Trophectoderm Quality Research Articles

Identifying key predictive features for live birth rate in advanced maternal age patients undergoing single vitrified-warmed blastocyst transfer

BackgroundInfertility affects one in six couples worldwide, with advanced maternal age (AMA) posing unique challenges due to diminished ovarian reserve and reduced oocyte quality. Single vitrified-warmed blastocyst transfer (SVBT) has shown promise in assisted reproductive technology (ART), but success rates in AMA patients remain suboptimal. This study aimed to identify and refine predictive factors for live birth following SVBT in AMA patients, with the goal of enhancing clinical decision-making and enabling personalized treatment strategies.MethodsThis retrospective cohort study analyzed 1,168 SVBT cycles conducted between June 2016 and December 2022 at the First Affiliated Hospital of Guangxi Medical University and Nanning Maternity and Child Health Hospital. Nineteen machine-learning models were applied to identify key predictive factors for live birth. Feature selection and 10-fold cross-validation were employed to validate the models.ResultsThe most significant predictors of live birth included inner cell mass quality, trophectoderm quality, number of oocytes retrieved, endometrial thickness, and the presence of 8-cell blastomeres on day 3. The stacking model demonstrated the best predictive performance (AUC: 0.791), followed by Extra Trees (AUC: 0.784) and Random Forest (AUC: 0.768). These models outperformed traditional methods, achieving superior accuracy, sensitivity, and specificity.ConclusionLeveraging advanced machine-learning models and identifying critical predictive factors can improve the accuracy of live birth outcome predictions for AMA patients undergoing SVBT. These findings offer valuable insights for enhancing clinical decision-making and managing patient expectations. Further research is needed to validate these results in larger, multi-center cohorts and to explore additional factors, including fresh embryo transfers, to broaden the applicability of these models in clinical practice.

P-552 Unveiling associated factors to in vitro embryonic ploidy: Insights from 8194 blastocysts by Next Generation Sequencing

Abstract Study question What factors are associated with an increased probability of having aneuploid or mosaic embryos in vitro? Summary answer Maternal age and biopsy day significantly influence aneuploidy, while trophectoderm quality impacts both aneuploidy and embryo mosaicism. What is known already Embryonic aneuploidy and mosaicism are critical factors affecting the success of in vitro fertilization. Several studies have demonstrated that advanced maternal age is the most important variable associated with chromosomal abnormalities. However, the influence of other factors, including the day of the blastocyst biopsy and the quality of specific embryonic components, such as the trophectoderm, on the likelihood of aneuploidy and mosaicism is less understood. Study design, size, duration This retrospective study involved the analysis of 8,194 blastocyst embryos derived from 2,180 IVF/ICSI cycles at a specialized assisted reproduction clinic. The study spanned 7 years, from 2017 to 2023, focusing on identifying factors influencing embryonic ploidy Participants/materials, setting, methods Retrospective analysis of 8194 blastocysts from 2180 IVF/ICSI cycles at a single fertility center. Evaluated maternal age, paternal age, day of embryo biopsy, inner cell mass and trophectoderm quality, embryo biopsy operator, fertilization technique, using logistic regression to assess impact on ploidy (euploid, aneuploid, mosaic). Analysis identified significant factors influencing ploidy, adjusting for confounders. Statistical significance at p < 0.05. Main results and the role of chance The analysis of 8,194 blastocyst embryos highlighted significant findings. Maternal age showed a considerable impact on aneuploidy with a p-value <0.001 and an odds ratio (OR) of 1.74, indicating a strong association. Similarly, the day of embryo biopsy was a significant factor (p < 0.001, OR = 1.51) emphasizing its influence on embryo aneuploidy. The quality of the trophectoderm was a crucial factor, with a notable correlation with both aneuploidy (p < 0.001, OR = 1.78) and mosaicism (p < 0.001, OR = 1.28). Further analysis was also performed to find the variables associated with mosaicism, and the only significant variable was quality of trophectoderm increasing the probability of mosaicism by 28%. These statistical values underscore the robustness of the study’s conclusions. The large sample size and the application of logistic regression models further minimized the role of chance, enhancing the reliability of these findings. The research provides significant insights into the factors influencing embryonic ploidy, extending beyond the well-recognized impact of maternal age. It underscores the importance of considering these variables in clinical assessments during fertility treatments, particularly in evaluating the risk of chromosomal abnormalities. Limitations, reasons for caution The study’s retrospective nature limits causal inferences. The findings need validation in prospective, multicenter studies to establish broader applicability and account for potential biases. Wider implications of the findings These results contribute to the understanding of the factors associated with aneuploidy and embryonic mosaicism, beyond maternal age. Integrating the day of embryo biopsy and trophectoderm quality as key factors into clinical evaluation that could improve clinical decision-making in fertility treatments, especially in assessing the risk of chromosomal abnormalities. Trial registration number Non-Clinical trials

P-149 A visual interpretability method to unbox ‘black-box’ deep learning image-based classification of embryo properties

Abstract Study question Can we decipher the underlying visual properties that drive image-based AI embryo classification models to assist clinical decisions and biological discovery? Summary answer Our framework interpreted which annotated and non-explicitly-annotated phenotypes impact model predictions and rank their importance. These discoveries were aligned with known blastocyst quality criteria. What is known already Deep learning models have shown great promise for complex pattern recognition when applied to embryo images. The success of these models relies on their ability to perform non-linear optimization of feature extraction during model construction. However, this involves their entanglement of multiple classification-driving image properties, thereby producing ‘black-box’ systems that lack user confidence, trust and interpretability. Therefore, there is an urgent need for an interpretability method that can uncover the semantic image properties that contribute to ‘black box’ embryo image-based AI classification model predictions to assist in blastocyst selection. Study design, size, duration 11,211 time-lapse videos were retrospectively collected from three IVF centers. A deep convolutional neural network is first trained to discriminate high-versus-low quality blastocysts. We then developed DISCOVER, a general-purpose interpretability method designed to discover underlying visual properties driving the classifier. DISCOVER encodes an image to an interpretable lower dimensional representation which is correlated to the classifier and encapsulates a different distinct phenotype in each one of the dimensions. Participants/materials, setting, methods The encoding of embryo images to low dimensional representations enables interpretability globally and locally. Globally, the embryo images are synthetically altered by amplifying subtle properties that affect the classification decision. With our method this can be done one property at a time, therefore separating confounding properties. By evaluating the altered images, embryologists can decipher their meaning. Locally, each one of the discovered properties can be ranked by its importance for a specific embryo instance. Main results and the role of chance Using DISCOVER, we interpreted the classification model driving features. We quantitatively linked the top two classification features as blastocyst size (as proxy to degree of expansion and development) and trophectoderm quality, by embryologists evaluation and annotations. We then asked whether DISCOVER can identify non-explicitly annotated latent features that encode morphologic properties not defined by ASEBIR/Gardner criteria. Expert embryologist interpreted the third top classification feature to be the blastocoel. DISCOVER interpreted high quality embryos as having denser and more granular blastocoelic regions, suggesting that this change in the blastocoel appearance is one of the encoded classification-driving morphologic properties. This visualization indicates that there are additional parameters of the blastocoel beyond its volume expansion associated with its quality. We showed how embryo properties can be weighted differently by the classifier on a per embryo basis, giving clinical insight to which properties influence the classification of a specific instance. These results indicate that DISCOVER enables expert-in-the-loop interpretation of the classification model both globally, discovering the overall main properties driving the classifier, and locally, showing a per instance explanation. Limitations, reasons for caution DISCOVER failed to interpret the inner cell mass (ICM) as a classification-driving feature in its latent representation, though it was explicitly used to label the data for training the classification model. It is possible that other properties collectively contained the discriminative information encoded in the ICM. Wider implications of the findings This deep analysis demonstrates the feasibility of providing interpretability for biomedical image-based classification models for clinical use in the IVF clinic. Trial registration number not applicable

