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Study question Are there specific morphokinetic time points which can be used to determine whether an embryo should be discarded? Summary answer Morphokinetic ranges where embryos will be discarded rather than transferred or cryopreserved, can be defined using time-lapse annotations automatically generated with artificial intelligence (AI). What is known already Time-lapse incubation has changed the way embryos are selected. Instead of static daily observations, continuous monitoring of embryos allows for generation of morphokinetic parameters which quantify the pace of development. However, annotations by humans have been shown to incur operator variations and are time-consuming to perform. AI can automatically annotate embryos with equivalence in accuracy to experienced embryologists. Although most embryo selection methods are designed to identify the embryo with the highest chance of becoming a healthy live birth baby, the ability to identify embryos that will not be suitable for treatment is equally important for clinical decision making. Study design, size, duration This is a prospective, observational, cohort study. Time-lapse videos from 142 embryos from a private fertility clinic in Spain were automatically annotated using CHLOE (Fairtility), an AI-based software. CHLOE automatically generated the following morphokinetic parameters: tPNa, tPNf, t2, t3, t4, t5, t6, t7, t8, t9+, tM, tSB, tB, tEB. Participants/materials, setting, methods Embryos analysed were from donor and own oocyte’s treatments. Selected embryos were analysed using CHLOE, to automatically identify morphokinetic parameters. The distribution for each morphokinetic parameter was compared between fates (data presented for transferred + frozen vs discarded as mean+-standard deviation, 2-sided t-test). Each continuous morphokinetic parameter was categorised according to the ranges where embryo utilisation was futile ( Main results and the role of chance For every morphokinetic parameter the difference in event time between frozen+transferred vs discarded embryos was statistically significant(p tPNa (7.68(2.03)vs22.04(27.15),p tPNf (21.71(2.86)vs34.63(24.11),p t2 (24.92(2.71)vs33.78(16.17),p t3 (34.62(4.03)vs42.58(22),p=0.0024), t4 (37.29(4.31)vs48.29(20.29,p t5 (47.03(6.47)vs55.32(22.63),p=0029), t6 (49.54(5.63)vs60.56(22.20),p t7 (53.1(7.86)vs69.13(24.54),p t8 (57.78(9.78)vs77.33(25.79),p t9+ (69.14(7.39)vs81.9(21.96),p tM (83.9(8.72)vs96.08(16.88),p tSB (97.89(7.55)vs105.38(11.38),p=0.0005), tB (105.74(7)vs113.25(15.53),p=0.0002), eEB (110.65(7.58)vs120.47(11.36),p=0.0031). When looking at the exact distribution of these embryos according to time, it became apparent that a goldilocks zone appeared whereby the proportion of embryos transferred or frozen peaked, and the number discarded was at its minimum. The converse was true when looking at the more extreme values of a particular parameter. Thus, we were able to determine the (optimal vs futile time ranges): tPNa (4.4-8.8 hours, where the utilization rate was at its maximum vs 13.7, where the utilization rate was at its minimum), tPNf (19.1-23.2vs28.9), t2 (23.-36.4vs33.6), t3 (32.1-37.4vs>24.6,>43), t4 (34-40.2vs55), t5 (42.7-52vs63.5), t6 (45-4-54.2vs63.70), t7 (47.8-56.7vs77.5), t8 (49.2-64.5 vs 82.5), t9+ (64.1-74.2vs90), tM (76.6-92.6 vs 104.2), tSB (91.2-105vs113.8), tB(97.2-111.2 vs 118.7), tEB (103.4-116.7,122.5). A 60% of the embryos were in the futile range in at least 1 parameter, from which only 1 in 3 were utilised. Limitations, reasons for caution This is a single centre study. Further work will (i) test the limits across different clinics, with different geographical demographic variations, and varied clinical practices, to understand how these factors affect the limits between futile and optimal ranges of morphokinetics, and (ii) assess clinical outputs (implantation, ploidy, live birth). Wider implications of the findings Identifying objective ranges for determining when an embryo is not suitable for treatment will help reduce variation between and within embryologists and clinics; will avoid overly optimistic decisions which waste time and resources and increase patient’s emotional burden, and increase professional confidence when selecting embryos for discarding, transfer or freezing Trial registration number not applicable

