Morphology Score Research Articles

P-302 Correlation of blastocyst shrinkage-pattern during vitrification and post-warming embryo performance

Abstract Study question Can blastocyst shrinkage-pattern during vitrification be predictive of post-warming survival and reproductive potential? Summary answer Blastocysts assuming a specific shrinkage-pattern during vitrification are liable to lower post-warming morphology and, potentially, degeneration. What is known already Blastocyst vitrification is a highly standardized technique. However, there are still possible factors than can compromise the outcome of the technique which should be minimized. For example, the degree of blastocoel expansion before vitrification may negatively influence embryo survival post-warming. There are studies which demonstrate that expanded blastocysts exhibit lower survival rate because of their higher blastocoel volume and amount of water, which is more prone to the detrimental ice crystal formation during vitrification. Moreover, shrinkage of expanded blastocysts during vitrification differs as some of them shrink in a uniform, spherical way while others shrink in a non-canonical way Study design, size, duration A retrospective study was conducted at IAKENTRO fertility clinic, Thessaloniki. A total of 2167 blastocysts vitrified between November 2018-December 2022, out of which 532 blastocysts warmed during the same period, were included in the present study. No biopsied or PGT-a tested embryos were included. Embryo vitrification was performed following the same protocol during the whole period of the study. For the clinical outcomes, 178 single-embryo transfers and 123 double-embryo transfers were analyzed Participants/materials, setting, methods During blastocyst exposure to the vitrification solution two shrinkage patterns were detected, allocating embryos into Group-1 for blastocysts presenting shrinkage without any detachment of the trophectoderm cells from the zona pellucida, leading to a ZP reforming which eventually resembled a “deflated” ball and into Group-2 for blastocysts that collapsed in a uniform way, having their TE cells fully detached from the ZP, which remained spherical throughout the exposure to the vitrification solution Main results and the role of chance In the total cohort of 2167 assessed blastocysts, 437 were allocated in Group-1 and 1730 in Group-2, which can be translated as 20,2% event occurrence. The Group-1 blastocysts were mostly of expansion degree 4 (76,7%) and degree 3 (23,3%), according to the Gardner system, while no smaller blastocysts were assigned in this group. As primary outcome was examined post-warming embryo survival with blastocysts in Group-1 presenting significantly higher degeneration rate (22/187, 11,7%) respect to blastocysts in Group-2 (7/345, 2%) (χ2 test, p < 0.0001, OR = 5,798, 95%CI = 2,511 to 14,55). Moreover, in order to assess any potential decline in embryo morphology post-warming, we corresponded Gardner’s system evaluation to a numerical score and we calculated the integrity in morphology before and after vitrification. Before vitrification, embryos’ morphology score was similar in both groups (74,8% and 73,1% for Group-1 and Group-2, respectively) while, after warming, Group-1 blastocysts exhibited significantly lower integrity of their morphology (86,52% integrity in score) respect to Group-2 blastocysts (93,82% integrity in score) (t-test, p < 0,0001). However, when analyzing PR and CPR no significant differences were observed between the two groups Limitations, reasons for caution The retrospective aspect of the study is per se a general limitation. Moreover, given that the analysis of the data was not limited to freeze-all cases, the embryos chosen for the fresh embryo-transfers, which means those with the highest implantation potential, were excluded from the study’s cohort Wider implications of the findings Our results show that blastocysts resembling a “deflated” ball during vitrification have compromised post-warming survival. Given that this phenomenon is exclusively observed in expanded blastocysts, whose ZP is thin and TE cells are stretched, identifying the expansion grade over which is happening could be beneficial in clinical practice Trial registration number Not applicable

P-190 Spontaneous blastocyst collapse during pre-vitrification equilibration is related to a lower live birth rate: a prospective cohort study