P-180 Trophectoderm biopsy technique and its effect on life birth rates: a prospective study

Abstract Study question Do life birth rates of euploid embryos vary based on factors such as laser intensity, number of laser shots, and the biopsy technique employed? Summary answer This study found no correlation between biopsy technique parameters and life birth rates of euploid embryos, underscoring the robustness of current trophectoderm biopsy methods. What is known already Embryo biopsy for Preimplantation Genetic Testing (PGT) must achieve two primary objectives: ensuring that the biopsied material accurately reflects the genetic makeup of the embryo and maintaining the viability of the embryo without compromise. The strategy that most closely aligns with accomplishing these dual goals is trophectoderm (TE) biopsy. However, it’s important to note that there is currently no standardized TE biopsy procedure, leading to the coexistence several techniques. Given the potential impact of TE biopsy on trophectoderm quality and integrity, and consequently on implantation, there is a critical need for studies to assess the effects of different biopsy techniques. Study design, size, duration This prospective observational study spanned from 2019 to 2021 and involved 298 Preimplantation Genetic Testing for Aneuploidy (PGT-A) treatments, incorporating a total of 1187 trophectoderm (TE) biopsies. Embriologyst were trained in both Pulling and Flicking techniques and choose between the two techniques according to their criteria. Pulling involved laser pulses weakening junctions, facilitating TE detachment with moderate tension. Flicking substituted tension with a cutting motion between holding and biopsy pipettes. Participants/materials, setting, methods Biopsy technique (pulling/flicking), number of laser shots (1-10), laser intensity (200µs-600µs), and the embriologyst responsible for the biopsy were annotated for every biopsied embryo. PGT indication, stimulation protocol, patient age (39.32±4.22), embryo quality, and sperm diagnosis were annotated and used as possible confounding factors in a logistic regression model to understand the impact of each independent variable on the log-odds of the outcome. Main results and the role of chance The 1187 trophectoderm biopsies resulted in a total of 410 (34.5%) euploid embryos. Among these, 237 euploid embryos underwent individual transfers, with 153 subjected to Pulling technique and 84 to Flicking technique leading to a total of 120 healthy babies (50.6% life birth rate). Logistic regression, considering potential confounding factors failed to identify any variable significantly influencing the likelihood of life births (p < 0.05). Both Pulling and Flicking techniques demonstrated comparable success rates (52.9% vs 46.4%), with no discernible impact from variations in laser shots (1-3=65.0%; 4-6=45.6%; >6=66.7%) or intensity (200µs=75.0%; 300µs=45.0%; 400µs=50.6%; 500µs=50.94%; 600µs=50.0%). Furthermore, when evaluating the embryologist conducting the biopsy, no significant differences were identified among operators (p < 0.05). This pattern persists for miscarriage rates, indicating no influence from biopsy technique, laser shots, or laser intensity (p < 0.05). Thus, our findings underscore that variations in trophectoderm biopsy variables, when performed by an experienced embryologist, do not adversely impact trophectoderm quality and do not compromise success rates. Limitations, reasons for caution This is a single-center, observational study, conducted from 2019 to 2021, and this may limit generalizability. The sample size variation between biopsy techniques and potential unmeasured confounders pose challenges. The assumption of technique homogeneity, and reliance on binary outcomes underscore caution in interpreting findings. Wider implications of the findings The lack of correlation between biopsy parameters and life birth rates in our study underscores the effectiveness and reliability of current biopsy techniques. This discovery offers reassurance regarding the proficiency of trophectoderm biopsy methods for achieving success in IVF treatments. Trial registration number Not Applicable

O-065 PGT-A reveals triploid embryos among normally fertilized oocytes: Frequency and blastocyst features

Abstract Study question Is there any embryo parameter that could differentiate 2 pronucleus (PN) triploid from 2 PN euploid embryos? Summary answer Accuracy in the fertilization check is still necessary as 2 PN but triploid embryos are due mostly to retention of the second polar body (PB). What is known already Since blastocyst culture has evolved and the development of genetic technologies enables to know the karyotipe of the embryos, the concept of normal fertilization is being revised. Commonly, correct fertilization is denoted by the appearance of 2 PN 16-18h after insemination. Deviation from 2PN is considered evidence of abnormal fertilization and ploidy anomalies. However, not all oocytes exhibiting 2 PN are euploids. After performing NGS in trophectoderm biopsies for preimplantation genetic testing for aneuploidy (PGT-A), a percentage of embryos are categorized as triploids. Study design, size, duration Retrospective study including 1862 cycles from 1605 patients aged 18-45 years old who underwent PGT-A with NGS of trophectoderm biopsies in our IVF center from 2019 to 2023. From 6526 analyzed blastocysts in an independent Genetics laboratory, 59 were categorized as triploids. These 59 blastocysts were obtained from 57 PGT-A cycles and belonged to 54 women. In total, these patients had 266 biopsied blastocysts. From the remaining 207 embryos, 42 were called as euploids. Participants/materials, setting, methods In embryos cultured in time-lapse, fertilization was re-evaluated to check the appearance of 2PN and the second PB extrusion. Genetic triploid embryos were analysed in relation to the day of biopsy (day 5 vs day 6), the trophectoderm quality and the timing of the first cell divisions (time of cleavage from 2 to 6 cells: t2, t3, t4, t5, t6). Categorical variables are shown as rate. T-student tests were performed. P-value<0.05 was considered statistically significant. Main results and the role of chance The re-evaluation of the fertilization of the 59 triploids categorized as 2 PN revealed 50 oocytes 2 PN, 8 embryos 3 PN and 1 embryo 2 PN + 1 micronuclei configuration. Therefore, the prevalence of real triploidy in 2PN embryos was 0,8% (n = 50/6526; CI95%:0,6-1,0%). These data showed that 2PN zygotes may give rise to embryos with altered ploidy. From 27 triploid 2PN embryos was possible to access to the whole development time-lapse images. Just 33,3% of triploid embryos exhibited the extrusion of the second PB, while 100% of euploid sibling embryos showed this extrusion (N = 9/27 vs N = 24/24,;p<0,005). Comparing 2 PN triploid and the rest of the embryos from the same cohorts, there were no differences regarding the day of biopsy (N = 31/176 day 5 biopsy, 17.6% vs N = 19/81 day 6 biopsy, 23.5%; p > 0.05). On the contrary, a worse blastocyst quality showed a significant relation with triploidy (N = 23/161,14,2% of good quality embryos are triploid vs N = 27/96, 38,6% of fair quality embryos are triploids; p:0.006).Embryos cleavage time points up to 6-cell stage did not show any significative difference between triploid and euploid embryos (t2: 27.36h vs 25.8h; t3: 38.5h vs 35.6h; t4 39.4h vs 37.2h; t5 52.8h vs 47.0h; t6 56.2h vs 52.1h; all comparisons p > 0.05). Limitations, reasons for caution The incidence of triplody from 2PN zygotes may vary among IVF laboratory settings. The small sample size may be responsible of no difference in morphokinetics. Wider implications of the findings Fertilization check should persist in IVF procedures. Special consideration should be given to the second PB extrusion as triploidy is the most common form of aneuploidy observed in the first trimester miscarriages. The role of diploid spermatozoa, the size of the pronucleus and location should be explored in further studies. Trial registration number NOT APPLICABLE