Study question What are the causes and consequences of blastocyst collapse? Summary answer ∼50% of blastocysts collapsed, especially if they are aneuploid and/or morphologically-poor. Yet, no impact on the live-birth-rate (LBR) per vitrified-warmed euploid single-embryo-transfer (SET) was reported. What is known already Time-lapse-microscopy (TLM) is a powerful tool to describe the peculiar dynamics of preimplantation development. Lately, artificial intelligence (AI) has been also implemented to automatize and standardize such description. Here, we adopted AI to comprehensively portray blastocyst collapse, namely the phenomenon of embryo contraction with an efflux of blastocoel fluid and the detachment of the trophectoderm (TE) from the Zona Pellucida (ZP). Although, the causes of this event are still undetermined, small blastocyst contractions have been reported beneficial for the hatching process, while a full collapse has been associated with lower competence. Study design, size, duration Observational study including 1943 blastocysts from 643 couples cultured in the Embryoscope between January-2013 and December-2020. TE biopsy without day3 ZP drilling and comprehensive-chromosome-testing were performed. The Fairtility® software automatically registered: (i)time of starting-blastulation (tSB), (ii)starting and ending time of each collapse (tSC and tEC), (iii)blastocysts’ areas, (iv)shrinkage% [(area at SC – area at EC)/area at SC)], (v)embryo:ZP ratio at EC (area of the collapsed embryo/area of the ZP), and (vi)time of biopsy (t-biopsy). Participants/materials, setting, methods Blastocyst quality was defined according to Istanbul Consensus (11, excellent; 12-21, good; 22-13-31, average; 33-23-32, poor) and with the Fairtility implantation score (IS) as well, i.e., a continuous variable from 0 to 1 generated by the KID+ software based on the TLM videos of preimplantation development. The main outcome was the LBR per euploid SET adjusted for confounders through logistic regressions. All couple and embryo features were also investigated for their association with blastocyst collapse. Main results and the role of chance 47.3% of the blastocysts collapsed 1- to 9-times (interval between collapses: 4-8hr), and 73% of the couples had ≥1 collapsed blastocyst (1.8±1.1, range:1-8). No couple feature, though, was associated with blastocyst collapse. The longest collapses lasted 1.5±1.1 (0.13-5.1)hr, while the largest shrinkage% and embryo:ZP ratio at EC were 35±14% (10-78%) and 81±9% (33-90%), respectively. In ∼50-60% of collapses a 20-40% blastocyst volume reduction was registered, 40-60% or 20-40% in ∼15-30%, 60-80% in 0-4%. In case of multiple collapses, the first three involved smaller shrinkages. Blastocysts undergoing ≥1 collapse showed similar tSB as not-collapsing blastocysts, but progressively longer tEB and t-biopsy. The earlier the first event, the more the consecutive collapses. Notably, the poorer the morphology, the higher the risk (excellent, good, average, and poor not-collapsing blastocysts were 64%,50%,44% and 37%), number (e.g.,≥4 collapses were 0.4%,2%,4% and 8%) and duration (1.2±1.0,1.4±1.0,1.6±1.1 and 1.9±1.3hr) of blastocyst collapse. Collapsing blastocysts were significantly less euploid than non-collapsing (35% vs 47%; multivariate-OR:0.75,95%CI 0.6-0.92,p Limitations, reasons for caution Gestational and perinatal outcomes were not assessed. Other culture strategies and media shall be assessed for their association with blastocyst collapse. Perhaps, future studies from other groups and with a larger sample size might unveil a significant impact on the clinical outcomes. Wider implications of the findings Collapse is common and delays blastocyst full-expansion. Moreover, poor morphology and aneuploidies involve a higher risk of collapse(s); however, no impact was reported on the clinical outcomes after euploid SET. AI appears to increase the throughput of the analysis, but additional data are required to research the causes of collapse. Trial registration number Not applicable

Study question Are automated blastocyst morphometric measurements combined with morphokinetic pattern associated with implantation rate? Summary answer Automated blastocyst morphometric measurements with morphokinetic pattern demonstrated that a larger blastocyst size and a shorter time interval tB-tSB are associated with higher implantation potential. What is known already Optimization of embryo selection is important for increasing implantation potential. Transfer of a high-quality blastocyst based on conventional morphological parameters has been shown to improve IVF clinical outcome. Novel parameters of blastocyst quality, including morphokinetics from time-lapse monitoring and manual analysis of morphometric parameters, have demonstrated promising results regarding implantation potential. However, manual measurement of morphometric parameters is a time-consuming task and is subjected to intra- and inter-observer variations. The introduction of automated morphometric measurements would remove subjective blastocyst analysis and further improve implantation rates. Study design, size, duration A nested retrospective case control analysis of 608 day-5 transferred blastocysts was conducted and included women who underwent IVF treatment in three public IVF units between 2014 and 2017. Participants/materials, setting, methods Automated morphometric blastocyst analysis was measured at the mean time of tEB-tPNf (85.82 h) by training a pixel-wise segmentation model (MaskRCNN) on time-lapse videos. Morphometric blastocyst parameters included the following: blastocyst size (μm), inner cell mass (ICM) size (μm), ICM to blastocyst size ratio, and ICM shape. Annotation variables included all the time intervals (hours) from time of pronuclei fading (tPNf) to the expanded blastocyst (tEB). Main results and the role of chance The mean blastocyst size for implanted embryos was significantly larger compared to non-implanted embryos (152.10 ±19.22μm vs 144.25±18.52μm, respectively, p Limitations, reasons for caution The study's limitations include its retrospective nature and the absence of some patient characteristics. Wider implications of the findings A blastocyst selection based on the combination of automated blastocyst size measurements and manual tB-tSB time interval may double the implantation rate. The inclusion of automated morphometric measurements to the blastocyst selection algorithm may reduce intra- and inter-observer variations and should be incorporated into models for implantation prediction. Trial registration number 0006-20-HMO

Study question Does an extra Gonadotropin (GT) dose on trigger day during a gonadotropin-releasing hormone (GnRH) antagonist cycle for oocyte cryopreservation result in improved cycle outcomes? Summary answer An extra GT dose on trigger day in patients undergoing elective egg freezing is not associated with increased oocyte yield. What is known already The most common controlled ovarian hyperstimulation (COHS) protocol used in elective oocyte cryopreservation cycles is a GnRH antagonist protocol with a GnRH agonist trigger. There is no consensus about the timing of the final gonadotropin dose administration. Some physicians add an extra GT dose during the ovulation trigger day and others advise to receive the last GT dose the day before. The yield of this extra GT dose was not previously evaluated. Study design, size, duration A retrospective cohort study, consisting of cycles of elective oocyte cryopreservation at Hadassah Medical Center from 1.2017 through 5.2021. Patients were divided into 2 groups- women who received their last GT dose a day before the GnRH agonist trigger, and women who received the last GT on the trigger day. Background and gynecologic characteristics, as well as COHS cycle parameters, were compared. The primary outcome was the average number of mature oocytes in each group. Participants/materials, setting, methods Women 30-41 years who were admitted for elective oocyte cryopreservation. Women who were referred due to medical reasons or cycles with a protocol other than GnRH antagonist were excluded. Ovarian stimulation with GT began on the 3rd day of the cycle, and five days later, a GnRH antagonist was added. When more than two mature follicles were demonstrated, a GnRH agonist trigger was administrated and approximately 36 hours later, the oocyte aspiration procedure was performed Main results and the role of chance During the study period, 448 elective egg freezing cycles were performed, 151 cycles with an extra dose on the trigger day and 297 cycles without an extra GT dose. The groups did not differ in their background characteristics, age, BMI, GT used, and the number of induction days. However, the extra GT group had significantly higher day 3 follicular stimulating hormone (FSH), lower anti-Mullerian hormone (AMH), and lower maximal Estradiol (E2) levels. The extra GT dose group had a significantly lower mean number of mature oocytes (11.1±7.1 vs. 6.4±4.5, p To adjust for the maximal E2 parameter, that differed between the groups, an additional analysis was performed, dividing the two groups into subgroups of cycles with maximal E2 above and below 10,000 pmol\l. Interestingly, even in cycles with maximal E2 above 10,000 pmol\l, the extra dose group had poorer results (10.3±4.3 vs. 12.7±7.2, p Limitations, reasons for caution The retrospective design of this study and the differences in cycle characteristics between patients that did or did not receive an extra GT dose on trigger day make it prone to selection bias. Wider implications of the findings Our study shows that administration of an extra GT dose on trigger day in oocyte cryopreservation cycles does not yields more mature oocytes. However, wider prospective studies are needed to thoroughly investigate the implications of this treatment’s effect on cycle outcomes. Trial registration number Not applicable

