuclear donor. Post-SCNT oocytes were inseminated and cultured up to the blastocyst stage. Embryo development and morphokinetics were assessed and compared among the three SCNT experimental cohorts. In some cases, blastocysts were transferred into pseudo-pregnant mice, post-natal development were monitored. Fertilization, embryo development, and offspring wellbeing were monitored. Participants/materials, setting, methods Oocytes from B6D2F1 mice were enucleated by micromanipulation. The ooplasts were allocated to 3 cohorts: 1) exposure to 45% PEG, 2) subzonal transfer of donor cell with HVJ-E, or 3) subzonal transfer the donor cell exposed to 1.75% lipofectamine. Reconstructed oocytes were monitored for fusion 30 minutes after exposure to fusogens and inseminated by spermatozoa from B6-EGFP mice to confirm paternal genome provenance. Fertilization and embryo development were monitored in a time-lapse system. Main results and the role of chance A total of 165 oocytes were enucleated and 160 (97.0%) survived and allocated to PEG-fusion (n = 50), Sendai-virus-fusion (n = 41) and lipofecamine-fusion (n = 80) cohorts. Fusion rate was 56.0% (28/50) for the PEG-fusion cohort, while Sendai-virus and lipofectamine-fusion cohorts yielded higher fusion rate at 92.7%(38/41) and 86.3%(69/80), respectively (P<0.0001). Control oocytes yielded 96.7% (145/150) fertilization rate and developed to 2-cell stage at 93.8% (136/145), and yielded an eventual blastocyst rate at 80.7% (117/145). Despite a lower oocyte reconstruction rate, PEG-fusion cohort fertilized at 89.3% (25/28), but cleavage rate to 2-cell stage was lower at 48.0% (12/25, P<0.0001) and did not yield any blastocyst. The HVJ-E cohort yielded a fertilization rate of 94.7% (36/38) and cleavage rate at 97.2% (35/36) similar to control. The blastocyst rate was observed at 22.9% (8/35, P<0.0001) when comparing to control. Lipofectamine-fusion cohort yielded 91.3% (63/69) fertilization rate and subsequent cleavage rate at 95.2% (60/63). Albeit at a lower blastocyst development rate comparing to control, the lipofectamine cohort yielded similar full-preimplantation rate at 19.0% (12/63) when comparing to HVJ-E cohort. All blastocysts (n = 20) were transferred into 2 pseudo-pregnant mice and 3 healthy female offspring were yielded to date. Limitations, reasons for caution HVJ-E mediated membrane fusion has been well established with proven safety record; however, its recent shortage has led to a temporary cessation of its utilization. Our lipofectamine-mediated cell fusion technique yielded satisfactory fusion, fertilization, blastocyst formation and some live offspring, however the safety and efficacy still remain to be validated. Wider implications of the findings This novel lipofectamine-mediated cell fusion has broader implications for advancing cloning technologies. Lipofection is a non-viral approach, overcomes shortage of HVJ-E and therefore propose itself as a more appealing cell fusion method for human SCNT. Trial registration number N/A

O-251 Generative artificial intelligence substantially enhances the accuracy of embryo selection models