P-213 Effectiveness and safety of single poor quality blastocyst transfer compared with single good quality blastocyst transfer in the first transfer cycle

Abstract Study question Comparison of pregnancy and perinatal outcomes in blastocysts with poor inner cell mass quality or poor trophectoderm quality to optimize non-quality single blastocyst transfer strategies. Summary answer Poor trophoblast quality blastocysts have better pregnancy outcomes than poor inner cell mass quality blastocysts, with no statistically significant difference in perinatal outcomes What is known already There is no doubt that good-quality blastocysts have better pregnancy outcomes than poor-quality blastocysts, but the preference for trophectodermal poor-quality blastocysts or inner cell mass poor-quality blastocysts in non-good-quality blastocyst cycles is still uncertain. Study design, size, duration A total of 46,433 first single blastocyst transfer cycles from March 1, 2014, to December 31, 2022 were retrospectively analyzed, and blastocyst transfer cycles were divided into 3 groups according to the grade of trophoderm and inner cell mass: A/B A/B (Group 0, N = 30748), A/B C (Group 1, N = 15455), and C A/B (Group 2, N = 230). Participants/materials, setting, methods 46,433 patients who underwent their first single blastocyst transfer at Reproductive and Genetic Hospital of CITIC-Xiangya were included. Multivariate log-binomial regression model with COPY methods were performed to estimate relative risk of Group 2/1 versus Group 0 and Group 2 versus Group 1 for reproductive and neonatal outcomes. Five propensity score weighting methods, namely IPW, processing, overlap, matching, and entropy, were used for sensitivity analysis to explore the robustness of the results. Main results and the role of chance After adjusting for female age, BMI, duration of infertility, number of cycles, AFC, AMH, endometrial thickness, causes of infertility (tubal disorders, ovulatory disorders, endometriosis, unexplained, more than one causes, male factors), culture days (D5, D6), expansion of blastocyst (3, 4, 5, 6), and type of blastocyst (fresh blastocyst, thawed blastocyst, thawed cleavage cultured to blastocyst, PGT blastocyst), we found that clinical pregnancy rate (CPR) and live birth rate (LBR) of Group 0, Group 1 and Group 2 were successively significantly decreased (CPR: Group 1 vs Group 0, RR = 0.83 (0.81, 0.84), Group 2 vs Group 0, RR = 0.43 (0.35, 0.53), Group 2 vs Group 1, RR = 0.53 (0.42, 0.64); LBR: Group 1 vs Group 0, RR = 0.79 (0.77, 0.81), Group 2 vs Group 0, RR = 0.36 (0.27, 0.47), Group 2 vs Group 1, RR = 0.45 (0.34, 0.59); respectively), while early miscarriage rate was successively significantly increased (Group 1 vs Group 0, RR = 1.3 (1.22, 1.38), Group 2 vs Group 0, RR = 2.25 (1.46, 3.15), Group 2 vs Group 1, RR = 1.73 (1.12, 2.42)). Late miscarriage, preterm birth, singleton live birth, sex ratio of singleton, low birth-weight of singleton, and birth weight of singleton were not statistically different between three groups. Above results were robust in our two sensitivity analysis. Limitations, reasons for caution As a retrospective study, there are inevitably unmeasured confounding factors. This study only included a small number of Group 2. It’s therefore possible that our study was under powered. Risk of gestational hypertension, gestational diabetes, cesarean section and macrosomia should also be evaluated with a more comprehensive assessment of safety. Wider implications of the findings High-quality blastocysts have better pregnancy and live birth rates than poor-quality blastocysts, but there are no significant differences in preterm birth, sex ratio, or birth weight. In the absence of good quality blastocysts for transplantation, poor quality blastocysts of the trophectoderm are preferred over poor quality inner cell mass. Trial registration number not applicable

Predictive factors of aneuploidy in infertile patients undergoing IVF: a retrospective analysis in a private IVF practice

BackgroundPGT-A has become an important part of IVF treatments. Despite its increased use, there are contradicting results on its role in improving reproductive outcomes of ART cycles. Given that aneuploidy is a main limiting factor for IVF success, we aimed to study the predictive factors of aneuploidy in infertile patients undergoing IVF and hence highlight the patients who would benefit the most from genetic testing.ResultsA retrospective analysis of 1242 blastocysts biopsied in the setting of PGT-A cycles was performed. The euploid group included 703 embryos, while the aneuploid group had 539 embryos. The factors included in the analyses were the couple’s history as well as the embryo characteristics. The primary outcome was the rate of aneuploid embryos per patient’s history as well as per embryo characteristics. The aneuploidy rate (AR) in our cohort was 43.4%. The woman’s age was found to be a significant predictor (OR 1.045, 95% CI 1.008–1.084, p = 0.016). Biopsy on day 5 as well as degree of expansion 3 was also found to affect significantly (OR 0.724, 95% CI .541–.970, p = 0.03 and OR 2.645, 95% CI 1.252–5.585, p = 0.011). Lack of consanguinity decreased the AR by an OR 0.274 with 95% CI .137–.547, p < 0.001. The number of blastocysts available, trophectoderm quality, embryo grade, gonadotropins as well as trigger used were not found to be significant predictors (p = 0.495, 0.649, 0.264, 0.717 and 0.659 respectively).ConclusionAdvanced female age, consanguinity, the day of embryo biopsy, and the degree of blastocyst expansion were all found to affect the incidence of AR. The age of the male partner, cause of infertility, and grade of embryo at biopsy were not found to correlate with aneuploidy.

Preimplantation genetic testing for aneuploidy: predictive embryonic factors.

In a preimplantation genetic testing for aneuploidy (PGT-A) cycle, does the blastocyst quality before biopsy, or the day of biopsy, or the embryo hatching status have an impact on either euploidy or the rate of embryo survival after freezing? This was a retrospective study including 6130 biopsied blastocysts coming from 1849 PGT-A cycles performed in our center (2016-2022). Embryos were categorized according to the inner cell mass and trophectoderm quality, using Gardner's scoring (excellent: AA; good: AB, BA, BB; poor: AC, CA, BC, CB, CC); the day of biopsy (5 or 6); and their hatching status (fully hatched blastocysts [FHB] or non-fully hatched blastocysts [nFHB]). The independent relationship between each group and both euploidy and survival rate was assessed. Excellent-quality embryos were more euploid than both good- and poor-quality embryos (52.69%, 39.69%, and 26.21%; p < 0.001), and day 5-biopsied embryos were more euploid than day 6-biopsied embryos (39.98% and 34.80%; p < 0.001). Survival rates of excellent-quality (92.26%) and good-quality (92.47%) embryos were higher than survival rates in the poor-quality group (84.61%) (p = 0.011 and p = 0.002). Day 5-biopsied embryos survived better than day 6-biopsied embryos (93.71% vs. 83.69%; p < 0.001) and FHB had poorer survival than nFHB (78.61% vs. 93.52%; p < 0.001). Excellent-quality and day 5-biopsied embryos are more prone to be euploid than good and poor or day 6-biopsied embryos, respectively. Poor-quality, day 6-biopsied embryos, and FHB have significantly lower survival after biopsy and vitrification.

