Blastocyst Expansion Research Articles

Oviduct epithelial spheroids during in vitro culture of bovine embryos mitigate oxidative stress, improve blastocyst quality and change the embryonic transcriptome

BackgroundIn vitro embryo production is increasingly used for genetic improvement in cattle but bypasses the oviduct environment and exposes the embryos to oxidative stress with deleterious effects on further development. Here we aimed to examine the effect of oviduct epithelial spheroids (OES) on embryo development and quality in terms of morphology and gene expression during two co-culture times (4 days: up to embryonic genome activation at 8–16 cell stage vs. 7 days: up to blastocyst stage) and under two oxygen levels (5% vs. 20%).MethodsBovine presumptive zygotes produced by in vitro fertilization (day 0) using in-vitro matured oocytes were cultured in droplets of synthetic oviductal fluid (SOF) medium with or without (controls) OES for 4 or 7 days under 5% or 20% oxygen (4 treated and 2 control groups). Cleavage rates were evaluated on day 2 and blastocyst rates on days 7–8. Expanded blastocysts on days 7–8 were evaluated for total cell numbers and gene expression analysis by RNA-sequencing.ResultsUnder 20% oxygen, blastocyst rates and total cell numbers were significantly higher in the presence of OES for 4 and 7 days compared to controls (P < 0.05), with no difference according to the co-culture time. Under 5% oxygen, the presence of OES did not affect blastocyst rates but increased the number of cells per blastocyst after 7 days of co-culture (P < 0.05). Both oxygen level and OES co-culture had a significant impact on the embryonic transcriptome. The highest number of differentially expressed genes (DEGs) was identified after 7 days of co-culture under 20% oxygen. DEGs were involved in a wide range of functions, including lipid metabolism, membrane organization, response to external signals, early embryo development, and transport of small molecules among the most significantly impacted.ConclusionOES had beneficial effects on embryo development and quality under both 5% and 20% oxygen, mitigating oxidative stress. Stronger effects on embryo quality and transcriptome were obtained after 7 than 4 days of co-culture. This study shows the impact of OES on embryo development and reveals potential molecular targets of OES-embryo dialog involved in response to stress and early embryonic development.

The kinetics of nucleolar precursor bodies clustering at the pronuclei interface: Positive correlations with the morphokinetic characteristics of cleaving embryos and euploidy in preimplantation genetic testing programs

Objective: This study investigated potential relationships between the kinetics of nucleolar precursor bodies (NPBs) in the pronucleus and developmental morphokinetics and euploidy in human preimplantation genetic testing for aneuploidy (PGT-A) cycles.Methods: The morphokinetic analysis of 200 blastocysts obtained from 53 PGT-A cycles was performed retrospectively in a time-lapse incubator. At the time of pronuclear breakdown (PNBD), we categorized the blastocysts into two groups based on the kinetic degree of clustering NPBs at the interface of the two pronuclei: clustered NPBs (CL) and non-clustered NPBs (NCL). We then compared morphokinetic parameters, abnormal behavioral events, and the rate of aneuploidy between the two groups.Results: Pronuclear fading and the first cleavage occurred earlier in the NCL group than in the CL group. However, the initiation of blastocyst formation and blastocyst expansion was delayed in the NCL group relative to the CL group. No differences were found in the rate of abnormal cleavage events, such as multinucleation at the 2-cell stage, direct cleavage from one to three cells, and from two to five cells between the CL and NCL groups. However, the fragmentation rate at the 8-cell stage was higher in the NCL group than in the CL group (10.3% vs. 1.9%, p&lt;0.05). Additionally, the euploid rate in the CL group was significantly higher than in the NCL group (37.9% vs. 12.4%, p&lt;0.05).Conclusion: These results demonstrate the effectiveness of combining NPB clustering at PNBD with morphokinetics as a parameter for selecting embryos with higher developmental potential in in vitro fertilization.

Cytoplasmic strings in human blastocysts: hypotheses of their role and implications for embryo selection.

What are the implications of the presence cytoplasmic strings (Cyt-S) and their quantity and dynamics for the pre-implantation development of human blastocysts? Cyt-S are common in human embryos and are associated with faster blastocyst development, larger expansion, and better morphological quality. Cyt-S are dynamic cellular projections connecting inner cell mass and trophectoderm (TE) cells, that can be observed during blastocyst expansion. Their prevalence in human embryos has been estimated to be between 44% and 93%. Data relevant to their clinical implications and role in development are lacking, limited, or controversial. Retrospective study conducted at a single IVF center between May 2013 and November 2014 and involving 124 pre-implantation genetic testing for aneuploidy cycles in a time-lapse incubator with ≥1 blastocyst biopsied and vitrified (N = 370 embryos assessed). These cycles resulted in 87 vitrified-warmed single-euploid blastocyst transfers. ICSI, continuous blastocyst culture (Days 5-7), TE biopsy of fully expanded blastocysts without Day 3 zona pellucida drilling, qPCR to assess uniform full-chromosome aneuploidies, and vitrification were all performed. Only vitrified-warmed euploid single-embryo-transfers were conducted. Blastocyst morphological quality was defined according to Gardner's criteria. The AI-based software CHLOE™ (Fairtility) automatically registered timings from time of starting blastulation (tSB) to biopsy (t-biopsy, i.e. blastocyst full-expansion) as hours-post-insemination (hpi), embryo area (including zona pellucida in µm2), and spontaneous blastocyst collapses. One senior embryologist manually annotated Cyt-S presence, quantity, timings, and type (thick cell-to-cell connections and/or threads). All significant associations were confirmed through regression analyses. All couples', cycles', and embryos' main features were also tested for associations with Cyt-S presence, quantity, and dynamics. About 94.3% of the patients (N = 117/124) had ≥1 embryo with Cyt-S. Out of a total of 370 blastocysts, 55 degenerated between blastulation and full-expansion (N = 55/370, 14.9%). The degeneration rate among embryos with ≥1 Cyt-S was 10.8% (N = 33/304), significantly lower than that of embryos without Cyt-S (33.3%, N = 22/66, P < 0.01). Of the remaining 315 viable blastocysts analyzed, 86% (N = 271/315; P < 0.01) had ≥1 Cyt-S, on average 3.5 ± 2.1 per embryo ranging 1-13. The first Cyt-S per viable embryo appeared at 115.3 ± 12.5 hpi (85.7-157.7), corresponding to 10.5 ± 5.8 h (0.5-31) after tSB. Overall, we analyzed 937 Cyt-S showing a mean duration of 3.8 ± 2.7 h (0.3-20.9). Cyt-S were mostly threads (N = 508/937, 54.2%) or thick cell-to-cell connections becoming threads (N = 382/937, 40.8%) than thick bridges (N = 47/937, 5.0%). The presence and quantity of Cyt-S were significantly associated with developmentally faster (on average 6-12 h faster) and more expanded (on average 2700µm2-larger blastocyst's area at t-biopsy) embryos. Also, the presence and duration of Cyt-S were associated with better morphology. Lastly, while euploidy rates were comparable between blastocysts with and without Cyt-S, all euploid blastocysts transferred from the latter group failed to implant (N = 10). Cyt-S presence and dynamics were assessed manually on seven focal planes from video frames recorded every 15 min. The patients included were mostly of advanced maternal age. Only associations could be reported, but no causations/consequences. Lastly, larger datasets are required to better assess Cyt-S associations with clinical outcomes. Cyt-S are common during human blastocyst expansion, suggesting their physiological implication in this process. Their presence, quantity and dynamics mirror embryo viability, and morphological quality, yet their role is still unknown. Future basic science studies are encouraged to finally describe Cyt-S molecular nature and biophysical properties, and Artificial Intelligence tools should aid these studies by incorporating Cyt-S assessment. None. N/A.

