Embryo Culture Research Articles

O-196 Men who fathered embryos with poor pre-implantation development rates exhibit an increased frequency of sperm with high DNA accessibility

Abstract Study question Do differing levels of DNA accessibility in sperm affect the ability of embryos generated by ART to undergo pre-implantation development? Summary answer Individuals whose sperm generated embryos that failed to efficiently form blastocysts tend to have more sperm with high levels of DNA accessibility. What is known already Chromatin is a complex composed of nucleic acids and proteins that serves to condense and regulate the genome in all eukaryotic cells. Sperm chromatin is packaged into a structure with higher density than that found in precursor germ cells or somatic cells. Samples from individuals with decreased normal semen parameters have previously been shown to possess an increased frequency of sperm with aberrant chromatin packaging. However, existing methodologies for measuring sperm chromatin packaging are indirect and non-quantitative. Study design, size, duration Prospective cohort study, registered January 2020. We adapted an enzymatic methodology for measuring DNA accessibility known as NicE-view (Nicking Endonuclease-mediated visualization), for use in human sperm. DNA accessibility was quantified in three millions of individual sperm from semen obtained from 60 individuals. Both total sperm and sperm selected for motility via swim-up were analyzed for each participant. Participants/materials, setting, methods Participants sought treatment for infertility at Universitätsspital Basel and possessed normospermic levels in conventional semen analysis. Participant partners had normal endocrine parameters and ovulation response to hormonal stimulation. 20 couples obtained pregnancies without the need for ART, while 40 individuals who were treated using ART and divided into two groups differing in pre-implantation development rates: (1) 20 with blastocyst development of > 50% of all zygotes and (2) 20 produced 0-1 blastocyst following embryo culture. Main results and the role of chance We quantified DNA accessibility and the presence of single stranded DNA nicks in more than 3 million sperm derived from 117 samples. We found that while DNA nicks were only present in a small fraction of the sperm analyzed, DNA accessibility levels varied dramatically with the data indicating a bimodal distribution. At a single sperm level, DNA accessibility and the presence of single stranded DNA damage both correlated with nuclear size. We found that DNA nicking was more abundant in total sperm samples compared to sperm selected for motility via swim-up preparation. In contrast, sperm with high levels of DNA accessibility were more abundant following swim-up, perhaps resulting from capacitation. We observed that individuals with a history of low blastocyst development were more likely to have high frequencies of sperm harboring DNA nicks and high DNA accessibility. We also measured increased frequencies of sperm with high DNA accessibility to negatively correlate with conventional semen parameter values. Limitations, reasons for caution Our study cohort consists of only 60 individuals. Variation is also observed between individuals within each participant category, so further analysis with increased participant numbers is required to strengthen these conclusions. Wider implications of the findings Our results suggest measuring DNA accessibility in sperm from normospermic individuals provide additional information about the efficiency of pre-implantation development following ART. Future work examining the sources of increased DNA accessibility in sperm will also be interesting to identify possible treatments for such individuals. Trial registration number SNCTP000003654 | NCT04256668

P-408 Monozygotic twins from in vitro fertilization: can bring the rate closer to that of natural conception?

Abstract Study question Whether the occurrence of monozygotic twins in human in vitro fertilization is associated with zona manipulations, and can we explore approaches to reduce its rates? Summary answer Monozygotic twinning is partially caused by zona manipulation, and it can be lowered by creating a larger opening in the zona before embryo transfer. What is known already While the rate of monozygotic twins in spontaneous conception is 0.5% of live births, the frequency of monozygotic twins after in vitro fertilization-embryo transfer can be a few times higher. Various factors have been suggested to contribute to this phenomenon, including ovarian stimulation, intracytoplasmic sperm injection, embryo cryopreservation, extended culture, embryo quality, maternal age, genetic factors, and micromanipulation of the zona pellucida, such as assisted hatching and biopsy. Due to the fact that the overall rate is low, it is difficult to pinpoint the main reason(s) contributing to it, and there are no effective approach(es) to lower this rate. Study design, size, duration We studied monozygotic twins in 8,063 clinical pregnancies resulting from single blastocyst transfers from 2016 to 2023. Blastocysts, whether fresh or frozen, were transferred with or without biopsy for preimplantation genetic testing and/or blastocyst collapse. Clinical pregnancy, embryo implantation, including twin implantation, and clinical outcomes such as live birth, ongoing pregnancy, and birth weight were further evaluated. Monozygotic twin rates were compared between partial zona removal (larger hole) and no zona removal (small hole) Participants/materials, setting, methods Monozygotic twins were initially evaluated in blastocysts with or without biopsy in 732 clinical pregnancies after fresh or frozen embryo transfer. Subsequently, monozygotic twins were further evaluated in 7331 clinical pregnancies after an approach to open a larger hole in the zona before blastocyst transfer. Additionally, 56 monozygotic twins were evaluated for live birth and birth weight outcomes. Main results and the role of chance In the context of fresh blastocyst transfer, the monozygotic twin rate following biopsy was significantly higher than that for blastocysts that did not undergo zona manipulation (3.4% vs. 0.29%; p = 0.025). After blastocysts underwent biopsy and cryopreservation/warming, the monozygotic twin rate was 2.79%, a similar rate as observed in fresh blastocyst transfer after biopsy. However, when frozen blastocysts (either biopsied and/or collapsed) underwent partial zona removal before transfer, the rate of monozygotic twins significantly decreased to 0.6% (p = 0.00001). When we further examined whether the biopsy procedure increased the monozygotic twin rate (28/4947) under the conditions that partial zona removal was performed by comparing those with blastocysts without biopsy (16/2384), we did not find a significant difference (0.57% vs. 0.67%, p = 0.587) between the two treatments. In this study, there were 34 twin live births (60.7%) including 5 ongoing twin pregnancies, and 9 (16.1%) singleton live births, contributing to an overall live birth rate of 76.8%. Ten cases (17.8%) experienced pregnancy loss with heartbeat cessation during the first trimester. Furthermore, 3 cases (5.4%) had abnormal pregnancies. When the birth weights were evaluated among the monozygotic twins, it was found that lower birth weight (< 1500g) was only observed in twin live birth. Limitations, reasons for caution This study only included data from one large clinic in the US under the conditions and protocols (embryo culture, embryo biopsy, and cryopreservation) used in this clinic. Different patients and protocols would affect the rate differently. Wider implications of the findings Zona manipulations are associated with an increased monozygotic twin rate in both fresh and frozen embryo transfers. This effect can be mitigated by creating a larger opening in the zona before embryo transfer, and this approach can bring the monozygotic twin rate closer to that of natural conception. Trial registration number NA

O-249 METAPHOR: METabolic imaging through AI-powered Phasor-based Hyperspectral analysis and Organelle recognition for the classification of human blastocysts

