Embryo Viability Research Articles

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-553 Embryo collection improves clinical pregnancy outcomes in next-generation sequencing(NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) euploid embryo transfers

Abstract Study question Is there still a significant impact of embryo-related characteristics on pregnancy rates in NGS based PGT-A euploid embryo transfers for advanced-age patients? Summary answer In single frozen euploid embryo transfers, priority of embryo transfer and grade significantly influence clinical pregnancy rates in women over 35 years old with PGT-A. What is known already It is widely recognized that better embryo grades and a shorter duration of blastulation are associated with favorable reproductive outcomes. Recent meta-analyses have affirmed these findings even in the context of transfer with euploid embryos. However, the impact of prioritizing the highest-ranked embryo among multiple vitrified euploid embryos on pregnancy rates remains unclear. The lack of research in this area implies a gap in understanding whether selecting embryos for transfer among multiple viable euploid embryos could enhance reproductive success. Study design, size, duration Throughout the year 2022, a retrospective review was conducted on 428 cycles of single frozen-thawed euploid embryo transfers performed at a single institution, targeting patients aged 35 and older. The study aimed to explore potential differences in clinical pregnancy rates based on embryo grade, embryo freezing date, and embryo selection priority. Participants/materials, setting, methods Women ≥ 35 years old with single frozen euploid blastocyst transfer cycles was categorized into three groups: (a) with one blastocyst (1/1), (b) with ≥ two blastocysts having the top-priority(½ or higher), and (c) lower-priority blastocysts (lower than ½) to examine potential differences in clinical pregnancy rates among groups based on embryo characteristics, including Gardner grading and day of embryo freezing. Results highlight the impact of both selection criteria and freezing conditions on pregnancy outcomes. Main results and the role of chance This study analyzed a total of 428 frozen embryo transfer (FET) cycles. In the context of frozen-thawed single embryo transfer, the clinical pregnancy rate was 52.2% (128/245) when only one euploid embryo was frozen and subsequently thawed for transfer (a). For cases where two or more euploid embryos were frozen, and embryos with a higher priority (½ or higher) were selected for transfer (b), the clinical pregnancy rate significantly increased to 71.3% (62/87). Conversely, when embryos with a lower priority (lower than ½ in order) were chosen for transfer (c), the clinical pregnancy rate was lower at 38.5% (15/38), indicating a statistically significant difference between the groups (p = 0.001). Categorizing embryos based on Gardner grading, “good” embryos (>3BB) demonstrated a clinical pregnancy rate of 66.8% (141/211), while embryos with lower quality (3BB or below) showed a significantly lower clinical pregnancy rate of 40.6% (88/217) (p < 0.001). The analysis of clinical pregnancy rates based on the embryo freezing day revealed significant differences among embryos freezing on 5 days (71.9%, 133/185), 6 days (39.9%, 71/178), 7 days (25%, 2/8), and thawing PGT-A embryos (38.2%, 21/55) (P < 0.001). Limitations, reasons for caution One limitation of this study is its retrospective nature, and the potential confounding effect of the close association between embryo selection and embryo grading. Further analysis will be conducted to confirm correlations and address this potential confounding effect. Wider implications of the findings In single euploid frozen embryo transfers, selecting the highest-quality embryo through careful embryo selection remains relevant. Morphological indicators of embryos significantly impact early pregnancy outcomes, highlighting the continued importance of strategic embryo accumulation for enhanced procedural success. Trial registration number not applicable

O-027 Efficacy results from the phase II randomized clinical trial: OXO-001 in infertile women undergoing egg donation IVF/ICSI

Abstract Study question Does OXO-001 increase implantation and pregnancy rates in women undergoing egg donation IVF/ICSI and embryo transfer (ET) compared to placebo? Summary answer This proof-of-concept (PoC) prospective randomized and controlled study shows that OXO-001 treatment increases implantation and pregnancy rates in infertile women undergoing IVF/ICSI with studied doses. What is known already OXO-001 is a drug being developed as an innovative treatment to enhance embryo implantation through a direct effect on the endometrium. Preclinical results show an increase in embryo implantation and viability. Moreover, they show that OXO-001 is safe for the mother, the fetuses, and the newborns without any deleterious effect on the development, viability, and implantation potential of OXO-001-exposed embryos. Neither conceptus development nor morphological disturbances have been observed in the deciduas of treated-pregnant mice. Furthermore, clinical trials confirmed that OXO-001 has the necessary wide safety margin and non-complex kinetics, allowing further research on its therapeutic potential in infertility. Study design, size, duration It was a randomized, double-blind, placebo-controlled, and multicenter phase II clinical trial with three parallel arms conducted at 28 European centers (September/2021-January/2023. EudraCT Num:2021-000001-25). A subset of women ≤40 years were analyzed. A total of 173 were randomized in the three study arms (Placebo, 200 and 300mg/day OXO-001). Treatment started one menstrual cycle before the ET cycle and continued until 5 weeks after ET. The results of this communication are focused on the 300mg/day dose. Participants/materials, setting, methods Patients were infertile women (18-40 years; BMI 18-30 kg/m2) eligible for a fresh-single blastocyst (day 5 and ≥3BB) ET resulting from a donor egg. Exclusion criteria include gynecological abnormalities and history of ≥ 2 failed ET, among others. The primary endpoint was ongoing pregnancy rate (OPR). Secondary included biochemical and clinical pregnancy (BPR and CPR), live birth rate (LBR), and safety and tolerability of OXO-001. Main results and the role of chance 111 women were randomized, and 96 performed a single ET (42 Placebo, 54 OXO-001 300mg/day). Higher BPR, CPR, OPR, and LBR per delivery after were observed in the OXO-001 group versus placebo (75.9% vs. 52.4%*, 50.0% vs. 37.5%, 46.3% vs. 35.7%, 42.6% vs. 35.7%, respectively. *p<0.05). The logistic regression analysis showed a statistically significant difference in the BPR for OXO-001 versus placebo (OR 3.03, 95% CI 1.17-7.85; p = 0.022), and a clinically meaningful difference for CPR, OPR, and LBR after a single ET. In safety evaluation, the frequency of treatment-emergent adverse events (TEAEs) assessed as at least possibly related to the study medication were similar between groups (14 for OXO-001 and 10 for placebo). Most TEAEs were mild or moderate in severity. The most common TEAES were headache, nausea and vomiting, gastrointestinal and dizziness. The assigned study medication was well tolerated, and relevant changes in laboratory analyses (hematology and biochemical profile) were not observed. Limitations, reasons for caution This PoC study was designed to define clinically meaningful differences in fertility outcomes and find patient profiles. It was not empowered to find statistical differences in the OPR. An empowered phase III study is needed to confirm the positive results from phase II. Wider implications of the findings his phase II PoC has achieved its objectives showing that OXO-001 increases more than 10% pregnancy rates in infertile women undergoing IVF/ICSI, with a safe and well-tolerated profile. This is a step forward toward the first therapeutic tool to increase embryo implantation and ET success. Trial registration number EudraCT Number: 2021-000001-25