Comparison of the developmental competence of in vitro-produced mouse embryos cultured under 5 versus 2% O2 with in vivo-derived blastocysts.

The prevalence of infertility in Canada has substantially increased over 30 years, and plateaued success rates of culture systems warrant further optimization for transfer outcomes. In clinical programs, embryos commonly undergo extended culture under 5% O2 until the blastocyst stage. The aim of this study is to characterize the developmental competence and stress-related responses of embryos cultured under 5 versus 2% O2 in comparison to in vivo-derived blastocysts. We hypothesized 2% O2 compromises developmental competence through altered embryonic stress responses and induction of apoptosis-related genes relative to those cultured under 5% O2 and in vivo-derived blastocysts. Quantitative measures of development and relative expressions of a cohort of stress-related genes in CD1 mouse zygotes cultured to blastocysts under 5 or 2% O2 were compared to in vivo-derived embryos. Apoptotic responses were evaluated using an immunofluorescence assay for Caspase-3. The mean percentage of blastocysts developed, and total cell number of embryos derived in vivo or cultured under 5% O2 was significantly higher than those cultured under 2% O2. Blastocyst expansion was greatest in embryos cultured under 5% O2. Stress response genes were significantly upregulated in embryos cultured under 2% O2, and expression of antioxidant-related genes was significantly lower in cultured versus in vivo-derived embryos. Caspase-3 immunofluorescence was significantly higher in cultured embryos versus in vivo-derived embryos. We inferred that 5% O2 systems better approximate physiologic oxygen availability for culture of mouse embryos, warranting re-evaluation of culturing embryos under threshold or sub-physiologic oxygen concentrations during clinical IVF programs.

High serum progesterone levels on the day of embryo transfer in patients undergoing artificial frozen-thawed blastocyst transfer: Is there a ceiling effect?

To evaluate the potential ceiling effect of high serum progesterone levels on the day of embryo transfer for pregnancy outcomes in patients undergoing artificial frozen-thawed blastocyst transfer (FET) cycles. This retrospective cohort study included 595 patients who underwent artificial FET cycles. We evaluated progesterone levels and found that 40.6 ng/mL corresponded to the 90th percentile and 23.9 ng/mL corresponded to the 50th percentile. Based on these findings, we categorized progesterone levels as <20 ng/mL (n=220, 37.0%), 20-40 ng/mL (n=312, 52.4%), and ≥40 ng/mL (n=63, 10.6%). The primary outcome measures were the clinical pregnancy rate (CPR) and live birth rate (LBR). Blastocyst morphology grades, including expansion, trophectoderm, and inner cell mass grades, were significantly associated with clinical pregnancy (p<0.001 for all). Progesterone levels between 20 and 40 ng/mL were associated with higher CPR (p=0.043). In the multivariate analysis, only blastocyst expansion and inner cell mass grades were independently and significantly associated with CPR [p=0.011, odds ratio (OR)=1.6, (confidence interval) CI 95%=1.13-2.39, and p=0.007, OR=1.65, CI 95%=1.14-2.39, respectively]. The progesterone level and trophectoderm grade were not statistically significant. Regarding LBR, only blastocyst expansion grades 4 and trophectoderm grades A or B were significantly associated. Based on these data, we speculate that if serum progesterone levels exceed 40 ng/mL on the day of embryo transfer in patients undergoing artificial FET cycles, there is no need to reduce the progesterone dose.

Use of federated learning to develop an artificial intelligence model predicting usable blastocyst formation from pre-ICSI oocyte images

Research questionCan Federated Learning be used to develop an artificial intelligence (AI) for evaluating oocyte competence using two-dimensional images of denuded oocytes in metaphase II prior to intracytoplasmic sperm injection (ICSI)? Design10,677 oocyte images with associated metadata were prospectively collected by 8 in vitro fertilization (IVF) clinics across 6 countries. AI training used Federated Learning, where data were retained on regional servers to comply with data privacy laws. The final AI required a single image as input to evaluate oocyte competence, which was defined by the formation of a usable blastocyst (≥expansion grade 3 by Day 5/6 post-ICSI). ResultsThe oocytes AI demonstrated area under the curve (AUC) up to 0.65 on two blind test datasets. A high sensitivity for predicting competent oocytes (83-88%) was offset by a lower specificity (26-36%). Exclusion of confounding biological variables (male factor infertility and maternal age ≥35 years) improved AUC up to 14%, primarily due to increased specificity. AI scores correlated with size of the zona pellucida and perivitelline space, and ooplasm appearance. AI scores also correlated with blastocyst expansion grade and morphological quality. The sum of AI scores from oocytes in group culture images predicted formation of ≥2 usable blastocysts (AUC=0.77). ConclusionAn AI for evaluating oocyte competence was developed using Federated Learning, representing an essential step in protecting patient data. The AI was significantly predictive of oocyte competence as defined by usable blastocyst formation, which is a critical factor for IVF success. Potential clinical utility ranges from selective oocyte fertilization, to guiding treatment decisions regarding additional rounds of oocyte retrieval.

P-128 Non-invasive morphometric assessment of embryo quality: Effectiveness of the number of trophectoderm cells at the equatorial region of expanded blastocysts

