Embryonic Development Research Articles

P-178 Putrescine supplementation enhances embryonic development in patients with Recurrent Implantation Failure (RIF)

Abstract Study question Does putrescine, a type of polyamine, affect embryonic development in patients with recurrent implantation failure (RIF)? Summary answer Putrescine supplementation on cumulus-oocyte complexes (COCs) was associated with better embryologic development from fertilization to blastulation in patients with RIF. What is known already Putrescine, a type of polyamine, plays a role in cell growth and proliferation. Several animal studies suggest the potential impacts of putrescine on embryo development, but its effects on human IVF have yet to be extensively studied. Study design, size, duration This retrospective study analyzed 442 patients who have undergone IVF cycles three or more times from January to December 2023. The average IVF cycle number of RIF patients was 7.4 times. A total of 1356 oocytes from the putrescine group and 1391 oocytes from the control group underwent aspiration and ICSI. Further analysis was conducted by classifying RIF patients into two groups: patients under 40 years of age and patients over 40 years of age. Participants/materials, setting, methods COCs were cultured for 4 hours in a culture medium supplemented with 0.5mM putrescine. 2 hours after oocyte collection, cumulus cells were denuded, and ICSI was performed. Embryonic development was evaluated based on fertilized oocytes (2PN), good quality embryos (GQE) on day 3, and blastulation rates on day 5/6. Group differences were analyzed using t-tests and chi-square tests (p < 0.05). Main results and the role of chance When maternal age was ≥40, the fertilization rate in the putrescine group was significantly higher than that in the control group (86.6% vs. 80.5%, p=0.002). The putrescine group had a numerically higher proportion of embryos with top-grade morphology on day 3 (47.8%, compared with 53.3% for control; p=0.057), without statistical significance. Additionally, the rate of blastulation on day 5 indicated a significant difference between the two groups (14.5% vs. 6.1%, p=0.0001). Further analysis was conducted in younger patients (<40 years old), and it also showed a significantly higher fertilization rate (89.7% vs. 71.1 p=0.0001) and blastocyst development rates on the day 5 in the putrescine group (22.3% vs. 11.1%, p=0.0001), respectively. This study suggests that putrescine supplementation can be utilized to improve embryo quality and facilitate blastocyst formation in IVF procedures for patients with RIF, irrespective of age. Limitations, reasons for caution Putrescine supplementation in IVF showed improved fertilization and blastulation rates, but caution is needed due to the small sample size used. Therefore, a larger sample size will help improve the robustness of the conclusions drawn with respect to the embryologic development in patients with RIF. Wider implications of the findings The study’s findings suggest that putrescine supplementation in IVF may enhance outcomes for patients with RIF, potentially leading to personalized treatments. Trial registration number not applicable

O-069 The first mitotic spindle instability impacts nucleation, fragmentation, and development in human embryos

Abstract Study question How multinucleation and fragmentation occur in the first mitosis in human embryos? Summary answer The first mitotic spindles had heterogeneity, and the shapes and movements of the spindles were strongly associated with multinucleation and fragmentation. What is known already Studies have shown that many problems occur during the first mitosis, such as fragmentation and multinucleation. However, the cell biologic mechanisms whereby these errors occur during the first mitosis in human embryos remain unknown. To clarify this aspect, it is necessary to analysis the dynamics of DNA and cytoskeleton using live human embryos. Although microinjections of DNA or mRNA into mammalian embryos were widely used for live imaging during early mitosis, the method into human poses the risk of ethical issues. Therefore, we established a method used for real-time imaging in human embryos using chemical fluorescent labeling. Study design, size, duration Frozen human two-pronuclear stage embryos were donated for research by couples who had completed fertility treatment. We obtained written informed consent from patients who donated human embryos. The mean age of patients at embryo freezing was 32.00 ± 3.03 (SD) years. We imaged thirty-one living human embryos using chemical fluorescent labeling and confocal microscopy to visualize DNA and tubulin. Participants/materials, setting, methods Thirty-one human embryos were cultured in medium containing the live-cell stain probes SPY555-DNA and SPY650-Tubulin. The Embryos were imaged using an LSM980 laser scanning confocal microscope every 10–20 min for a period of eight hours. Three-dimensional (3D) visualizations of human embryos were performed using the Imaris software. The first spindle shapes were quantified by image analysis and calculated the aspect ratio of the spindles using ImageJ. Main results and the role of chance We discovered that over 81% of two-pronuclear zygotes became multinucleated 2-cell stage embryos. We demonstrated that there was variation in the first mitotic spindles. There was a significant difference between the aspect ratio (AR) of the spindles leading to the mononuclear and multinuclear types at the 2-cell stage (p < 0.005). In addition, the low-AR spindles were unstable and often had more defocused poles than the high-AR spindles, which lead to multinucleation at the 2-cell stage (55 % vs. 0%, p < 0.05). More embryo having high-aspect ratio spindles tend to develop blastocysts than those having low aspect-ratio spindles (p < 0.05). Moreover, the movement of the central spindles varied after chromosome segregation. The embryos with more fragments at two cell stage had a significantly longer period for the central spindles to transform into midbodies compared to those with fewer fragments. The central spindles of embryos with more fragments moved a longer distance from the beginning of furrow ingression to the completion of the first division (p < 0.05). These results suggest that the first mitotic spindle instability and heterogeneity cause many errors such as multinucleation, fragmentation, and embryonic development. Limitations, reasons for caution Imaged human embryos were donated from a fertility clinic, and detailed patient information, such as underlying disease and method of ovarian stimulation, was not available. It is possible that the human zygotes used in this study had a bias in patient characteristics. Wider implications of the findings Our study showed that there were instability and heterogeneity in the first mitotic spindle, which was strongly associated with multinucleation, fragmentation, and embryonic development. Revealing the mechanism underlying spindle formation in cleavage-stage embryos will contribute to improving the success rate of assisted reproduction technologies in humans. Trial registration number not applicable

O-072 The canonical Hippo signalling pathway controls the first lineage segregation in the human preimplantation embryo

Abstract Study question What is the role of the canonical Hippo signalling pathway during the first lineage segregation in the human preimplantation embryo? Summary answer Canonical Hippo signalling regulates the first lineage segregation by controlling the expression of trophectoderm (TE) and inner cell mass (ICM) associated genes. What is known already The Hippo signalling pathway is a conserved regulatory network that plays a critical role in the first lineage segregation in mouse, rat, bovine and human preimplantation embryos. This pathway consists of two distinct branches (canonical, non-canonical). Despite their shared role in lineage segregation, the respective functions of canonical and non-canonical Hippo signalling branches remain largely unexplored. Since these branches are regulated by distinct upstream mechanisms, they may have different roles in embryonic growth, function, and survival. Unravelling the distinct roles of these branches is essential for understanding the intricate mechanisms that govern early human embryonic development. Study design, size, duration This experimental study involved the functional characterization of the canonical Hippo signalling pathway by pharmacologically inhibiting the core kinase MST1/2. Eight-cell stage human embryos donated for research were warmed and incubated with or without the inhibitor, till completion of the first lineage segregation days post fertilization (dpf) 5. The control (n = 20) and treated embryos (n = 17) were evaluated based on embryonic development (kinetics, morphology), and expression of lineage-specific markers. Participants/materials, setting, methods The optimal concentration (1μΜ) of the highly selective XMU-MP-1 inhibitor targeting the MST1/2 core kinase of the canonical Hippo signalling pathway was established in titration experiments. Time-lapse imaging (Miri TL, Esco Medical) captured effects on embryonic development (kinetics, morphology). Changes in the main effectors of the Hippo signalling pathway (YAP1, p-YAP1 and WWTR1) and the expression of lineage-specific markers (ICM: NANOG, SOX2; TE: GATA3) were assessed through immunofluorescence imaging and confocal microscopy (LSM800, Zeiss). Main results and the role of chance For the first time, we provide a complete spatiotemporal analysis of the main effectors of the Hippo signalling pathway YAP1 and WWTR1. They are co-localised in the nuclei of outer cells in compacting (dpf3) and compacted (dpf4) embryos. In the dpf5 blastocyst, YAP1 and WWTR1 are detected in the nuclei of the TE cells. Phosphorylated effector p-YAP1, indicative of active Hippo signalling, was first detected in the inner cells of the compacted embryo (dpf4) and remained in the ICM of the dpf5 blastocyst, supporting the role of the Hippo signalling pathway in the first lineage segregation. Treatment of human embryos with XMU-MP-1 reduced p-YAP1 levels, confirming its specificity for MST1/2. Inhibition of the MST1/2 kinase had a significant effect on the developmental kinetics as treated embryos had lower blastocyst formation rates. Embryo morphology was significantly compromised as treated embryos formed ICMs with less cells compared to control embryos. Immunofluorescence analysis revealed that the XMU-MP-1 treatment led to the upregulation of YAP1 and upregulation of TE marker GATA3, while the ICM markers SOX2 and NANOG were downregulated. Overall, our data suggest that active Hippo signaling promotes ICM formation, while TE formation requires inactive Hippo signaling. Limitations, reasons for caution Caution is warranted due to potential non-specific effects of the XMU-MP-1 inhibitor, although this compound has been shown in other models to be very specific. Further studies should explore off-target impacts to ensure result reliability. Wider implications of the findings Understanding the signalling pathways that regulate human preimplantation embryo development is fundamental for understanding embryonic growth, function, and survival, and can also help improve IVF success rates by addressing deficiencies that lead to embryo developmental arrest. Trial registration number Not applicable