Abstract Study question Can we classify human blastocysts non-invasively using hyperspectral imaging? Summary answer HS imaging can significantly enhance our classification of human embryos and reveal numerous cellular features present in the embryo invisible to brightfield imaging. What is known already Non-invasive measurement of metabolism is a safe and powerful tool to reliably predict oocyte and embryo quality. We have previously shown that our METAPHOR technology, based on HS imaging coupled to artificial intelligence, was able to separate mouse oocyte categories according to maternal age with an average AUC of 96.2% and predict blastocyst formation (AUC 82.2%). Moreover, it was able to classify mouse embryos deprived from different nutrients with an average AUC of 93.7% showing clear superiority to grading provided by experienced embryologists (51%). Study design, size, duration A total of 74 human embryos donated for research at the blastocyst stage have been imaged to date. Embryos were warmed and imaged according to our established protocol and survival was statistically indistinguishable from control embryos. Participants/materials, setting, methods Embryos are imaged using multiphoton illumination near the infra-red. The HS detection covered the simultaneous measurement of the whole visible spectrum. Embryos are then allowed to implant in our proprietary 3D ex vivo platform. Implantation is monitored up until day 9 before immunostaining is performed to quantify presence and prevalence of lineage and implantation markers. Main results and the role of chance After imaging, embryos were placed on the 3D ex vivo implantation platform and let implant. Successful implantation (75.41%) was scored by observation of the characteristic deformation of collagen fibers within the matrix and staining for relevant cell lineages (OCT4, pMLC2). Interestingly, we observed that embryos with a higher trophectoderm (TE) grading tended to implant with a higher efficiency and penetrate deeper into the matrix than embryos with lower grading. HS images have unveiled a profound cellular diversity within embryos, elucidating distinctions between the Inner Cell Mass (ICM) and TE, as well as revealing different metabolic states defined by NADH dominance, FAD dominance, protoporphyrin levels, and apoptosis. HS raw images are processed using our HS-phasor approach which allows quick computing by converting the multidimensional HS data into comprehensive visual representation of bidimensional histograms and phasor plots. In summary, our analysis pipeline encompasses the algorithms to detect and segment the embryos, the computation of histogram and phasor-plot, and the classifier. AI allows us to classify embryos in distinct spectral spaces based on their metabolic features, paving the way to the establishment of a new grading system based on HS imaging. Limitations, reasons for caution The cohort size is still limited, and ongoing experiments will increase the dataset needed for more accurate prediction of embryo implantation. The implantation platform is an in vitro test that will require future clinical validation. Wider implications of the findings The safety and speed of METAPHOR warrants its incorporation at the IVF clinic, adding an additional layer of information to the current embryo selection methods. Moreover, the non-invasive measurement of the metabolic function opens new alleys of research such as the effect of media and culture supplements on embryo culture. Trial registration number Not applicable

P-109 Non-invasive Genetic Testing (niPGTA) and Laser Assisted Hatching (LAH) may improve pregnancy and ongoing pregnancy rates in patients of advanced maternal age

Abstract Study question Does niPGTA and LAH improve pregnancy and ongoing pregnancy rates in patients of advanced maternal age? Summary answer niPGTA increases pregnancy and clinical pregnancy rates in patients aged ≥38 years compared to those without tested embryos, while LAH may further improve pregnancy outcomes. What is known already Embryos release cell-free DNA (cf-DNA) in the IVF culture media, which can be analyzed for chromosomal constitution. The benefit that niPGTA holds over PGT-A is that it is a non-invasive test and could potentially produce comparable results. Therefore, the time to pregnancy and the risk of miscarriage could be reduced in comparison to an IVF treatment with non-tested embryos. Previous studies have shown high concordance with trophectoderm biopsies in PGT-A patients and improved outcomes with LAH in frozen-thawed cycles. However, there is a lack of studies investigating clinical pregnancy rates with niPGTA. Study design, size, duration Retrospective cohort study including 74 patients aged ≥38 years, who underwent Single Frozen Embryo Transfer (FET) from May 2021 to February 2023. Patients who had Endometrial Receptivity Assay (ERA) were excluded. Participants/materials, setting, methods 32 couples had niPGTA; Spent Blastocyst Media (SBM) for each blastocyst were collected on Day 6 and analyzed using the EMBRACE test. Embryos were vitrified. 42 couples had their blastocysts vitrified on Day 5/6 without testing. LAH was introduced to all patients in October 2021. Before that, no LAH was performed. Embryos were transferred to patients 2 hours post-warming after LAH. Pregnancy test was performed 10 days after FET and clinical pregnancy was established. Main results and the role of chance There was a statistical significant increase of pregnancy rate (p = 0.017, Pearson’s Chi-Squared test) and a trend towards higher clinical pregnancy rate for niPGTA patients versus those who had a transfer of not tested embryos. Regarding LAH, there was a trend towards higher pregnancy and clinical pregnancy rates in both niPGTA and not tested group. Results for pregnancies and clinical pregnancies are presented in Table 1. Limitations, reasons for caution More patients are required to confirm clinical significance of pregnancy and clinical pregnancy rates of niPGTA over not tested embryos and if LAH can improve the outcomes. A review of patients who had previous implantation failures and miscarriages in addition to ERA could yield clearer conclusions. Wider implications of the findings The results of this study are encouraging for niPGTA combined with LAH for patients aged ≥38 years. Further studies on niPGTA and modifications of the protocol for increased concordance rates and shorter time in embryo culture should be explored. Trial registration number Not applicable

P-046 Ejaculatory abstinence: is there any association with embryo developmental competence? A Retrospective Study in time-lapse incubators

Abstract Study question Is ejaculatory abstinence (EA) associated with fertilization and blastocyst development in ICSI cycles with embryo culture in time-lapse incubators? Summary answer There is no association between length of EA and fertilization, blastocyst development or morphology. What is known already Semen analysis is pivotal in medically assisted reproduction (MAR), serving as a significant indicator of male partners’ health. The period of EA is a relevant factor that can affect sperm quality. Numerous studies described EA association with clinical outcomes, while its impact on embryological results remains inconclusive. Recent meta-analyses, despite a conflicting literature, highlighted positive effects of shorter abstinence periods. A recent study suggested that prolonged abstinence might reduce fertilization rate in ICSI cycles without time-lapse imaging, thereby reducing usable zygotes’ rate Study design, size, duration Retrospective study including all oocytes (N = 7164) from 1148 ICSI cycles (N = 902 couples) cultured in time-lapse incubators at a single IVF center between January 2020 and December 2022. Only non-donor fresh gametes were included (Mean Maternal age: 38.5±4 years; Paternal Age: 41±5.6 years). The primary embryological outcomes were fertilization rate and type, and blastocyst development per inseminated oocyte and per 2PN-zygote. Secondary outcomes were euploidy, day of blastocyst development and Gardner’s morphology. Participants/materials, setting, methods Semen analysis was conducted according to WHO. All oocytes were cultured up to day7 in EmbryoScope or GERI incubators with continuous media and refresh in day5 (if needed). If requested, PGT-A was performed to assess full-chromosome uniform aneuploidies. Univariate and multivariate logistic regressions were conducted. Possible confounders were assessed among ovarian stimulation, maternal, paternal, sperm, and IVF cycles characteristics. Associations were confirmed with Generalized estimating equations (GEE) to account for repeated measurements among oocytes’ cohort. Main results and the role of chance Logistic regressions and GEE were conducted to assess associations between possible confounders and blastocyst development per inseminated metaphase-II oocyte. Maternal age, sperm concentration, and motility A+B were associated. The same confounders were reported on blastocyst development among 2PN-zygotes. The days of EA (1-8 days) did not show any association with fertilization (p = 0.198) or blastocyst development per metaphase-II oocyte (p = 0.495). No association was reported with abnormal fertilization, neither combining all abnormalities in a single group, nor clustering them as 1PN, 3PN, 3.1PN, and multi-PN. An increasing trend was reported in blastocyst development among 2PN-zygotes according to the length of EA (1-day: 17/37,45.9%; 2-days: 190/424,44.8%; 3-days: 1405/2574,54.6%; 4-day: 746/1410,52.9%; 5-days: 450/827,54.4%; 6-days: 70/133,52.6%; 7-days: 46/101,45.5%; 8-days: 85/162,52.5%), but this was not significant in a multivariate logistic regression (p = 0.887). Length of EA showed no association with euploidy (p = 0.424), nor with day of development (p = 0.966) and Gardner’s inner cell mass (p = 0.204) or trophectoderm (p = 0.428) morphology. Limitations, reasons for caution This study is inherently limited by its retrospective design. The limited number of cycles included was balanced using time-lapse incubators to improve the accuracy of fertilization check and benefit from an undisturbed embryo culture environment. Artificial Intelligence will be implemented to automate developmental timings’ annotation and objectify embryo quality assessment. Wider implications of the findings The length of EA seems not associated with embryological outcomes, adding to the controversy on this topic. Here, we included only ICSI cycles with blastocyst culture conducted in time-lapse to enhance the quality of the evidence. Larger datasets and diverse settings are now required, especially enriched with poor prognosis patients. Trial registration number not applicable