Abstract Study question Does the number of trophectoderm cells at the equatorial region of expanded blastocysts (eTEs) affects pregnancy rate in single vitrified blastocyst transfer (SVBT)? Summary answer The number of eTEs highly correlated with the total number of TEs. The greater number of eTEs was higher pregnancy rate became. What is known already The increased use of blastocyst transfer has emphasized the importance of selecting high-quality embryos. Morphological grading: Gardner’s criteria, or time-lapse imaging of embryo morpho kinetics is established a tool for selection of high-quality blastocysts. The Gardner’s criteria is one of the most used evaluation according to grading scores of developmental stage, trophectoderm (TE) and inner cell mass (ICM). Although, it cannot completely exclude subjectivity, and it varies from observer to observer. A quantitative evaluation method for blastocyst selection is necessary to solve these problems and to improve the pregnancy rate of SVBT. Study design, size, duration First, we checked the correlation between the number of eTEs and the total number of TEs using 23 discarded blastocysts by immunostaining of CDX2. Then, we analyzed 1103 vitrified-warmed blastocysts from July 2017 to June 2021 for pregnancy rates. Expanded blastocysts which reached the blastocoel cavity of 160 µm or more with obvious ICM were vitrified and cryopreserved. Relationship between the number of eTEs and pregnancy rate after SVBT was analyzed by logistic regression. Participants/materials, setting, methods CDX2-positive cells were defined as TE, and the difference between total cells by Hoechst staining and CDX2-positive cells as ICM. The correlation between the number of eTEs and the total number of TEs was examined. In SVBT, all embryos were monitored by a time-lapse system. eTEs were counted in the time-lapse image just before cryopreservation. Pregnancy and abortion rates were examined for each eTEs. Also, logistic regression analysis was performed on eTEs and pregnancy. Main results and the role of chance There was a strong positive correlation between the number of eTEs and the total number of TEs (CDX2-positive cells) in the whole blastocysts (r = 0.945). In addition, there was a significant correlation between the number of eTEs and the total cell number of blastocysts (r = 0.73). On the other hand, there was no correlation between the number of eTEs and the cell number of ICM (r = 0.008). The number of eTEs was presented as quartiles: Pregnancy rates for &amp;lt;5, 5-6, 6-7 and ≥7 eTEs were 25.9, 29.4, 38.4, and 64.2% (P &amp;lt; 0.05), respectively, and the miscarriage rates were 50.0, 28.3, 14.1 and 19.2% (P &amp;lt; 0.05), respectively. Pregnancy rate for ≥7 eTEs was significantly higher than that for the other groups (P &amp;lt; 0.05), and miscarriage rate was lower than that for &amp;lt;6 eTEs. That is, increased eTEs significantly increased the pregnancy rate and decreased the miscarriage rate. Significant relationship between the number of eTEs and pregnancy was shown by logistic regression analysis (P &amp;lt; 0.0001), with an AUC of 0.68 and a cutoff value of 6.5. Limitations, reasons for caution In this study, expanded blastocysts at 160 µm or more in diameter were analyzed and cryopreserved. The different size of blastocysts may be different number of eTEs, and the relationship between the number of eTEs and pregnancy rate will be needed to confirm in individual clinic. Wider implications of the findings Quantitative analysis of eTEs makes it possible to select highly fertile blastocysts. This assessment is simply and easy. It can be the additional possible evaluation before cryopreservation to allows for quantitative treatment of continuous variables by classifying them in more detail than Gardner’s criteria. Trial registration number N/A

P-198 Time to maximum blastocyst expansion (tmBE) may represent a novel metric for assessing embryo competence

Abstract Study question Can the time to achieve maximum blastocyst expansion (tmBE) and the frequency of spontaneous collapses serve as useful indicators of embryo competence? Summary answer Compared to the frequency of spontaneous collapses, tmBE was a better indicator of embryo competence, potentially serving as a valuable metric for embryo selection. What is known already Identifying the most viable blastocyst for transfer is essential for success in assisted reproduction. In scenarios with good prognosis, such as oocyte donation, blastocyst selection may require a more nuanced approach, given the high number of available good quality embryos. Exploring indicators of embryo competence beyond traditional morphology-based criteria may be beneficial, however an in-depth understanding is still lacking. Blastocyst expansion and contraction dynamics have been suggested to play a role in developmental and chromosomal fitness. However, their predictive value has not been investigated across a wide range of patient populations. Study design, size, duration This is a single-center retrospective study of 287 blastocysts transferred in autologous (n = 88) and heterologous (n = 199) cycles, performed between September 2019 and September 2022. Only fresh single embryo transfers were considered. We compared blastocysts across two groups: advanced maternal age (AMA) patients (&amp;gt;35 years old, n = 88) and oocyte donors (&amp;lt;35 years old, n = 199). We evaluated well-established indicators of embryo quality, as well as novel metrics, including tmBE and the frequency of spontaneous collapses. Participants/materials, setting, methods Conventional indicators of embryo quality included blastocyst grade and morphokinetic timings (tPNf, t2, t3, t4, t5, t8, tSB and tB). tMBE was defined as the maximum time (hpi) required for the blastocyst to reach its largest diameter, extending beyond tB. Blastocyst contractions were measured as a decrease and subsequent recovery of the blastocoel area over time. ImageJ was employed for size measurements. p&amp;lt;0.05 was considered statistically significant after Two-tailed Student’s t-test or Chi-square test. Main results and the role of chance We observed a significant difference in live birth rates between oocyte donors and AMA patients (40% vs 18%, p=0.0004). Nevertheless, there were no differences across blastocyst expansion scores between oocyte donors and AMA patients (5= 58% vs 65%; 4= 38% vs 30% and 3= 4% vs 5%, p &amp;gt; 0.05), nor in their morphology score (Excellent= 57% vs 61%; Good= 24% vs 18%; Average= 15% vs 16% and Poor= 4% vs 5%). In terms of morphokinetics, when comparing oocyte donor and AMA embryos, the only significant difference was observed at tPNF (22.0±2.4 vs 22.6±2.3; p= 0.04). No differences were found at t2, t3, t4, t5, t8, tSB and tB. Additionally, the frequency of spontaneous collapses showed no significant differences between the oocyte donor and AMA group (1.4±1.2 vs 1.5±1.0, p=0.66). However, blastocysts from oocyte donors reached tmBE significantly faster than AMA embryos (115.0±4.8 vs 117.2±3.4; p = 0.0003). When these variables were further correlated to clinical outcomes, the frequency of spontaneous collapses did not appear to influence live birth in either group. Notably, however, tmBE was significantly shorter for embryos resulting in a live birth, across both the oocyte donor (113.5 vs 115.4, p = 0.04) and AMA group (115.6 vs 117.6, p = 0.04). Limitations, reasons for caution This is a single center retrospective study with a relatively small sample size, especially in the AMA group. Caution should be made when generalizing results to other patient populations, as confounding were not taken into consideration. No data on the chromosomal status of embryos was available. Wider implications of the findings Our results suggest that tMBE may serve as a reliable indicator of embryo potential, allowing a more nuanced embryo selection, particularly in cases with a high number of good quality embryos. This study opens avenues for further research into novel metrics for assessing embryo competence Trial registration number not applicable

O-127 Blastocyst expansion speed assay: a putative artificial intelligence-powered tool for embryo selection. Retrospective study involving 2184 blastocysts and 786 PGT-A cycles