O-215 Copper oxide nanoparticles (CuONPs) impairs mitophagy and promotes DNA methylation to suppress mouse embryonic pluripotency

Abstract Study question Does exposure to copper oxide nanoparticle (CuONPs) have adverse effects on early embryonic development? What is the potential mechanism? Summary answer CuONPs exposure can damage mitophagy and lead to α-KG reduction, resulting DNA hypermethylation, thereby reducing the pluripotency of pre-implantation embryos in mice. What is known already Environmental nanomaterials contamination is a great concern for organisms including human. Exposure to nanomaterials have been associated with fecundity and fertility in couples conceiving via assisted reproduction technology. Among these bio-effects, nanomaterials induced epigenetic changes are of particular interest. Copper oxide nanoparticles (CuONPs) are widely used in a huge range of applications which might pose potential risk to organisms. Recent studies reported that CuONPs exposure resulted in transgenerational toxicity in Caenorhabditis elegans associated with possible epigenetic regulation. However, whether CuONPs exposure affect mammalian embryonic development, and if so the underlying mechanisms, are unclear. Study design, size, duration For the human experiments, we measured the copper ion content in women’s urine, and assessed the expression of pluripotent genes in 3PN embryos treated with CuONPs. For the embryo experiments, we collected zygotes from ICR mice and cultured them in KSOM medium with CuONPs (10 μg/mL) until blastocyst stage. For the cell experiments, we treated mouse embryonic stem cells (mESCs) with CuONPs (2 μg/mL) for four days to detect cell proliferation and pluripotency. Participants/materials, setting, methods The ICP-MS method was used to detect copper ion content in women’s urine. Time–lapse incubator was used to document the developmental potential of embryos. To quantify the metabolites of embryo, we employed metabolomics. Single-cell multiomics sequencing that enables profiling of genome-wide chromatin accessibility, DNA methylation and RNA expression were performed to detect genes expression and epigenetic modification. We used real-time PCR, immunofluorescence, and western blot to measure the expression of pluripotency related genes. Main results and the role of chance In human, we discovered a significant negative relationship between the urine’s copper ion content and clinical pregnancy rate. In embryo experiments, CuONPs exposure has been shown to have adverse effects on embryo developmental potential, resulting morula and blastocyst transformation arrest, as well as reduction in blastocyst rate and live birth rate. Furthermore, CuONPs treatment led to a reduction in blastocyst quality, indicating a decrease in blastocyst cell count, particularly in ICM cells. CuONPs also inhibited the pluripotency of blastocysts and mESCs, showing down-regulated expression of pluripotent genes. Mechanistically, CuONPs aggregated in lysosomes and decreased the mitophagic flux of embryos, which caused damaged mitochondria to accumulate and ultimately caused mitochondrial dysfunction. Notably, in embryos treated with CuONPs, there was a decrease in α-ketoglutarate (α-KG) due to the disruption of the tricarboxylic acid (TCA) cycle. α-KG is an essential cofactor for α-KG dependent dioxygenases such as the ten-eleven translocation (TET) family of DNA demethylase. The lack of α-KG in CuONPs-treated embryos led to DNA hypermethylation, which reduced chromatin accessibility, and suppressed the expression of pluripotent genes like sox2, klf2, and klf4. The poor pluripotency ultimately leads to abnormal embryonic development in CuONPs-treated embryos. Limitations, reasons for caution The molecular mechanism by which CuONPs arrest the development of pre-implantation embryos remains to be determined. In addition, future work should be extended to human embryos to determine whether this mechanism is conserved between mice and humans. Wider implications of the findings The findings of this work indicated the relationship between CuONPs and epigenetic modification. The results will support a more thorough understanding into the biosafety of CuONPs, and offer fresh perspectives on the potential uses of nanomaterials in the field of reproductive medicine in the future. Trial registration number Not Applicable

P-036 Female age affects the combined impact of DNA fragmentation and high DNA stainability on embryo development in intracytoplasmic sperm injection cycles

Abstract Study question To investigate the role of female age in regulating the co-effect of DNA fragmentation index (DFI) and high DNA stainability (HDS) on ICSI embryological outcomes. Summary answer When female age exceeded 35, co-exposure high DFI and middle HDS might delay embryonic development after ICSI treatment. What is known already Sperm DNA damage had a negative influence on embryonic development. Oocytes and embryos have adopted specific response and repair mechanisms to repair sperm DNA damage. However, the DNA repair capacity in the oocyte is limited and likely decline with increasing age. Study design, size, duration This historical cohort study was included 1334 couples underwent ICSI from January 2017 to December 2022. An unsupervised K-means clustering method was used to divide participants into three clusters base on DFI value and HDS value, as follow: cluster 1 with low DFI values and low HDS values (lDFI-lHDS group), cluster 2 with middle DFI values and high HDS values (mDFI-hHDS group), and cluster 3 with high DFI values and middle HDS values (hDFI-mHDS group). Participants/materials, setting, methods All participants were further stratified into female age<35 subgroup and female age≥35 subgroup. The univariate and multivariate linear regression model was used for statistical analysis. Multivariate model was adjusted for female factor (including age, AMH, basal FSH and the numbers of retrieved oocyte) and male age which were significantly correlated with embryo development in univariate models. The main outcome measure was D5 blastocyst development rate and D6 blastocyst development rate. Main results and the role of chance In the female age<35 subgroup, D5 blastocyst development rate in the mDFI-hHDS group (48.53% [25.00%, 66.67%] and the hDFI-mHDS group (50.00% [28.57%, 71.07%]) was significantly higher than that in the lDFI-lHDS group (40.00% [0.00%, 63.96%]). No significant differences were observed between the three groups regarding D6 blastocyst development rate. And univariate model and multivariate model showed that the mDFI-hHDS group (adjusted B value=0.36, 95% CI: 0.08∼0.64, P=0.010) and hDFI-mHDS group (adjusted B value=0.41, 95% CI: 0.12∼0.70, P=0.006) had higher D5 blastocyst development rate compared with lDFI-lHDS group. In the female age≥35 subgroup, D5 blastocyst development rate in the hDFI-mHDS (20.00% [0.00%, 50.00%]) group was significantly lower than that in the lDFI-lHDS group (36.93% [0.00%, 66.67%]) and the mDFI-hHDS group (41.43% [0.00%, 63.54%]). Accordingly, D6 blastocyst development rate in the hDFI-mHDS group (66.67% [33.33%, 100.00%]) was significantly higher than that in the lDFI-lHDS group (50.00% [16.67%, 80.00%]) and the mDFI-hHDS group (50.00% [33.33%, 66.67%]). And both model showed that hDFI-mHDS group (adjusted B value=0.73, 95% CI: 0.36∼1.11, P<0.001) had higher D6 blastocyst development rate compared with the lDFI-lHDS group. Although not statistically significant, hDFI-mHDS had negative correlation with D5 blastocyst development rate. Limitations, reasons for caution The main weakness of this study was the participants were not randomized. Wider implications of the findings hDFI-mHDS group were negatively associated with D5 blastocyst development rate and positively related with D6 blastocyst development rate in female age≥35 subgroup. These results suggested that co-exposure high DFI and middle HDS might be recommended as a predictor for the detection of delayed embryo development when female age exceeded 35. Trial registration number not applicable