Abstract Study question This report provides updated data from a study investigating the association between the spontaneous collapse patterns of blastocysts during pre-vitrification equilibration and pregnancy success. Summary answer Live birth rates were lower for completely collapsed blastocysts, suggesting the possibility for including blastocyst collapse pattern as a criterion for selecting embryos for transfer. What is known already Previous time-lapse studies have found that blastocysts exhibiting strong contraction during culture have a low probability of hatching in animals and implanting in humans, suggesting blastocyst contraction has a negative impact on reproductive outcomes. Conversely, artificial shrinkage of human blastocysts before vitrification is considered to improve embryo survival and/or pregnancy rates by promoting cryoprotectant permeation inside the blastocoel. However, shrinkage is not induced in some vitrification protocols, including ours, as the viability is high; this demonstrates the potential of existing protocols to achieve sufficient cryoprotectant permeation. Moreover, the Alpha consensus meeting on cryopreservation did not issue recommendations regarding artificial shrinkage. Study design, size, duration This study included 798 patients who were undergoing their first autologous IVF/ICSI cycle followed by a freeze-all strategy, including blastocyst vitrification, at our clinic between June 2018 and November 2021. For patients with multiple vitrified blastocysts, embryos for transfer were selected hierarchically based only on morphological scoring during culture. To reduce bias, only data from a single blastocyst (day 5, Gardner score 4, excluding CC) transfer from each patient’s first warmed cycle was analyzed. Participants/materials, setting, methods Blastocysts were vitrified-warmed in in-house-prepared solutions using Rapid-i carriers. Prior to vitrification, intact blastocysts were equilibrated in 10% ethylene glycol (15 min, 37 °C). Their spontaneous collapse patterns were assessed under an inverted microscope before they were vitrified in 15% ethylene glycol + 15% dimethylsulfoxide + 0.5 M sucrose. Blastocyst collapse was defined as the separation of the trophectoderm cells from the zona pellucida. Collapsed blastocysts with/without a blastocoel cavity were defined as partially/completely collapsed blastocysts. Main results and the role of chance Non-collapsed (Nc), partially collapsed (Pc), and completely collapsed (Cc) blastocyst transfers (embryo survival rate, 99.6%) constituted 27.1%, 54.6%, and 18.3% of the 798 cycles, respectively. Live birth rates differed significantly between groups (Nc, 54.2%; Pc, 46.6%; Cc, 34.2%; P < 0.001). Regarding perinatal outcomes of 366 singletons, the female rate tended to differ between groups (Nc, 35.3%; Pc, 44.3%; Cc, 53.1%; P = 0.084). Significant differences in maternal age (years: Nc, 34.6 ± 4.0; Pc, 35.4 ± 3.7; Cc, 36.4 ± 4.0; P < 0.001), MII oocyte number (Nc, 13.0 ± 6.2; Pc, 12.0 ± 6.6; Cc, 10.8 ± 6.9; P = 0.009), blastocyst diameter (μM: Nc, 184 ± 18; Pc, 181 ± 20; Cc, 173 ± 23; P < 0.001), and good-quality (4AA/4AB/4BA) blastocyst percentage (Nc, 70.4%; Pc, 61.2%; Cc, 47.9%; P < 0.001) were found between groups. Upon logistic regression analysis including these potential confounders, only maternal age (odds ratio (OR), 0.89; 95% confidence interval (CI), 0.86–0.93; P < 0.001) and collapse pattern (Cc/Nc: OR, 0.59; 95% CI, 0.37–0.93; P = 0.023) significantly impacted the live birth rate, whereas blastocyst morphology (good/not: OR, 1.37; 95% CI, 0.97–1.94; P = 0.069) tended to impact it. Limitations, reasons for caution The cryoprotectant concentration of our pre-vitrification equilibration solution is relatively low. Thus, caution is required when assessing the collapse pattern, as the solution osmolarity can influence blastocyst shrinkage. Follow-up studies with more participants are warranted to confirm these results and the health of the children born after vitrified-warmed embryo transfers. Wider implications of the findings A negative relationship was found between spontaneous blastocyst collapse during pre-vitrification equilibration and live birth rate. Studying blastocyst collapse pattern may assist selection of the most viable blastocysts after vitrification in ethylene glycol and dimethylsulfoxide—a widely used permeable cryoprotectant combination—thus, increasing IVF success rates. Trial registration number Not applicable