Abstract Study question Is blastocyst expansion-speed, examined through Artificial-Intelligence (AI) between time of blastulation (tB) and time of expanding blastocyst (tEB), associated with embryo blastulation and reproductive competence? Summary answer Blastocyst expansion-speed-assay (ESA) was significantly associated with euploidy and live-birth rates, being discordant with clinical embryologists’ ranking in 50% of embryonic cohorts. What is known already Blastocyst expansion is one of the earliest morphogenetic events in mammalian development. It is essential for the establishment of the blastocyst layout and requires trophectoderm integrity. Several studies suggested that expansion is a valuable feature for ranking and prioritizing blastocysts for transfer based on their developmental competence. Time-lapse technology (TLT) implementation in IVF allows a deeper understanding of blastocyst expansion dynamics. In this study, we combined TLT and AI to develop a blastocyst-ESA and investigate its association with embryo blastulation and reproductive competence. Study design, size, duration Retrospective study including 2184 blastocysts cultured in EmbryoScope incubators during 786 PGT-A cycles across 2013-2020. Videos were analyzed through an AI-powered tool (CHLOETM, Fairtility). The expansion-speed was calculated as [(Δ embryo proper area at tEB – embryo proper area at tB) / (Δ tEB - tB)]. ESA was tested for its association with embryo quality, euploidy and live-birth rate (LBR) in 548 vitrified-warmed single euploid transfers. A simulation of ESA putative clinical effectiveness was conducted. Participants/materials, setting, methods ICSI, trophectoderm biopsy of fully-expanded blastocysts without day3 zona-drilling, and qPCR/NGS to assess full-chromosome uniform aneuploidies were performed. tB and tEB, expressed as hours-post-insemination (hpi), embryo proper area (in µm2) at both these stages, and blastocyst quality (EQ-Score from 0 to 1) were all automatically recorded through CHLOE. Possible confounders (e.g., maternal age, male factor, and cause of infertility) were considered. Regression analyses were conducted to adjust the data. Main results and the role of chance The average ESA was 705±458 µm2/hour. Higher ESA was associated with higher EQ-Score. Euploid blastocysts showed higher ESA than aneuploid (761±465 µm2/hour versus 667±449 µm2/hour; p &amp;lt; 0.01). ESA was also associated with LBR per euploid blastocyst transfer (LB: 873±438 µm2/hour versus 736±467 µm2/hour; p &amp;lt; 0.01). Of note, maternal age showed no association with ESA. Multivariate regressions outlined a + 5.1%-increase in the chance of euploidy and a + 6.3%-increase in chance of LB, every +100 µm2/hour-increase in ESA. ESA and embryologists were discordant in grading top-quality embryos in each cohort in 50% of the cases. In 59% of the 352 cohorts where both euploid and aneuploid blastocysts were obtained, ESA would have ranked the former embryos as top-quality. In the 216 cycles with ≥2 euploid blastocysts obtained and ≥1 transferred, ESA would have prioritized (i) a competent embryo in 46% of the cases, (ii) an incompetent embryo in 20%, but (iii) in 33% its value could not be assessed (i.e., the top embryo according to ESA has not been transferred yet). When compared to the embryologists, ESA would have been (i) equally-effective in 49% of the cases, (ii) more effective in 12-24%, and (iii) less effective in 5%-26%. Limitations, reasons for caution Retrospective single-center study. Both continuous and sequential media were used, although they were not associated with the outcomes under investigation. To assess the clinical value of ESA, a prospective study among first transfers is warranted. Wider implications of the findings ESA is an automated, unbiased, and easily-applicable measurement that certainly deserves further appraisal. If validated in prospective studies, AI-based selection algorithms could include this assessment to increase their predictive power. Trial registration number None

P-116 Relationship between exposure in equilibration solution pre-vitrification for human collapsed blastocysts and miscarriage rate in ART treatments

Abstract Study question To explore the relationship between equilibration time pre-vitrification of human collapse blastocyst and clinical pregnancy and miscarriage rate in patients undergoing ART treatments Summary answer The miscarriage rate was significantly lower in the group with a short equilibration time of 7-8 minutes compared to the group of 9-10 minutes What is known already The introduction of vitrification represents one of the most important advancement in ART. However, protocols currently applied to cryopreserve human embryos still have some weak points that might be improved. A critical aspect is represented by the high concentration of cryoprotectants (CPAs) used during the vitrification and some of this CPAs might impact negatively cellular metabolism and function. Thus, this study investigated whether a shorter time in the equilibration solution before vitrification of artificially collapsed blastocysts might have an impact on survival and pregnancy outcomes, as well as live birth rate and risk of miscarriage in patients undergoing ART. Study design, size, duration This prospective study was performed at the Centre for Reproductive Medicine, Haikou Mary Hospital, China from March 2018 to May 2022. Informed consent for experimentation with human subjects was obtained before the patients start the ovarian stimulation. Female age &amp;lt;35 years, and causes of infertility included male factors, female infertility, and unexplained infertility. Following the fresh embryo transfer, supernumerary good quality blastocysts ≥ 2, according to Gardner’s score were vitrified and allocated as discussed below Participants/materials, setting, methods The study included a total of 831 expanded blastocysts, which were divided into two groups according to the equilibration time before vitrification: group (A) 7-8 minutes (413); and group (B) 9-10 minutes (418). Expanded blastocysts (grade 3 or more) were artificially shrunk by applying one or two laser pulses. Patients were included in the study only when at least two blastocysts were available for vitrification, in order to be allocated one in each group Main results and the role of chance Results: A total of 831 vitrified-warmed blastocysts were analysed in this study, of which 825 survived at the warming step (99.3%: 825/831). All surviving blastocysts were transferred in 585 embryo transfers. Overall, the clinical pregnancy rate per transfer was CPR: 68.5% (401/585), and live birth and miscarriage rates: 61.0% (357/585) and 11.0% (44/401). No significant differences were observed between the two groups regarding the mean age of patients, the average number of blastocysts transferred, the basal FSH, BMI, and infertility duration. Results show the same survival rate after warming for group A (99.3%) and group B (99.3%), as well as similar CPR (A: 69.1% versus B: 68.0%).The live birth (A: 63.8% versus B: 58.3%) and multiple gestation rates (A: 20.8% versus B: 23.5%) were comparable in the two groups. When analysing the overall miscarriage rate, data displayed a statistically significant difference (P &amp;lt; 0.05) in favour of group A (7.6%) compared to group B (14.2%). There were no differences between the two groups concerning the prevalence of male babies (A: 57.1% and B: 55.4%), average gestational length (A: 38.67±1.37 versus B: 38.33±1.21), preterm birth rate (A: 19.2% versus B: 20.6%), and birth weight (A: 2.95±0.58 versus B: 3.05±0.63 kg). Limitations, reasons for caution This is not a randomized controlled trial, it is a prospective observational cohort study aiming to calculate the effect of a shorted ES time on the risk of miscarriage. Also, potential confounding factors due to the heterogeneous nature of the sample investigated may impair the validity of our conclusions. Wider implications of the findings Results demonstrate that a shorter equilibration time resulted in optimal survival, clinical pregnancy, and live birth rates compared to exposure to ES for 9-10. Thus, suggesting that a longer exposure to the ES is not needed, and might negatively effect cellular metabolism and function, increasing the risk of miscarriage. Trial registration number Not applicatione

P-146 The importance of cytoplasmic strings between inner cell mass and trophectoderm after blastulation on embryonic development and pregnancy outcome