P-028 Pregnancy and perinatal outcomes in male patients with temporary ejaculation failure undergoing assisted reproductive technology

Abstract Study question When temporary ejaculation failure occurs on the oocyte retrieval day, choose surgical sperm retrieval or oocyte cryopreservation? Summary answer TESA-ICSI and oocyte cryopreservation are both relatively efficient and safe way. Embryos have better developmental potential through TESA-ICSI. What is known already Nearly 9% of men among couples who use assisted reproductive technology have temporary ejaculation failure (TEF) on the oocyte retrieval day. If they fail to provide semen sample, the couples may face three choices (1. surgical sperm retrieval; 2. oocyte cryopreservation; 3. canceling the oocyte retrieval). The aim of the study was to compare the impact of testicular sperm aspiration (TESA) or oocyte cryopreservation on the clinical outcomes of intracytoplasmic sperm injection (ICSI) patients with TEF. Study design, size, duration The retrospective study included 345 male patients with TEF in our University-affiliated IVF center from 2015 to 2021. Participants/materials, setting, methods Three hundred and forty-five male patients were divided into two groups. Two hundred patients chose TESA-ICSI (the TESA group), while another 145 patients chose oocyte cryopreservation, waiting for the man’s next successful sperm ejaculation and subsequent ICSI (the frozen oocyte group). We investigated the embryo development, pregnancy and perinatal outcome, and included the data from fresh embryo transfer (ET) cycle. Herein, all patients in the frozen oocyte group received hormone replacement and fresh ET. Main results and the role of chance Firstly, there were no significant difference in the female BMI, FSH, antral follicle count (AFC), AMH, and male semen quality between the two groups. For embryo development, both two groups have the similar normal fertilization rate (71.5% vs 69.7%, P = 0.242). However, the frozen oocyte group has lower rate of Day3 high quality embryo (31.1% vs 49.3%, P < 0.001), available embryo rate (49.8% vs 55.8%, P = 0.043), and available blastocyst rate (18.6% vs 33.7%, P < 0.001). For pregnancy outcome, the clinical pregnancy rate (44.2% vs 50.0%), pregnancy loss rate (4.8% vs 2.9%) and live birth rate (38.5% vs 46.1%) were similar. And there were no significant differences in the rates of Gestational diabetes mellitus, hypertensive disorders of pregnancy, placenta previa and fetal malformation between the two groups. Limitations, reasons for caution This study only included data from fresh embryo cycles, not yet from thawed embryo cycles. Although we presented complete embryo development data for each couple, there may be some bias in pregnancy and perinatal outcomes due to the lack of data from thawed embryo cycles. Wider implications of the findings The present study suggested that TESA-ICSI and oocyte cryopreservation are both relatively efficient and safe way to help the patients encountering TEF. Due to the embryos with better developmental potential through TESA-ICSI, choosing the surgical sperm retrieval might have more have more advantages, and this has to be further verified. Trial registration number REC No. (2023(S142))

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-009 Impact of sperm preparation methods on embryo development rate and embryo ploidy status: ICSI egg donation as a model

Abstract Study question Do different sperm separation techniques result in distinguishable contributions to embryo development rates and ploidy status? Summary answer CA0 sperm separation device, devoid of centrifugation, yielded a higher euploidy rate than conventional swim-up (50% vs. 74%, p = 0.02), notably observed in abnormozoospermic cases. What is known already Sperm DNA fragmentation (SDF) emerges as a key paternal risk factor associated with embryo aneuploidy, increasing the risk of ART failures. Factors like advanced male age, prolonged ejaculatory abstinence, and the detrimental effects of centrifugation during sperm separation jeopardize sperm DNA integrity. A recent centrifugation-free sperm separation device (CA0), employing live sperm sorting technology, exhibits efficiency in producing highly motile sperm with minimized SDF. This retrospective cohort study solely focusing on ICSI egg donation cycles to assess the influence of sperm separation methods (conventional swim-up vs. CA0) on early embryo development, and embryo ploidy. Study design, size, duration This retrospective study included 83 couples who underwent ICSI egg donation cycles at Chachava Clinic, Tbilisi, Georgia. Thirty-three semen specimens were processed using conventional swim-up (SU) and 427 Mii were inseminated. Forty-four semen samples were separated by CA0 and 519 Mii were inseminated. Clinical outcome measures included fertilization rate (number of 2PN over number of inseminated oocyte), good embryo rate (embryo number with more than 8 cells over inseminated oocyte), and euploidy rate using PGT-A. Participants/materials, setting, methods Male age and basic semen parameters via CASA (CEROS II, Hamilton-Thorne) were recorded. SU involved layering 1 mL neat semen to 1.2 mL HTF medium, inclining the tube at 45° for 60 min at 37 °C, collecting 1 mL uppermost, adding 2 mL HTF for centrifugation at 500×g for 5 min, and resuspending sperm pellet with 0.5 mL HTF. Semen samples were sorted using LensHooke® CA0 device following the manufacturer’s instructions. Main results and the role of chance In comparing demographic characteristics between SU and CA0 arms, CA0 exhibited higher median total motility (SU: 40% vs. CA0: 54%, p = 0.02), with no significant differences in female age and Mii oocyte count. This study assessed the impact of sperm separation methods on early embryo development, finding no significant differences in fertilization rate (SU: 79% vs. CA0: 77%) and good embryo rate (SU: 64% vs. CA0: 62%). However, CA0 demonstrated a significantly higher euploidy rate than SU (SU: 59% vs. CA0: 69%, p = 0.03). Further analysis within subpopulations, including normozoospermic and asthenozoospermic (total motility <40%), revealed equivalent euploidy rates between SU and CA0 for normospermic specimens (SU: 66% vs. CA0: 68%). However, a significant differentiation in euploidy rate emerged for asthenozoospermic cases (SU: 59% vs. CA0: 74%, p = 0.02). Specifically, comparing outcomes in normozoospermic, a significant reduction was observed in asthenozoospermic cases using SU (66% vs. 50%, p = 0.02), whereas CA0 provided improved euploidy rates regardless of semen quality (68% for normozoospermic and 74% for asthenozoospermic). In conclusion, this study suggests that the non-invasive live sperm sorting device enhances embryo euploidy rates, serving as a reliable sperm preparation method, ensuring selection consistency despite sample variations. Limitations, reasons for caution This study’s limitations include its retrospective nature and single-center trial design. To more effectively assess CA0 efficiency, a randomized controlled trial targeting diverse patient eligibility criteria is warranted. Wider implications of the findings Attributed to the improved embryo euploidy with CA0, we anticipate enhanced ART outcomes, such as increased embryo transfer rate and prevention of ART failures related to embryo aneuploidy. Additionally, CA0 offers practical benefits by streamlining and standardizing the sperm separation process with only three steps involved. Trial registration number not applicable