P-128 Morphokinetics and blastocyst biomarkers of twin and triplet monozygotic pregnancy using artificial intelligence

Abstract Study question Can AI reduce the rate of twin and triplet monozygotic pregnancies following elective single embryo transfer (eSET)? Summary answer AI can automatically annotate morphokinetic developments and other biomarkers. Embryos leading multiple monozygotic pregnancies have slower cell divisions and larger ICM than monoamniotic embryos. What is known already Monozygotic twin (MZT) and monozygotic triplet (MTP) pregnancies are a rare phenomenon in spontaneous pregnancies, but the incidence increases significantly when pregnancies are achieved by assisted reproductive technology (ART); 0.4% vs. 1.56% MZTs, and 0.004% vs. 0.048% MTPs. It is unclear what mechanisms cause an embryo to split into two or three, although several have been proposed such as culture to blastocyst, decompacting ICMs, frozen-warmed embryo transfers, assisted hatching and even ICSI. In order to study this split phenomenon, time-lapse imaging has been used to discover any signs of embryo division. Study design, size, duration This is a retrospective assessment of a total of 8 embryos from 2018 to 2022 that led to single pregnancy (n = 4), twin pregnancy (n = 3) and triple pregnancy (n = 1) following a fresh single embryo transfer. All embryos were inseminated using the ICSI technique and assisted hatching was performed before fresh transfer. Participants/materials, setting, methods Using CHLOE (Fairtility, Tel Aviv) we automatically assessed the morphokinetics, blastocyst biomarkers and scores. Singletons and multiple pregnancies were compared in terms of morphokinetics (t-test) and surface area of ICM, ICM diameter, ICM area/embryo area ratio, ICM shape and CHLOE embryo quality score were compared using the ANOVA test. Main results and the role of chance Embryos that led to multiple pregnancies had slower embryo development than embryos that led to singletons (single vs multiple: t2: 22.1+-2 vs 25.2+-2, p = 0.04; t3: 32.56+-2.65 vs 36.98 +-1.48, p = 0.01; t5: 44.09+-4.84 vs 50.37+−0.69, p = 0.02; t6: 47.26+-3.87 vs 53.59+-2.40, p = 0.01; t7: 48.76+-4.04 vs 55.15+-3, p = 0.02; t8: 50.18+-4.27 vs 57.55+-1.5, p = 0.008; t9: 63.27+-3.76 vs 73.04+-5.33, p = 0.03). Embryos leading to triplets were slower than twins which were, in turn, slower than singletons (single vs twins vs triplets: t4: 33.86+-3.41 vs 37.69+-1.61 vs 48.95, p = 0.007; t8: 50.18+-4.27 vs 58.07+-1.29 vs 55.96, p = 0.04; t9: 63.27+-3.76 vs 71.23+-4.81 vs 78.44, p = 0.01). Embryos that led to multiple pregnancies had a larger ICM to embryo surface area ratio (single vs multiple: 0.14+−0.08 vs 0.27+−0.06, p = 0.04) and smaller embryo diameter (single vs multiple: 177.2+-16.5 vs 138.15+-8.25, p = 0.003). We didn’t find statistically significant differences between the groups in CHLOE EQ score (single vs multiple: 0.97 vs 0.76, p = 0.36), Blast score (single vs multiple: 0.87+−0.06 vs 0.78+−0.13, p = 0.26), CHLOE Rank, Trophectoderm quality, ICM Area (single vs multiple: 3629+-1361 vs 4151 +- 569, p = 0.48) and ICM shape (single vs multiple: 1.64+−0.45 vs 1.27+−0.12, p = 0.15). Limitations, reasons for caution The main limitation of this study is the number of cases included in the study, as we studied one triplet, 3 twins and 4 single pregnancies. Wider implications of the findings This might be the first time that AI has been used to analyse the behaviour of embryos resulting in multiple pregnancies. The transfer of a slowly dividing embryo and/or with a large ICM could result in a multiple pregnancy. Trial registration number not applicable

Live birth rates in day 5 fresh versus vitrified single blastocyst transfer cycles: A cross-sectional analysis.

The use of frozen embryo transfers (FET) in assisted reproduction has increased worldwide. Controlled ovarian hyperstimulation in a fresh transfer may impair endometrial-embryo synchronicity. However, there is conflicting evidence on live birth rates (LBR) and clinical pregnancy rates (CPR). To compare LBRs and CPRs between single autologous day 5 fresh vs. vitrified blastocyst transfer cycles, to investigate the impact of controlled ovarian hyperstimulation on embryo-endometrium asynchrony. A large cross-sectional analysis of 6002 embryo transfers (ET) comprised 3774 fresh and 2228 FET cycles from 2016 to 2019. Multivariate and subgroup analysis were performed for high responders ( 20 oocytes). Univariate analysis showed no difference in LBR (28.3% vs. 27.4%, p = 0.43) and CPR (32.2% vs. 30.9%, p = 0.30); however, multivariate analysis demonstrated significantly lower LBR (OR 0.864, p = 0.046, 95% CI 0.749-0.997) and CPR (OR 0.852, p = 0.024, 95% CI 0.742-0.979) in FET compared to fresh ETs. Younger participant age, previous in vitro fertilization pregnancy, advanced blastocyst expansion, higher trophectoderm quality, and lower cumulative number of ETs all improved the odds of LBR and CPR. Conventional in vitro fertilization, rather than intracytoplasmic sperm injection, improved CPR but not LBR. Body mass index affected neither LBR nor CPR. In the subgroup, multivariate analysis of high responders showed no difference in LBR or CPR. This study demonstrates relatively higher LBR and CPR of nearly 14% for fresh ETs compared to FETs, in multivariate analysis. A universal freeze-all strategy, without appropriate indication, may lead to suboptimal outcomes. In high responders, freeze-all cycles may be beneficial, as outcomes appear similar.

A Cross-Sectional Analysis of Live Birth Rates in Day 5 Fresh Versus Vitrified Single Blastocyst Transfer Cycles

Background: The use of frozen embryo transfers (FETs) in assisted reproduction has increased worldwide. Proponents of FETs suggest that controlled ovarian hyperstimulation (COH) in a fresh transfer impairs endometrial-embryo synchronicity, however there has been conflicting evidence on live birth rate (LBR) and clinical pregnancy rate (CPR) outcomes. Aim: To compare LBRs and CPRs between single autologous day 5 fresh versus vitrified blastocyst transfer cycles, in order to investigate the impact of COH on embryo-endometrium asynchrony. Method: A large cross-sectional analysis of 6,002 embryo transfers (ETs), comprising of 3774 fresh and 2228 FET cycles from 2016-2019. Inclusion criteria: first two stimulation cycles, single ET, and no pre-implantation genetic testing for aneuploidy (PGT-A). Multivariate analysis performed, also sub-group analysis for high-responders (&gt;20 oocytes collected). Results: Univariate analysis showed no absolute difference in LBR (28.3% vs 27.4%, p=0.43) and CPR (32.2% vs 30.9%, p=0.30), however multivariate analysis demonstrated significantly lower LBR (OR 0.864, p&lt;0.05, 95% CI 0.749-0.997) and CPR (OR 0.852, p&lt;0.05, 95% CI 0.742-0.979) in FET compared to fresh ETs. Younger patient age, previous IVF pregnancy, advanced blastocyst expansion, higher trophectoderm quality and lower cumulative number of ETs all improved the odds of both LBR and CPR (p&lt;0.001). Conventional IVF, rather than ICSI, improved CPR (p&lt;0.05) but not LBR (p=0.138). BMI affected neither LBR (p=0.492) nor CPR (p=0.359). In sub-group multivariate analysis of high-responders, no significant difference in LBR (p=0.439) or CPR (p=0.450) noted. Conclusion: This study demonstrates a relative higher LBR and CPR for fresh ETs compared to FETs, in multivariate analysis. Having a FET decreased the odds of LBR by nearly 14% compared to fresh ET, when adjusted for confounders. Our results suggest that universal freeze-all strategy, without appropriate indication, may lead to suboptimal outcomes. In high-responders, freeze-all cycles may be beneficial, as adjusted outcomes appear to be similar.