Abstract Study question Can generative artificial intelligence (AI) produce high-fidelity human embryo images to improve AI-based embryo selection models? Summary answer Generative AI models exhibit the capability to generate high-fidelity human embryo images and can substantially improve the performance of AI models through extensive training data. What is known already The integration of AI into in vitro fertilization (IVF) procedures holds the potential to enhance objectivity and automate embryo selection for transfer. However, the effectiveness of AI is limited by data scarcity and ethical concerns related to patient data privacy. Generative Adversarial Networks (GAN) have emerged as a promising approach to alleviate data limitations by generating synthetic data that closely approximate real images. However, the advantage of applying GAN in IVF embryo images is yet to be determined. Study design, size, duration Embryo images were retrieved as training data from time-lapse microscopy (TLM) videos (n = 328). Embryo key morphokinetic variables including blastomere division from 1-cell stage to 9 (and more) cells-stage, compaction of morula, and blastocyst formation were manually annotated by six embryologists. A style-based GAN was fine-tuned as the generative model and a residual network as the morphokinetic classification model. Participants/materials, setting, methods We configured generative models with data augmentation (AUG) and pretrained weights (Pretrained-T/R) to test their refinement to synthetic image quality. We analyzed quantitative metrics including Fréchet Inception Distance (FID) and Kernel Inception Distance (KID) to assess the quality and fidelity of the generated images. Subsequently, we evaluated qualitative performance through a visual Turing test. Then, we compared the performance of a classifier by training real images alone and by integrating real images with generated data. Main results and the role of chance During the training process, we observed consistent improvement in image quality that was measured by FID and KID scores. The AUG+Pretrained-R model showed the highest performance of the evaluated five configurations with FID and KID scores of 15.2 and 0.004 following 5,000 training iterations. Subsequently, we carried out a visual Turing test, such that a total of 60 individuals including IVF embryologists (Group I, n = 25), IVF laboratory technicians (Group II, n = 15), and non-experts (Group III, n = 20) evaluated the synthetic blastocyst-stage embryo images. Group I displayed accuracy of 55.7% (±6.2), sensitivity of 65.9% (±14.1), and specificity of 45.1% (±17.8). Group II showed accuracy of 54.2% (±4.7), sensitivity of 65.9% (±17.6), and specificity of 42.0% (±19.6). Group III had the accuracy of 50.1% (±3.8), sensitivity of 49.3% (±6.3), and specificity was 50.9% (±10.4). Statistical analysis indicated significant differences in accuracy (Kruskal-Wallis test, P=1.2x10-3) and sensitivity (Kruskal-Wallis test, P=1.6x10-4) among the three groups. In the embryonic stage classification model, integrated with generated data outperformed the model using real data solely, with an increase of validation accuracy from 72.6% to 90.6%. The classifier was tested on external TLM video data (n = 100) where the area under the curve was 0.92. Limitations, reasons for caution Further research is needed to involve a more extensive and diverse dataset, e.g. video generation, and more detailed annotations including polar body appearance, pronuclei appearance and disappearance, and blastocyst expansion and hatching. Wider implications of the findings Generative AI offers potential in generating high-fidelity human embryo images. This approach ensures sufficient training datasets while addressing class imbalances for robust AI-based methods, ultimately transforming embryo selection and enhancing IVF outcomes. A set of 5,000 generated images (1024x1024-pixel) at each embryonic developmental stage was open-source for future AI studies. Trial registration number not applicable

O-212 A need to standardize: the use and posterior recall of toxic consumables could be avoided with the appropriate testing protocols

Abstract Study question Could the recall of toxic products have been avoided if the samples had been tested with a more sensitive Mouse Embryo Assay (MEA)? Summary answer Standardizing MEA protocols with sensitive methodologies could prevent the potential harm imparted on human embryos and avoid the need to recall initially unidentified toxic products. What is known already Dozens of consumables are used during an IVF cycle, with a potential effect on gametes/embryos in culture. Bioassays, such as the MEA, are commonly used to pinpoint embryo-toxic products/batches before their release. No strict regulations exist on how to perform these tests, although several variables have been described to play a major role on the assay’s sensitivity and its capacity to detect embryo-toxicity. Toxic products/batches that are not identified may need to be recalled. However, recalls are typically issued after the products/batches are suspected of hindering embryo development in clinical laboratories, with the loss of irreplaceable biological samples. Study design, size, duration Only in 2023, four important batch recalls have been issued by regulatory bodies or manufacturers, concerning such diverse products as oil, media, capillaries, or follicle aspiration needles (FAN). Reportedly, thousands of couples undergoing IVF cycles may have been affected by this undetected toxicity in samples that were already MEA-tested by the manufacturers or external testing laboratories, following an undisclosed methodology. Eight of these lots (2xmedia, 3xcapillaries, 2xFAN) were retested with a standardized sensitive MEA protocol. Participants/materials, setting, methods The samples were retested following a high-sensitivity and previously published protocol, using 1-cell stage embryos from a sensitive hybrid mouse strain (B6CBAF1), supplementing with 5mg/ml HSA, and maintaining a low embryo density per droplet. A total of 462 embryos were cultured, allocated to the different test groups (21 + 42 embryos for Medium 1 and 2; 63 + 84+147 embryos for Capillaries 1, 2 and 3; 42 + 42 embryos for FANs 1 and 2, respectively). Main results and the role of chance Embryos were cultured at 37.3oC, 6%CO2 and 7%O2 for 5 days (96h). The expanded blastocyst formation rate (BFR) in each group was assessed as the end-point of the assays, and an 80% was considered as the toxicity-marking threshold. When cultured in Medium 1 and Medium 2, supplemented with 5mg/ml HSA, only 0/21 (0%) and 7/63 (11.1%) of embryos reached the expanded blastocyst stage, respectively. Capillaries and FANs were tested with an indirect method, in which the samples were first incubated with a small volume of control culture medium, and the extract was used to culture the embryos. With this approach, Capillaries 1, 2 and 3 yielded a 23.8% (15/63), 22.6% (19/84) and 0.7% (1/147) BFR, respectively. Similarly, when testing FANs 1 and 2, only 66.7% (28/42) and 19.1% (8/42) of the embryos were able to form a blastocyst. Unfortunately, the recalled oil batches were not available for retest. In parallel to each test, a control group was cultured with standard culture medium without contact with any of the samples; the BFR in all control groups was above 80%. These surprisingly low results suggest that toxicity was present in these samples in high concentrations, evidencing the need for their recall. Limitations, reasons for caution The methodology used in this project successfully identified all the recalled samples as embryo-toxic. However, the same protocol might not prove equally sensitive when testing other products, as the sensitivity of the MEA has been shown to be highly dependent on numerous external factors and to vary between different toxins. Wider implications of the findings Recent events could lead to the belief that the MEA is an insensitive bioassay to assess embryo-toxicity. Far from true, attention should be paid to the specific methodology used in each case. The lack of regulations and standardization translates into huge differences in sensitivity, even leading to completely contrary results. Trial registration number not applicable