P-043 Preliminary clinical outcome using the Q300™ device in a reproductive laboratory environment

Abstract Study question How does the Q300™ Device impact clinical outcomes in a reproductive laboratory environment compared to established standards? Summary answer The device showed favorable clinical outcomes and was user-friendly. More research is needed to identify the patient subgroups that can benefit the most from system What is known already Embryologists struggle with accurately assessing sperm morphology without staining, prompting interest in emerging technologies like AI. These methods evaluate motility parameters, offering non-invasive sperm selection. A previous study comparing Q300 to manual staining methods showed high agreement and repeatability, indicating Q300’s accuracy in measuring live sperm cells. It aids in selecting cells per WHO2021 guidelines without staining, potentially revolutionizing sperm assessment. Study design, size, duration The study is an open-label, non-randomized study. Semen samples intended for ICSI were imaged by the Q300™ device before micro-injection to aid in selecting morphologically compliant sperm cells. The data collected was compared to the Key Performance Indicators (KPI) for ART laboratories according to the Vienna Consensus. Up to 5 sites will be enrolled in the study, with up to 100 couples in total from all sites. Study duration: 18 months Participants/materials, setting, methods The Q300™ device is an optical system using quantitative phase microscopy and holographic imaging to analyze human semen morphology. The device aids embryologists in selecting live and motile sperm cells for ICSI by providing automatic, objective analysis. Couples meeting inclusion criteria, including female age ≤40 and availability of motile sperm, were recruited for ICSI procedures. There were no restrictions on previous IVF/ICSI cycles. Endpoints: fertilization, day-3 embryo development, good blastocyst development rates, and implantation rates. Main results and the role of chance 14 couples were recruited from Barzilay Medical Center’s IVF unit in Ashkelon, Israel. The average fertilization rate is 79.1% (Vienna consensus competency is ≥ 65%), the day 3 embryo development rate is 55% (Vienna consensus competency is ≥ 45%), and the good blastocyst development rate is 42.5% (Vienna consensus competency is ≥ 30%). The implantation rate (day 3+day5) is 36.8% (Vienna consensus competency is ≥ 25% for day 3 embryo transfers and ≥ 35% for blastocyst transfers). The intended users performed the procedures with no reported malfunctions. 270 sperm imaging procedures were performed using the Q300 device to select 109 morphologically suitable cells for ICSI. The utilization of the product resulted in approximately a 40% yield of correct selections by the embryologist. Consequently, out of every 10 cells deemed compliant by the embryologist, only 4 were confirmed by the device, with roughly 3 falling within a margin of 10% around the WHO measurement range. Moreover, only about 1 of these selections fell within the boundaries defined by the WHO standards. This observation offers insights into the characteristics of cells routinely chosen for injection into oocytes. Limitations, reasons for caution The study recruited 14 couples from a single site; it is important to validate and statistically power the outcome measures and compare them with clinical control groups in the future. In the next generation of the product, incorporating an objective selection based on sperm motility can further improve its functionality. Wider implications of the findings The findings offer insights into optimizing sperm selection processes, potentially improving fertilization, embryo development, and implantation rates in couples undergoing ICSI. Moreover, the study highlights the importance of continued research and refinement of technological innovations in ART to better serve patient needs and improve treatment outcomes. Trial registration number NCT06232720

P-550 Monoallelic CCNB2 variants may cause oocyte maturation defects and embryo developmental arrest

Abstract Study question Have all the genes that may cause embryo development arrest and oocyte maturation defects been identified in female patients? Summary answer We identified a variant in CCNB2 in our patient. To our knowledge, this is first time that meiotic arrest can be caused by mutated-CCNB2. What is known already Successful reproduction involves the maturation of gametes, fertilization, and early development of the embryo. Oocyte maturation involves a sequence of both morphological and molecular transformations, progressing from germinal vesicle oocytes to metaphase I oocytes and ultimately to metaphase II oocytes. Any disruption in the process of oocyte maturation can result in infertility. With the widespread use of whole exome sequencing (WES), several genes that influence oocyte maturation at different stages have been recently identified. Study design, size, duration We here report the case of repeated ART failure due to oocyte maturation defect and embryo developmental arrest. Participants/materials, setting, methods A 30-year-old female and 38-year-old male applied to our clinic due to unexplained infertility for 4 years. The first ART attempt resulted in 2 of 13 oocytes with late MII. During ART performed in our center, germinal vesicle and meiotic arrest were observed in all oocytes. No mature oocytes were obtained. Her grandfather’s sister was also infertile. WES and Sanger analysis were performed to detect any mutation in known/candidate genes and show familial segregation Main results and the role of chance WES analysis revealed no mutation in genes that were previously reported as a cause of oocyte maturation defect or early embryo development in human. We do a further analysis of genes highly or specifically expressed in oocytes or expressed at low levels yet previous studies have suggested its role in embryo development. We identified a c.787C>T, p.Arg263* heterozygous nonsense variant in the CCNB2 gene that was extremely rare in both multiple databases and our in-house database. Multiple lines of computational evidence support the deleterious effect of the variant on the gene product. This variant was transmitted from her healthy father. CCNNB2 is not expressed in any cells other than oocytes in human. The activity of the M phase-promoting factor (MPF) is crucial for advancing through both mitosis and meiosis. CCNB2 plays a vital role by contributing to the activity of pre-MPF during prophase, necessary for generating enough MPF to proceed through maturation. Furthermore, CCNB2 is significantly involved in mouse oocyte meiosis, playing a key role in both the G2/M and MI/anaphase I transitions. Limitations, reasons for caution New disease-causing variants in this gene should be demonstrated in larger cohorts with female infertility. In addition, further functional studies should be carried out both in CCNB2-mutated human and animals. Wider implications of the findings This study may provide a better understanding background of female infertility and the failure of the ART process. CCNB2 may have a crucial role in the oocyte maturation process and can be utilized as a female infertility genetic marker. Trial registration number NA

P-260 In Vitro Fertilization (IVF) using magnetotactic sperm cells and their prospects for assisted in vivo reproduction