Study question To identify challenges in choosing a robust AI following comparative validation with data already pre-selected with established embryos selection tools: blastulation, morphology, time-lapse, PGTA. Summary answer Challenges included: bias; assessment against outcomes AI models were not trained on; performance metrics prioritisation; statistical methodology; continuous data cutoffs for binary clinical decision making. What is known already AI is commercially available to be incorporated into routine practice to support embryo selection decision-making. Different clinical practices and demographics are used to train AI models, potentially impacting the prediction efficacy of the same model when used in different clinics. Fertility professionals require robust methods of validation to responsibly implement AI-based tools. Unbiased and robust frameworks for comparing AI systems in the same dataset are needed. Validating AI in a dataset of time-lapse selected euploid blastocysts using all the current methods of embryo selection currently available is the toughest assessment possible and has not previously been performed. Study design, size, duration This study uses a retrospectively timelapse dataset collected from 2018-2021 at a single private fertility clinic. The dataset included 915 blastocysts which underwent PGTA (913 results: 381 euploids, 528 aneuploids, 4 mosaics) and 46 euploids transferred with known bhcg and ongoing clinical outcome (of which 40 resulted to live birth). Following a prospective, comparative, observational, cohort study design, blastocysts were blindly scored using the CHLOE(FAIRTILITY) and another commercially available AI system, referred to as ‘AI-2’. Participants/materials, setting, methods Patients aged 24-47years (average 35.4). Blastocysts selected for biopsy and transfer based on morphology and KIDScore(Vitrolife). Both AI systems were tested in the data set blindly, without any training. Correlation Regression analysis assessed correlation with KIDSCORE and relative to each AI system. Efficacy of prediction (using metrics AUC, Accuracy, Sensitivity, Specificity and Informedness) of outcomes (ploidy, biochemical and clinical pregnancy) were assessed for both AI models (CHLOEvsAI-2) by two independent statisticians to establish significance. Main results and the role of chance Regression analysis demonstrated no correlation between KIDSCORE and AI-2(r2=0.3%,p=0.5) or between CHLOE(FAIRTILITY) and AI-2(r2=0.03%,p=0.9). CHLOE(FAIRTILITY) correlated with KIDSCORE(r2=29%,p AI-2 was not predictive of ploidy (Euploids vs Aneuploids+mosaic: AUC=0.5,p=0.6). CHLOE(Fairtility) was predictive of ploidy(AUC=0.66, p Neither AI-2 or CHLOE(Fairtility) predicted which embryo the human embryologist prioritised for transfer (AI-2 vs CHLOE:accuracy:0.31vs0.49, p CHLOE(Fairtility) was more specific than AI-2 for predicting selection for transfer(0.44/0.80vs0.17/0.93,p Informedness was positive for both CHLOE(Fairtility) and AI-2 in predicting all outcomes assessed. Informedness was greater for AI-2 for predicting morphology(AI-2vsCHLOE:0.16vs0.31,p Limitations, reasons for caution In this single clinic study, both algorithms were assessed against outcomes (live birth following transfer of time-lapse cultured euploid blastocysts) for which they were not trained on: AI-2(designed for ploidy prediction) and CHLOE(FAIRTILITY, implantation prediction of non-PGTA embryos) and no clinic data was used for training. Wider implications of the findings The only way to decide which AI model is more useful is by a direct comparison of two or more models on the same dataset with same outcomes and metrics, as recommended by TRIPOD. To date, this is the first publication comparing multiple commercial AI models on the same dataset. Trial registration number NA

Study question Which is the clinical value of day 7 blastocysts? Summary answer Ending embryo culture at 144 hours-post-insemination (hpi) would involve7.3%- and 4.4%-relative reductions in the patients obtaining euploid blastocysts and live birth(s)(LBs), respectively. What is known already Many studies showed that day 7 blastocysts are clinically valuable although less euploid and less competent than faster growing embryos. Nevertheless, a large variability exists in: (i) the definition of “day 7”; (ii) the criteria to culture embryos to day 7; (iii)the clinical setting; (iv) the local regulation; and/or (v) the culture strategies and incubators. Here,we aimed at ironing out these differences and portray day 7 blastocysts with the lowest possible risk of bias. To this end, we have also adopted an artificial intelligence (AI)-powered software to automatize developmental timings annotations and standardize embryo morphological assessment. Study design, size, duration Observational study including 1966 blastocysts obtained from 681 patients cultured in a time lapse incubator between January 2013 and December 2020 at a private Italian IVF center. Participants/materials, setting, methods Trophectoderm biopsy without hatching and comprehensive-chromosome-testing were performed. Blastocysts were clustered in six groups based on the time-of-biopsy every 12hr from 168hpi. Blastocyst quality, time-of-expanding-blastocyst (tEB) and duration of expansion were annotated through AI and confirmed manually. The main outcomes were euploidy-rate and LB-rate (LBR) per transfer. Lastly, patients obtaining (euploid) blastocysts, LBs, and supernumerary blastocysts, were