P-165 Benefits of ultra-low oxygen tension (2%) in patients with low quality embryos or previous arrest: the patient as its own control

Abstract Study question Is reduction in oxygen tension from 5% to 2% after D3 of extended culture beneficial for blastocyst development in patients with previous failed culture? Summary answer Ultra-low oxygen tension in extended culture improves blastocyst development (overall blastulation rate OBR), quality (top blastulation rate TPR) and increases the pregnancy rate (PR). What is known already The practice ESHRE guidelines recommends human embryo culture at low oxygen concentrations (2-8%). Traditionally atmospheric conditions (20% O2) were used but better outcomes were observed at low monophasic 5% O2. Human embryo leaves the oviduct (5-7% O2) to cross the uterus-tubal junction around D3-4 and reaches the uterin ultra-low O2 tension (2%). This decreasing gradient coincides with the shift from pre-compaction oxidative phosphorylation in the oviduct, to post-compaction glycolysis in the uterus. Few studies with ultra-low 2% O2 incubation suggested that the best O2 level may be stage-dependent and its reduction after D3 may be a more physiological approach. Study design, size, duration Prospective observational study in a single private hospital during 2023. 71 patients experienced a failed cycle of extended culture (n = 29 cIVF, n = 37 ICSI and n = 5 Sibling oocytes) with a poor embryo quality and low OBR, under standard monophasic 5% O2 incubation. In the subsequent cycle for these 71 patients (n = 24 cIVF and n = 46 ICSI and n = 1 Sibling oocytes) the O2 level was reduced to 2% from D3 onwards. Participants/materials, setting, methods After a failed extended culture, considered as the control cycle, the patients were enrolled for the study. Blastocysts were rated by Gardner’s classification. In the control cycle embryos were cultured under monophasic 5% O2 until D6. In the subsequent cycle embryos were cultured under 5% O2 until D3 and in ultra-low 2% O2 from D3 to D6. Sage 1-Step™ media was used for both cycles. The statistical analyses were done by chi-square and fisher test. Main results and the role of chance 71 patients were included in the study regardless of age, indication or technique. Each patient was its own control. The mean age was not different between the 2 groups (36.5±4 vs 37.2±4) but the attempt rank was logically statistically higher in the study cycle (2.1±1.4 vs 3.4±1, p<0.001). Oocyte maturity (75% vs 76.4%) and fertilization rate (66.8 vs 70.3%) were similar between groups. The average embryo quality at D3 was poor in both groups. OBR and TBR (40.7% vs 52.4%, p=0.004 and 15.8% vs 37.5%, p<0.001) were statistically higher after culture in ultra-low O2 conditions. Extended culture failure with absence of usable blastocyst (57.7% vs 19.7%, p<0.001) was statistically lower in ultra-low incubation group and the pregnancy rate PR per cycle (3.6% vs 25%, p=0.004) was seven fold higher after ultra-low culture. Limitations, reasons for caution The small size of the sample needs to be expanded in order to confirm the encouraging findings in a larger population. The study will continue during 2024 and will compel the live birth rate and the cumulative live birth rate. Wider implications of the findings Providing embryos with the most physiologic culture system would improve IVF cost-effectiveness and a better infertility management. Embryo energetic adaptation to suboptimal conditions results in impaired viability and lower pregnancy rates. In our study extended culture of low-quality embryos under 2% O2 after D3 yielded better culture and pregnancy outcomes. Trial registration number NA

P-440 Idiopathic infertility does not increase rates of placental abnormalities among singleton pregnancies conceived with either in-vitro fertilization (IVF) or ovulation induction±intrauterine insemination (OI±IUI)

Abstract Study question Is there an adverse impact of idiopathic infertility diagnosis on placental pathology among singleton pregnancies conceived with fertility treatments? Summary answer Idiopathic infertility does not increase rates of placental abnormalities and exhibits distinctive associations within the inflammatory and vascular spectrum of placental pathology. What is known already Pregnancies resulting from fertility treatments are often considered to be at higher risk for placenta-mediated obstetric complications and subsequent adverse perinatal outcomes. Treatment induced hormonal changes altering the endometrial milieu, along with embryo manipulation and culture conditions, may impact the processes of implantation, decidualization, trophoblast invasion, and placental vascularization. Nevertheless, the impact of infertility diagnoses on placental pathology among singleton pregnancies conceived with fertility treatments has not been elucidated, and whether idiopathic infertility alters the risk of placental abnormalities remains unknown. Study design, size, duration Retrospective review of placental pathology data from 1205 singleton livebirths conceived with fertility treatments (899 IVF, and 306 OI±IUI cycles) between 01/2004 and 04/2022. Placenta pathology was reviewed by one expert pathologist and classified as anatomic, inflammatory, infectious, and vascular [including any features of fetal (FVM) or maternal (MVM) vascular malperfusion], using the Amsterdam Workshop Consensus definitions. Placental abnormalities were compared between idiopathic infertility (IdI, n: 269) and all other, non-idiopathic infertility (non-IdI, n:936) diagnoses. Participants/materials, setting, methods Primary outcomes: anatomic, inflammatory, infectious, and vascular placental abnormalities. Parametric, and non-parametric tests were used as appropriately; odds ratios (OR) with 95% confidence intervals (95%CI) were assessed through logistic regression, adjusting for maternal age, body mass index (BMI, kg/m2), race, parity, gestational age, neonatal gender, treatment type, gestational diabetes, and hypertensive disorders. Analyses were further stratified by OI±IUI vs IVF treatments [the latter further stratified into fresh and frozen embryo transfers (FET)]. Main results and the role of chance Mean age, BMI, and AMH did not differ between groups, and neither did placental weight at term. Overall, unadjusted rates of inflammatory, infectious, and vascular abnormalities did not differ between groups (12.6% vs 11.4%, p 0.568; 22.0% vs 19.1%, p 0.256; 54.5% vs 58.6%, p 0.186, respectively). However, a higher rate of anatomic abnormalities was noted among IdI patients (32.8% vs 27.1%, p 0.044, IdI vs. non-IdI, respectively). When adjusting for potential confounders, no differences were noted between groups regarding inflammatory, infectious, and vascular abnormalities, and the observed difference in anatomic abnormalities lost its significance [adjOR(95%CI); inflammatory: 0.98(0.94-1.03), infectious: 1.03(0.98-1.09), vascular: 0.96(0.90-1.02), and anatomic: 1.02(0.96-1.08), non-IdI: ref]. Similarly, when separating OI±IUI from FET and fresh IVF cycles, adjOR(95%CI) revealed no differences in anatomic, infectious, and vascular abnormalities between groups [OI±IUI: 0.97(0.85-1.1), 0.97(0.87-1.08), 0.99(0.87-1.12); FET: 1.08(0.94-1.24), 1.07(0.94-1.23), 1.04(0.98-1.11) and, fresh IVF: 1.06(0.98-1.15), 1.04(0.96-1.13), 0.96(0.87-1.07), for anatomic, infectious, and vascular, respectively]. However, lower odds of inflammatory abnormalities were noted among fresh IVF but not among FET or OI±IUI cycles in IdI [adjOR (95%CI); fresh IVF: 0.95(0.91-0.99), FET: 1.09(0.98-1.21), OI±IUI: 0.97(0.87-1.08), non-IdI: ref]. Interestingly, among vascular abnormalities studied in the programmed FET cycles, MVM showed a difference between the groups [adjOR(95%CI) 0.90(0.85-0.96), non-IdI: ref]. Limitations, reasons for caution This study is limited by its retrospective design. The reported findings are from an infertile population undergoing treatments and thus not easily generalizable to natural conceptions. Other factors, such as environmental, nutritional, and lifestyle, might alter a patient’s individual pregnancy risk for placental abnormalities. Wider implications of the findings Overall, idiopathic infertility does not appear to increase the risk for placental abnormalities. However, it may impact placental pathology in a unique way, within the vascular and inflammatory spectrum, which might differ from that of other infertility diagnoses, and might be further altered by the treatment protocol. Trial registration number Not Applicable