P-681 LH priming before ovarian stimulation in POSEIDON-4 patients: effects on oocyte recovery and post ICSI/IVF pregnancy rates

Abstract Study question Can a seven-day long LH priming treatment preceding ovarian stimulation (OS) improve follicular responses, oocyte yields and ICSI/IVF outcomes of POSEIDON-4 patients? Summary answer A seven-day long LH priming can substantially increase pregnancy rates through specific benefits in different subgroups of POSEIDON-4 patients. What is known already The combination of advanced maternal age (AMA) with diminished ovarian reserve (DOR) has become one of the most relevant and complex topics in the field of reproductive medicine. AMA/DOR patients appear to be refractory to increased gonadotropin dose and may benefit from strategies specifically capable of increasing the population of FSH-responsive small follicles before the onset of ovarian stimulation (OS). Androgen mediated LH intra-ovarian activity stimulates early follicular development. Pre-treatment with LH has been suggested to improve ICSI/IVF outcomes in normal responders but has not been assessed in AMA/DOR patients, despite the prevalence and relevance of this population. Study design, size, duration Retrospective cohort study from 2022 to 2023, including 235 POSEIDON-4 patients stimulated with FSH+LH in a flexible antagonist protocol with oestradiol pre-treatment (n = 129) or with the same stimulatory treatment preceded by a seven-day LH priming (150 IU/day) combined with GnRH agonist downregulation (n = 106). Follicular responses, oocyte recovery and ICSI/IVF outcomes were compared between treatment-groups in overall POSEIDON-4 patients and in a subgroup of patients with AMH <0.75 ng/mL (n = 77/Control, n = 59/LH priming). Participants/materials, setting, methods All participants were POSEIDON-4 caucasian patients aged 35 to 44 years undergoing a single homologous ICSI/IVF cycle in our clinic. Outcomes in the form of percentages were compared with the Fisher’s exact test, whereas differences in continuous variables were assessed with the Wilcoxon sum rank test. A multivariate analysis was performed to control for the interference of unequal variations in maternal age, AMH, insemination method and presence of male subfertility. Main results and the role of chance Patients in different treatment groups did not differ (p > 0.05) regarding maternal age (39.2±2.5 vs. 39.3±2.5), basal FSH serum concentrations (11.0±4.6 vs. 10.1±4.3) and antral follicle count (6.3±3.0 vs. 6.4±3.3, for patients stimulated with and without LH priming, respectively), but patients treated with LH priming presented slightly lower AMH serum levels (0.56±0.3 vs. 0.63±0.3; p = 0.05). Nevertheless, patients treated with LH priming achieved a twice higher clinical pregnancy rate per cycle (19.8% vs. 9.3%; p = 0.02), accompanied by a 40% reduction in cycle cancellation (32.1% vs. 45.0%; p = 0.03) and increased production of viable embryos (transferred fresh + frozen) per cycle (1.22±1.09 vs. 0.94±1.07; p = 0.02) and per oocyte inseminated (48.3% vs. 38.5%; p = 0.01), but did not differ regarding follicular outputs or oocyte yield. The multivariate analysis indicated a robust association between LH priming and pregnancy achievement, independently of potential confounding variables (OR = 2.52; p = 0.02). A two-fold increase in pregnancy achievement was also observed in severely restricted POSEIDON 4 patients (AMH <0.75 ng/mL) subjected to the LH priming (p = 0.05), which in this case was accompanied by higher oocyte yields per cycle (3.3±2.8 vs. 2.2±2.0; p = 0.04) and per pick (3.9±2.6 vs. 2.3±2.0; p < 0.01). Limitations, reasons for caution This study is limited by its retrospective nature and the data may be potentially influenced by the use of different downregulation strategies in the treatment-groups, even if the literature indicates equivalent performance for these strategies in the population under analysis. Wider implications of the findings The present data strongly suggest that a seven-day LH priming preceding OS can markedly improve ICSI/IVF outcomes of POSEIDON-4 patients, while indicating that this strategy benefits in different ways subgroups of this increasingly prevalent patient category. These findings thus represent new valuable parameters for the progress of ovarian stimulation. Trial registration number NA

P-269 Three-dimensional assessment of the inner cell mass is more effective at predicting implantation and live birth than Gardner grading