P-093 Single sperm morphokinetic variables during ICSI at the time of sperm aspiration into the microneedle

Abstract Study question Is it possible to classify sperm morphology as normal or abnormal based on their kinetic characteristics? Summary answer Using different classifiers, it was found that balanced dataset the classification scores of individual sperm morphology can be optimized during their motility just before ICSI What is known already Sperm selection plays a crucial role during ICSI. This selection by the embryologists is accompanied by two factors: motility parameters of the sperm to be injected, as well as the morphology. Motility affects morphological decision-making which may be subjective due to spermatozoa not being in a narrow vertical space in a PVP droplet, i.e. counting chamber. Machine/Deep Learning models -as classifiers- are gaining popularity in IVF as it could minimize subjectivity in gamete selection. SiD software is an algorithm that from a group of spermatozoa characteristics can support the selection of a single sperm during real time ICSI. Study design, size, duration In this prospective study 1699 individual spermatozoa were video-recorded (resolution of 200 X 200 pix.) during sperm selection in a 7%PVP solution. Motility variables (VSL, VCL, LIN, VAP, ALH, WOB, STR, MAD) were obtained from each video using the software SiD. Each sperm was classified as normal or abnormal. Based on motility variables, different classification models were used to make a morphokinetic association of the variables with the classification of each sperm. Participants/materials, setting, methods 1699 individual spermatozoa were classified and labeled by three senior embryologists into two categories: normal or anormal. To belong to any category, at least two embryologists had to agree. A normalization was applied to the motility data obtained with SiD. Machine learning classification models were applied. The three classification models with the best results were selected to optimize the hyper-parameters and improve their performance. Main results and the role of chance A set of motility variables were obtained using the software SiD1 (IVF2.0 Ltd., UK) these were used as features for each of the sperm samples and were tagged either as normal or abnormal having a total of 257 normal samples and 1442 abnormal samples. Different classification algorithms were used to perform sperm classification as normal and abnormal. Then hyperparameter tuning algorithms such as GridSearch were used to compute the optimum values, finding KNN algorithm to achieve the best results to classify normal and abnormal spermatozoa with 80% accuracy. Given the nature of the unbalanced dataset, the F1 score was calculated, achieving 0.77, other algorithms such as Decision-Tree and Random-Forest were used achieving similar results in the F1 score. We will continue growing the dataset until it is balanced and run the same algorithms expecting the performance to improve. Limitations, reasons for caution Each clinic's laboratory setup, including the camera used to record the ICSI procedure, is a clinic-specific configuration. Sperm abnormality detection is sensitive to camera resolution, showing the classifiers are resolution-dependent observers. To replicate these results, it is important to consider the quality of the camera. Wider implications of the findings Using the selected kinematics features, it is possible to classify individual spermatozoa during motility as having normal or abnormal morphology. Results may improve using standard morphological examination of each population and having more normal sperm samples. Trial registration number Not applicable