O-297 Prediction of pregnancy outcomes of single vitrified-warmed blastocyst transfer using combination of an automatic classification algorithm applied on cleavage stage embryos and blastocyst morphology

Abstract Study question Does combination of embryo assessment algorithm on cleavage-stage based on the automatic annotation of morphokinetic and blastocyst morphology in embryo selection improve implantation outcomes? Summary answer Combination of embryo assessment algorithm on cleavage-stage based on the automatic morphokinetic annotation and blastocyst morphological evaluation was showed significantly predictive for implantation outcomes. What is known already Conventional morphological assessment is still the gold standard for embryo selection. Timelapse monitoring of embryo development may represent a superior way to culture and select embryos in vitro. Nowadays, time-lapse culture systems are becoming more popular, many algorithms have been developed for embryo selection based on embryo morphokinetics, providing additional information for morphological assessment. However, manual annotations of developmental events and application of algorithms can be time-consuming and subjective processes. Automation of morphokinetic annotation can be a potential approach that reduces subjectivity in embryo selection. Study design, size, duration The observational, retrospective study was conducted in a single centre between 2020-2023 and included 511 single vitrified-warmed blastocyst transfer cycles. Blastocyst selected for transfer on Day 5,6 based on conventional morphological evaluation. Embryos were evaluated on Day 3 with a score from 1 (best) to 5 (worst) (from highest to lowest developmental potential) by the automatic embryo assessment algorithm. The correlation of the algorithms scoring and pregnancy outcomes was qualified by generalized estimating equations (GEEs). Participants/materials, setting, methods Embryos were cultured in timelapse GERI incubator and classified on day 3 by automatic embryo assessment algorithm depending on four parameters: P2 (t3-t2), P3 (t4-t3), oocyte age, and number of cells. The correlation of the algorithms scoring and pregnancy outcomes was qualified by generalized estimating equations (GEEs). Three GEE model using embryo assessment algorithm, conventional morphological assessment, and a combination of both classification system as the predictor were compared. Main results and the role of chance The implantation rate was higher with lower the scores generated by the embryo assessment algorithm. A GEE model showed the negative association between higher embryo score and lower odds of implantation. The OR of Score 3; 4; 5 vs 1 were 0.335; 0.259; 0.245 (95% CI 0.194-0.580; 0.138-0.485; 0.111-0.544, P = 0.000), respectively, for implantation. The AUC of the model using embryo assessment algorithm for implantation potential = 0.641. The AUC of the model combining both embryo assessment algorithm and conventional morphological assessment for implantation potential = 0.733. The differences were statistically significant (z-statistic = 3.896, p = 0.0001). Limitations, reasons for caution The sample size was enough to confirm the ability of the model for embryo selection. However, the retrospective nature of this study may be a limitation. Wider implications of the findings Using combination of timelapse technology with automated embryo assessment and conventional morphological assessment can predict the success rates of assisted reproduction cycles. To our knowledge, this is the first study using embryo dataset from single vitrified-warmed blastocyst transfer cycles with this embryo assessment algorithm. Trial registration number not applicable

P-192 Correlation between the ratio of mitochondrial DNA to genomic DNA copy number in spent culture media and embryo quality in IVF

Abstract Abstract withdrawn by the authors

P-138 Effect of biofeedback from the blastocysts on the cumulative pregnancy rate

Abstract Study question The study is to compare cumulative pregnancy rates of embryos transferred with and without spent culture media to confront biofeedback’s role in clinical outcomes Summary answer Transferring the blastocysts with spent culture media improved the pregnancy rates. Biofeedback was observed from the blastocyst stage. The blastocyst freezing and transfer approach improved. What is known already Biofeedback is often encountered in regulating menstrual cycles and other events in continuing gestations with estrogen, progesterone, beta hCG..etc. A similar biofeedback is possible with growing blastocyst for implantation. Since the in-vitro culture of embryos is devoid of its occurrence, it may lead to asynchrony of subsequent events and implantation. There is no scientific evidence from metabolomics on the characterization of these substances or secretions from spent culture medium that includes cell-free DNA giving feedback and its role in improving clinical outcomes. Study design, size, duration This retrospective multi-centric study included the 3591 frozen embryo transfers from 1st Apr 2022 to 31st Nov 2023 out of 4623 women who underwent oocyte retrieval from 1st Jan 2022. Patients were divided into study (group A) and control (group B). In group A, 1035 women received the transfers from spent culture media; in group B, 2556 women received transfers after shifting the blastocysts to fresh culture media before loading into the ET catheter. Participants/materials, setting, methods All the transfers considered in this study were day-5 blastocysts, irrespective of their frozen state. Day-5 frozen-thawed were incubated short-time (∼1 hour) before the transfer. Whereas, the frozen embryos and morulas were cultured till day 5. In the study group, all cultures were conducted in 100ul of culture media under oil per embryo to maintain consistency amongst the group. The rest of the laboratory and clinical procedures were consistent between the two groups. Main results and the role of chance The cumulative pregnancy rate was significantly higher in group A than in group B (60.9% Vs 55.6%; OR = 1.23; 95% CI, 1.07 -1.44, Z-score 2.901, P < 0.003). The effect of spent culture media was further observed by regrouping the day-3 or 4 frozen (group A1) and day-5 frozen patients (group A2). Day-5 thawed blastocysts transferred after short incubation to allow the secretions to directly deposit on the endometrium. Group A1 might have a high concentration of secretions due to its accumulation from the embryo or morula stages. Pregnancy rates in group A1 were higher than A2 but not statistically significant (61.7% Vs 59.7%; OR = 1.08, 95% CI, 0.84-1.4, Z-score 0.654, P = 0.51). The less impact indicates that the feedback came from blastocysts rather than embryos. Limitations, reasons for caution It is not evident how these secretions contribute to biofeedback. Quantification, identifying their time of release and recovery from the qualitative tests for their therapeutic use are the limitations. Nevertheless, spent media can be employed if found beneficial. Wider implications of the findings We have taken a large possible data set for control against the test group and took cumulative pregnancy rate as the primary outcome indicator to prove biofeedback from blastocysts can improve the clinical outcomes irrespective of the age and other factors of infertility. Trial registration number Not applicable

P-735 Is Artificial Intelligence (AI) currently able to provide evidence-based scientific responses on methods that can improve the outcomes of embryo transfers?