Abstract Study question 3-dimensional quantitative assessment of the inner cell mass (ICM) is more effective at selecting viable embryos than the widely used Gardner grading system? Summary answer 3-dimensional quantitative assessment of the ICM is significantly more predictive of implantation and live birth than the widely used Gardner grading of the ICM What is known already ICM assessment forms a key part of most widely used blastocyst grading systems. Grading under these systems, however, is often qualitative, subjective, and subject to inter/intra-operator variability. To reduce subjectivity in ICM grading, it has been suggested that quantitative criteria are used. Previous studies have focused on the assessment of such parameters (e.g. ICM area) in 2 dimensions. However, such approaches fail to account for the various embryo orientations which could lead to different measurements. Therefore, quantitative 3-dimensional analysis of the ICM is needed to improve the reliability and consistency of grading. Study design, size, duration This was a retrospective analysis of 153 randomly selected tEB embryos from two clinics. All data was captured on Embryoscope between 2018 and 2020. Data for maternal age, blastocyst grade, blastocyst ploidy, implantation and live birth rates were also recorded. Participants/materials, setting, methods Embryos were imaged at 7+ focal planes and in-focus sections of each ICM were annotated using ImageJ. These sections were combined into a 3D model using Blender, and sphericity (ψ) computed. Embryos were categorised as spheroid (ψ ≥ 0.8), ellipsoid (ψ < 0.8), crescent and irregular. Chi-squared tests were performed, comparing classification and clinical outcome. The predictive power of the system was compared to the Gardner scale (spheroid/ellipsoid classifications and A grades considered predictive of a positive outcome respectively). Main results and the role of chance Most ICM were either ellipsoid (48%) and spheroid (37%) rather than crescent (6%) or irregular (8%, p < 0.001). ICM 3D classification was associated with implantation, X2(3, N = 119)= 11.10, p = 0.01 and live birth, X2(3, N = 119)= 10.78, p = 0.01. Blastocysts with ellipsoid (70%, 28/40) and spheroid (58%, 18/31) shaped ICM had the greatest chances of implantation, whilst those with crescent (40%, 6/15) and irregularly shaped ICM (33%, 11/33) had lower chances of implantation (p < 0.011). Similarly, blastocysts with ellipsoid (58%, 23/40) and spheroid (48%, 15/31) shaped ICM had the greatest chances of live birth, whilst those with crescent (33%, 5/15) and irregularly shaped ICM (21%, 7/33) had lower chances of live birth (p < 0.013). 3D assessment of ICM outperformed Gardner ICM grading with regards to prediction of implantation (accuracy 64.7% vs 49.6%, area-under-the-curve 0.642 vs 0.486, precision 64.8% vs 51.9%, sensitivity 73% vs 65.1%, specificity 55.4% vs 32.1%, F1 score 68.7% vs 57.8%, 3D vs Gardner) and live birth (accuracy 62.2% vs 50.4%, area-under-the-curve 0.641 vs 0.531, precision 53.5% vs 44.3%, sensitivity 76% vs 70%, specificity 52% vs 36.2%, F1 score 62.2% vs 54.3%, 3D vs Gardner). Limitations, reasons for caution Manually rendering the 3D images for each ICM was time consuming, making it impractical for clinical application. Further work is now focusing on automating this process using image recognition and augmented reality. Wider implications of the findings This is a novel ICM assessment that provides an improved perspective of the 3-dimensionality of the ICM which is more consistent and effective than traditional methods of embryo assessment, potentially contributing towards improving embryo selection efficacy and time to pregnancy. Trial registration number none

P-140 Does assessment of time-lapse videos result in improved prediction of clinical pregnancy compared to assessment of single images of blastocysts?

Abstract Study question Is there a difference in performance between artificial intelligence(AI) algorithms that predict the likelihood of pregnancy from a single blastocyst image compared to time-lapse videos? Summary answer The performance of AI to predict pregnancy from a single blastocyst image was similar to the performance of an AI using time-lapse video. What is known already Vitrolife’s “iDAScore (iDA)” and Presagen’s Life Whisperer Viability (LW) are AI systems trained on videos (iDA) or images (LW) of blastocysts, with corresponding pregnancy outcomes (fetal heartbeat). iDA requires a dedicated time-lapse incubator and uses multiple images to predict pregnancy. LW requires only one image taken with a camera that meets image quality requirements. However, as tools for selecting viable embryos for transfer, these methods adopt different approaches: iDA relying on multiple morphokinetic timepoints to achieve prediction based on past embryo development, and LW relying on predictive analytics from morphological assessment at the decision point prior to transfer. Study design, size, duration 359 embryos cultured in EmbryoScope +/8 that reached blastocyst stage (Gardner classification ≥1) on Day 5 were utilised for first single vitrified blastocyst transfer (between 2018-2021). Pregnancy prediction was scored from EmbryoScope’s embryo images using iDA and LW, and a dataset was created with fetal heartbeat as the outcome. Participants/materials, setting, methods iDA data was classified into quartiles (Low, Medium, High, Very High) and LW into the manufacturers’ recommended four categories (Low, Medium, High, Very High). The trend of pregnancy outcome and respective scores were evaluated using a trend test (Cochran-Armitage test). Additionally, the Quadratic Weighted Kappa coefficient was used to evaluate the concordance of the categories. The Area Under the Roc Curve (AUC) for each prediction accuracy was also determined and compared using Delong’s test. Main results and the role of chance A trend of increasing pregnancy rate was observed for both methods as the score category increased (iDA: Low 35.2%, Medium 46.9%, High 52.1%, Very High 65.4%; LW: Low 36.0%, Medium 46.3%, High 60.5%, Very High 63.4%). The respective AUCs were iDA: 0.635 LW: 0.627, (not significantly different; P = 0.732). The Quadratic Weighted Kappa coefficient was 0.622, indicating that the categories of evaluation were consistent. Limitations, reasons for caution This is a retrospective study performed in a single clinic. In addition, the study was limited to transfer cycles of embryos where blastocyst formation was reached on day 5 of culture. Wider implications of the findings These results indicate that an AI that predicts pregnancy from a single blastocyst image can perform equally well compared to AI that predicts pregnancy from time-lapse videos. This suggests that tracking of morphokinetic timepoints during embryo development may be less important than end-point morphology for predicting pregnancy at blastocyst stage. Trial registration number not applicable

O-035 Euploidy’s hidden potential: emerging insights from abnormal fertilized embryos

Abstract Study question Can pregnancies be achieved from abnormally fertilized embryos? Summary answer After conducting parental origin PGTA testing to confirm ploidy status, achieving pregnancy and live births becomes feasible. What is known already A major limitation in IVF is the availability of viable embryos per cycle. Particularly relevant for patients with poor prognosis in response to controlled ovarian stimulation. In these couples, the limited number of oocytes results in a significant reduction of the chance to achieve a live birth. In IVF, zygotes displaying successful extrusion of the 2PB and 2PN are considered “normally fertilized” and cultured further to be used for treatment. In contrast, zygotes showing a single or more than 2PN are deemed abnormally fertilized. However, some can ultimately develop into normal embryos, which may lead to live birth. Study design, size, duration This is a single centre retrospective observational study of 125 blastocysts classified as abnormal through standard fertilization check post-IVF (50 embryos) and ICSI (75 embryos) Embryos were subjected to genetic testing utilizing a validated targeted next-generation sequencing-based PGT-A protocol. The protocol's proven efficacy lies in distinguishing between haploid, diploid and triploid samples, a distinction made possible by the parallel analysis of genotyping data and chromosome copy number variations. Participants/materials, setting, methods The participants in this study were couples who underwent IVF cycles using PGT-A between 2021-2024. Embryos were divided into two groups based on their fertilization method Group A involved blastocysts derived from In vitro fertilization (IVF) = 50. Classified 1PN, micro 3PN, 3PN, and 4PN. Group B blastocysts derived from Intracytoplasmic sperm injection Intracytoplasmic sperm injection (ICSI) =75 Classified 1PN, micro 3PN, 3PN, and 4PN. Main results and the role of chance Group A: Involved embryos resulting from IVF; 54% were diploid, with 15% being euploid from 3PN. From 1PN, 92% were diploid, and 54% were euploid. Micro PN had a 100% diploid rate, of which 33% were euploid. No diploid embryos were reported from 4PN fertilization. Group B: Involved embryos resulting from ICSI; 36% were diploid, and 14% were euploid from 3PN. From 1PN, 53% were diploid, and 40% were euploid. Micro PN had a 100% diploid rate, with 23% being euploid. 4PN had a 50% diploid rate and no euploid embryos. On the other hand, 0.4% (17/4822) of 2PN-classified embryos were reported as haploid, and 1.4% (68/4822) of the 2PN-classified embryos were reported as polyploid. Eighty-nine percent of haploidy and polyploidy in ICSI originated maternally, while 71% of polyploidy in IVF originated paternally and only one embryo was haploid resulted from IVF was maternally originated. Remarkably, three live births were reported: one from 1PN, one from Micro PN, and the third from 3PN. Limitations, reasons for caution The data are observational and were retrospectively analyzed, so unknown confounders could not be assessed. Wider implications of the findings To our knowledge, this is the most extensive cohort study analyzing embryos resulting from abnormal fertilization. It has implications for clinical practice and gives an extra chance for patients, particularly those with a low number of eggs, in response to controlled ovarian stimulation. Trial registration number n/a