O-261 Frozen gametes differ in post-insemination dynamics to their fresh sibling counterparts in cycles using testicular sperm

Abstract Study question Are there differences in post-insemination events between fresh and frozen-derived gametes when testicular sperm extraction (TESE) is used? Summary answer Frozen-TESE leads to decreased fertilization, but not blastocyst euploidy rates; 2PN-lag time is significantly longer when frozen gametes are utilized compared to their fresh counterparts What is known already Studies indicate that the origin of testicular sperm influences the development potential of cleavage-stage embryos into blastocysts. Recent morphokinetic analysis suggest that embryos from frozen-TESE exhibit prolonged pronuclear stages and more frequent uneven cleavage compared to those from ejaculated sperm. Notably, no comparison with fresh-TESE was performed. To date, no studies have explored analysis in embryo morphokinetic parameters comparing fresh and frozen-TESE, especially when derived from the same patient.Such comparisons could provide a more accurate understanding of potential differences. Given that TESE occasionally involves the use of vitrified oocytes, it is essential to thoroughly examine outcomes across all combinations. Study design, size, duration Retrospective cohort study in a tertiary referral IVF centre including 72 cycles of 30 couples between January 2017 to September 2023. Following insemination by TESE-sperm, embryos were cultured in time-lapse (TL) incubator. Blastocysts ≥BL3CC (Gardner’s) underwent trophectoderm (TE) biopsy for Preimplantation Genetic Testing for aneuploidy (PGT-A) by next generation sequency (NGS). Participants/materials, setting, methods Gamete sources were fresh (fresh-TESE) or frozen-thawed-TESE (frozen-TESE) from the same patients with non-obstructive azoospermia (NOA), and fresh or vitrified (frozen) oocytes from the same female partner. Fertilization, usable blastocysts, and euploidy rates were recorded. Morphokinetics were annotated for time (t) of PB2 (tPB2), tPNa (PN-appearance), tPNf (PN-fading), t2-t9, CP (compaction), tM (Morula), tSB (start blastulation), tB (blastocyst) and timings between each developmental event. Main results and the role of chance Among included 980 oocyte/TESE dyads, 253 (25.8%) used fresh oocyte/sperm, 141 (14.4%) frozen oocyte/fresh sperm, 558 (56.9%) fresh oocyte/frozen sperm and 28 (2.9%) frozen oocyte/sperm. In oocyte/sperm dyads using fresh-TESE, normal fertilization (2PN) (52.0% vs. 44.5%) were higher compared to dyads using frozen-TESE (P < 0.01). Mixed-effects logistic-regression accounting for dependency structure between oocytes/sperm dyads of the same couple, showed an association of frozen-TESE with lower fertilization (OR: 0.64, 95%CI: 0.45-0.89, P = 0.008) and lower blastulation (³BL3CC)/MII (OR: 0.53, 95%CI: 0.37-0.77, P = 0.001), but similar blastulation (³BL3CC)/2PN (OR: 0.69, 95%CI: 0.45-1.07, P = 0.09). After accounting for female age, euploidy per tested blastocyst was similar whether fresh or frozen sperm was used (OR: 0.91, 95% CI: 0.46-1.79, P = 0.788). Frozen oocyte use on the other hand was not significantly associated with these outcomes (P > 0.05 for all). Analysis of morphokinetic parameters revealed that use of frozen dyads was associated with longer duration of 2PN after tPNa to tPNf: in frozen-TESE, 2.10h longer (95%CI: 0.55-3.64, P = 0.008); and in frozen oocytes, 2.34h longer (95%CI: 0.31-4.36, P = 0.024). Among embryos reaching blastulation, frozen-TESE embryos reached 6-cell stage earlier (tPNf to t6, 2.4h earlier, P = 0.009) but they were somewhat delayed in reaching expansion from there on (t6 to tEB, 4.4 hours later, P = 0.059). Limitations, reasons for caution In certain oocytes, insemination was conducted using either fresh or frozen immotile sperm that showed unresponsiveness to activation methods. However, the samples size hinders a comprehensive comparison. Wider implications of the findings Use of frozen-thawed TESE from NOA-patients may decrease fertilization and consequently lower blastocyst yield, but not euploidy rates. Synchronizing fresh-TESE with oocyte retrieval appears safer to maximize fertilization. Regardless of the gamete type, frozen-sourced sperm or oocyte impact 2PN-lag time, possibly indicating nuclear repair mechanisms post-freezing, requiring further investigation. Trial registration number 2310-ABU-020-VF