P-141 The good, the bad and the ugly – The fate of collapsed blastocysts in frozen embryo transfer

Abstract Study question What is the clinical outcome of collapsed blastocysts without signals of re-expansion in frozen embryo transfers (FET)? Summary answer FET of non-re-expanded blastocysts after thawing showed a live-birth rate in 11.5% of the cases. Embryos vitrified on day 5 revealed the best results. What is known already Embryo viability is crucial for FET to be successful. Since the introduction of vitrification techniques, outcomes have improved substantially and are even comparable to fresh transfers. Nevertheless, some indicators may help predict the outcome of frozen-thawed blastocysts. Blastocoele re-expansion after thawing has been widely studied and is a strong predictor of clinical pregnancy outcome. However, accurate data on non-re-expanded blastocysts after FET is still not available. Hence, this study aims to evaluate the clinical outcome of non-re-expanded blastocyst after FET. Study design, size, duration The retrospective study was designed and conducted at the Kinderwunsch Institut Schenk GmbH (Dobl, Austria). Data was collected from FET performed between 2017 and 2021. In total, 104 FET cycles of collapsed blastocysts were analysed. All the transfers performed were single embryo transfers (SET). Patient’s average age was of 33.6 years at the time of embryo vitrification. Embryos were excluded if they showed more fragmentation than before vitrification. Participants/materials, setting, methods Patients were divided into two groups following the time of blastocyst cryopreservation: day 5 (44 SET) and day 6 (60 SET); which was dependant on the time of blastocoel expansion. They were further divided according to the trophectoderm quality: good (42 SET) and poor (62 SET) and the overall embryo quality, considering trophectoderm and inner cell mass (ICM): good (37 SET) and poor (67 SET). In both cases quality was assessed before vitrification. Main results and the role of chance A total of 104 collapsed blastocysts were transferred (SET). 16.3% of the patients had a positive biochemical pregnancy. 11.5% of the pregnancies resulted in a live birth and 4.8% of the pregnancies ended in abortion. FET with day 5 non-re-expanded blastocysts after thawing showed better results than day 6 thawed blastocysts regarding biochemical pregnancy (29.5% vs 6.6%, p = 0.02) and live birth rate (20.4% vs 5%, p = 0.017). Good embryo quality before vitrification showed a trend for better clinical outcome, however, results were not significant. A good trophectoderm quality before vitrification revealed a live birth rate of 16.7% compared to 8.1% in poor trophectoderm quality. Regarding overall embryo quality (trophectoderm and ICM quality) the results were 14.3% deliveries for good quality blastocysts vs 7.6% for poor quality blastocysts. Limitations, reasons for caution The sample size may be seen as a study limitation. However, statistically significant results were obtained. Nonetheless, results should be confirmed with a bigger sample size. Wider implications of the findings The present study revealed a lower chance of live birth for non-re-expanded blastocysts after thawing, which should nevertheless not be overlooked; specially in cases with good quality embryos frozen and thawed on day 5. Transfer of collapsed embryos after thawing would be recommended taking into consideration the study results. Trial registration number Not Applicable

P-258 Impact of Direct Unequal Cleavage (DUC) on embryo development, blastocyst formation and ploidy - artificial intelligence (AI) analysis

Abstract Study question Do DUCs significantly impact embryo development? In particular, morphokinetics, grading, and Pre-implantation Genetic Testing for Aneuploidy (PGT-A) outcome? Is this analysis corroborated by artificial intelligence? Summary answer DUC embryos develop slower, have lower rates of blastulation and lower CHLOE (Fairtility) scores for blastulation and implantation. However, occasionally euploid blastocysts form from DUCs. What is known already Time-lapse technology enables the identification of DUCs during embryo development. Previous research associates DUCs with poorer blastulation, implantation, and ploidy outcomes. However, DUCs are not routinely annotated in all clinics. Some algorithms, deselect embryos with short second cell cycles; hence, DUCs are rarely transferred. Whether DUC embryos should be automatically discarded or deprioritised is an ongoing debate which leads to inconsistency in clinical practices across fertility centres. AI image processing algorithms may assist embryologists in the identification of DUCs. Study design, size, duration A retrospective single-centre study of normally-fertilised embryos cultured in time-lapse incubators throughout 2019--2021. We reviewed 9284 time-lapse videos using an AI image processing tool (CHLOE, Fairtility), and assessed DUC embryo outcomes (ploidy, blastulation, and blastocyst quality). Additionally, we analysed pronuclei data searching for possible causes of DUCs. Participants/materials, setting, methods CHLOE (Fairtility) software analysed time-lapse videos identifying pronuclei, DUCs, and blastulation; recording all morphokinetic time points (tPNa,tPNf,t2,t3,t4,t5,t6,t7,t8,t9,tM, tSB,tB,tEB), morphological grades for the inner cell mass (ICM) and trophectoderm, blastocyst size at 116hpi; and assessing the likelihood of blastulation (at 30hpi) and implantation. We evaluated the statistical significance for all variables using t-tests (continuous variables) and chi-squared tests (categorical variables). We quantified the two pronuclei (2PN) detection efficacy using four metrics: accuracy, sensitivity, specificity, and informedness. Main results and the role of chance Of all the embryos analysed (n = 9284), 35% showed DUCs (n = 3269). Blastulation was significantly higher in non-DUC versus DUC embryos (76% and 49%, p &amp;lt; 0.0001). Of the embryos that blastulated, ICM quality (A,B,C,D: 24%,13%,19%,21% and 3%,4%,16%,47%, p &amp;lt; 0.001) and trophectoderm quality (20%,21%,15%,23% and 2%,7%,14%,52%, p &amp;lt; 0.0001) were significantly higher in non-DUC than in DUC embryos. As defined, DUC embryos were significantly quicker at reaching t3 than non-DUC [Mean(SD): 34(15) and 39(10), p &amp;lt; 0.0001], with the minimum times being 4hpi and 13hpi respectively. Interestingly, there was no significant difference in achieving t5 [52(21) and 51(12), NS]. For all other morphokinetic milestones, DUC embryos were 6 hours slower than non-DUC embryos. DUC embryos had an euploidy rate of 27.2% (12/44). Only one DUC embryo was transferred in a double embryo transfer cycle leading to a negative outcome. Implantation score [0.14(0.24) and 0.46(0.36), p &amp;lt; 0.0001] and blastulation score [0.4(0.46) and 0.75(0.4), p &amp;lt; 0.0001] were lower for DUC embryos than for non-DUC embryos. CHLOE automatic PN assessment agreed with human annotation in 92% of cases (TP = 388,TN=5,FP=29,FN=7). CHLOE Blastocyst prediction at 30hpi had an AUC of 0.89. The embryologist agreed on 97% of all 483 embryos that CHLOE classified as DUC. Discrepancies arose from CHLOE misclassifying fragments as blastomeres. Further studies warranted. Limitations, reasons for caution Differentiating between fragments and blastomeres within the 5 hours from the first division proves challenging for embryologists and, especially, AI algorithms. Hence, some embryos’ DUC status may be misclassified. Additionally, our sample sizes are limited and larger sizes are needed to corroborate our findings, especially those pertaining to ploidy status. Wider implications of the findings DUC embryos are associated with poorer outcomes and DUC status should be integrated into embryo classification frameworks. Nevertheless, some DUC embryos prove to be euploid. Hence, DUC embryos should not excluded from culture at cleavage stage and instead be allowed to reach blastocyst stage before assessing their suitability for transfer/vitrification/PGT-A. Trial registration number IRB-001C01-01-22

The higher the score, the better the clinical outcome: retrospective evaluation of automatic embryo grading as a support tool for embryo selection in IVF laboratories.