Abstract Study question Could chatbots potentially be used as tools for both patients’ and physicians’ education in an infertility journey? Summary answer Artificial Intelligence (AI) is not yet in a position to give clear, evidence-based recommendations in the field of fertility, particularly concerning embryo transfer. What is known already The rapid development of AI has raised questions about its potential uses in different sectors of everyday life. Individuals from diverse fields have tried to incorporate its usage in their personal but also in their professional lives, with varying levels of success. Specifically in medicine, ChatGPT has been studied intensely with more than 400 related articles having been indexed in PubMed until May 2023, while AI has reportedly also succeeded in several standardized medical tests and passed several Medical Board examinations. Hence the question arises whether chatbots could be used as tools for clinical decision-making or patients’ and physicians’ education. Study design, size, duration We used nine of the most popular free AI chatbots available (ChatGPT, Bard, Writesonic, You, Perplexity, Learnt, Bing, Magickpen, and Rytr) and entered the following command: “Write me a 300-word scientific essay about evidence-based methods that can improve the outcomes of embryo transfer” in May 2023. We collected the responses and extracted the methods each chatbot suggested. When sufficient similarity among answers was present we categorized the answers under one category to facilitate the study. Participants/materials, setting, methods We calculated descriptive statistics and the prevalence of each response. We used as a comparator for widely acceptable practices that are proven to improve embryo transfer outcomes the 2017 ASRM guideline on performing embryo transfer taking also into consideration more current literature. Data was compared using chi-squared tests and a level of p < 0.05 was used as the threshold of statistical significance. Main results and the role of chance The range of the recommendations was from one to nine per chatbot (median = 4, IQR = 2) with an average of 4.78 suggestions per chatbot. Out of a total of 43 recommendations, which could be grouped into 19 similar categories, only 3/19 (15.8%) were evidence-based practices, those being “ultrasound-guided embryo transfer”, which was also the most commonly appearing response, appearing in 7/9 (77.8%) chatbots, “single embryo transfer” appearing in 4/9 (44.4%) chatbots and “use of a soft catheter” in 2/9 (22.2%) chatbots. Some controversial responses appeared even more often than the two latter evidence-based ones with “preimplantation genetic testing (PGT)” being the second most common response with 6/9 chatbots suggesting it (66.7%) and the vague suggestion of “optimal endometrium preparation” being in the third place with 5/9 chatbots (55.6%), both non-evidence-based practices to improve embryo transfer. The majority of the recommendations were unique, with 10 answers appearing only once (1/9; 11.1%), those being “endometrial scratching”, “natural cycle embryo transfer”, “use of GnRH antagonist protocol”, “blastocyst transfer”, “use of specialized catheter”, “preimplantation genetic screening (PGS)”, “mock embryo transfer”, “uninterrupted embryo culture”, “minimizing transfer time”, and “maintaining temperature and pH of the culture media”. Limitations, reasons for caution Firstly, we only used some of the most popular chatbots and not all existing ones. Additionally, chatbots tend to give different answers when repeatedly asked the same questions. However, we believe that our study effectively captures the prevailing practices of chatbot users, who typically pose a question only once. Wider implications of the findings Both patients and physicians should be wary of guiding care based on chatbot recommendations in infertility since the majority of responses consists of scientifically unsupported recommendations. Chatbot results might improve with time especially if trained to obtain information from validated medical databases, however, this will have to be verified scientifically. Trial registration number N/A

O-039 Con - the faster the better

Abstract Late o’clock: Are slow growing embryos clinically usable? Con - the faster the better Reproductive medicine and, within it, embryology, have experienced milestones in the understanding of pre-implantation embryonic development over the last quarter of a century. The development of blastocyst culture media for embryo culture even after embryonic genome activation and the possibility of continuous monitoring of embryonic morphokinetics throughout the pre-implantation period have been major contributors. The development of molecular genetic techniques has also played an important role. The introduction of new cryopreservation techniques has also improved the success rate of the clinical use of surplus embryos, including slow-growing embryos not otherwise selected for fresh cycle transfer. Two questions are most frequently encountered in clinical embryology today: which embryo in a cohort has the best chance of resulting in a live-born child, and how slow and morphologically suboptimal can an embryo be to still be considered clinically useful? In particular, the latter question does not have an easy answer, as embryos demonstrate varying pace of development. A further problem arises if there are no optimal embryos available in the cohort and this answer has to be given on day 2, 3 or 5, as this is when the embryo transfer is scheduled to take place. Some studies suggest that longer timing of any stage of embryonic development from pronuclear fading onwards means that the embryo is late in development due to certain abnormalities and is likely to be energetically depleted by the activation of its repair mechanisms. Such embryos not only have a lower implantation potential compared to fast embryos, but also diverge in time from the optimal receptivity of the endometrium. Additional testing and/or storage of slow-growing embryos brings the question of their clinical usability to the time of frozen embryo replacement cycles and quiets the couple's expectation of a “successful” completion of the stimulated cycle. Whether embryos are to be selected for fresh ET or FER, the “the faster - the better” principle is likely to be applied. In daily clinical practice, decisions on the use of slow-growing and/or morphologically sub-optimal embryos and the timing of embryo transfer are made on an individual basis, taking into account various medical as well as non-medical (e.g. economic, logistical) factors. These may also include portraying a positive image of the centre's performance. However, patients expect infertility specialists to make realistic assessments not only of the viability of slow growing embryos, but also of the outcome of the transfer of them. The assessment must be based not only on medical and ethical but also on rational grounds.

P-274 Humid or dry culture with medium renewal: impact on A-Grade Day 5 blastocysts. Randomized prospective cohort study

Abstract Study question Could the humid embryo culture impact the development of top-graded blastocysts? Summary answer While no differences were observed in the overall blastocyst development rate, humidity conditions emerged as a significant factor influencing the achievement of A-Grade D + 5 blastocysts. What is known already In recent years, several publications have highlighted the advantages of humid culture (HC) over dry culture (DC) (<45% relative humidity, RH). HC, with its lower risk of media evaporation and consequent osmolality changes. Nevertheless, to the best of our knowledge, this is the first prospective study addressing this aspect through oocytes from the same cohort. Study design, size, duration We have been conducting a randomized control trial (RCT) consisting on 379 MII oocytes from 30 patients from August 2022 until January 2024 (and ongoing). After ICSI procedure, sibling oocytes were divided following 1:1 ratio into both humid (>70% HR) and dry (0% HR) chambers of a Geri incubator (Genea Biomedx, Australia). Ejaculated sperm ICSI cycles with >5 fresh MII were included in the analysis. Time-lapse cleavage timings were manually gathered. Participants/materials, setting, methods Extended culture to the blastocyst stage (D + 5, D + 6 or D + 7) was performed in 80μL of single step G-TL medium (Vitrolife, Sweden). HC remained undisturbed while medium was renewed on D + 3 in DC by changing the culture dish. Fertilization, overall blastocyst development and A-Grade Day 5 blastocysts rates (ASEBIR’s morphology criteria) were compared. Statistical analysis consisted on Chi-squared test, t-student test and logistic regression for computation of Odds Ratio (OR, 95%CI). Main results and the role of chance While no statistically significant differences were observed in the fertilization rate (DC: 76.4%; HC: 69.1%, NS) or the overall usable blastocyst development rate (DC: 48.6%; HC: 53.1%, NS), noteworthy distinctions emerged in the proportion of top-graded embryos based on ASEBIR’s morphology criteria. Specifically, the A-Grade Day 5 blastocyst development rate displayed significant disparities when comparing all fertilized embryos in each culture type (DC: 19.9%; HC: 32.3%; p < 0.05) and also among all usable blastocysts (DC: 40.8%; HC: 60.9%, p < 0,05). The Odds Ratio for achieving A-Grade Day blastocysts depending on type of humidity conditions was 1.87 (1,099 – 3,235) for the humid culture. Moreover, time-lapse imaging facilitated the detection of direct cleavage presence, which was similarly distributed between both incubation types (DC: 27/146; HC: 23/130, NS). However, only 3 embryos showing direct cleavage and cultured in HC reached the blastocyst stage at Day 6, while in dry incubator, 10 embryos with direct cleavage were able to develop to usable blastocysts, three of them being A-Grade on Day 5. Limitations, reasons for caution This study is currently ongoing, aiming to increase the sample size. Future analyses will explore clinical outcomes, especially for underrated embryos, day 6 blastocysts, and those with morphodynamic phenomena. While A-grade blastocyst rate variations may not impact pregnancy outcomes directly, they could influence cumulative success rates. Wider implications of the findings These data highlight that day+3 medium renewal doesn’t equalize conditions to humid culture. Ongoing efforts involve validating these insights through an expanded sample size and extensive outcome analysis. Investigating the long-term impact on clinical outcomes and refining culture protocols can amplify the relevance of these findings in assisted reproductive technologies. Trial registration number pending_protocolOK