O-065 PGT-A reveals triploid embryos among normally fertilized oocytes: Frequency and blastocyst features

Abstract Study question Is there any embryo parameter that could differentiate 2 pronucleus (PN) triploid from 2 PN euploid embryos? Summary answer Accuracy in the fertilization check is still necessary as 2 PN but triploid embryos are due mostly to retention of the second polar body (PB). What is known already Since blastocyst culture has evolved and the development of genetic technologies enables to know the karyotipe of the embryos, the concept of normal fertilization is being revised. Commonly, correct fertilization is denoted by the appearance of 2 PN 16-18h after insemination. Deviation from 2PN is considered evidence of abnormal fertilization and ploidy anomalies. However, not all oocytes exhibiting 2 PN are euploids. After performing NGS in trophectoderm biopsies for preimplantation genetic testing for aneuploidy (PGT-A), a percentage of embryos are categorized as triploids. Study design, size, duration Retrospective study including 1862 cycles from 1605 patients aged 18-45 years old who underwent PGT-A with NGS of trophectoderm biopsies in our IVF center from 2019 to 2023. From 6526 analyzed blastocysts in an independent Genetics laboratory, 59 were categorized as triploids. These 59 blastocysts were obtained from 57 PGT-A cycles and belonged to 54 women. In total, these patients had 266 biopsied blastocysts. From the remaining 207 embryos, 42 were called as euploids. Participants/materials, setting, methods In embryos cultured in time-lapse, fertilization was re-evaluated to check the appearance of 2PN and the second PB extrusion. Genetic triploid embryos were analysed in relation to the day of biopsy (day 5 vs day 6), the trophectoderm quality and the timing of the first cell divisions (time of cleavage from 2 to 6 cells: t2, t3, t4, t5, t6). Categorical variables are shown as rate. T-student tests were performed. P-value<0.05 was considered statistically significant. Main results and the role of chance The re-evaluation of the fertilization of the 59 triploids categorized as 2 PN revealed 50 oocytes 2 PN, 8 embryos 3 PN and 1 embryo 2 PN + 1 micronuclei configuration. Therefore, the prevalence of real triploidy in 2PN embryos was 0,8% (n = 50/6526; CI95%:0,6-1,0%). These data showed that 2PN zygotes may give rise to embryos with altered ploidy. From 27 triploid 2PN embryos was possible to access to the whole development time-lapse images. Just 33,3% of triploid embryos exhibited the extrusion of the second PB, while 100% of euploid sibling embryos showed this extrusion (N = 9/27 vs N = 24/24,;p<0,005). Comparing 2 PN triploid and the rest of the embryos from the same cohorts, there were no differences regarding the day of biopsy (N = 31/176 day 5 biopsy, 17.6% vs N = 19/81 day 6 biopsy, 23.5%; p > 0.05). On the contrary, a worse blastocyst quality showed a significant relation with triploidy (N = 23/161,14,2% of good quality embryos are triploid vs N = 27/96, 38,6% of fair quality embryos are triploids; p:0.006).Embryos cleavage time points up to 6-cell stage did not show any significative difference between triploid and euploid embryos (t2: 27.36h vs 25.8h; t3: 38.5h vs 35.6h; t4 39.4h vs 37.2h; t5 52.8h vs 47.0h; t6 56.2h vs 52.1h; all comparisons p > 0.05). Limitations, reasons for caution The incidence of triplody from 2PN zygotes may vary among IVF laboratory settings. The small sample size may be responsible of no difference in morphokinetics. Wider implications of the findings Fertilization check should persist in IVF procedures. Special consideration should be given to the second PB extrusion as triploidy is the most common form of aneuploidy observed in the first trimester miscarriages. The role of diploid spermatozoa, the size of the pronucleus and location should be explored in further studies. Trial registration number NOT APPLICABLE