P-148 The importance of filopodia during early human embryonic development

Abstract Study question The aim of this study was to examine the relation between the presence of filopodia and the embryo viability in human in vitro fertilised embryos. Summary answer The presence of filopodia related to a faster blastocyst development, a better blastocyst quality and higher morphokinetic scores which indicates a higher embryo viability. What is known already Filopodia are involved in numerous cellular processes, for example cell migration, neuronal extension growth and during embryonic development. There is no uniform name to describe these cytoplasmic string structures in the literature. Beside the frequent presence in different cell functions, we have limited information about its function in human embryos. In the early 2000s, the occurrence of filopodia or cytoplasmic strings at the blastocyst stage was classified as a negative sign of embryo viability, but more recently, it has been described as a positive feature, however, the importance and clinical significance of this structure is still controversial. Study design, size, duration Evaluation of the morphokinetic data on the development of 208 embryos from 78 IVF cycles. Examination was carried out at the Division of Assisted Reproduction, Department of Obstetrics and Gynaecology, Semmelweis University, Budapest between December 2020 and March 2021. Participants/materials, setting, methods Embryos from both conventional IVF (cIVF) and intracytoplasmic sperm injection (ICSI) treatments were analysed and evaluated. The dynamics of embryo development, embryo morphology and morphokinetic scores generated by time-lapse system was compared between the embryos with filopodia (FP+) and embryos without filopodia- (FP-) groups. Main results and the role of chance 81.2% of the embryos had filopodia in blastocyst stage. Embryos created by cIVF had 77%, while embryos fertilised by ICSI had 86% of filopodia presence (p = 0.08). FP+ embryos developed in a greater number into a higher quality blastocyst (52.1% vs 20.5%, p = 0,02), their KIDScore was higher (6.1 ± 2.1 vs 4,7 ± 2,07, p < 0.001) and they showed a tendency of higher implantation rate (39.7% vs 14.3% p = 0.16) than the FP- group. The dynamic of the early embryo development was similar between the two groups, however, FP+ embryos reached blastocyst stage significantly earlier (tB: 103.9 hours vs. tB: 107.6 hours; p = 0.007). Based on our results, there was a higher number of embryos with filopodia than without, and the presence of it is not related to the fertilisation method. These embryos developed faster into a blastocyst stage, their morphokinetic parameters were better. KIDScore and iDAScore embryo evaluation systems also recommend in a greater number the FP+ blastocysts for ET. We could also observe vesicle like transport along the filopodia, which leads us to believe that there might be a molecular communication between the differentiating cell populations. Limitations, reasons for caution Due to the low number of cases, the higher implantation rate is not significant. Wider implications of the findings The examination of this feature improves our knowledge of the early embryonic development and may help us to make decisions about the embryos with higher implantation potential. Trial registration number Not applicable

O-158 Prediction of implantation of vitrified-warmed blastocysts using a deep learning algorithm on a single post-warming image of each embryo

Abstract Study question Can a deep learning algorithm determine the implantation likelihood of vitrified blastocysts from single time-point post-warming time-lapse images obtained from EmbryoScope? Summary answer A significant positive correlation was found between the score given by the deep learning algorithm and the implantation rate of the frozen blastocysts analyzed. What is known already Despite the safety and success of the vitrification technique, many of the best morphologically classified embryos prior to vitrification do not maintain their characteristics when returned to room temperature. Since artificial intelligence (AI) has been shown to be useful in assessing the quality of fresh embryos at different stages, it could also be used to predict the outcome of frozen embryo transfers. This is the first attempt to distinguish vitrified blastocysts that will implant from those that will not by applying deep learning on a single image taken in the period between warming and transfer. Study design, size, duration This retrospective single-centre study included 689 blastocysts with known implantation data. All of them were vitrified and warmed using the Cryotop method (Kitazato, Biopharma, Japan). Warmed embryos were assessed by experienced embryologists according to the ASEBIR morphological scoring system and the degree of expansion. Immediately after warming, blastocysts were placed in EmbryoScope (Vitrolife, Denmark) time-lapse incubators for 2-5 hours to let them re-expand until embryo transfer. Participants/materials, setting, methods A pre-transfer embryo image was provided to the algorithm (Life Whisperer Viability), which returned scores from 0 to 10. Data were analyzed by chi-square test or one-way ANOVA. Multivariate logistic regression analysis was performed including embryo score, oocyte origin (donated vs. autologous), oocyte age, patient age, oocyte handling (fresh vs. vitrified eggs), and day of vitrification (5 vs. 6). The area under the receiver operating characteristic (ROC) curve (AUC) was used to calculate evaluation performance. Main results and the role of chance There was a significant difference between the scores given to vitrified implanted and non-implanted embryos. The mean value was 4.98 [95% CI 4.70-5.26]* for non-implanted embryos and 6.16 [95% CI 5.84-6.48]* for implanted embryos. When dividing the embryo score by quartiles, the implantation rate for each quartile was 27.7% for Q1, 33.7% for Q2 (Odds ratio (OR)=1.31 [0.82–2.09]), 44.8% for Q3 (OR = 2.05 [1.30–3.22]*) and 48.3% for Q4 (OR = 2.46 [1.56–3.87]*). The contribution of each increased unit of the algorithm score to the implantation (OR = 1.16 [95% CI: 1.09–1.23]*) was statistically significant. The deep learning algorithm score successfully predicted implantation with an AUC of 0.65 [95% CI: 0.60–0.69]*. Moreover, the algorithm performed better than the ASEBIR morphological classification, which achieved an AUC of 0,62 [95% CI: 0.58–0.66] in predicting implantation. *P < 0.001. Limitations, reasons for caution Our clinic was not involved in the development of the algorithm. Furthermore, it was designed to assess fresh embryos prior to vitrification but not thawed ones, so this study should be considered an external trial. A post-warming full video analysis by AI could possibly provide additional information. Wider implications of the findings The use of predictive models on vitrified cycles may help to select the best frozen embryo for transfer and predict which embryos will result in implantation failure early enough to thaw another one with a better chance of success. It also indicates that AI can generalize to thawed embryo assessment. Trial registration number -