O-214 The effect of constant 5% versus gradient 8%, 5%, 2% oxygen concentration on the development of sibling human embryos in vitro to the blastocyst stage

Abstract Study question Does a physiological gradient of oxygen concentration in embryo culture atmosphere yield a higher percentage of fertilized oocytes developing into morphologically optimal blastocysts? Summary answer Study favours sustaining a static 5% oxygen level, instead of a gradient, during extended embryo culture, resulting in more viable embryos for cryopreservation and transfer. What is known already To date, there’s ongoing research about the optimal oxygen concentration for embryo culture. The available data from studies based on animal models show that the O2 level in the fallopian tube is around 8%, and the in-utero O2 level is at around 2% (Wale & Gardner, 2010). Data on oxygen measurement in the human uterus is scarce, yet aligns with similar findings in other mammalian species (Ng et al., 2018). These findings support the use of sequential O2 levels for embryo culture. Our study stands as the first to implement such dynamic oxygen culture conditions within an in vitro setting. Study design, size, duration A prospective sibling-split oocyte study, analyzing 44 ICSI cycles from January 2022 to January 2023, with alternate allocation into a static 5% (Control group) during entire culture period or under an oxygen gradient (intervention group), starting with 8% from day-0 to day-3, continuing with 5% on day-3 and following with 2% of oxygen from the end of day-3 to day-5/6. Participants/materials, setting, methods 44 ICSI cycles, which produced a total of 521 metaphase II oocytes. A total of 353 2PN embryos were cultured and annotated in time-lapse for morphokinetic and morphometric analysis. Main results and the role of chance While morphological outcomes slightly favored the intervention group for day 2 and day 3 embryos, the trend reversed by day 5. The control group (5% O2) showed a notably higher proportion of zygotes developed to clinically utilized blastocysts (47% vs. 37%, p = 0.016) and consequently more of them were used for transfer on day 5 (10% vs. 4%, P = 0.008) compared to the intervention group. Additionally, though not statistically significant, the control group showed a higher percentage of morphologically optimal blastocysts on day 5 (10.3% vs. 14.7%, p = 0.1233). Morphokinetic timings, as revealed by time-lapse analysis, indicated a delayed development in the intervention group, notably seen in the initiation of blastulation (p = 0.0024), initiation of expansion (p < 0.0001), and full blastocyst formation (p < 0.0001). The speed of blastulation, however, does not necessarily reflect differences in implantation potential, as implantation (58.6% vs 57.1% p > 0.92) and clinical pregnancy rates (55.2% vs 57.1%, p > 0.92) after transferring blastocysts from either group were comparable. Limitations, reasons for caution Measuring the in vivo O2 levels in female reproductive tract should take into account possible oscillations and dynamic nature of gas and nutrient exchange. It’s also important to acknowledge the complexity of embryo’s adaptive response, making it challenging to attribute observed outcomes solely to the impact of targeted oxygen concentration. Wider implications of the findings Based on these clinical results, we will analyze the metabolomic footprint in spent embryo culture under static 5% or gradient 8-5-2% oxygen concentration, employing non-invasive NMR and chromatography methods. A randomized control trial should be conducted to explore the effect of different oxygen regimes on clinical outcomes. Trial registration number NCT05898178

P-664 Insulin-like peptide 3 as a potential biomarker in predicting IVF success and ovarian reserve in women with unexplained infertility and diminishing ovarian reserve