CRISPR-based genome editing of a diurnal rodent, Nile grass rat (Arvicanthis niloticus)

BackgroundDiurnal and nocturnal mammals have evolved distinct pathways to optimize survival for their chronotype-specific lifestyles. Conventional rodent models, being nocturnal, may not sufficiently recapitulate the biology of diurnal humans in health and disease. Although diurnal rodents are potentially advantageous for translational research, until recently, they have not been genetically tractable. The present study aims to address this major limitation by developing experimental procedures necessary for genome editing in a well-established diurnal rodent model, the Nile grass rat (Arvicanthis niloticus).ResultsA superovulation protocol was established, which yielded nearly 30 eggs per female grass rat. Fertilized eggs were cultured in a modified rat 1-cell embryo culture medium (mR1ECM), in which grass rat embryos developed from the 1-cell stage into blastocysts. A CRISPR-based approach was then used for gene editing in vivo and in vitro, targeting Retinoic acid-induced 1 (Rai1), the causal gene for Smith-Magenis Syndrome, a neurodevelopmental disorder. The CRISPR reagents were delivered in vivo by electroporation using an improved Genome-editing via Oviductal Nucleic Acids Delivery (i-GONAD) method. The in vivo approach produced several edited founder grass rats with Rai1 null mutations, which showed stable transmission of the targeted allele to the next generation. CRISPR reagents were also microinjected into 2-cell embryos in vitro. Large deletion of the Rai1 gene was confirmed in 70% of the embryos injected, demonstrating high-efficiency genome editing in vitro.ConclusionWe have established a set of methods that enabled the first successful CRISPR-based genome editing in Nile grass rats. The methods developed will guide future genome editing of this and other diurnal rodent species, which will promote greater utility of these models in basic and translational research.

Characterization of bovine long non-coding RNAs, OOSNCR1, OOSNCR2 and OOSNCR3, and their roles in oocyte maturation and early embryonic development

In mammals, early embryogenesis relies heavily on the regulation of maternal transcripts including protein-coding and non-coding RNAs stored in oocytes. In this study, the expression of three bovine oocyte expressed long non-coding RNAs (lncRNAs), OOSNCR1, OOSNCR2, and OOSNCR3, was characterized in somatic tissues, the ovarian follicle, and throughout early embryonic development. Moreover, the functional requirement of each transcript during oocyte maturation and early embryonic development was investigated using a siRNA-mediated knockdown approach. Tissue distribution analysis revealed that OOSNCR1, OOSNCR2 and OOSNCR3 are predominantly expressed in fetal ovaries. Follicular cell expression analysis revealed that these lncRNAs are highly expressed in the oocytes, with minor expression detected in the cumulus cells (CCs) and mural granulosa cells (mGCs). The expression for all three genes was highest during oocyte maturation, decreased at fertilization, and ceased altogether by the 16-cell stage. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes was achieved by microinjection of the cumulus-enclosed germinal vesicle (GV) oocytes with siRNAs targeting these lncRNAs. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 did not affect cumulus expansion, but oocyte survival at 12 h post-insemination was significantly reduced. In addition, knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes resulted in a decreased rate of blastocyst development, and reduced expression of genes associated with oocyte competency such as nucleoplasmin 2 (NPM2), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and JY-1 in MII oocytes. The data herein suggest a functional requirement of OOSNCR1, OOSNCR2, and OOSNCR3 during bovine oocyte maturation and early embryogenesis.