P-755 Effect of female age and specific cycle characteristics on success in modified natural frozen-thawed single euploid embryo transfer cycles (mNC/FET)

Abstract Study question Does the age of woman and specific cycle characteristics impact the success of modified natural frozen-thawed single euploid embryo transfer cycles (mNC/FET)? Summary answer The success rate of mNC/FET cycles is similar in all age groups. As the LH level increased ( above 23.5ng/dL) the success was significantly increased. What is known already The success of frozen embryo transfer can depend on various factors such as the patient’s age, the number of transferred embryos, the duration of embryo culture, and the method used to prepare the endometrium (Holschbach et al. 2023). However, there is currently insufficient evidence in the literature about the effect of age and cycle characteristics on the effectiveness of mNC/FET cycles in patients who have received single top and good quality euploid embryos. Study design, size, duration This study, conducted at Istanbul Memorial Hospital, ART and Reproductive Genetics Center, is a retrospective, single-centre analysis of 583 cycles of euploid mNC/FET in women aged 22-44 years. The study period was from July 2017 to January 2024. The cycles were divided into two groups based on the age of the women. Group A (n = 408) included women under the age of 38, while Group B (n = 175) included women aged 38 or above. Participants/materials, setting, methods The study investigated success predictors in mNC/FET with top and good-quality embryos. Excluded were women with recurrent pregnancy losses, recurrent implantation failure, high BMI (>35kg/m2), uterine/endometrial problems, severe male infertility, and autoimmune diseases. The mNC/FET success was a pregnancy lasting 12 weeks or more. The analysis included patient demographics and cycle outcomes. Chi-square, t-tests, and Mann-Whitney U tests assessed variables with a p-value < 0.005, indicating statistical significance. Main results and the role of chance No significant demographic differences were observed between the two groups, except for AMH levels. AMH levels were significantly higher in younger group than older group (3.02 ng/ml vs. 1.96 ng/ml, p = 0.000). The mNC/FET success rates were similar, with 76% in the younger group and 73% in the older group. Estradiol levels the day before trigger day were significantly higher in older group (278.02 pg/ml vs. 237.84pg/ml, p = 0.028). Both groups had similar mean LH levels (32.50 IU/L and 32.41 IU/L, respectively), estradiol levels and endometrial thickness on the trigger day. In the younger group, LH rise was observed later, follicular phase was longer, and embryo transfer day was later than the older group (p = 0.001). Univariate regression analysis revealed that increased LH level was an important independent variable in achieving mNC/FET success, with a 1.001-fold increase (p = 0.043). The LH levels of 23.5 IU/L or higher predicted high mNC/FET success with 60% sensitivity and 43% specificity based on the Receiver Operating Characteristic curve. (AUC: 0.606, %95 CI 0.540-0.671, p = 0.002). Prolonged infertility duration and previous ET failures reduced mNC/FET success by 0.889 and 0.544-fold, respectively (p = 0.002, p < 0.001). Higher β-hCG levels on days 14 and 16 increased mNC/FET success by 1.038-fold and 1.008-fold, respectively (p < 0.001). Limitations, reasons for caution Our study has limitations as it was retrospective. Prospective randomized studies are needed to evaluate the differences between the young (age <38) and old patient groups (age ≥38) based on mNC/FET success. The association between LH rise and mNC/FET success needs verification with a larger sample. Wider implications of the findings The mNC/FET success rates are similar in young (age <38) and old patient groups (age ≥38). However, prolonged infertility duration and previous failed ET cycles decreased the success of mNC/FET. Additionally, It is important to schedule trigger time after the optimum LH rise, which is 23.5 IU/L or higher. Trial registration number not applicable

O-209 Identifying toxicity trends: analysis of over 6700 Mouse Embryo Assays performed on 16 categories of IVF supplies

Abstract Study question What type of disposables and solutions exhibit a higher frequency of Mouse Embryo Assay (MEA) failure and where should manufacturers put special attention during production? Summary answer Flexipet-pipette types, protein supplements, ovum-pick-up needles, catheters, steripettes, culture media, and mineral oils showed higher MEA-failure frequencies and/or lethality levels due to embryotoxicity among IVF-supplies. What is known already The MEA is considered the gold standard assay for quality control of IVF products. It is based on the analysis of in vitro mouse embryonic development under direct or indirect presence of a to-be-tested product, and it requires over 80% expanded blastocyst formation rates (EBFRs) for a product to be considered non-toxic and be released to the market. Recent episodes reported toxicity issues found in embryo culture supplies and IVF industry devices that were recalled by manufacturers. This underscores the necessity for more specific characterization of products, guidelines, and rigorous quality control protocols to prevent potentially adverse clinical outcomes. Study design, size, duration MEAs were conducted over 5 years on 6759 batches encompassing 16 categories of IVF products from various manufacturers. Results were retrospectively analysed based on MEA test outcomes for each category. A total of 217 dishes, 610 catheters, 1182 culture media, 135 steripettes, 1907 mineral oils, 167 glass-pasteur pipettes, 66 protein supplements, 207 flexipet-pipettes, 191 pipette tips, 96 PVPs, 68 syringes, 126 tubes, 1294 vitrification kits, 182 micropipettes, 98 disinfectants, and 213 needles lots were analysed. Participants/materials, setting, methods All procedures adhered to protocols approved by an Ethical Committee. Samples underwent classification as toxic or non-toxic based on MEAs results, and the proportion of samples that tested positive for embryotoxicity was determined for each category. Statistical comparisons were made among categories to identify those most prone to be identified as toxic. Additionally, in failed assays, mean EBFRs were categorized into 0-30%, 30%-70%, and 70%-80% ranges to assess extreme, mild and low sample-toxicity levels, respectively. Main results and the role of chance Out of 16 studied categories, only 5 of them exceeded a 5% of toxic lots. Flexipet-pipette types were reported as the highest toxic-samples rate category, with 72 out of the 207 tested batches (34.8%) failing the MEA. Protein supplements were found to be the second highest toxicity-ranked category, with 14/66 noxious lot samples (21.2%). They were followed by needles and catheters with 36/213 (16.9%) and 68/610 toxic lot samples (11.2%), respectively. Finally, a considerably lower toxicity rate was obtained with steripettes, with 10/135 samples reported as toxic (7.4%). Among these five top toxic-samples rate categories, protein supplements were found to possess a mild lethal effect on embryos, with a 43.6% mean EBFR in failed assays. However, extreme embryotoxic effects were reported for the remaining highest toxic-samples rate categories, based on their mean EBFR in failed assays: 20.8% for flexipet-pipette types, 16.2% for needles, 10.8% for steripettes, and 10.3% for catheters. Interestingly, although culture media and mineral oils were not among the top MEA-failure frequencies, as only 48/1182 (4.1%) and 23/1907 (1.2%) toxic lot samples were registered for each category, respectively, both categories showed a nearly-extreme lethality caused by their toxic-assessed samples, reflected in 31.7 and 35.1% mean EBFRs. Limitations, reasons for caution Increasing the number of sample-providing manufacturers per category might strengthen the wider market representativeness of the results. Moreover, it should be noted that the identification of specific sources of toxicity results highly difficult, hindering the implantation of corrector modifications within the manufacturing process. Wider implications of the findings These findings indicate that protein supplements, flexipet-pipette types, steripettes, catheters and needles exhibit a heightened toxicity tendency. Additionally, toxic mineral oils and culture media-linked high-embryotoxicity levels were observed. Therefore, special attention should be given to the production of these demonstrated potentially embryotoxic products to identify and mitigate clinical toxicity risks. Trial registration number Not applicable