Is the automatic embryo grading function of specific time-lapse systems clinically useful as a decision support tool for IVF laboratories? Blastocyst grading according to the automatic scoring system is directly associated with the likelihood of implantation and live birth, at least in treatments without preimplantation genetic testing for aneuploidy (PGT-A). Several embryo selection algorithms have been described since the introduction of time-lapse technology in IVF laboratories, but no one algorithm has yet been sufficiently consolidated for universal use. Multicentric models based on automated grading systems offer promise for standardization of embryo selection. A retrospective cohort study was performed including 1678 patients who underwent IVF treatments between 2018 and 2020 and whose embryos (n = 12468) were cultured in time-lapse systems. After obtaining the required parameters (division time to 2, 3, 4 and 5 cells; time of blastocyst formation; inner cell mass quality; and trophectoderm quality), the automatic embryo score was calculated using the software included in the appropriate workstation. First, embryo score was compared with conventional morphological quality and the subsequent clinical outcomes of 1952 single blastocyst transfers. Second, we quantified the contribution of the automatic embryo score and conventional morphological grade to implantation and live birth outcome with multivariate logistic regression analysis in different patient populations. A higher embryo score was associated with a better clinical outcome of IVF treatment. The mean of the automatic embryo score varied significantly (P < 0.001) among embryos with different morphological categories, between euploid and aneuploid embryos, between embryos resulting in positive versus negative pregnancy, between implanted and non-implanted embryos, and between embryos resulting in positive and negative live birth. Embryo score was related to the odds of implantation and live birth in the oocyte donation program (odds ratio (OR)=1.29; 95% CI [1.19-1.39]; P < 0.001 for implantation and OR = 1.26; 95% CI [1.16-1.36]; P < 0.001 for live birth) and in conventional treatments with autologous oocytes (OR = 1.38; 95% CI [1.24-1.54]; P < 0.001 for implantation and OR = 1.47; 95% CI [1.30-1.65]; P < 0.001 for live birth). There was no significant association of embryo score with implantation or live birth in treatments involving PGT-A. This study is limited by its retrospective nature. Further prospective randomized trials are required to confirm the clinical impact of these findings. The single-center design should be taken into account when considering the universal application of the model. Evidence of the clinical efficiency of automated embryo scoring for ranking embryos with different morphological grade and potential in order to achieve higher implantation and live birth rates may make it a decision support tool for embryologists when selecting blastocysts for embryo transfer. This research has been funded by a grant from the Ministry of Science, Innovation and Universities FIS (PI21/00283) awarded to M.M. There are no competing interests to declare. N/A.

Sex ratio imbalance following blastocyst transfer is associated with ICSI but not with IVF: an analysis of 14,892 single embryo transfer cycles.

Assisted reproductive technology (ART) has an impact on secondary sex ratio (SSR), which is seemed to be elevated after blastocyst transfer (BT) but decreased following ICSI procedure. We aim to assess whether the higher SSR associated with BT could be influenced by fertilization method used. All consecutive IVF/ICSI cycles (fresh and frozen) involving single embryo transfer (SET) resulting in a live birth between 2015 and 2019 were retrospective analyzed. Logistic regression was used to model the effect on the SSR of maternal and specific ART characteristics. Six thousand nine hundred twenty-two women were included with the crude SSR of 54.8%. The impact of BT on SSR is influenced by the fertilization method used. After adjustment for potential confounders, the SSR in the ICSI BT group was significantly higher when compared to ICSI cleavage-stage embryo SET (aOR 1.24; 95% CI 1.10-1.40, P < 0.001). However, this effect was not detected among SBT with IVF treatment (aOR 1.04; 95% CI 0.97-1.12, P = 0.260). Assessing blastocyst morphological parameters, high trophectoderm quality was significantly associated with elevated SSR (aOR 1.76, 95% CI 1.34-2.31 [A vs. C], and aOR 1.28, 95% CI 1.14-1.44 [B vs. C]). No significant difference was shown in expansion, inner cell mass, or days of blastocyst formation between male and female blastocysts. The impact of BT on SSR could be influenced by the fertilization method used. The higher SSR was observed after BT with ICSI procedures but not with IVF. Interpretation of the findings is limited by the potential for selection and confounding bias.

Inter-centre reliability in embryo grading across several IVF clinics is limited: implications for embryo selection

Research questionWhat is the intra- and inter-centre reliability in embryo grading performed according to the Istanbul Consensus across several IVF clinics? DesignForty Day 3 embryos and 40 blastocysts were photographed on three focal planes. Senior and junior embryologists from 65 clinics were invited to grade them according to the Istanbul Consensus (Study Phase I). All participants then attended interactive training where a panel of experts graded the same embryos (Study Phase II). Finally, a second set of pictures was sent to both embryologists and experts for a blinded evaluation (Study Phase III). Intra-centre reliability was reported for Study Phase I as Cohen's kappa between senior and junior embryologists; inter-centre reliability was instead calculated between senior/junior embryologists and experts in Study Phase I versus III to outline improvements after training (i.e. upgrade of Cohen's kappa category according to Landis and Koch). ResultsThirty-six embryologists from 18 centres participated (28% participation rate). The intra-centre reliability was (i) substantial (0.63) for blastomere symmetry (range –0.02 to 1.0), (ii) substantial (0.72) for fragmentation (range 0.29–1.0), (iii) substantial (0.66) for blastocyst expansion (range 0.19–1.0), (iv) moderate (0.59) for inner cell mass quality (range 0.07–0.92), (v) moderate (0.56) for trophectoderm quality (range 0.01–0.97). The inter-centre reliability showed an overall improvement from Study Phase I to III, from fair (0.21–0.4) to moderate (0.41–0.6) for all parameters under analysis, except for blastomere fragmentation among senior embryologists, which was already moderate before training. ConclusionsIntra-centre reliability was generally moderate/substantial, while inter-centre reliability was just fair. The interactive training improved it to moderate, hence this workflow was deemed helpful. The establishment of external quality assessment services (e.g. UK NEQAS) and the avant-garde of artificial intelligence might further improve the reliability of this key practice for embryo selection.