Abstract Study question Could insulin-like peptide 3 (INSL3) predict ovarian reserve and in vitro fertilization (IVF) success in women with unexplained infertility (UI) and diminishing ovarian reserve (DOR)? Summary answer In women with DOR, reduced intrafollicular and serum INSL3 levels correspond with low AMH and high FSH levels, similar to established ovarian reserve markers. What is known already INSL3, belonging to the insulin-like peptide family, is produced by theca interna cells within antral follicles and the corpora lutea. It is hypothesized that INSL3 is integral to the initial development and function of antral follicles, specifically through its regulatory effect on androgen biosynthesis in the thecal cells of these follicles. Moreover, INSL3 is implicated in the modulation of the ovarian microenvironment, which is essential for facilitating the maturation of oocytes. Study design, size, duration This research was carried out at the IVF centers of two tertiary university hospitals, assessing 49 infertile women with either UI (n = 24) or DOR (n = 25) during IVF treatment. A control group (n = 26) comprised women with normal ovarian reserve undergoing IVF due to male or tubal factors. Outcome parameters are ovarian reserve markers (serum FSH, estradiol, AMH, INSL3 concentration and antral follicle count [AFC]), positive pregnancy rate and live birth rate (LBR) (gestational age ≥28 weeks). Participants/materials, setting, methods Women aged between 20-40 with a body mass index (BMI)<30 kg/m² were included. Serum FSH, estradiol, AMH, INSL3 levels and AFC were evaluated on menstrual cycle days 2 or 3, and INSL3 in follicular fluid from dominant follicles (>14 mm) were evaluated during oocyte retrieval. Exclusions included endocrine abnormalities (e.g., polycystic ovary syndrome, hyperprolactinemia, hypo and hyperthyroidism) cycle cancellation due to lack of a viable embryo, uncorrected Mullerian anomalies, recurrent miscarriage history and hydrosalpinx presence. Main results and the role of chance The mean age in the study groups was 28.41±3.5 (UI), 32.40±4.21 (DOR), and 29.65±3.91 (control). In the DOR group, female age (p = 0.001), BMI (p = 0.049), duration of infertility (p = 0.006), previous IVF attempts (p < 0.001), basal FSH (p < 0.001), and basal estradiol (p < 0.001) were higher, while AMH (p < 0.001), AFC (p < 0.001), and follicular fluid INSL3 (p < 0.001) were lower. Serum INSL3 in DOR was lower but not statistically significant. No correlation was found between serum INLS, follicular fluid INSL3, ovarian reserve markers, positive pregnancy rates, miscarriage, and LBR. DOR group had longer stimulation durations (p = 0.017), but lower max estradiol (p < 0.001), fewer oocytes (p < 0.001), mature follicles (p < 0.001), and PN (p < 0.001). Pregnancy rates were 54.1% (UI), 24% (DOR), and 53.84% (control); LBRs were 54.1% (UI), 20% (DOR), and 42.3% (control), with significant differences in pregnancy tests (p = 0.04) and LBR (p = 0.03), both lower in DOR. Univariate analysis identified duration of infertility (p = 0.019) and basal FSH (p = 0.009) as significant LBR predictors. Multivariable analysis confirmed basal FSH (p = 0.033) as significant, with increased duration of infertility and basal FSH level reducing LBR probability. Limitations, reasons for caution The limited sample size of our study potentially undermines the robustness of circulating INSL3 as a predictive biomarker for DOR, evidenced by the non-significant decrease in serum INSL3 levels in the DOR group. Moreover, the lack of INSL3 level assessment during oocyte retrieval further constrains our findings. Wider implications of the findings Our research explored INSL3 as a viable biomarker for predicting ovarian reserve and IVF success in UI and DOR cases. Reduced INSL3 levels in circulation and follicular fluid in DOR patients could be beneficial in clinical practice. Trial registration number not applicable

O-187 Real-word data on effectiveness of ICSI over conventional IVF according to infertility factors based on human fertilisation and embryology authority dataset

Abstract Study question Can the analysis of Human Fertilisation and Embryology Authority (HFEA) dataset provide real-word data on effectiveness of ICSI over conventional IVF according to infertility factors? Summary answer A statistically significant reduction in live birth rate was shown to be associated with ICSI use in cycles with female infertility factors. What is known already The adoption of ICSI has seen a considerable increase over the years, even in cases without an evident male-factor infertility. Available studies including large retrospective studies and meta-analyses evaluating the effectiveness of the two strategies tend to support the idea that in the absence of a male factor, ICSI does not offer a significant advantage over conventional IVF(cIVF). However, some uncertainties still exist regarding live birth rates and in relation to various infertility factors. Study design, size, duration Retrospective study of 275.825 first cycles treated with cIVF or ICSI between 2005-2018, included in the HFEA database. Infertility indications were simplified into three groups: 1) female only (endometriosis, tubal, ovulatory and cervical factors); 2) male (male only or with male and female factors together); 3) unexplained infertility. The primary outcome was live birth rate in the first fresh cycle. Secondary outcomes included fertilization, implantation, miscarriage rates, cycle cancellation rate and neonatal outcomes. Participants/materials, setting, methods Outcomes were evaluated through binomial logistic regression including the following predictors: female age, inseminated oocytes, number/stage of transferred embryos (condensed with principal component analysis), subtype of female infertility, year of treatment. A paired-analysis was also performed matching couples in a 1:1 ratio between ICSI and conventional IVF based on female age, indication, inseminated oocytes, year of treatment. Results were reported according to infertility cause using crude and adjusted Odds Ratios (OR) with 95%CI. Main results and the role of chance The study revealed a consistent balance in treatment distribution between conventional IVF and ICSI, with a slight increase in ICSI cycles in recent years. Based on logistic regression analysis, a significant reduction in live birth rate was demonstrated using ICSI compared to conventional IVF in cycles with female factors (adjusted OR = 0.95, 95%CI: 0.91-0.99). No significant differences emerged in unexplained factor cycles. With the use of conventional IVF, the overall odds of having a total fertilization failure increased by a factor of 1.84 (95%CI: 1.75-1.94). No significant differences in implantation rates between conventional VF and ICSI were seen after adjustment. The OR for male sex in newborns compared to female sex from single pregnancies was significantly influenced by ICSI use across the three groups of infertility causes and ranged between 0.84 and 0.87. The matched dataset confirmed a reduced occurrence of live birth per cycle in case of female factors (aOR=0.91, 95%CI: 0.86-0.96) and an overall significant lower proportion of cycles ending in cancellation for absence of viable embryos using ICSI compared to conventional IVF. Limitations, reasons for caution Some biases are intrinsic in the availability and quality of retrospective data obtained from registries and unmeasured confounders may influence the generalizability of the results. The study does not provide information on cumulative live birth rates. Wider implications of the findings ICSI introduces an additional, invasive step in assisted reproduction with a potential reduction in live birth rates for specific patients’ groups. These findings emphasize the importance of tailoring treatments to specific patient profile.The routine use of ICSI for all cases should not be considered an appropriate clinical practice. Trial registration number not applicable