P-208 Culture media affect gender after IVF treatment: A detailed analysis of explanatory variables

Abstract Study question Does embryo culture media affect gender at birth and why? Summary answer More male children were born after culture in the Sage 1-Step media compared to the G-TL media. What is known already ART increases the proportion of male children at birth. Laboratory techniques and culture conditions is known to impact secondary sex ratio (SSR). Physiological development differs in male and female embryos, leading to different metabolic requirements at the preimplantation stage. The composition of commercial culture medias varies and thus, the different medias may have different impact on embryo development and quality according to gender. This, in turn, may influence SSR, as embryos are selected based on morphological scoring systems and developmental speed. Study design, size, duration The study was designed as a retrospective registry-based study with all data collected at a single clinic. All embryos resulting in live birth from frozen and fresh single blastocyst transfers, in the period from 1. January 2017 to 31. December 2020 were included. Donor embryos and embryos cultured in more than one culture media were excluded. A total of 1371 SET embryos were included. Participants/materials, setting, methods Embryos were cultured in either the G-TL media, Vitrolife (n = 686) or the Sage 1-Step media, Origio (n = 685). All embryos were monitored for 5-6 days in a time-lapse incubator, Vitrolife, before single blastocyst transfer. Multivariate logistic analysis of cycle and embryo characteristics was performed to identify all factors associated to sex. The association between culture media and embryo morphokinetic according to sex was evaluated using a mixed model analysis. Main results and the role of chance Significantly more male singletons were born after culture in 1-Step media compared to G-TL media (risk ratio (RR) 1,1 95% CI [1,0, 1,3], P = 0,01). The female/male ratio was 42,3/57,7% in 1-Step media and 48,5/51,5% in G-TL media. The multivariate regression analysis displayed a higher chance of a male child with higher expansion grade (grade 5) (RR, 1,2 95% CI [1,0, 1,4] P = 0,04), lower inner cell mass grade (grade B) (RR 1,13 95% CI [1,0, 1,3] P = 0,02) and second embryo transfer (RR 1,2 95% CI [1,0, 1,3] P = 0,03). Trophectoderm (TE) grade B reduced the probability of male child compared with TE grade A (RR 0,8 95% CI [0,8, 0,9] P > 0,05). Male embryos developed significantly faster in the 1-Step media compared to the G-TL media for the stages of; blastocyst (tB) (-1,1 hours 95% CI [-2,1, -0,1]), expanded blastocyst (tEB) (-1,3 hours 95% CI [-2,3, -0,3]) and hatched blastocyst (tH) (-1,7 hours 95% CI [-3,0, -0,5]). Timing of development were the same for female embryos in the G-TL media compared to the 1-Step media. There was no significant difference in timing of development between female and male embryos in identical culture media. Limitations, reasons for caution This study was a retrospective study with data collection from only a single clinic, and as such in risk of confounding. Apart from culture media, culture conditions were similar during the two time periods. Paternal factors and type of infertility was not included. Wider implications of the findings Our observations suggest that culture media impact male embryo quality selectively, thus favoring selection of male embryos. As the impact appears to vary between different culture media, and has a measurable impact on gender after birth, the influence of culture media on embryo quality deserves more attention. Trial registration number not applicable