Abstract Study question Whether the cytoplasmic strings between inner cell mass and trophectoderm during blastocyst expansion have a significant impact on embryo development and pregnancy outcomes? Summary answer The presence of cytoplasmic strings is related to morphological quality, but it has no impact on embryo ploidy or clinical outcome after euploid embryo transfer. What is known already Cytoplasmic strings are a dynamic cellular structure, and their width and length vary with the degree of embryonic expansion. The presence of cytoplasmic strings is considered a characteristic of embryos with poor vitality. In recent years, researchers have found the presence of cytoplasmic strings may indicate a higher embryonic developmental potential. However, the specific function of cytoplasmic strings remains unclear, and their impact on embryonic development is still controversial. Study design, size, duration A retrospective cohort analysis was conducted on clinical data of 530 patients who underwent single blastocyst transfer after pre-implantation genetic testing for aneuploidy (PGT-A) in our center from June 2019 to December 2021. Two embryologists annotated the presence of cytoplasmic strings, the morphological quality of blastocyst. Participants/materials, setting, methods A total of 2132 biopsied blastocysts were obtained from the 530 patients, with 534 blastocysts having cytoplasmic strings (+) and 1598 blastocysts having cytoplasmic strings (−). The quality and ploidy differences between the two groups were compared. After PGT-A, single euploid blastocysts were transferred, and the difference in pregnancy outcomes between cytoplasmic strings (+) (n = 115) and cytoplasmic strings (−) (n = 415) cycles was compared. Main results and the role of chance There was no significant difference in the euploidy rate between the two groups (44.6% vs. 45.3%). As the morphological quality of the embryos decreased, the rate of cytoplasmic strings (+) significantly decreased (Good-quality vs. Average-quality: 30.19% vs. 24.62%, P = 0.008; Average-quality vs. Low-quality: 24.62 vs. 12.63, P = 0.000). After adjusting for patient age, anti-Müllerian hormone, and gonadotropin dose, multivariate regression analysis showed that cytoplasmic strings were associated with embryo quality, but not with embryo ploidy. There were no significant differences in implantation rate (72.17% vs. 66.02%, P = 0.213), miscarriage rate (10.43% vs. 8.43%, P = 0.504), and live birth rate (61.74% vs. 57.59%, P = 0.424) between blastocysts with cytoplasmic strings (+) and without cytoplasmic strings (−). Limitations, reasons for caution Larger datasets are required to confirm putative associations between cytoplasmic strings and blastocyst ploidy, and the pregnancy outcome. Wider implications of the findings The presence of cytoplasmic strings is related to the embryological quality of the blastocyst, but it has no impact on embryo ploidy or clinical outcome after euploid embryo transfer. Trial registration number not applicable

O-040 Pro -better late than never

Abstract In IVF, blastocysts developed on Day5-6 are typically selected for transfer, cryopreservation, or biopsy, while embryos that do not reach this developmental stage before 144 hours post-insemination (hpi) are often discarded. Recently, there has been an increased adoption of blastocyst culture aimed at enhancing embryo selection potential and supporting a single embryo transfer approach. Consequently, the clinical significance of Day7 blastocysts (embryos reaching this stage beyond 144 hpi) has become a prominent topic of discussion, particularly in the context of treating patients with poor prognosis. Numerous studies worldwide have reported lower competence for these embryos, as evidenced by poorer morphological quality, higher aneuploidy rates, and lower implantation rates following both untested and euploid transfer. Nonetheless, a notable proportion of these blastocysts are euploid and result in healthy live births. While their clinical utilization may entail lower efficiency per transfer, it ultimately increases the cumulative live birth rate per cycle. However, most studies have encountered challenges due to diverse definitions of Day7, varying clinical settings, culture strategies, incubators, and local regulations. Presently in IVF, leveraging time-lapse technology (TLT), artificial intelligence (AI), and preimplantation genetic testing for aneuploidies (PGT-A) could standardize and enhance Day7 blastocyst characterization and clinical assessment. Consequently, we designed a study exclusively in freeze-all PGT-A cycles with vitrified-warmed euploid single embryo transfer after continuous culture in time-lapse incubators, trophectoderm biopsy without zona pellucida drilling on day3, comprehensive chromosome testing, confirming embryo morphological assessments through an AI-powered software, and utilizing this tool also to automate developmental timing annotations. Our analysis confirmed that all blastocysts were biopsied only after reaching an expansion level suitable for the procedure (approximately 24,000 µm²) and consistent across all embryos clustered every 12 hours in 6 groups (&amp;lt;120, 120-132, 132-144, 144-156, 156-168, and &amp;gt;168 hpi). Day7 blastocysts accounted for approximately 15% of all blastocysts obtained over 8 years, derived from older oocytes, exhibiting poorer quality according to both Gardner’s score and AI score, slower development from early stages, and accumulating delays across various intermediate processes like blastocyst expansion from tEB to the time of biopsy. They were less euploid and resulted in a lower live birth rate per euploid transfer. However, only the latter association was significant when adjusted for confounders (e.g., blastocyst quality). Comparing only embryos biopsied before 144 hpi versus embryos biopsied after this threshold, neither the euploidy nor the live birth rate were significantly different, supporting the extension of embryo culture beyond Day6. Overall, we estimated that Day7 embryos contribute to a relative increase of 4.4%, 13.7%, and 5.2% in the number of patients who achieved a live birth, who were not pregnant after the transfer of faster embryos, and who have supernumerary transferable embryos after a live birth, respectively. The main limitations of our investigation were its retrospective nature and the fact that retrospective associations do not provide evidence of causation. There might be a bias towards poor prognosis patients being subject to the transfer of Day 7 embryos, for instance. Lastly, more data are required regarding the putative impact of extended embryo culture including post-natal outcomes (reassuring to date), as well as the cost-effectiveness of this clinical choice. Considering the increasing personalization and patient-centeredness of IVF, where permitted by local regulations, the decision to culture embryos to Day7 should be based on a careful evaluation of couples’ reproductive history and future prognosis, and should be subject to a patient-centered decision-making process. In the future, AI can be harnessed to support IVF practitioners by objectifying the assessment of poor-quality and slow-growing embryos with measurable information and reliable predictions, thereby mitigating the inherent subjectivity of embryologists’ decisions.