O-321 A foundation model for embryology trained on time-lapse images

Abstract Study question Can a generalizable computer vision foundation model be trained using self-supervised learning on time-lapse embryo images to perform multiple clinically relevant downstream tasks? Summary answer We developed FEMI, a foundation model trained on eight million time-lapse images, to perform multiple clinical tasks, including blastocyst quality scoring, ploidy prediction, and segmentation. What is known already In vitro fertilization success critically depends on choosing viable embryos, a process hampered by current limited diagnostic tools, high costs, and ethical concerns. In recent years, various medical fields have increasingly explored foundation models using vision transformer architectures. These models, trained in a self-supervised manner on vast unlabeled image datasets, perform various clinically relevant tasks. Once trained on a massive unlabeled dataset, the encoder portion of the foundation model can be extracted and subsequently fine-tuned with a labeled dataset to perform various clinically relevant tasks like classification, regression, and segmentation. Study design, size, duration The foundation model was trained on 8 million Embryoscope® (E-SD) and Embryoscope+® (E+) images spanning clinics in the United States, Canada, and Europe. Datasets contained various additional information, such as ploidy status, blastocyst scores (numerical values from 3 to 14 based on Zhan et al. 2020), and segmentation masks (for trophectoderm [TE], inner cell mass [ICM], and zona pellucida [ZP]) that were used for training downstream tasks. Participants/materials, setting, methods A masked autoencoder architecture is trained on time-lapse images to create a foundation model (FEMI). The encoder from the autoencoder is extracted and fine-tuned on three image-based downstream tasks: ploidy prediction, blastocyst scoring, and embryo component segmentation. The performance of the fine-tuned foundation model is compared to VGG16 architectures, specifically trained for each downstream task. We evaluated performances using the area under the receiver-operating-characteristic (AUROC), mean absolute error (MAE), and mean intersection over union (mIoU). Main results and the role of chance The training dataset for the downstream tasks consisted of image data and labels, including PGT-A ploidy results (euploid or aneuploid), blastocyst scores (3-14), and segmentation masks. No clinical data like maternal age was used in order to assess FEMI’s learning capabilities from only time-lapse images against VGG16 model baselines. Both FEMI and baseline models used the same data splits for consistent comparisons. For ploidy prediction, models trained on a Weill Cornell Medicine (WCM) dataset were validated using WCM, Florida, and Spain data. FEMI achieved a 0.610 ± 0.004 AUROC, outperforming the baseline’s 0.590 ± 0.005. In blastocyst score prediction, FEMI attained a superior MAE of 0.099 ± 0.002 compared to the baseline’s 0.118 ± 0.001. For embryo segmentation (TE, ICM, ZP), a publicly available dataset from Simon Fraser University was split for training and validation. FEMI exceeded the baseline in mIoU for TE segmentation (0.738 ± 0.002 vs. 0.726 ± 0.002) and was comparable in ICM (0.832 ± 0.008 vs. 0.821 ± 0.011) and ZP segmentation (0.779 ± 0.002 vs. 0.778 ± 0.010). Limitations, reasons for caution The current version of FEMI is trained on a subset of our available datasets and can be improved through further training. Moreover, the downstream ploidy and blastocyst score models may be biased by errors in PGT results and the subjectivity of embryologists, respectively. Wider implications of the findings A generalizable foundational model for IVF time-lapse imaging aids clinicians in embryo selection by providing various clinical insights for better decision-making. FEMI, once fully trained, will be publicly accessible to the scientific community allowing for researchers to fine-tune FEMI on their own clinic-specific applications with their own image datasets. Trial registration number not applicable

P-547 Impact of SARS-COV-2 infection on embryo euploidy rate: a retrospective cohort study of 498 PGT-A cycles in China

Abstract Study question Does SARS-CoV-2 infection before oocyte retrieval have any impacts on embryo euploidy rate? Summary answer SARS-CoV-2 infection before oocyte retrieval had no detrimental impact on embryo euploidy rate, indicating that cycle cancellation due to SARS-CoV-2 infection may not be necessary. What is known already SARS-CoV-2 infection has significant effects on the health of people worldwide and poses a serious threat to reproductive health. According to the previous studies, SARS-CoV-2 infection had negative impacts on the blastocyst formation rate, top-quality embryos rate. However, the association between the infection and the embryo euploidy rate is indeterminate. It is still worth exploring the impact of SARS-CoV-2 infection on embryo euploidy rate. Especially this is the key question to be addressed for providing appropriate counseling to couples seeking for preimplantation genetic testing for aneuploidy (PGT-A) treatments during the ongoing pandemic. Study design, size, duration This was a retrospective cohort study of 498 couples who underwent PGT-A cycles with next-generation sequencing technology from November 1st, 2022 to June 30th, 2023 at a tertiary-care medical center in China. Participants/materials, setting, methods Women tested positive for SARS-CoV-2 before oocyte retrieval were included in the COVID-19 group, while patients who were asymptomatic and tested negative during the same period were included in the non-COVID-19 group. The primary outcome was the euploidy rate. Subgroup analysis was conducted according to the time interval between infection and oocyte retrieval. Univariate and multivariate logistic regression models were applied to adjust for potential confounders. Main results and the role of chance A total of 278 and 220 women were included in the COVID-19 and non-COVID-19 group, respectively. In the COVID-19 group, the maturated oocyte rate (79.5% vs. 83.7%, p＜0.001), number of total viable embryos (2 vs. 3, p=0.038), and number of euploid blastocyst (1 vs. 1, p=0.028) were significantly decreased compared with non-COVID-19 group. The euploidy rate (38.5% vs.40.3%, p = 0.451), aneuploidy rate (45.0% vs. 43.8%, p=0.616), and mosaicism rate (16.5% vs. 15.9%, p=0.746) were all comparable between the two groups. Results of multivariate logistic regressions revealed no association between SARS-CoV-2 infection and euploidy rate, aneuploidy rate, or mosaicism rate. Similarly, no association was detected between the time interval of infection and oocyte retrieval (≤30 days, 31-60days, 61-90 days, 91-120 days, or ≥ 120 days prior to oocyte retrieval) and euploidy rate. Limitations, reasons for caution The retrospective nature of the study and limited sample size for subgroup analysis limits the generalizability of the findings. Other potential confounders, such as the level of SARS-CoV-2 antibody, symptoms of infection, are needed to be taken into consideration as well. Large-scale prospective studies are warranted to confirm the conclusion. Wider implications of the findings Our study demonstrated that SARS-CoV-2 infection before oocyte retrieval had no detrimental impact on embryo euploidy rate, indicating that cycle cancellation due to SARS-CoV-2 infection may not be necessary. Trial registration number Not Applicable