Shape of the first mitotic spindles impacts multinucleation in human embryos

During human embryonic development, early cleavage-stage embryos are more susceptible to errors. Studies have shown that many problems occur during the first mitosis, such as direct cleavage, chromosome segregation errors, and multinucleation. However, the mechanisms whereby these errors occur during the first mitosis in human embryos remain unknown. To clarify this aspect, in the present study, we image discarded living human two-pronuclear stage zygotes using fluorescent labeling and confocal microscopy without microinjection of DNA or mRNA and investigate the association between spindle shape and nuclear abnormality during the first mitosis. We observe that the first mitotic spindles vary, and low-aspect-ratio-shaped spindles tend to lead to the formation of multiple nuclei at the 2-cell stage. Moreover, we observe defocusing poles in many of the first mitotic spindles, which are strongly associated with multinucleation. Additionally, we show that differences in the positions of the centrosomes cause spindle abnormality in the first mitosis. Furthermore, many multinuclei are modified to form mononuclei after the second mitosis because the occurrence of pole defocusing is firmly reduced. Our study will contribute markedly to research on the occurrence of mitotic errors during the early cleavage of human embryos.

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.

Selection and application of small non-coding RNAs for normalizing RT-qPCR data of bovine preimplantation embryo conditioned medium

Small non-coding RNAs (sncRNAs) present in the conditioned medium (CM) of bovine preimplantation embryos are potential noninvasive biomarkers for assessing embryo quality. Accurate quantification of sncRNA levels in the spent CM is of utmost importance in this regard. RT-qPCR is considered as the gold standard for quantifying RNA. In order to standardize RT-qPCR data in the sample type under investigation, the use of suitable stable sncRNAs is essential. Here, we selected 10 sncRNAs from small RNA sequencing of CM samples derived from both bovine blastocysts and degenerate embryos, and evaluated their expression stability together with that of cel-miR-39 as a spike and the often-used U6 small nuclear RNA at different embryo developmental stages. In CM of 2-cell embryos, rsRNA-1044 showed the most stable expression, while tDR-1:32-Gly–CCC–1 was the most stable expressed sncRNA in CM of the stages beyond the 2-cell stage. Next, tDR-1:32-Gly–CCC–1 was used for normalizing the RT-qPCR data from the CM of blastocysts and degenerate embryos. Bta-miR-155 and tDR-39:75-Arg-CCG-2 were found to be significantly up-regulated in the CM of blastocysts compared to that of the degenerated embryos (P = 0.028 and P = 0.017, respectively), suggesting their expression levels are related to embryo development stage. In conclusion, tDR-1:32-Gly–CCC–1 can serve as a suitable reference sncRNA for normalization of RT-qPCR data of the CM from bovine blastocysts.

Escherichia coli self-organizes developmental rosettes

Rosettes are self-organizing, circular multicellular communities that initiate developmental processes, like organogenesis and embryogenesis, in complex organisms. Their formation results from the active repositioning of adhered sister cells and is thought to distinguish multicellular organisms from unicellular ones. Though common in eukaryotes, this multicellular behavior has not been reported in bacteria. In this study, we found that Escherichia coli forms rosettes by active sister-cell repositioning. After division, sister cells "fold" to actively align at the 2- and 4-cell stages of clonal division, thereby producing rosettes with characteristic quatrefoil configuration. Analysis revealed that folding follows an angular random walk, composed of ~1 µm strokes and directional randomization. We further showed that this motion was produced by the flagellum, the extracellular tail whose rotation generates swimming motility. Rosette formation was found to require de novo flagella synthesis suggesting it must balance the opposing forces of Ag43 adhesion and flagellar propulsion. We went on to show that proper rosette formation was required for subsequent morphogenesis of multicellular chains, rpoS gene expression, and formation of hydrostatic clonal-chain biofilms. Moreover, we found self-folding rosette-like communities in the standard motility assay, indicating that this behavior may be a general response to hydrostatic environments in E. coli. These findings establish self-organization of clonal rosettes by a prokaryote and have implications for evolutionary biology, synthetic biology, and medical microbiology.