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

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

P-121 Post-warming morphokinetics as a predictive tool for embryo implantation

Abstract Study question Does frozen-thawed blastocyst morphokinetics observed using time-lapse imaging provide an indicator for implantation in intracytoplasmic sperm injection (ICSI) cycles with deferred embryo transfers? Summary answer The blastocyst morphokinetic behavior after warming may provide an indicator for implantation in ICSI cycles. What is known already Embryo cryopreservation has become a pivotal component in assisted reproduction, applicable both to surplus embryos and in freeze-all cycles. Throughout vitrification and warming processes, embryos face considerable challenges. Historically, the evaluation of fresh blastocysts has relied on the examination of morphological features and the assessment of blastocoel expansion, trophectoderm, and inner cell mass. However, for vitrified and warmed blastocysts, traditional evaluation may not suffice to predict implantation. The emergence of a TLI culture system has facilitated a comprehensive examination of embryo development. Hence, post-warming culture in TLI incubators may provide a more precise assessment of vitrified and warmed blastocyst behavior. Study design, size, duration From January to October 2022, an observational cohort study was conducted at a private university-affiliated IVF centre. This included 411 vitrified/warmed blastocysts, from freeze-all cycles, with known implantation data (no implantation and full implantation per transfer), which were evaluated by TLI. Blastocyst morphological dynamics variables after warming were recorded, and their potential association with clinical pregnancy was investigated using Generalized linear models (GzLM) adjusted for oocyte age, followed by Bonferroni post hoc test. Participants/materials, setting, methods After warming, the recorded factors were: (i) initial blastocyst area (IBA); (ii) maximum blastocyst area (MBA); (iii) Blastocyst area delta (DBA); (iv) initial ZP thickness (IZP); (v) minimum ZP thickness (MZP); (v) ZP thickness delta (delta ZP area); and (iv) blastocyst expansion (BE). Embryos received a final score from 0 to 9, according to the sum of the scores from each parameter and were grouped as: Low total-score, medium total-score), and high total-score. Main results and the role of chance No significant difference was observed when maternal age (37.8 ± 5.3, 37.2 ± 5.2, and 37.4 ± 5.7, for low, medium and high total-score groups respectively, p = 0.743) and oocyte age (34.9 ± 4.9, 34.1 ± 4.7, and 33.7± 5.2, for low, medium and high total-score groups respectively, p = 0.229) were compared between the score groups. A significant increase in the implantation rate was noted among embryos presenting higher scores, followed by those with median scores, while embryos with lower scores presented the lowest implantation rate (34.8 ± 5.3, 35.9 ± 5.2, and 39.7 ± 5.2, for low, medium and high total-score groups respectively, p &amp;lt; 0.001). No significant differences in the miscarriage rate were observed between the three score groups (10.0 ± 4.2, 13.2 ± 4.1, and 5.1 ± 2.9, for low, medium and high total-score groups respectively, p = 0.239.) The logistic regression analysis showed that embryos in the low total-score group presented a 12.3% decreased chance of implantation when compared with embryos in the high total-score group (OR: 0.877, CI: 0.843-0.912, p &amp;lt; 0.001), while embryos in the medium total-score group presented a 9.6% decreased implantation chance when compared with embryos in the high total-score group (OR: 0.904, CI: 0.870-0.939, p &amp;lt; 0.001). Limitations, reasons for caution The use of historical cohort groups is a drawback. Wider implications of the findings The behavior of blastocysts after warming, as indicated by blastocyst area, ZP thickness, re-expansion, along with the pre-vitrification embryo classification, may provide crucial information for selecting the optimal embryo for transfer and managing patient expectations. Trial registration number N/A

P-113 Comparison of mitochondrial function between expanded and hatching human blastocysts

Abstract Study question Is there a difference in mitochondrial function between expanded and hatching fresh and vitrified blastocysts? Summary answer Freezing and thawing may lead to mitochondrial dysfunction. What is known already Mitochondria, as crucial cellular organelles, play role in energy production through the electron transport chain, cell motility and organelle movement. They also have a significant impact on embryonic development. Furthermore, during the process of embryonic hatching, the energy generated by these organelles can affect ion pump activity, blastocyst expansion, and collapse. Therefore, factors that stress embryonic mitochondria may contribute to embryogenesis and hatching. Cooling and warming, as stress factors, maybe result in organelle damage, protein denaturation, membrane disruption, and DNA fragmentation. Additionally, they can cause changes in mitochondrial function, leading to decreased ATP levels and increased ROS levels. Study design, size, duration In this study, vitrified and fresh human 8-cell embryos were collected from April 18, 2021, to July 19, 2023. The embryos were cultured until the blastocyst stage, and then four samples were assessed in four groups. These groups included vitrified expanded blastocysts, vitrified hatching blastocysts, fresh expanded blastocysts, and fresh hatching blastocysts. Participants/materials, setting, methods Vitrified embryos were sourced from the embryo bank of the Royan Institute, while fresh embryos were obtained from good-quality 3PN embryos or excess embryos from couples who did not wish to freeze them. A stock solution containing lipophilic cationic JC-1 dye in dimethyl sulfoxide was prepared. Blastocysts were then exposed to a concentration of 1 μg/ml of JC-1 diluted in HamsF10-HSA. After washing with HF-HSA, imaging was performed using a 2-photon confocal microscope. Main results and the role of chance Our results showed that mitochondrial membrane potential in expanded blastocysts of both fresh and vitrified eight-cell embryos was significantly higher than in hatching blastocysts of fresh and vitrified embryos (P &amp;lt; 0.05). However, there was no significant difference in mitochondrial function between the vitrified and fresh groups, neither in expanded nor hatching blastocysts. Limitations, reasons for caution Due to the human origin of the samples, the minimum necessary replicates were considered for each experimental group. Wider implications of the findings In this study, it appears that plump zona breakers can be detected in hatching blastocysts using confocal microscopy. Previous studies, such as the one conducted by Sathananthan, identified these cells using a TEM microscope. Trial registration number -

P-144 Utilizing artificial intelligence to uncover Day 5 morphokinetic features associated with single blastocyst transfer implantation outcome

Abstract Study question Can AI aid in studing embryos’ continuous development in Day5 and identifying morphokinetic features (blastocyst growth and spontaneous collapse etc.), to improve implantation potential prediction? Summary answer The AI system utilized time-lapse images to measure newly identified morphokinetic features, establishing a significant correlation with clinical pregnancy outcomes. What is known already In Single Blastocyst Transfer (SBT), the conventional Gardner evaluation system may inadequately differentiate between embryos of comparable morphology scores in specific cases. To address this, integrating new morphokinetic features is essential for enhanced blastocyst evaluation. Spontaneous collapse (SC) during blastocyst expansion is a phenomenon that was revealed relatively recently following the clinical application of time-lapse monitoring in IVF laboratories. Previous studies link embryo size and SC to quality, yet these correlations require further validation. In the critical selection process utilizing time-lapse imaging (TLI) systems, AI technology is indispensable for developing automated and interpretable tools to ensure precise quantitative measurements. Study design, size, duration 255 patients underwent IVF with Day5 single blastocyst transfer in fresh cycles at the Center for Reproductive Medicine, Nanjing Drum Tower Hospital, from April 2021 to August 2022, and were analyzed in this retrospective study. Embryos were cultured in time-lapse incubators (CCM-iBIS, ASTEC). On Day 3 embryos were transferred to sequential media (G2, Vitrolife, Sweden) and cultured in the same incubator until Day5. In this cohort, there were 167 clinical pregnancies and 88 non-pregnant cases. Participants/materials, setting, methods An AI system was developed to calculate the development curves of areas in each part of the embryo (ZP,blastocel, ICM and TE) based on TLI images from 92 to 116 phi(±2). T-test and correlation analysis were used to select effective features. The dataset was divided into training and testing subset in an 80:20 ratio. The performance of different prediction models were compared by Delong test. Main results and the role of chance In this study, an AI system automatically generated blastocyst development curves, from which 35 morphokinetic features were extracted. These features were carefully defined to comprehensively capture the dynamics of embryo size development and spontaneous collapse (SC). Six parameters, including maximum size, max/min ratio of blastocyst area (with and without ZP region), speed of expansion after tSB, and number of minor SC, exhibited statistical correlation with clinical pregnancy outcomes. Using these parameters, an XGBoost model achieved an AUC of 0.71 for pregnancy prediction, surpassing (p = 0.0307, p &amp;lt; 0.05) the AUC of 0.48 from the model trained with manual Gardner evaluation. These findings underscore the potential of AI-generated morphokinetic features of D5 in enhancing blastocyst evaluation systems. Limitations, reasons for caution The limited sample size used in this retrospective experiment may lead to an underrepresentation of certain significant features. Wider implications of the findings In contrast to prior findings, most SC-based features in our study did not significantly correlate with clinical pregnancy outcome. Further research is needed to understand their causes and implications. Integrating AI with TLI offers promise for improving single blastocyst transfer outcomes, emphasizing quantifiability and interpretability in approach. Trial registration number NA