O-171 Apicobasal RNA asymmetries regulate cell fate in the early mouse embryo

Abstract Assessing the morphological appearance of an embryo as it progresses from zygote to blastocyst is the primary method used by embryologists to evaluate embryo viability and developmental potential. Standard embryo grading techniques, using time-lapse microscopy, analyse morphological characteristics such as size, shape, number, and appearance of blastomeres. The aim of this study was to determine how individual cells of the embryo function on a subcellular level to build a healthy embryo. RNA localisation plays indispensable roles in establishing asymmetries and coordinating cell fate decisions during early embryogenesis across various non-mammalian species. To direct the spatiotemporal distribution of RNA within the cells of an embryo, the microtubule cytoskeleton provides highly sophisticated trafficking pathways. Yet, it remains unknown whether subcellular heterogeneities exist during mammalian preimplantation development and how they contribute to cell fate determination. Using advanced live imaging, we visualised global RNA transcripts at high spatiotemporal resolution from fertilisation to the blastocyst stage in living preimplantation mouse embryos. We discovered apicobasal RNA asymmetries specific to outer cells of the 16-cell stage mouse embryo, coinciding with cell fate decisions and the emergence of the pluripotent inner cell mass. Highly clustered RNAs accumulated proximal to the basal membrane, while more dispersed RNA foci were identified apically as the embryo reaches the late 16-cell stage. The targeted distribution of membrane-less RNA molecules was facilitated by the microtubule cytoskeleton, associated with lysosomes which serve as RNA transport vehicles. We discovered that apically located RNA foci were more dynamic and accompanied an enrichment of translation components. Furthermore, we uncovered that the established RNA asymmetries enhanced translation capacity in apical regions compared to basal regions. These spatiotemporal RNA heterogeneities were unevenly inherited by outer and inner daughter cells during subsequent cell divisions. Notably, embryos that were unable to establish RNA asymmetries failed to allocate cells to the trophectoderm and inner cell mass. Our study provides insights into a subcellular mechanism driving asymmetric RNA localisation and compartmentalised translational regulation in outer cells of the 16-cell stage embryo, which may contribute to cell fate decisions during mammalian preimplantation embryogenesis. We envision that when paired with classical morphological assessment criteria of the embryo, these findings may assist with selecting the embryo with the highest developmental potential.

O-117 Advances in PGT diagnostics - technologies beyond copy number

Abstract Genetic abnormalities are well recognised as the main factor contributing to the high rate of infertility, pregnancy loss and congenital abnormalities in humans. A prime example of this can be seen from recent studies showing that embryos identified as uniformly aneuploid following PGT-A have little to no reproductive potential. However, embryos shown to have no gross chromosomal abnormalities after PGT are not a guarantee of healthy pregnancy and many other undetected genetic issues are likely to influence embryo competence. Developments in both sample preparation and methods for DNA analysis are driving ever-increasing data yield from PGT platforms, permitting not only DNA quantitation, but also allowing qualitative evaluation of genotyping data in parallel. This opens the door to the possibility of overcoming some of the previous limitations of PGT, leading to a more comprehensive assessment of the genetic health and reproductive potential of embryos. How to use this increasing amount of information, and if it can tangibly improve test accuracy and clinical outcomes, remains open to debate. There is, for instance, no consensus on how to interrogate genotyping data for better distinction of clinically relevant aneuploidy as well as genome wide (ploidy) abnormalities and exogenous DNA contamination, which could impact the correct reporting of PGT results. There is also no consensus on the potential benefit of genotyping data to be used for the accurate distinction of meiotic recombination by linkage analysis, nor for fingerprinting of embryos to identify rare, but catastrophic, lab error events as a routine part of treatment. Finally, there is no clear path on how to utilise these improvements in embryo genotyping, to detect sub-chromosomal de novo genetic abnormalities that could impact embryo viability beyond the scope of current testing for chromosomal abnormalities alone. The aim of this presentation is therefore to focus on the current state of the ART and address the current and potential future uses of genotyping in PGT. Topics covered will include identification of biopsy sample contamination to determine the truth from the noise, through to how we can rescue embryos by utilising PGT as a molecular fertilisation check and how fingerprinting of embryo can be an invaluable tool in the clinic. The origin of aneuploidy will be explored and how genotyping is reshaping our understanding of aneuploidy landscape of the preimplantation embryo. From a more basic science perspective, the potential of genotyping to identify meiotic recombination will be discussed and how this fundamental element of human reproduction could be an important biomarker for reproductive competence. Lastly future perspectives on how the evolution of PGT technologies can bring us closer to the accurate detection of de novo genomic changes will be explored and how this is taking PGT-A beyond just aneuploidy detection. It is no secret that PGT for embryo selection continues to be a controversial area of reproductive medicine, and the field of PGT-A especially continues to show disquieting inconsistency in data analysis and reporting. This new era of genotyping brings with it information that can be used as a standardization tool helping to focus and facilitate improvements in the field of PGT for better and more consistent clinical reporting and outcomes.

Unique algorithm for the evaluation of embryo photon emission and viability

Living cells have spontaneous ultraweak photon emission derived from metabolic reactions associated with physiological conditions. The ORCA-Quest CMOS camera (Hamamatsu Photonics, Japan) is a highly sensitive and essential tool for photon detection; its use with a microscope incubator (Olympus) enables the detection of photons emitted by embryos with the exclusion of harmful visible light. With the application of the second law of thermodynamics, the low-entropy energy absorbed and used by embryos can be distinguished from the higher-entropy energy released and detectable in their environment. To evaluate higher-entropy energy data from embryos, we developed a unique algorithm for the calculation of the entropy-weighted spectral fractal dimension, which demonstrates the self-similar structure of the energy (photons) released by embryos. Analyses based on this structure enabled the distinction of living and degenerated mouse embryos, and of frozen and fresh embryos and the background. This novel detection of ultra-weak photon emission from mouse embryos can provide the basis for the development of a photon emission embryo control system. The ultraweak photon emission fingerprints of embryos may be used for the selection of viable specimens in an ideal dark environment.

Beet curly top virus affects vector biology: the first transcriptome analysis of the beet leafhopper.

Curly top disease, caused by beet curly top virus (BCTV), is among the most serious viral diseases affecting sugar beets in western USA. The virus is exclusively transmitted by the beet leafhopper (BLH, Circulifer tenellus) in a circulative and non-propagative manner. Despite the growing knowledge on virus-vector interactions, our understanding of the molecular interactions between BCTV and BLH is hampered by limited information regarding the virus impact on the vector and the lack of genomic and transcriptomic resources for BLH. This study unveils the significant impact of BCTV on both the performance and transcriptome response of BLHs. Viruliferous BLHs had higher fecundity than non-viruliferous counterparts, which was evident by upregulation of differentially expressed transcripts (DETs) associated with development, viability and fertility of germline and embryos in viruliferous insects. Conversely, most DETs associated with muscle movement and locomotor activities were downregulated in viruliferous insects, implying potential behavioural modifications by BCTV. Additionally, a great proportion of DETs related to innate immunity and detoxification were upregulated in viruliferous insects. Viral infection also induced notable alterations in primary metabolisms, including energy metabolism, namely glucosidases, lipid digestion and transport, and protein degradation, along with other cellular functions, particularly in chromatin remodelling and DNA repair. This study represents the first comprehensive transcriptome analysis for BLH. The presented findings provide new insights into the multifaceted effects of viral infection on various biological processes in BLH, offering a foundation for future investigations into the complex virus-vector relationship and potential management strategies for curly top disease.