Abstract Study question Are bovine Magnetotactic Sperm Cells (MSC) able to fertilize an oocyte in-vitro and Which would be the main requirements for translating this technology to in-vivo? Summary answer IVF using highly purified MSC result in embryo development until blastocyst stage. We demonstrate different alternatives to control multiple MSC, individually and as a swarm. What is known already Medical microrobots have appeared as the new generation of drug delivery vehicles. They are tiny devices in the order of a few micrometers, able to move in biological environments by different means, physical, chemical, or biohybrid. They are functionalized to deliver targeted therapies for drugs, cells, or other payloads. In assisted reproduction, sperm-hybrid microcarriers have been pioneered by our group, and various in vitro demonstrations have been done to assist sperm cells with motion deficiencies or low count and to deliver anticancer drugs for treating, for example, cervical and ovarian cancer, but so far in-vitro fertilization has never been shown. Study design, size, duration Experimental quantitative study design. A total of 520 bovine cumulus-oocyte complexes were used for in vitro fertilization. The analyzed sample groups were using bovine frozen sperm cells, MSC, or by evaluating parthenogenetic activation. A power of 0.98 with an a-error of 0.05 was used for the IVF experiments. Sperm samples in the control as well as the micromotor groups have been analyzed for different sperm parameters. Participants/materials, setting, methods Bovine MSC are created by thawing frozen sperm cells and adding polystyrene microparticles. Different sperm parameters were evaluated to check the effects of the particles in the cells. After developing a purification method for the MSC, bovine ovaries were collected from a slaughterhouse nearby and, after in vitro maturation, were used for the IVF experiments. Ultrasound, and photoacoustic imaging setups with a set of electromagnets were employed for real-time manipulation and visualization of MSC. Main results and the role of chance Sperm viability was significantly higher in the MSC group compared to the control group 6h after formation (72.3 ± 5.0% vs 35.0 ± 4.1%), and after 24 hours, both viabilities decreased drastically to 16.6 ± 3.9% and 7.7 ± 1.7% respectively, probably a possible local antioxidant effect by the microparticles through the formation of a protein corona. In the case of motility, the MSC group presented significantly higher motility up to 6h (64.0 ± 1.5%), and in all the measured times, the motility of the MSC group was higher than the control group. Purification of the micromotor sample was performed, but an average of 63 free sperm was still on the MSC sample. IVF experiments were performed, and blastocyst development was observed in IVF control (2x106 sperm/ml), medium concentration (same concentration as MSC group), and the MSC groups (275.000 sperm/ml). No blastocyst formation was observed in the parthenogenetic, parthenogenetic + particles, and low-concentration groups (number of free sperm). The MSC group was significantly different in cleavage and blastocyst rates compared to the low concentration groups, with p-values of 0.04552 and 0.005865, respectively, which suggest that fertilization may have occurred by the micromotors instead by the free sperm in the sample. Limitations, reasons for caution More studies of these MSC in the different gynecological tissues and on embryo development are needed to ensure the safety of future human translation. In addition, developing a detection system for micromotors in deep tissue is needed to perform in vivo experiments. Wider implications of the findings MSC show an alternative to efficiently guide efficiently sperm cells in different biological environments and can be a promising alternative method for assisted fertilization, and the treatment of a variety of gynecological diseases. They can also be used as exploratory tools to learn more about sperm migration in-vivo, among others Trial registration number European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program No. 853609

P-371 It’s worth the freeze: outcomes from thawing electively cryopreserved oocytes are comparable in patients with reduced vs normal ovarian reserve at the time of cryopreservation

Abstract Study question For patients coming back to use oocytes derived from elective cryopreservation, does their initial ovarian reserve predict thaw survival, embryos’ developmental milestones and pregnancy success? Summary answer Thaw survival, fertilization rate, embryo development and pregnancy success rates do not vary significantly according to AMH level at initial cryopreservation. What is known already Increasing numbers of patients are electing to cryopreserve oocytes for future use. AMH (Anti Mullerian Hormone) is an ovarian reserve marker often assessed prior to initiation of treatment that predicts response to fertility treatment and the resulting oocyte yield. However, it is not known whether oocytes derived from a patient with compromised ovarian reserve (perhaps over multiple treatment cycles) will fare as well as that of a patient with normal ovarian reserve and a higher per cycle oocyte yield. Our study seeks to shed light on whether a lower initial AMH level negatively impacts the prognosis of the thawed oocytes. Study design, size, duration We included in our retrospective study all oocyte thaw cycles resulting from electively cryopreserved oocytes since 1996 at a single academically affiliated fertility center. 325 patients completed a total of 510 thaw cycles. 315 cycles were excluded from analysis for no AMH information. 6376 oocyte cryopreservation cycles were performed over that timeframe. The primary outcomes were laboratory statistics including oocyte survival, fertilization and embryo blastulation rates, while the secondary outcome was cumulative live birth rates. Participants/materials, setting, methods AMH categories of < 1, 1-2 and > 2 were selected to stratify reduced, low-normal, and normal ovarian reserves, respectively. 70.7% inseminated oocytes with partner sperm while the remaining 29.3% used donor sperm. Mean age at time of oocyte cryopreservation was 36.5 (range 27.5 to 46.2) while mean age at time of oocyte thaw was 39.2 (range 27.7 to 47.5). The statistical analysis was performed using ANOVA for mean comparisons and chi-squared for pregnancy data. Main results and the role of chance Average oocyte yield per cryopreservation cycle was significantly different across AMH groups (7.7 oocytes in AMH < 1, 10.5 oocytes in AMH 1-2, and 17.2 oocytes in AMH > 2, p < 0.01). The number of treatment cycles completed was highest in patients with AMH < 1 at 1.26 cycles. Oocyte survival (mean 89.0%), fertilization rate (mean 75.3%), and blastocyst conversion rate (mean 62.4%) did not vary significantly across AMH groups. Time to oocyte use was not significantly impacted by AMH at the time of initial testing. Those with reduced AMH < 1 came back earlier to utilize the oocytes (787 days), vs 1141 days for patients with AMH 1-2 and 1021 days for patients with AMH > 2. However, this difference was not statistically significant, p = 0.4. The rate of PGT-A euploid embryos did not vary between groups (38.8% in AMH < 1, 45.3% in AMH 1-2, 53.0% in AMH > 2, p = 0.07). Clinical pregnancy, miscarriage rate, and live birth rate in the first transfer cycle was not impacted by AMH. Total cumulative live birth rate was not correlated with AMH (31.6% in AMH < 1, 44.4% in AMH 1-2, 47.8% in AMH > 2, p = 0.48). Limitations, reasons for caution This data was collected at a single fertility center and thus has limited generalizability. 8.0% of patients came back to utilize their oocytes, which varies widely across the published literature. 60.6% of patients did not have AMH assessed as part of their pre-treatment testing and were excluded from analysis. Wider implications of the findings Although reduced ovarian reserve impacts oocyte yield, many studies find no impact on resulting oocyte quality, embryo development and pregnancy success rates. Those with compromised AMH have comparable oocyte thaw outcomes to those with normal ovarian reserve, though more oocyte cryopreservation cycles may be needed to achieve a successful outcome. Trial registration number N/A

O-194 Cumulative live birth and neonatal outcomes according to different male factors in IVF/ICSI cycles

Abstract Study question What are the impacts of different male factors on cumulative live birth and neonatal outcomes of IVF/ICSI cycles? Summary answer Severe male infertility factors may be negatively associated with fertilization and egg utilization, embryo development and cumulative live birth rate, but not with neonatal outcomes. What is known already Several studies have explored the effect of semen quality on IVF/ICSI clinical outcomes and have drawn contradictory conclusions because the semen quality of male partners could be fluctuated by many factors and there are many confounding factors from women partner. However, an all-round and definitive conclusion has not been reached yet, and clinicians have not yet determined whether men with abnormal semen parameters should be treated to improve their semen parameters and IVF clinical outcomes. Study design, size, duration The retrospective cohort study involved 6,580 males with abnormal sperm parameters and 17,254 males with normozoospermia who underwent their first IVF/ICSI cycle between January 2018 and September 2022 in the reproductive medicine center at a university hospital. They were divided into five different study groups: normozoospermia (N), moderate male factor (MMF), severe oligoasthenoteratozoospermia (OAT-S), azoospermia-husband (azoospermia-H), and azoospermia-donor (azoospermia-D). We compared fertilization, embryo development, cumulative live birth and neonatal outcome. Participants/materials, setting, methods Reproductive and neonatal outcomes of men with abnormal sperm parameters (MMF, OAT-S, azoospermia-H, and azoospermia-D) were studied through four propensity score matching (PSM) comparisons along with corresponding control groups(N) (match factors: including female age, BMI, Gn time, controlled ovarian stimulation protocol, number of oocytes obtained, endometrial thickness and duration of infertility). Fertilization outcomes were also compared stratified by IVF or ICSI. Main results and the role of chance The median ages of the females in the azoospermia, OAT-S, MMF, and N groups were 29, 30, 32 and 32 years old, respectively, which were comparable among groups after PSM. The fertilization rates were significantly reduced in OAT-S and azoospermia-H compared with N in ICSI cycles (67.05% and 65.05% versus 71.50%, p < 0.001, respectively). The oocyte utilization rates were also significantly declined in OAT-S and azoospermia-H compared with N in IVF/ICSI cycles (28.39% and 29.64% versus 34.83%, p < 0.001, respectively). Azoospermia-H group was divided into obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). The conservative cumulative live birth rate (CLBR) in the OAT-S group with poor spermatogenic function decreased (63.6% vs. 72.9%, P < 0.001), and was also declined in the NOA group with the poorest spermatogenic function (62.4% vs. 72.9%, P < 0.001), compared with that in the OA group with mostly normal spermatogenic function. After binary multifactorial logistic regression, we found that conservative CLBR was lower in the OAT-S (aOR 0.675, 95%CI 0.536-0.850, P = 0.001) and NOA (aOR 0.574, 95%CI 0.384-0.857, P = 0.007) groups than in the OA group. No impact of sperm factor on obstetrical/perinatal outcomes was observed. In IVF/ICSI cycles, reproductive and neonatal outcomes were similar between MMF, azoospermia-D and normal semen groups. Limitations, reasons for caution The main limitation of this study is that it was a single-center, observational and retrospective study. However, the large sample size, use of PSMs and use of a multivariate regression model for a wide array of possible confounding factors rendered the conclusion relatively reliable. Wider implications of the findings This is the first study involving long-term outcomes (obstetrical and neonatal outcomes, cumulative pregnancy and live rates) from IVF/ICSI cycles for different male factors, which can help clinicians identify paternal influence in an all-round way. Trial registration number not applicable