P–752 Embryo morphological grading across several IVF centers is not consistent but an interactive training is useful to improve its consistency

Abstract Study question Are the embryologists across several IVF clinics concordant when evaluating embryo morphology? Summary answer Embryo morphological grading is sufficiently consistent among embryologists from the same center, while an interactive training was essential to improve its accuracy across several clinics. What is known already Embryo morphology, mostly at the blastocyst stage, is the strongest non-invasive embryological feature that associates with implantation potential. This association is confirmed also when euploid blastocysts are transferred. At present, several embryo grading schemes exist but is still unclear which is the most effective among them. Moreover, many IVF clinics adopt internal embryo grading scores, further limiting the transferability of this crucial prognostic information across different laboratories. With the aim of assessing the level of concordance in embryo grading within and between IVF clinics, the Italian Society of Embryology, Reproduction and Research (SIERR) conceived this study. Study design, size, duration We photographed 40 cleavage-stage and 40 blastocyst-stage embryos (3 focal-planes=240 photos). Two embryologists (senior and junior) from 65 Italian IVF clinics were invited to grade them. Their evaluations were blindly collected as Phase-I (January2020-March2020). Phase-II consisted of an interactive-training on Google-Classroom during which 6 selected experts found a Consensus on the morphological evaluation of the 80 embryos (April2020). As Phase III (May2020-July2020), a second set of 240 pictures was sent to senior participants and experts. Participants/materials, setting, methods Eighteen centers agreed to participate, and 36 embryologists were included. The embryo grading scheme adopted was the Alpha-ESHRE Istanbul Consensus (parameters: cleavage-stage blastomeres’ symmetry and fragmentation, blastocyst’s expansion, inner-cell-mass and trophectoderm quality), conventionally used in 50% of the centers (N = 9/18). The concordance within (junior versus senior) and between (senior versus experts) centers was calculated through the Cohen’s-k. The concordance between centers was compared before and after the interactive training on the two sets of pictures. Main results and the role of chance The centers and embryologists included were representative of the Italian IVF scenario: oocyte-retrievals per year:711±636,range100–2200; cycles with cleavage-stage embryo-transfer:322±339,0–1300; cycles with blastocyst-stage embryo-transfer:390±403,0–1100; operators per center:5.6±4.0,2–13; senior embryologists’ experience:14.8±7.4yr,7–30; junior embryologists’ experience:2.7±0.6yr,1–3. The intra-center concordance was (i)for blastomeres’ symmetry 82±15% (38–100%), k 0.59±0.27 (0.02–1), (ii)for blastomeres’ fragmentation 88±9% (65–100%), k 0.71±0.2 (0.29–1), (iii)for blastocysts’ expansion 80±16% (48–100%), k 0.66±0.27 (0.19–1), (iv)for inner-cell-mass quality 73±16% (35–95%), k 0.58±0.24 (0.07–0.92), (v)for trophectoderm quality 71±19% (38–95%), k 0.54±0.32 (0.01–0.97). Linear regressions showed no association of centers’ and embryologists’ characteristics with all k-values. Among clinics with the highest mean number of cycles per year and intra-center concordance, we selected 6 experts for the interactive-training. We then calculated the inter-center concordance as the agreement rate between senior embryologists and the experts for phase-I and phase-III: (i)for blastomeres’ symmetry 67±15% (30–85%) and 73±17% (15–90%;Wilcoxon-signed-ranks-test=0.06), k 0.33±0.22 (–0.29–0.58) and 0.42±0.33 (–0.56–0.77); (ii)for blastomeres’ fragmentation 81±17% (23–95%) and 83±14% (50–95%;Wilcoxon-signed-ranks-test=0.8), k 0.54±0.22 (–0.05–0.84) and 0.55±0.22 (0.17–0.81); (iii)for blastocysts’ expansion 59±16% (35–85%) and 67±17% (23–90%;Wilcoxon-signed-ranks-test=0.04), k 0.35±0.20 (0.06–0.73) and 0.44±0.22 (–0.10–0.7); (iv)for inner-cell-mass quality 60±14% (33–80%) and 69±11% (48–85%;Wilcoxon-signed-ranks-test=0.02), k 0.40±0.20 (0.01–0.69) and 0.51±0.18 (0.18–0.77); (v)for trophectoderm quality 55±12% (23–70%) and 63±10% 48–78%;Wilcoxon-signed-ranks-test&amp;lt;0.01), k 0.29±0.15 (–0.08–0.52) and 0.42±0.15 (0.21–0.66). Limitations, reasons for caution Only 28% (N = 18/65) of the Italian IVF centers invited to participate responded to the survey. The conventional adoption of grading schemes other than Istanbul-Consensus by 50% of the embryologists might have biased their evaluation. The experts were not fully-concordant in grading 13.8% of the embryos (N = 22/160), which required active discussions. Wider implications of the findings: Blastocyst-grading concordance was significantly improved after the training phase. Therefore, interactive consensus meetings and training platforms are keenly needed to standardize this practice across the centers. The “avant-garde” of artificial intelligence applied to embryo image analysis might help overcoming this issue in the future. Trial registration number N.A.

P–203 Applying artificial intelligence for ploidy prediction: The concentration of IL–6 in spent culture medium, blastocyst morphological grade and embryo morphokinetics as variables under consideration

Abstract Study question Would it be possible to predict embryo ploidy by taking into account conventional morphological and morphokinetic parameters together with IL–6 concentration in spent culture medium? Summary answer Our artificial neural network (ANN) trained with blastocyst morphology, embryo morphokinetics and IL–6 concentration distinguished between euploid/aneuploid embryos in 65% of the testing dataset. What is known already The analysis of spent embryo culture media represents the protein and metabolic state of the embryo and could be a non-invasive method of obtaining information about embryo quality. The impact of the presence/absence of several proteins in embryo culture samples over clinical results has been widely studied. The IL–6 is one of the most mentioned protein for its effect on embryo development, implantation and likelihood of achieving a live birth. In this initial attempt, we examined the predictive value for euploidy of a model that took into account the concentration of IL–6 in the spent culture medium. Study design, size, duration This prospective study included 319 embryos with PGT-A results. Out of the total, 127 were euploid and 192 aneuploid embryos. Concentration of IL–6 in spent embryo culture media (collected on the day of trophectoderm biopsy-fifth/sixth day of development), morphokinetic parameters (division time to 2 cells-t2; to 3 cells-t3, to 4 cells-t4; to 5 cells-t5 and time of blastocyst formation-tB) and blastocyst morphological grade (according to ASEBIR criteria) were considered to predict the embryo ploidy. Participants/materials, setting, methods Embryos were cultured in EmbryoScope. The chromosome analysis was performed using next-generation sequence technology. The concentration of IL–6 was measured in 20µL of spent embryo culture media with ELISA kits. Morphokinetic parameters were automatically annotated and the blastocyst morphology was evaluated by senior embryologists based on blastocele expansion, inner cell mass and trophectoderm quality. All the embryos were divided into 70% for training, 15% for validating and 15% for testing our ANN model with MatLab®. Main results and the role of chance The general description for the euploid embryo population was the following: 2% of the embryos were graded as A, 71% were graded as B and 28% were graded as C; the means and standard deviations were 25.32±2.97 hours (h) for t2, 35.33±5.15h for t3, 37.30±5.43h for t4, 48.24±6.62h for t5 and 103.93±12.8h for tB; and the average of IL–6 concentration was 1.51±0.70 pg/ml. The general description for the aneuploid embryo population was the following: 1% of the embryos were graded as A, 48% were graded as B and 51% were graded as C; the means and standard deviations were 26.13±3.51h for t2, 36.70±4.29h for t3, 38.20±4.24h for t4, 49.86±6.89h for t5 and 107.10±8.29h for tB; and the average of IL–6 concentration was 1.47±0.71 pg/ml. Our ANN model showed a higher general success rate as we increased the variables considered in the final prediction of euploid embryos. The accuracy, sensitivity and specificity for the testing dataset were: 0.60, 0.12 and 0.87 with morphokinetic parameters; 0.63, 0.24 and 0.93 with morphokinetics and IL–6 concentration; and 0.65, 0.16 and 0.96 with morphokinetics, IL–6 concentration and blastocyst morphological grade. Limitations, reasons for caution The low sensitivity and high specificity achieved in our models indicated that they were more capable of detecting aneuploid than euploid embryos. As this was a preliminary study, the small number of embryos included in the test (n = 48) was also a limitation. Wider implications of the findings: The results showed that our model tended to classify the embryos as aneuploid. More euploid embryos would be necessary to train our model and achieve better results in the prediction of chromosomally normal embryos. Further studies with large number of embryos and additional variables could improve the non-invasive ploidy prediction. Trial registration number Not applicable