P-196 Early blastocyst formation on day 4 is associated with improved clinical outcome and advocates for embryo quailty assessment on day 4

Abstract Study question Is early cavitation on day 4 associated with improved clinical outcome? Summary answer Early blastulation on day 4 is a positive predictor of implantation after single blastocyst transfer on day 5. What is known already In order to select the blastocyst with the highest implantation potential for transfer or freezing, several morphological and/or morphokinetic parameters have been proposed. For instance, early compaction on day 3 (D3) or blastocyst expansion on day 5 (D5) have been reported to be relevant when selecting embryos for transfer. However, day 4 has often been overlooked and very few studies focused on early blastulation on day 4 (D4). Indeed, data on early blastulation on day 4 is scarce. It was however recently demonstrated that embryos transferred on day 4 could provide acceptable pregnancy rates. Study design, size, duration This monocentric retrospective cohort study was conducted in all consecutive couples undergoing IVF cycles in 2017-2022 with single blastocyst transfer and observation on day 4. Participants/materials, setting, methods Morphological and morphokinetic assessment was performed by 2 operators at 93±2h, 116±2h (and 140±2h) post insemination, on day 4, 5 (and 6) respectively. A total of 26 507 D4 embryos were included in this study and grouped according to the observation of early cavitation or not : 23 244 were in the D4 control group (absence of early cavitation on D4) and 3263 were in the D4 blastocyst group (observation of early cavitation on D4). Main results and the role of chance The proportion of embryos used clinically for either transfer or freezing on day 5 or 6 was significantly higher in the D4 blastocyst group than in D4 control group (86.7% vs 50.1% respectively, p &amp;lt; 0.05). Concerning pregnancy rate, cinical pregnancy rate was significantly higher in the D4 blastocyst group than in D4 control group (p &amp;lt; 0.05). In the D4 control group, 5051 embryos were transferred and clinical pregnancy rate is 28.7%. In the D4 blastocyst group, 1286 embryos were transferred (fresh or frozen) and clinical pregnancy rate is 40.2%. Limitations, reasons for caution The retrospective and monocentric design calls for caution when considering the generalizability of our findings. Wider implications of the findings We postulate that D4 embryo quality assessment should be widely implemented in any blastocyst program in order to improve embryo quality assessment and subsequently clinical outcome. Trial registration number not applicable

O-094 Novel stem cell-based twin embryo model reveals inner cell mass division and enhanced endometrial adhesion in monochorionic twin development

Abstract Study question How do monochorionic twin embryos arise and develop during the pre- and peri-implantation stage? Summary answer Twinning involves inner cell mass division during blastocyst expansion and heightened endometrial adhesion, with twin blastoids showing greater implantation potential. What is known already The occurrence of monozygotic twins is between 0.4 and 0.5% of livebirths globally from natural conception, of which the majority are monochorionic. Monochorionic twins are associated with increased risk of complications and higher mortality rates during pregnancy due to twin-to-twin transfusion syndrome, twin anemia-polycythemia sequence, developmental malformations and increased incidence of pregnancy-related diseases in the mother. The rarity of monochorionic twinning, combined with ethical constraints in human embryo research, renders the study of twinning practically infeasible, which presents in a significant knowledge gap regarding the origins and implications of early developmental monochorionic twin embryos development. Study design, size, duration In a stem cell-based model of the blastocyst-stage embryo (i.e., blastoid) conditions were identified that favor the generation of monochorionic twin blastoids. To obtain sufficient statistical power, screening experiments were performed using 3 culture wells per condition, each well containing at least 90 blastoids. Experiments were repeated 3 times and results were pooled. Blastoids were cultured for at least 3 days followed by 2 days on endometrium monolayers on-chip. Participants/materials, setting, methods Human naïve induced pluripotent stem cells and embryonic stem cells were used to form twin blastoids within custom microwell array platforms. Patient-derived endometrium epithelial cells were expanded as organoids and seeded as monolayers in microfluidic chips. Blastoids were infused into the chip, allowed to adhere for 2 days, followed by a controlled stepwise increase of flow rate after which the blastoid adhesion rate to the endometrium layer for both singleton and twin blastoids was quantified. Main results and the role of chance Twin blastoids contained a cystic GATA3+ single layered trophectoderm-like epithelium encasing two distinct inner cell masses (ICMs). Morphological and morphokinetic analyses revealed that twinning occurs during the blastocyst cavitation phase via division of the OCT4+ pluripotent cell mass. After three days of culture, twin blastoids measured 228±32 µm, which is in the upper range of the cyst diameter of a blastocyst. In addition, both sibling ICM-like clusters preserved similar epiblast and hypoblast cell proportions and were consistently positioned nearly 150° (146.6±26°) apart along the blastocoel lining (measured as 2D-projections and in 3D reconstructions), which suggests a minimal angular distance for establishing two separate condensed ICM clusters. Notably, each ICM in twin blastoids contains its own NR2F2+ polar trophectoderm-like region, indicative of implantation readiness. Under defined flow regimes, twin blastoids show increased adhesion capacity to endometrium monolayers compared to singleton blastoids (60% vs 38% at highest flow rate), suggestive of increased implantation potential possibly due to a larger adhesive surface area. Twin blastoids cultured on endometrium monolayers could develop into structures with two distinctive OCT4+ pluripotent cell clumps both delineated by SOX17+ hypoblast-like cells and as a whole encompassed by GATA3+ trophoblast-like cells, suggestive of post-implantation organization. Limitations, reasons for caution While twin blastoids adhered to the endometrium monolayer and maintained two separate OCT4 compartments after 48 hours in post-implantation culture, both singleton and twin blastoids did not establish organized 3D structures associated with later post-implantation development, including amniotic and yolk sac-like cavities. Wider implications of the findings Twin blastoids open new avenues for studying the various aspects of twinning, ranging from their formation processes to the effects of multiple ICMs on trophectoderm maturation and implantation potential. In the future, with more sophisticated endometrium platforms, this model may inform on how clinical complications arise in twin pregnancies. Trial registration number not applicable

O-216 The impact of maternal age on cumulative life birth rates after single euploid embryo transfer: differences between autologous and donor oocyte cycles