O-294 Human oocyte survival, early embryo development, metabolic fingerprinting, and pregnancy outcomes following ultra-rapid or standard vitrification and thawing

Abstract Study question Is ultra-rapid vitrification and thawing superior to standard vitrification in terms of preserving oocyte viability, embryo competence and enhancing pregnancy outcomes? Summary answer Ultra-rapid vitrification and thawing preserves oocyte viability and embryo quality as confirmed by their euploidy, non-invasive biomarkers in the culture media, and favorable pregnancy outcomes. What is known already Cryopreservation and vitrification protocols for oocytes and embryos have varying cooling rates, cryoprotectant chemical composition and concentration. While vitrification avoids intracellular ice crystal formation, the elevated concentrations of cryoprotectants may be cytotoxic. Over the past 20 years, parameters for vitrification protocols were optimized to improve efficiency and viability of human oocytes and embryos, however few studies have evaluated the metabolic and secretome profiles in addition to embryology and pregnancy outcomes, particularly following ultra-rapid vitrification and thawing. Mid-infrared spectroscopy is a great option for characterizing environmental components and metabolic profiles using the more spectrally complex fingerprint region from 1450-500 cm-1. Study design, size, duration This prospective study included 1,214 donor oocytes collected between January 2022 and November 2023. Sibling oocytes were assigned to standard or ultra-rapid vitrification and thawing. Corresponding blastocysts underwent the same type of vitrification following trophectoderm biopsies for PGT-A. Spent embryo culture fluid was collected on day 1, 3, and 5 for spectroscopy and PCR analysis, as well as on day 6 for metabolic fingerprinting. Clinical pregnancy was assessed 5 days following single embryo transfer. Participants/materials, setting, methods Vitrified oocyte survival was assessed one hour after thawing. Secretome profiling of day 1, 3, and 5 embryos was conducted by Attenuated Total Reflection Fournier Transform Infrared (ATR-FTIR) spectroscopy. Metabolic profiling of blastocysts (day 6) was conducted by Fournier Transform Infrared (FTIR) spectroscopy. Embryology and pregnancy data together with lipid, protein, and nucleic acid expression profiles were evaluated using the Shapiro-Wilk test followed by the student t-test (parametric variables) or the Mann-Whitney U-Test (non-parametric variables). Main results and the role of chance There was 100% survival in oocytes subject to ultra-rapid vitrification-thawing (n = 531) compared to 94.5% in oocytes that underwent standard vitrification (n = 634). There were no significant differences in the fertilization rates following intracytoplasmic sperm injection (81.3% with ultra-rapid and 83.2% with standard vitrification) or potential to reach day 3 of embryo development[RL1]. Ultra-rapid vitrification-thawing produced significantly more blastocysts than standard vitrification-thawing (67.3% vs. 53.0%, respectively) but both techniques had similar euploidy rates (61.8% vs. 61.4%, respectively; p > 0.05). Spectra in the 4000-450 cm-1 region revealed there were no significant differences in lipid and protein expression profiles of day 1, 3, 5 and 6 embryos. The similar result obtained for the culture media for day 1,3,5 and 6 day embryo development with no significant difference. Day 3 blastomere biopsy samples and Day5, Day 6 trophectoderm biopsy sample showed no significant differences for total protein concentration, lipid and nucleic acid experssion profiles in both group.This finding was corroborated by PCR analysis showing no significant difference in nucleic acid profiles. Finally, clinical pregnancy rates were 65.22% with ultra-rapid and 56.5% [RL2] with standard vitrification. (p < 0.05) Limitations, reasons for caution While both our ultra-rapid and standard vitrification-thawing protocols appear to preserve embryo competence to the blastocyst stage and euploidy, future studies should evaluate whether RNA expression levels are altered with these approaches. Wider implications of the findings This study shows competent and euploid embryos can be generated following ultra-rapid vitrification and provides embryo metabolites that can serve as non-invasive biomarkers for the identification of healthy embryos before embryo transfer. Trial registration number NA

P-264 Analysis of embryo development based on time of pronuclei appearance (tPNa) and aspects of overcoming delayed pronuclei appearance through morphokinetic patterns

Abstract Study question How much delay in the appearance of 2PN can be considered normal when predicting embryo transfer potential? Summary answer Embryos with tPNa <13hpi post-insemination develop into good-quality blastocysts; embryos with tPNa ≥13hpi develop blastocysts by shortening the time of tPNf-tPNa. What is known already Oocytes generally exhibit 2PNs at 17–20 hours, with pronuclei fading (time of pronuclei fading; tPNf) at 23–25 hours and developing into two cells (time of 2-cell division; t2) at 25–33 hours. Fertilization confirmation is typically performed 17–18 hours after insemination. However, a few oocytes with no visible PN (0PN) at the time of fertilization confirmation develop morphologically normal blastocysts, ultimately leading to pregnancy. The failure to identify PNs can be attributed to two scenarios: rapid fading or delayed appearance. Notably, there is still a lack of research on the normal range for delayed PN appearance. Study design, size, duration This study was conducted with 2153 embryos obtained from 390 intracytoplasmic sperm injection cycles (August 2021 to June 2023). All embryos were incubated for 5 days using a time-lapse system (EmbryoScopeTM+, Vitrolife). The blastocyst development rate and morphokinetic parameters according to the tPNa of embryos were analyzed using KIDScore D5 v3 and iDAScore v2.0 (VTH server+, Vitrolife). Clinical pregnancy was also analyzed. Participants/materials, setting, methods Morphokinetic parameters were analyzed from time of second polar body (tPB2) ∼ time of expanded blastocyst (tEB). The times taken for PN to appear after the second polar body release (tPNa–tPB2), for PN to fade (tPNf–tPNa) and for 2-cell division (t2–tPNf) were calculated and compared. Blastocysts were graded using the Gardner system, a grade of BB or higher divided into good quality blastocysts (GQ-BL). Main results and the role of chance tPNa was observed as 8.09±2.11hpi [1.93hpi∼32.46hpi; <5hpi (n = 49), 5∼6hpi (n = 474), 7∼8hpi (n = 1220), 9∼10hpi (n = 266), 11∼12hpi (n = 91), 13∼14hpi (n = 29), 15∼20hpi (n = 18) and >20hpi (n = 6)]. The rate of blastocysts was highest at 5∼6hpi (64.98%) and significantly lower at 9∼10hpi (54.14%), 11∼12hpi (42.86%) and 13∼14hpi (31.03%) (p < 0.005). Similarly, the rate of GQ-BL was also highest at 5∼6hpi (29.11%) and significantly lower at 9∼10hpi (19.17%), 11∼12hpi (10.99%) and 13∼14hpi (3.45%) (p < 0.005). No embryos developed into GQ-BL at 15∼20hpi, and no embryos developed into blastocysts at > 20hpi. The iDAScore was significantly different at < 13hpi and ≥13hpi (6.00±1.86 vs. 4.24±2.26, p < 0.005). Similarly, KIDScore D5 showed the same patterns (6.33±1.93 vs. 4.05±1.66, p < 0.005). At ≥ 13hpi, no blastocysts led to pregnancy. Morphokinetic parameters were analyzed to identify the factors influencing the development of blastocysts. The analysis revealed that tPNf-tPNa tended to gradually become shorter with delayed tPNa. Notably, there were significant differences in tPNf-tPNa between <13hpi and ≥13hpi (16.37±4.10 vs. 13.72±6.28, p < 0.005). At ≥ 13hpi, tPNf-tPNa was shorter in blastocysts than in cases of cleavage arrest (10.68±5.74 vs. 15.28±5.97, p < 0.05). It was particularly observed that tPNf-tPNa of GQ-BL in ≥ 13hpi was 7.90hpi, and t2 was not significantly different from blastocysts and GQ-BL in < 13hpi (25.06 vs. 25.66±3.18 vs. 25.19±2.97). Limitations, reasons for caution More data are needed for conclusive pregnancy results due to the small number of samples. Additionally, the maturation of oocytes had yet to be considered; further detailed studies related to oocyte maturity are needed. Wider implications of the findings Embryos with a tPNa of < 13hpi develop into GQ-BL. Embryos with delayed tPNa tend to overcome this by shortening the tPNf-tPNa, resulting in develop into blastocysts. Predicting blastocyst development can be achieved by considering factors such as tPNa∼t2. This can aid in improving the selection of embryos for cleavage-stage ET. Trial registration number N/A