P-770 Health outcomes of taiwanese infants after different embryo stages in both fresh and frozen transfer procedures

Abstract Study question Does freezing embryos have any effects on neonatal and postnatal health? Does extended embryo culture have any impact on these health outcomes? Summary answer frozen embryo transfer (FET) infants were more likely be large gestational age, but with lower odds of recurrent upper respiratory infection and dermatitis. What is known already Infants born from frozen embryo transfers tend to have higher birth weights and are less likely to be small for gestational age. There are no notable variations in congenital malformations or child development. However, there’s a lack of long-term follow-up data. Study design, size, duration In this retrospective cohort study conducted in Taiwan, data from the national assisted reproductive technology (ART) database spanning from January 1st, 2013, to December 31st, 2017, was utilized. The study categorizes cases into fresh and frozen embryo transfer groups, with additional subdivisions based on cleavage and blastocyst stage transfers. Singletons born in each group were then linked to the National Health Insurance Database for follow-up on health conditions until December 13th, 2018. Participants/materials, setting, methods We collected data on singleton births resulting from assisted reproductive technology procedures performed in Taiwan from January 1st, 2013, to December 31st, 2017. Our analysis included 10,669 eligible infants. We investigated factors such as gestational age, birth weight, and various systemic diseases, encompassing neurological disorders, facial and eye abnormalities, cardiovascular diseases, respiratory disease, gastrointestinal disorders, renal system disorders, musculoskeletal disorders, skin and submucous disorders, chromosomal abnormalities, infections, and birth injuries. Main results and the role of chance Compared to infants conceived from fresh embryos, those born to mothers who underwent frozen embryo transfer (FET) were more likely to be large for gestational age (OR 1.63, 95% CI 1.30-2.04 for blastocyst and OR 1.69, 95% CI 1.36-2.10 for cleavage stage), and to have a neonatal birth weight of ≥ 4000g (OR 1.88, 95% CI 1.15-3.07 for blastocyst and OR 2.64, 95% CI 1.63-4.28 for cleavage stage). However, they were less likely to be small for gestational age (OR 0.68, 95% CI 0.56-0.82 for blastocyst and OR 0.68, 95% CI 0.56-0.82 for cleavage stage). Infants born from frozen embryo transfers also experienced lower odds of recurrent upper respiratory infection (OR 0.72, 95% CI 0.60-0.86 for blastocyst and OR 0.60, 95% CI 0.52-0.69 for cleavage stage), as well as eczema and dermatitis (OR 0.75, 95% CI 0.62-0.91 for blastocyst and OR 0.75, 95% CI 0.62-0.91 for cleavage stage). There was no observed effect on infant health between blastocyst and cleavage stage embryo transfers. Limitations, reasons for caution Our retrospective study design may not possess the same level of rigor as a randomized controlled trial (RCT). The follow-up period in our study ranged from 1 to 5 years, and some diseases may not manifest within this short-term timeframe, necessitating longer-term tracking for more comprehensive observation. Wider implications of the findings The result highlights the potential benefits of FET in reducing the risk of low birth weight. The observed decrease respiratory infections and skin disorders among FET infants suggests a possible advantage in immune system development or environmental adaptability. Further research with longer-term tracking is warranted. Trial registration number KMUHIRB-E(I)-20210222

P-148 Humid versus dry culture: impact on euploidy, live birth sex ratio and clinical outcomes

Abstract Study question Does humidity have an impact on top-quality blastocyst rate, ploidy, live birth sex ratio and clinical outcomes? Summary answer Humid culture conditions in time-lapse incubators may enhance clinical outcomes, particularly in autologous cycles, without biasing euploidy or newborn sex. What is known already Recent advances in assisted reproductive technology have focused on improving embryo culture conditions and incubators are becoming more sophisticated. Although culture conditions such as oxygen and temperature are well-studied, the optimal humidity level remains uncertain. Dry atmospheres favour higher evaporation of culture media compared to humid conditions, increasing its osmolality. This appears to be detrimental to several animal model embryos: developmental arrest, changes in gene expression and epigenetic alterations in the embryos. Previous studies have shown than incubator humidification improves clinical outcomes and embryonic development. However, other studies showed no significant improvements. Study design, size, duration A single center retrospective study was performed. Data included a total of 793 cycles of IVF/ICSI between January 2019 and May 2023. Participants/materials, setting, methods 793 cycles of IVF/ICSI from 734 patients were studied. Study population was categorized in autologous (n = 544) and egg-donation (n = 249) cycles. A total of 5909 embryos were included in the study. Patients were randomly distributed in two distinct cohorts at the moment of their treatment: dry condition (DC) and humid condition (HC). Blastocyst quality (BR), biochemical pregnancy (BPR), implantation rate (IR), live birth rate (LBR) were studied. Ploidy rate and LB sex ratio was also studied. Main results and the role of chance In egg donation cycles, no differences were observed between HC and DC groups in the rates of top-quality blastocysts (61.5 HC vs 64.4% DC), BPR, (71.1% HC vs 71.2% DC), IR (53.8% HC vs. 59.2% DC) or LBR (51.9% HC vs. 44.8% DC). However, autologous cycles showed an improvement when humid conditions were used. BR and IR were higher in humid conditions (BR: 58.8% HC vs. 55,6% DC, p-value 0.03; IR:57.1% HC vs. 39.7% DC, p-value 0.011)). Humid and dry culture conditions yielded similar rates of BPR (65.0% HC vs 53.0% DC, p-value: 0.11) and live birth rate (44.3% HC vs 34.5% DC, p-value: 0.16). Although there is a tendency towards a 20% of benefit in presence of humidity in both rates, no significance was reached. No differences were found in euploidy between dry and humid groups. When stratifying patients by age, all age groups met the ploidy expected rates in both culture conditions. Humid culture conditions enhance clinical outcomes, particularly in autologous cycles. Donated oocytes may take advantage of their better quality to compensate the detrimental effect of dry ambients. Our study, moreover, demonstrates that humidity does not introduce any bias towards chromosomal alterations or newborn sex. Limitations, reasons for caution This is a retrospective study and should be further confirmed with a prospective randomized study. Sample size should be increased, especially for ploidy Specific humidity values could also be tested. Wider implications of the findings Humid culture conditions may highly improve ART outcomes for particular groups of patients. If tested with stratified groups by age, a specific age group might be especially benefited of humidity, without any bias in sex ratios when best embryo is transferred in fresh cycles. Trial registration number not applicable