reported based on a hypothetical 144hpi cut-off, and all relative reductions calculated. Main results and the role of chance 14.6% of the blastocysts reached full expansionbeyond 144hpi (5.9% between 144-156hpi, 7.9% between 156-168hpi, and 0.8% >168hpi). Slower blastocysts were of a worse quality based on the evaluation of both embryologists and AI. Both longer tEB and a longer duration of expansion concurred to day7 development, quite independently of embryo quality. The lower euploidy rate among day7 blastocysts is due to their worse morphology and more advanced oocyte age, rather than to a slower development per se. Conversely, the lower LBR was significant even after adjusting for confounders, with a first relevant decrease for blastocysts biopsied in the range 132-144hpi (N = 76/208, 36.5% versus N = 114/215, 53.0% in the control, multivariate-OR: 0.61, 95%CI 0.40-0.92, adjusted-p=0.02), and a second step for blastocysts biopsied in the range 156-168hpi (N = 3/21, 14.3%, multivariate-OR:0.24, 95%CI 0.07-0.88, adjusted-p=0.03). Nevertheless, when the cut-off was set at 144hpi, no significant difference was reported. In this patient population, ending embryo culture at 144hpi would have caused 10.6%-, 7.3%-, 4.4%-, 13.7%-, and 5.2%-relative reductions in the number ofpatients obtaining blastocysts, euploid blastocysts, LBs, supernumerary blastocysts without a LB and after a LB, respectively. Limitations, reasons for caution Gestational and perinatal outcomes were not assessed, and a cost-effectiveness analysis was not performed. We encourage the production of these data in other clinical settings and regulatory contexts. Wider implications of the findings Day7 culture shall be supported following a careful case-by-case evaluation. Patients shall be aware of their lower competence, yet day7 blastocysts are valuable for poor-prognosis couples, couples less compliant towards other attempts in case of failures, and couples wishing for second children. AI may improve the generalizability of these evidence. Trial registration number Not applicable

Study question Does high ovarian response to controlled ovarian stimulation (COS) have a negative effect on oocyte quality? Summary answer High ovarian response is associated with reduced oocyte quality manifested as higher fraction of immature oocytes and higher rate of direct uneven cleavage (DUC) embryos. What is known already The literature regarding the effect of ovarian hyperstimulation on oocyte quality is limited and controversial. Results from several studies suggest that hyper response to controlled ovarian stimulation has a detrimental effect on oocyte and embryo quality, while others failed to confirm the existence of a direct toxic effect on oocyte and embryo quality. The association between temporal embryonal milestones and implantation rate has been previously demonstrated ,offering an additive tool by which oocyte quality, represented by embryo dynamics, can be evaluated. None of the aforementioned studies, however, used time lapse monitoring (TLM) system to evaluate oocyte and embryo quality. Study design, size, duration This study included a retrospective assessment of morphokinetic parameters performed by TLM from three university affiliated medical centers between January 2014 and December 2019. The developmental process and kinetics of 1863 embryos obtained from the study group, referred as the “ high ovarian response” (HOR) group, was compared to 4907 embryos from the control group - the “normal ovarian response” (NOR) group. Participants/materials, setting, methods The study included patients younger than 38 years who underwent COS with consecutive aspiration of either more than 15 oocytes (the HOR group) or 6–15 oocytes (the NOR group). A comparison was made between the groups regarding morphokinetic parameters, including the rate of embryos manifesting direct uneven cleavage (DUC) at first cleavage (DUC–1), as well as implantation and pregnancy rates. Logistic regression was conducted to assess the association between patients’ characteristics and implantation rate. Main results and the role of chance Oocyte maturation rate was significantly lower, and the DUC–1 embryos rate was significantly higher in the high ovarian response group compared to the normal ovarian response group (56.5% Vs 90.0%, p Limitations, reasons for caution The groups were not homogenous in their basic characteristics. Important information regarding the maximal dose of GT obtained, previous IVF response and ovarian reserve testing was lacking Wider implications of the findings: Higher oocyte quantity might have an effect on oocyte quality manifested as higher fraction of incompetent oocytes and higher rate of DUC–1 embryos. Once beyond the preliminary developmental stages, embryos from both groups reach the morphokinetic milestones at a similar rate and display similar implantation and pregnancy rates. Trial registration number Not applicable