P-222 Can embryo morphokinetics act as early warning key performance indicators in relation to consumable batching

Abstract Study question Can morphokinetics be used as an early warning indicator of a batch-related effect of oil currently in use in the laboratory on implantation rate? Summary answer Where morphokinetic timings were slower for a batch of oil, implantation rate was also reduced, suggesting they could be used as an early warning system. What is known already The commercialisation of oil means that there are strict manufacturing guidelines leading to consistency between batches. However, this does not mean that minor differences do not exist. Furthermore, oil can undergo peroxidation if stored incorrectly, causing it to become toxic to embryos. The introduction of timelapse incubation has allowed extensive research into several morphokinetic parameters. This research has established time-frames within which each developmental milestone should occur to indicate the likelihood of implantation. Implantation rate (IR) is often used as a key performance indicator in the laboratory however, potential systemic issues may be identified sooner if morphokinetic parameters were used. Study design, size, duration The following morphokinetic parameters were compared to IR to determine if a batch of oil was affecting embryo development; time of division to two cells (t2), five cells (t5), eight cells (t8), time to start of blastulation (tSB) and cell synchronicity between two and eight cells (CS2-8). Data from January 2019 to October 2019 from a single centre were used, and included data from 2008 embryos and 333 patients. Participants/materials, setting, methods Data was exported from the EmbryoScope + [Vitrolife, Sweden]. Data from RIWitness Laboratory Manager [CooperSurgical, Denmark] was utilised to identify when different batches of oil (Ovoil™, Vitrolife) were used within the specified time period. The data from the EmbryoScope+ was then separated by date to signify when a new batch of oil was open. A Kruskal-Wallis test was then used to identify whether the morphokinetic timings differed between batches of oil. Main results and the role of chance There was no significant difference in patient age (p = 0.178) or the number of oocytes collected (p = 0.097) between batches of oil used. There was no significant difference between the morphokinetic timings for each batch of oil used. However, the morphokinetic timings follow the same trend line as the IR for both t2 and t8; where t2 and t8 are higher, the IR for the corresponding batch of oil is reduced. The trend can also be seen in CS2-8. The batch of oil with the lowest IR (25.56%) also had the highest mean time to t2, t8 and tSB compared to the other batches of oil. This corresponds with previous research that identified a correlation between slower embryo development and reduced IR (Meseguer et al., 2011). A clear difference could be seen between the average time taken to reach each developmental milestone and each batch of oil used, perhaps suggesting oil can have an effect, however small, on embryo development. This study suggests that morphokinetic timings could be used as an early warning indicator that a new batch of oil may be affecting development allowing a rapid response by the laboratory team and possible cessation of use pending further investigation. Limitations, reasons for caution Different batches of culture medium were also used throughout this time period and were not introduced at the same time as oil batches. Therefore, the results of this investigation cannot be associated entirely to the introduction of a different batch of oil. Wider implications of the findings This information could be incorporated into an early warning algorithm that could be assessed after a batch of oil was introduced. The use of the batch could then be ceased immediately without waiting for a decrease in IR by which time it would have been used for many more embryos. Trial registration number Not applicable

P-515 The morphokinetic signature of high-level mosaic embryos in time-lapse ART analysis

Abstract Study question Do high-level mosaic embryos exhibit differences in developmental speed compared to euploid or low-level mosaic embryos? Summary answer High-level mosaic embryos exhibit slower development than euploid and low-level mosaic embryos during the blastulation and blastocyst formation stages. What is known already Time-lapse technology (TLT) has facilitated the real-time observation of human embryo development without disrupting culture conditions, generating a wealth of morphokinetic data linked to outcomes such as implantation potential, blastocyst development, and ploidy status. However, the developmental patterns of mosaic embryos based on their aneuploidy level using TLT remain not well understood. Consequently, we aimed to assess the clinical significance of morphokinetic parameters in human embryo development, with a specific focus on embryos categorized by their mosaic status. Study design, size, duration This retrospective study examined 385 blastocysts undergoing preimplantation genetic testing for aneuploidy (PGT-A) between January and December 2022. Out of the total, 191 expansion blastocysts underwent PGT-A biopsy on day 5. The PGT-A results, which categorized embryos into euploid, aneuploid, and mosaic based on their genetic composition, were determined through next-generation sequencing (NGS) by an analysis company. Mosaic embryos were further categorized into two groups: high-level mosaicism (>30% aneuploidy cells) and low-level mosaicism (≤30%). Participants/materials, setting, methods Differences in development rates were assessed through the examination of morphological and morphokinetic parameters. These parameters included the time to second polar body extrusion (tPB2), time to pronuclei appearance and fading (tPNa, tPNf), time to reach 2–8 cells (t2, t3, t4, t5, t6, t7, t8, t9), and time to the onset of blastulation and blastocyst formation (tSB, tB). Statistical analyses, incorporating ANOVA and post-hoc tests, were employed to compare developmental timings across different groups. Main results and the role of chance When categorizing mosaic embryos based on their level, high-level mosaic embryos showed a significantly slower developmental rate than low-level mosaic embryos, evident at both the blastulation stage (tsB, high-level vs. low-level: 101.80h vs. 97.3h, p=0.003) and blastocyst stage (tB, high-level vs. low-level: 109.98h vs. 105.92h, p=0.005). In comparison to euploid embryos, low-level mosaic embryos demonstrated comparable outcomes. Conversely, high-level embryos exhibited a significant difference in both blastulation (tsB, euploidy vs. high-level: 98.85h vs. 101.80h, p=0.026) and blastocyst (tB, euploidy vs. high-level: 106.73h vs. 109.98h, p=0.011) stages. These findings suggested that the mosaic level of embryos might influence developmental kinetics, proposing this approach as an additional tool for the selection of mosaic embryos in the context of embryo transfer. Limitations, reasons for caution This study focused on morphokinetic parameters captured using time-lapse imaging, which may not fully represent the complex cellular dynamics within embryos. Furthermore, the categorization of mosaic embryos into high-level and low-level groups based on the percentage of mosaicism (>30% and ≤30%, respectively) might not capture all embryo developmental potential. Wider implications of the findings The findings suggest valuable predictive potential in assessing the development of aneuploid and mosaic embryos. Further research into the development of euploid, aneuploid, and mosaic embryos, as well as high-level and low-level mosaic embryos, could provide valuable insights for ART practices. This could significantly impact ART decision-making and patient outcomes. Trial registration number not applicable