P-203 Applying artificial intelligence for ploidy prediction: The concentration of IL-6 in spent culture medium, blastocyst morphological grade and embryo morphokinetics as variables under consideration

Abstract Study question Would it be possible to predict embryo ploidy by taking into account conventional morphological and morphokinetic parameters together with IL-6 concentration in spent culture medium? Summary answer Our artificial neural network (ANN) trained with blastocyst morphology, embryo morphokinetics and IL-6 concentration distinguished between euploid/aneuploid embryos in 65% of the testing dataset. What is known already The analysis of spent embryo culture media represents the protein and metabolic state of the embryo and could be a non-invasive method of obtaining information about embryo quality. The impact of the presence/absence of several proteins in embryo culture samples over clinical results has been widely studied. The IL-6 is one of the most mentioned protein for its effect on embryo development, implantation and likelihood of achieving a live birth. In this initial attempt, we examined the predictive value for euploidy of a model that took into account the concentration of IL-6 in the spent culture medium. Study design, size, duration This prospective study included 319 embryos with PGT-A results. Out of the total, 127 were euploid and 192 aneuploid embryos. Concentration of IL-6 in spent embryo culture media (collected on the day of trophectoderm biopsy-fifth/sixth day of development), morphokinetic parameters (division time to 2 cells-t2; to 3 cells-t3, to 4 cells-t4; to 5 cells-t5 and time of blastocyst formation-tB) and blastocyst morphological grade (according to ASEBIR criteria) were considered to predict the embryo ploidy. Participants/materials, setting, methods Embryos were cultured in EmbryoScope. The chromosome analysis was performed using next-generation sequence technology. The concentration of IL-6 was measured in 20µL of spent embryo culture media with ELISA kits. Morphokinetic parameters were automatically annotated and the blastocyst morphology was evaluated by senior embryologists based on blastocele expansion, inner cell mass and trophectoderm quality. All the embryos were divided into 70% for training, 15% for validating and 15% for testing our ANN model with MatLab®. Main results and the role of chance The general description for the euploid embryo population was the following: 2% of the embryos were graded as A, 71% were graded as B and 28% were graded as C; the means and standard deviations were 25.32±2.97 hours (h) for t2, 35.33±5.15h for t3, 37.30±5.43h for t4, 48.24±6.62h for t5 and 103.93±12.8h for tB; and the average of IL-6 concentration was 1.51±0.70 pg/ml. The general description for the aneuploid embryo population was the following: 1% of the embryos were graded as A, 48% were graded as B and 51% were graded as C; the means and standard deviations were 26.13±3.51h for t2, 36.70±4.29h for t3, 38.20±4.24h for t4, 49.86±6.89h for t5 and 107.10±8.29h for tB; and the average of IL-6 concentration was 1.47±0.71 pg/ml. Our ANN model showed a higher general success rate as we increased the variables considered in the final prediction of euploid embryos. The accuracy, sensitivity and specificity for the testing dataset were: 0.60, 0.12 and 0.87 with morphokinetic parameters; 0.63, 0.24 and 0.93 with morphokinetics and IL-6 concentration; and 0.65, 0.16 and 0.96 with morphokinetics, IL-6 concentration and blastocyst morphological grade. Limitations, reasons for caution The low sensitivity and high specificity achieved in our models indicated that they were more capable of detecting aneuploid than euploid embryos. As this was a preliminary study, the small number of embryos included in the test (n = 48) was also a limitation. Wider implications of the findings The results showed that our model tended to classify the embryos as aneuploid. More euploid embryos would be necessary to train our model and achieve better results in the prediction of chromosomally normal embryos. Further studies with large number of embryos and additional variables could improve the non-invasive ploidy prediction. Trial registration number not applicable

Advanced trophectoderm quality increases the risk of a large for gestational age baby in single frozen-thawed blastocyst transfer cycles.

Does trophectoderm (TE) quality affect birthweight after single frozen-thawed blastocyst transfer? Transfer of single blastocyst with advanced TE quality was associated with higher birthweight and increased risk of a large for gestational age (LGA) baby. Transfer of blastocysts with advanced TE quality results in higher ongoing pregnancy rates and a lower miscarriage risk. However, data on the relationship between TE quality and birthweight are still lacking. This retrospective cohort study at a tertiary-care academic medical center included 1548 singleton babies born from single frozen-thawed blastocyst transfer from January 2011 to June 2019. Babies were grouped into four groups according to embryo expansion (Stages 3, 4, 5 and 6), three groups according to inner cell mass (ICM) quality (A, B and C), and three groups according to TE quality (A, B and C). Main outcomes included absolute birthweight, Z-scores adjusted for gestational age and gender, and adverse neonatal outcomes. Multivariable linear and logistic regression analyses were performed to investigate the association of neonatal outcomes with expansion stage, ICM quality and TE quality. As TE quality decreased, birthweight (3468.10 ± 471.52, 3357.69 ± 522.06, and 3288.79 ± 501.90 for A, B and C, respectively, P = 0.002), Z-scores (0.59 ± 1.07, 0.42 ± 1.04, and 0.27 ± 1.06 for A, B and C, respectively, P = 0.002) and incidence of LGA (28.9%, 19.7% and 17.4% for A, B and C, respectively, P = 0.027) decreased correspondingly. After adjusting for confounders, compared with the Grade A group, blastocysts with TE Grade B (standardized coefficients (β): -127.97 g, 95% CI: -234.46 to -21.47, P = 0.019) and blastocysts with TE grade C (β: -200.27 g, 95% CI: -320.69 to -79.86, P = 0.001) resulted in offspring with lower birthweight. Blastocysts with TE grade C brought babies with lower Z-scores than TE Grade A (β: -0.35, 95% CI: -0.59 to -0.10, P = 0.005). Also, embryos with TE Grade B (adjusted odds ratio (aOR):0.91, 95% CI: 0.84 to 0.99, P = 0.033) and embryos with TE Grade C (aOR : 0.89, 95% CI: 0.81 to 0.98, P = 0.016) had lower chance of leading to a LGA baby than those with TE Grade A. No association between neonatal outcomes with embryo expansion stage and ICM was observed (all P > 0.05). The retrospective design, lack of controlling for several unknown confounders, and inter-observer variation limited this study. The study extends our knowledge of the down-stream effect of TE quality on newborn birthweight and the risk of LGA. This study was funded by National Key R&D Program of China (2018YFC1003000), National Natural Science Foundation of China (81771533 to Y.P.K. and 31200825 to L.S.) and Innovative Research Team of High-level Local Universities in Shanghai (SSMU-ZLCX20180401), Shanghai Sailing Program(21YF1423200) and the Fundamental research program funding of Ninth People's Hospital affiliated to Shanghai Jiao Tong university School of Medicine (JYZZ117). The authors declare no conflict of interest in this present study. N/A.