Superovulating cattle with corifollitropin-alpha, a long-acting recombinant human FSH (rhFSH): Dose-response, half-life, effects on the ovaries, and embryo outcomes

The aim of this study was to evaluate the superestimulatory and superovulatory responses of cattle treated with corifollitropin-alpha, a long-acting human recombinant FSH (rhFSH). In the first and second experiments, we used Nelore (Bos indicus) heifers previously submitted to follicular wave suppression by active immunization against GnRH. In Experiment 1 (a dose-response study), heifers (n = 20) were randomly allocated into five groups, which received placebo (saline) or a single sc dose of 7.5, 15.0, 22.5 or 30.0 μg rhFSH. The heifers were subjected to daily ovarian scan and blood sampling during 11 days. We observed group, time, and group x time effects (P<0.0001) for both average follicle size and circulating FSH concentrations, with a strong correlation (R = 0.82, P<0.0001) between the area under curve (AUC) for both parameters. The peak concentration of FSH 24h after treatment and average follicle size at all timepoints, however, were similar (P>0.05) between groups 22.5 and 30.0 μg. In Experiment 2, heifers (n = 18) were allocated into three groups, which received (0h) either placebo (control), 25 μg rhFSH or 130 mg pFSH (Folltropin). There was no difference (P>0.05) in average follicle size at any moment, as well as in intrafollicular E2 at 120h or in plasma P4 seven days later between groups rhFSH and pFSH. In Experiment 3, cycling Nelore heifers (n = 20) were subjected to a wave synchronization protocol and superovulated (day 0) using a standard pFSH protocol (120 mg split in eight decreasing im doses) or with a single sc injection of 20 μg rhFSH. The number of follicles >7 mm on day 4 did not differ (P=0.4370). Heifers receiving rhFSH had greater average follicle size on day 4 (P=0.0005), ovulation rate (P<0.0001), and number of CL (P=0.0155), as well as a trend towards a greater number of ova (P=0.07) and viable embryos (P=0.0590). In Experiment 4, superovulation was induced with a single sc injection of 25 μg rhFSH in Girolando and Nelore cows and heifers (n = 20). None of the embryo yield endpoints differed between the two breeds (P>0.05). In conclusion, cattle superstimulation and superovulation can be successfully induced with a single dose of a long-acting rhFSH (corifollitropin-alpha).

Meiotic maturation failure in primary ovarian insufficiency: insights from a bovine model.

Oocytes from women presenting primary ovarian insufficiency (POI) generate viable embryos at a lower rate than non-POI women, but the mechanisms responsible for the lower oocyte quality remain elusive. Due to the scarcity of human oocytes for research, animal models provide a promising way forward. We aimed at investigating the molecular events characterizing final maturation in POI oocytes in a well-defined POI-like bovine model. Single-cell RNA-sequencing of bovine control and POI-like, GV, and MII oocytes (n = 5 per group) was performed. DEseq2 was used to identify differentially expressed genes. Further, a Gene set enrichment analysis and a transcriptomic meta-analysis between bovine and human oocytes were performed. In control cows, we found 2223 differentially expressed genes between the GV and MII stages. Specifically, the affected genes were related to RNA processing and transport, protein synthesis, organelle remodeling and reorganization, and metabolism. The meta-analysis with a set of young human oocytes at different maturation stages revealed 315 conserved genes through the GV-MII transition in cows and humans, mostly related to meiotic progression and cell cycle. Gene expression analysis between GV and MII of POI-like oocytes showed no differences in terms of differentially expressed genes, pointing towards a substantial failure to properly remodel the transcriptome in the POI model, and with the clustering analysis indicating that the cow's genetic background had a higher impact than the oocyte's maturation stage. Overall, we have identified and characterized a valuable animal model of POI, paving the way to identifying new molecular mechanisms involved in POI.

Targeted bisulfite sequencing of Scots pine adaptation-related genes

During environmental changes, epigenetic processes can enable adaptive responses faster than natural selection. In plants, very little is known about the role of DNA methylation during long-term adaptation. Scots pine is a widely distributed coniferous species which must adapt to different environmental conditions throughout its long lifespan. Thus, epigenetic modifications may contribute towards this direction. We provide bisulfite next-generation sequencing data from the putative promoters and exons of eight adaptation-related genes (A3IP2, CCA1, COL1, COL2, FTL2, MFT1, PHYO, and ZTL) in three Scots pine populations located in northern and southern parts of Finland. DNA methylation levels were studied in the two seed tissues: the maternal megagametophyte which contributes to embryo viability, and the biparental embryo which represents the next generation. In most genes, differentially methylated cytosines (DMCs) were in line with our previously demonstrated gene expression differences found in the same Scots pine populations. In addition, we found a strong correlation of total methylation levels between the embryo and megagametophyte tissues of a given individual tree, which indicates that DNA methylation can be inherited from the maternal parent. In conclusion, our results imply that DNA methylation differences may contribute to the adaptation of Scots pine populations in different climatic conditions.

Hexyltrimethylammonium ion enhances potential copper-chelating properties of ammonium thiomolybdate in an in vivo zebrafish model

Ammonium and hexyltrimethylammonium thiomolybdates (ATM and ATM-C6) and thiotungstates (ATT and ATT-C6) were synthesized. Their toxicity was evaluated using both in vitro and in vivo approaches via the zebrafish embryo acute toxicity assay (ZFET), while the copper-thiometallate interaction was studied using cyclic voltammetry, as well as in an in vivo assay. Cyclic voltammetry suggests that all thiometallates form complexes with copper in a 2:1 Cu:thiometallate ratio. Both in vitro and in vivo assays demonstrated low toxicity in BALB/3T3 cells and in zebrafish embryos, with high IC50 and LC50 values. Furthermore, the hexyltrimethylammonium ion played a crucial role in enhancing viability and reducing toxicity during prolonged treatments for ATM and ATT. In particular, the ZEFT assay uncovered the accumulation of ATM in zebrafish yolk, averted by the incorporation of the hexyltrimethylammonium ion. Notably, the copper-thiometallate interaction assay highlighted the improved viability of embryos when cultured in CuCl2 and ATM-C6, even at high CuCl2 concentrations. The hatching assay further confirmed that copper-ATM-C6 interaction mitigates inhibitory effects induced by thiomolybdates and CuCl2 when administered individually. These results suggest that the incorporation of the hexyltrimethylammonium ion in ATM increase its ability to interact with copper and its potential application as a copper chelator.