P-021 Development of recombinant Phospholipase C Zeta protein as a therapeutic intervention to overcome certain types of male infertility

Abstract Study question Developing recombinant Phospholipase C Zeta (PLCζ) protein using a bacterial expression system to be utilized for potential future replacement of Ca2+ ionophores during IVF treatments. Summary answer Our findings support the use of MBP-PLCζ recombinant protein as a therapeutic tool for treating certain types of male factor infertility associated with fertilization failure What is known already The long-term safety of calcium ionophores, which are exclusively used for treating cases of fertilization failure, is still questionable. PLCζ is an endogenous agent that may represent a better replacement for treating male infertility cases related to fertilization failure Study design, size, duration Maltose binding protein (MBP)-tagged PLCζ was produced by cloning wild-type PLCζ into a pET41MM vector and purified using affinity chromatography techniques. Circular dichroism (CD) was employed to evaluate the secondary structure and the thermal denaturation of PLCζ protein. Moreover, the PIP2 hydrolysis assay was used to test the enzymatic activity of PLCζ protein over time and under different storage conditions. Furthermore, the embryonic toxicity of the protein was tested using a chick embryo model. Participants/materials, setting, methods Several methods were used in this study including Molecular cloning, circular dichroism, and chick embryo model to investigate the embryonic viability. Main results and the role of chance Our findings demonstrated that PLCζ recombinant protein displayed a properly folded secondary structure mainly composed of alpha-helices. Moreover, the protein was effective in maintaining the enzymatic activity and Ca2+ sensitivity for 90 days when stored at -80oC. Moreover, exposing chicken eggs to MBP-PLCζ did not alter the embryonic viability when compared to the control group. Kaplan-Meier survival curve showed that the rate of survival did not differ between the treatment, control and sham groups until the endpoint of the study. Limitations, reasons for caution The toxicology assays used are not sufficient to confirm the safety of the protein. Further in vivo studies are required to investigate the effect of MBP-PLCζ protein on early embryonic development before being used as a replacement for calcium ionophores in cases of oocyte activation failure. Wider implications of the findings These findings can help develop a new therapeutic tool for treating cases of male infertility associated with oocyte activation failure. Trial registration number not applicable

P-527 Low sperm motility and elevated sperm DNA fragmentation index (DFI) are associated with increased rates of embryo aneuploidy in patients with advanced reproductive age

Abstract Study question Is there a correlation between the paternal age, sperm characteristics, and the prevalence of chromosomal abnormalities in embryos generated through in vitro fertilization (IVF)? Summary answer A correlation exists between advanced paternal age, suboptimal sperm parameters, and elevated rates of embryo aneuploidy in individuals of advanced reproductive age. What is known already The exact mechanisms underlying the relationship between paternal age, sperm quality and embryo ploidy, although a subject of considerable research in assisted reproduction, are not fully elucidated. Some reports, including our group’s, suggest that paternal age may influence sperm quality and segmental embryo aneuploidy, while others suggest a potential association between high DFI(DNA Fragmentation Index) and an increased likelihood of embryo aneuploidy. Studies also show that sperm with optimal motility and fertilization competence are more likely to contribute to the development of euploid embryos, while data on the effect of abnormal sperm parameters on embryo ploidy is scarce and conflicting. Study design, size, duration This was a retrospective cohort study performed in the Reproductive Genetics Department, CReATe Fertility Centre, Toronto, Canada, between January 2019-December 2023. A total of 7628 blastocysts from 1512 cycles (1392 patients) were analyzed using high resolution NGS PGT-A. Sperm motility, concentration and DFI data, as well as, IVF cycle parameter data were collected. In addition, age of gamete providers and any relevant clinical, laboratory and demographic data were obtained for statistical analysis. Participants/materials, setting, methods Embryos were allocated into two groups by oocyte-provider age: OP-≤33 years and OP-≥34 years. Each group was divided into two sub-groups by paternal age: SP-≤39 years (n = 4570); SP-≥40 years (n = 3058). Multiple logistic/ordinal regressions were conducted to evaluate Segmental abnormalities and Segmental mosaicism between these age groups in relation to sperm DFI (GOOD ≤15%; FAIR 15-30%; POOR ≥30%), Motility (HIGH >40%; LOW <40%) and Count (LOW <5 mil/ml; MEDIUM 5-15 mil/ml; HIGH >15 mil/ml). Main results and the role of chance PGT-A analysis of 7628 embryos showed 51.5% euploidy, 36.7% aneuploidy and 11.8% mosaicism. In the OP-≤33 group, there were no significant differences between the two paternal age groups for euploidy, aneuploidy or mosaicism rates. In the OP-≥34 group, decreasing euploidy rates (p < 0.0001 and increasing aneuploidy rates (p < 0.0001) were observed between the two paternal age groups, while mosaicism rates were not different, an expected oocyte age-effect result. No significant differences in aneuploidy rates were observed for each of the sperm parameters (DFI, motility, count) in the groups of the OP-≤33 with SP-≤39, OP-≤33 with SP-≥40 and OP-≥34 with SP-≤39. In the group of OP-≥34 with SP-≥40, in patients with LOW motility vs. HIGH motility the aneuploidy rates were higher (56.8% vs.50.6%, p = 0.005) and euploidy rates were lower (33.8% vs.38.4%, p = 0.03). Each sperm parameter in each age group was further analyzed for chromosomal segmental abnormalities and segmental mosaicism. Higher rates of segmental abnormalities were seen in LOW motility groups with POOR-DFI compared to GOOD DFI in OP-≤33 with SP-≤39 (3.2% vs. 7.4%, p = 0.03). Limitations, reasons for caution The main limitation was the retrospective nature of this study; however, multiple regression analyses were conducted to limit potential bias by adjusting for sperm and oocyte characteristics. In addition, PGT-A analysis cannot provide information on origin of the aneuploidy and all conclusions are deduced from indirect associations to paternal age. Wider implications of the findings The identified associations could stimulate further research into exploring the molecular/cellular processes involved in fertilization, early embryonic development, and the potential impact of sperm-related factors on the chromosomal stability of developing embryos. The broader implications of these findings extend to clinical practice, patient counseling, technological advancements, and ethical considerations. Trial registration number not applicable

Twin Embryos in Arabidopsis thaliana KATANIN 1 Mutants.

Regulation of microtubule dynamics is crucial during key developmental transitions such as gametogenesis, fertilization, embryogenesis, and seed formation, where cells undergo rapid changes in shape and function. In plants, katanin plays an essential role in microtubule dynamics. This study investigates two seed developmental mutants in Arabidopsis thaliana, named elk5-1D (erecta-like 5, ELK5) and loo1 (lollipop 1), which are characterized by round seeds, dwarfism, and fertility defects. Notably, elk5-1D exhibits a dominant inheritance pattern, whereas loo1 is recessive. Through positional cloning, we identified both mutants as new alleles of the KATANIN 1 (KTN1) gene, which encodes a microtubule-severing enzyme critical for cell division and morphology. Mutations in KTN1 disrupt embryo cell division and lead to the emergence of a twin embryo phenotype. Our findings underscore the essential role of KTN1 in fertility and early embryonic development, potentially influencing the fate of reproductive cells.