P-774 Conventional-IVF versus NC-IVF: unravelling the origins of fetal cardiac programming in ART

Abstract Study question Do fetuses from natural cycle IVF (NC-IVF) present signs of cardiac remodeling and suboptimal function similar to those observed in fetuses from conventional IVF (cIVF)? Summary answer Fetuses from both NC-IVF and cIVF present signs of fetal cardiac remodeling and suboptimal function as compared to spontaneously conceived fetuses (SC) from fertile couples. What is known already Previous publications have demonstrated that fetuses conceived by IVF present cardiac remodeling and suboptimal function. These fetuses show dilated atria, more globular and thicker ventricles, reduced longitudinal motion, and impaired relaxation. However, the pregnancies included in these studies were obtained following cIVF. In IVF cycles, ovarian stimulation has an impact on the oocyte quality and the endometrial milieu that may potentially affect perinatal results and fetal cardiovascular programming. Our aim was to compare cardiac morphometry and myocardial function in fetuses conceived by cIVF with those obtained after NC-IVF, without ovarian stimulation. Study design, size, duration This is a nested case-control study of 120 singleton pregnancies within a prospective cohort recruited from 2017 to 2021, including 40 SC from fertile couples, 40 following NC-IVF and 40 after cIVF with fresh embryo transfer, matched by maternal age, ethnicity, and gestational age at scan. IVF pregnancies were recruited from a single assisted reproduction center, ensuring homogeneity in ovarian stimulation protocols, laboratory procedures, and embryo culture conditions. Participants/materials, setting, methods The study protocol included prospective collection of parental demographics, reproductive and ART data, a fetal ultrasound scan at 28-34 weeks assessing biometry, feto-placental Doppler and morphological and functional echocardiography, and perinatal outcomes. Comparisons between groups were adjusted by gestational age and fetal weight at scan, and birthweight centile. Oocyte donation and PGT cycles were exclusion criteria. All the laboratory procedures including gamete manipulation, fertilization methods and embryo culture conditions were identical for NC-IVF and cIVF. Main results and the role of chance There were no significant differences in demographic data, perinatal outcomes, biometry and feto-placental Doppler parameters between the study groups. Reproductive data and IVF indications were also similar among the IVF groups. As compared to the SC, both IVF groups showed significant signs of fetal cardiac remodeling (higher cardio-thoracic ratio: cIVF mean 0.31 [SD 0.04]* versus NC-IVF 0.31 [0.03]* versus SC 0.28 [0.04], adjusted P-value <0.001; larger right atria-to-heart ratio: cIVF 19.1% [3.0]*† versus NC-IVF 18.8% [3.3]* versus SC 17.4% [2.9], adjusted P-value <0.001; increased relative wall thickness: c IVF 0.80 [0.2]* versus NC-IVF 0.85 [0.3]* versus SC 0.72 [0.3], adjusted P-value <0.001) and suboptimal function (tricuspid ring displacement (mm): cIVF 7.1 [0.8]* versus NC-IVF 7.3 [0.9]* versus SC 7.5 [1.4], adjusted P-value 0.003; left myocardial performance index: cIVF 0.50 [0.06]* versus NC-IVF 0.49 [0.06]* versus SC 0.48 [0.08], adjusted P-value <0.001). * Adjusted P value <0.05 as compared with SC. † Adjusted P value <0.05 as compared to NC- IVF. Limitations, reasons for caution The sample size was limited by the difficulty in recruiting viable pregnancies obtained by NC-IVF from a single center. The reported cardiac changes are subclinical and, although they are recognized as potential cardiovascular risk factors, their association with the long-term cardiovascular disease in these subjects remains to be proven. Wider implications of the findings Our findings contribute to assess the safety of different ART procedures in terms of cardiac performance in fetal life, before the influence of postnatal factors, in order to unravel the origins of fetal cardiac remodeling in ART. Studies evaluating the long-term health in this population are needed. Trial registration number Not applicable

P-789 Trophoblast organoids as a 3D model for the study of oxygen concentration during in vitro preimplantation embryo culture

Abstract Study question To determine if trophoblast organoids could represent a relevant robust model to study the impact of oxygen concentration on human preimplantation embryo development. Summary answer Transcriptomic analysis of trophoblast organoids cultured in monophasic (5%) or biphasic (5-2%) O2 concentration identified 1,184 RNAs differentially expressed. What is known already Our team has previously demonstrated that a biphasic (5-2%) O2 concentration strategy during preimplantation embryo culture (from Day 0 to Day 6) significantly improved blastulation and live birth rates in in vitro fertilization (IVF). We also identified differentially expressed RNAs between monophasic and biphasic O2 strategies through whole transcriptome analysis on human embryos donated for research. However, the number of embryos donated to research is limited, they are all genetically different, and their use iethically controversial, representing a significative drawback to experimentation. Latest studies on trophoblast organoids indicate that they could represent a relevant model for studying preimplantation embryo development. Study design, size, duration A prospective monocentric study approved by the Institutional Review Board (I.R.B.) was conducted between February 2022 and September 2023: four early first-trimester human placentas were collected after surgical abortions and used for derivation of organoids in a 3D culture system. The protocol for organoid culture is described in the study of Sheridan and al, 2020. Organoids were cultured with either a monophasic or a biphasic oxygen concentration strategy. Participants/materials, setting, methods Trophoblast organoids were either exposed to 5% oxygen concentration during 5 days or to 5% O2 for 3 days and to 2% O2 for 2 days. Total RNA extraction of 21 organoids for each condition was performed. After whole transcriptome analysis using human Clariom D arrays (ThermoFisher Scientific®), microarray data were normalized and differentially expressed RNAs were identified using the Transcriptome Analysis Console software; the genetic network was generated with Ingenuity Pathway Analysis (Qiagen®). Main results and the role of chance Transcriptomic analysis of trophoblast organoids cultured in monophasic (5%) or biphasic (5-2%) O2 concentration identified 1,184 RNAs that were differentially expressed depending on the O2 strategy (fold change >2 or <-2, p < 0.05), with most of them (75.1%, n = 889/1,184) being downregulated in the biphasic (5-2%) O2 group. IPA analysis highlighted downregulation of transcripts involved in cell survival and differentiation as well as upregulation of transcripts involved in embryo angiogenesis and vascularization of extraembryonic tissue. The transcript HIF1a was upregulated in organoids cultured in biphasic strategy compared to monophasic strategy (fold change 2, p = 0.0129). Comparison between differentially expressed transcripts in monophasic and biphasic strategies highlighted 22 transcripts in common between organoids and embryos donated to research from our previous study. Limitations, reasons for caution Our study was limited by a low sample size. Wider implications of the findings This innovative model of trophoblast organoids could help us to overcome the rarity as well as the genetic/epigenetic heterogeneity associated with IVF human embryos donated to the research, thus leading to the realization of large-scale experimentations and promoting the identification of new non-invasive predictive biomarkers for IVF. Trial registration number not applicable

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

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