Abstract Study question How much of an impact has the maternal age on cumulative life births after euploid Single Embryo Transfer (SET) in autologous and donor oocyte cycles? Summary answer A maternal age ≥ 42 years old affects negatively the cumulative Life Birth Rates (cLBRs) even after euploid SET, but only in the autologous cycles. What is known already Despite the improvements in assisted reproduction technologies (ART) in the last decades, the LBRs remain suboptimal, especially when advanced maternal age is considered. Although several factors could explain these outcomes (i.e. infertility history; uterine pathologies/surgery; advanced paternal age), maternal age-related aneuploidies remain the most limiting factor for a favorable clinical outcome. Thus, the Genetic Testing for Aneuploidies (PGT-A) has been integrated into ART as a strategy to evaluate chromosomal status before transfer. Interestingly, recent studies show that, even after the transfer of euploid blastocyst, the maternal age negatively influences the LBRs. Study design, size, duration This study, carried out in Villa Mafalda Reproductive Medicine Department (Italy), included 568 euploid SET: 542 from autologous cycles (divided in four subgroups according to maternal age: ≤ 34 y.o. (n = 207); 35-38 y.o. (n = 193); 39-41 y.o. (n = 106); ≥ 42 y.o. (n = 36)) and 26 from oocyte donation (OD) cycles (average recipient age is 44.2). All cycles were performed from May 2020 to February 2023. PGT for Monogenic disorders (PGT-M) cycles were excluded. Participants/materials, setting, methods All blastocysts included in this study were analyzed by Next Generation Sequencing (NGS) after a day 5/6 trophectoderm biopsy. For each subgroup was annotated the embryo morphology- divided into good (AA-AB-BA); average (BB-BC-CB) and poor (CC) according to Gardner/Schoolcraft blastocyst grading system- and the day of expanded blastocyst formation (grade 4 of Gardner/Schoolcraft blastocyst grading system). Primary analysis included the cumulative LBRs. Secondarily, blastocyst morphology and blastocyst expansion were examined for all embryo categories considered. Main results and the role of chance The pairwise comparison between groups was performed by Chi-square analysis and p &amp;lt; 0.05 was considered statistically significant. The cLBRs for each subgroup were respectively: ≤ 34 (63.09%); 35-38 (58.47%); 39-41 (56.84%); ≥ 42 (48.57%); OD (69.23%). Data showed that cLBRs decreased with the increase of maternal age, although statistical significance was observed only among women ≤ 34 y.o. and ≥ 42 y.o. (P=0.029). Notably, there was no statistical significance in cLBRs comparing women ≤ 34 y.o. and OD patients (P=0.086). The good-quality blastocyst rate was respectively: ≤ 34 y.o. (54.58%); 35-38 y.o. (47.66%); 39-41 y.o. (60.37%); ≥ 42 y.o. (50%); OD (59.37%). The pairwise comparison showed no significant difference between groups. Furthermore, there was not found significant difference about the day of expanded blastocyst formation: ≤ 34 y.o. (D5: 78.92%; D6 21.07%); 35-38 y.o. (D5: 78.49%; D6: 21.50%); 39-41 y.o. (D5:75.70%;D6: 24.29%); ≥ 42 y.o. (D5:69.44%; D6: 30.55%); OD (D5: 87.5%; D6: 12.5%). Data showed that maternal age ≥ 42 y.o. affects negatively cLBRs even after euploid SET, suggesting that other factors age-related, rather than aneuploidies, influence implantation. Interestingly, these factors do not affect OD recipients, with advanced maternal age, who, instead, reach successful LBRs like patients ≤ 34. Limitations, reasons for caution These results should be evaluated carefully because of the small sample size of the OD patients. A greater number of OD cycles are needed to verify the results obtained. Wider implications of the findings Our findings suggest that embryonic age-related factors - changes in gene expression, metabolism or epigenetics - could influence the embryo capability to, successfully, support implantation other than the ploidy status. This thesis was supported by the OD recipient’s behavior, who had cLBRs comparable to young autologous patients. Trial registration number Not Applicable

P-194 Developing a classification system for euploid embryos: leveraging morphology grades and biopsy day to accurately rank them based on ongoing pregnancy potential

Abstract Study question Can morphology grades and day of trophectoderm (TE) biopsy be utilized to accurately rank euploid blastocysts according to their potential for ongoing pregnancy (OP)? Summary answer The proposed ranking system categorized euploid blastocysts accurately in terms of their ongoing pregnancy potential. What is known already Implementing a ranking for embryo selection within a pool is a valuable optimization strategy. Many studies have documented the importance of inner cell mass (ICM) and trophectoderm (TE) grading, as well as the day (D) at which blastocysts are biopsied on clinical outcomes. However, literature sparks controversy on clinical findings in relation to blastocyst quality. This is primarily because it reports a diversity in blastocyst grade-ranking systems, and none of them accounting for the day of biopsy. Enhancing consistency and comparability across various research studies could be achieved by establishing a standardized approach for qualitatively classifying and ranking euploid blastocysts. Study design, size, duration This retrospective cohort study included 2209 single euploid frozen embryo transfers (SeFET) between 2017 and 2023. Blastocyst (BL) expansion, TE and ICM were assessed before biopsy according to Gardner’s criteria. Only blastocysts graded ≥BL3CC were biopsied on D5, D6 or D7. Classification models (in-house, logistic regression, gradient boost) were assessed for their calibration, i.e., correct prediction of ongoing pregnancy (OP) probability. Participants/materials, setting, methods In-house ranking system categorized euploid transferred blastocysts (BL3-BL6) as ‘Top-quality’ for blastocysts AA/AB/BA on D5; ‘Good-quality’ for blastocysts BB on D5 and AA/AB/BA/BB on D6; ‘Fair-quality’ for AC/BC/CA/CB on D5, BC on D6 and AA/AB/BA/BB on D7; and ‘Poor-quality’ for blastocysts CC on D5, AC/CA/CB/CC on D6 and CA/CB/AC/BC/CC on D7. The in-house classification ranking system was compared to other developed models to evaluate whether performance could be increased. Main results and the role of chance A total of 406 blastocysts were ranked as Top-quality (100% D5), 1323 as Good-quality (51.3% D5 and 48.7% D6), 190 Fair-quality (57.9% D5, 36.3% D6 and 5.8% D7) and 290 as Poor-quality (12.8% D5, 73.8% D6 and 13.4% D7). The ongoing pregnancy rate was significantly higher for blastocysts ranked as Top compared to Good, Fair and Poor-quality (58.9%, 52.2%, 35.8% and 26.6%, respectively). Miscarriage rates were significantly associated with ranking quality (20.9%, 24.3%, 30.6% and 39.4% for Top, Good, Fair and Poor-quality blastocysts, respectively). We further developed two new classification ranking systems using logistic regression model and XGBoost machine learning model. But the new models provided similar AUC and calibration slope (CS) values as the in-house ranking system (AUC [CS]: 0.604 ± 0.0167 [0.962 ± 0.213] and 0.601 ± 0.0177 [0.949 ± 0.219] vs.0.597 ± 0.015 [0.980 ± 0.230] for logistic regression, XGBoost versus in-house model, respectively). Limitations, reasons for caution The present embryo classification ranking system may not be extrapolated to non-PGT-A cycles. Wider implications of the findings Different grade-ranking systems of blastocysts keep being reported in the different publications. Reaching a consensus to rank euploid blastocysts qualitatively, as the hereby proposed classification, may improve consistency and comparability across different research studies. Crucially, the present ranking-system aids embryo selection for transfer from available pool. Trial registration number not applicable