P-264 Correlations between the artificial intelligence scoring system (iDAScore v1.0) and live birth outcomes in preimplantation genetic testing for aneuploidy cycles

Abstract Study question Is the iDAScore v1.0 score correlated with pregnancy outcomes in single embryo transfer (SET) cycles following preimplantation genetic testing for aneuploidy (PGT-A)? Summary answer We demonstrated that elevated iDAScore v1.0 scores are positively correlated with the probabilities of pregnancy and live birth (LB) in SETs following PGT-A. What is known already To select the blastocysts with high development potential in advance, several groups have proposed the implementation of artificial intelligence (AI)-based software is capable of predicting implantation in women undergoing in vitro fertilization (IVF). It has also been known that using high resolution next generation sequencing preimplantation genetic screening (hr-NGS) for PGT-A can enable the exclusion of aneuploid embryos before embryo transfers and thus improve pregnancy outcomes of SETs. However, the capacity of AI-based assessments has remained unclear in IVF cycles with PGT-A. Study design, size, duration This retrospective study, approved by the Institutional Review Board of Chung Sun Medical University, was performed to assess the dataset of 317 SET cycles after PGT-A from January 2017 to September 2019. A single euploid or mosaic blastocyst with an AI score calculated using the iDAScore software (v1.0) was selected for frozen embryo transfer according to the given priority of blastocyst morphology. The iDAScore groups were categorized by quartiles of AI scores. Participants/materials, setting, methods Embryos were cultured in a time-lapse incubator and qualified blastocysts underwent next generation sequencing (NGS)-based PGT-A. The confounding factors associated with LB were evaluated using logistic regression analysis in generalized estimating equations (GEEs). The differences between iDAScore groups were assessed using the Mann–Whitney U test or Fisher’s exact test, as applicable. The receiver operating characteristic (ROC) curve analysis was used to estimate the predictive powers. A P value < 0.05 was considered statistically significant. Main results and the role of chance The results revealed the patient age, anti-Müllerian hormone, body mass index, oocyte sources (autologous or donor), PGT-A results (euploidy, low-level mosaicism, or high-level mosaicism), aberrant chromosome types (none, whole chromosome, segmental chromosome, or whole with segmental chromosome), and aberrant chromosome sites (0, 1, 2, or > 2) were not correlated with LB. However, embryo biopsy days (day 5 vs. day 6, OR = 2.271, 95% CI = 1.534–14.824), blastocyst morphology scores (OR = 1.185, 95% CI = 1.034–1.359), and AI scores (OR = 1.640, 95% CI = 1.231–2.183) were significantly correlated with LB. The AI scores were then divided into quartiles (group 1: 5.0–7.9; group 2: 8.0–8.5; group 3: 8.6–8.9; and group 4: 9.0–9.5). The ongoing pregnancy (29.1% vs. 54.2%–57.5%) and live birth (27.8% vs. 52.8%–56.3%) rates of group 1 were significantly lower than other groups. The blastocyst morphology score (5.3 ± 1.2), and KIDScore D5 scores (v1 = 3.0 ± 1.7; v3 = 4.6 ± 1.5) were also lowest in group 1. The ROC curve analysis confirmed a significant but limited LB prediction capability for iDAScore v1.0 (AUC = 0.6), which was similar to the KIDScore D5 (AUC = 0.61–0.62). Limitations, reasons for caution Because of the retrospective nature, the major limitation was the lack of randomization, which may present a risk of selection bias. According to the criteria of blastocyst selection for SETs, the embryos with low (< 5.0) or high (> 9.5) AI scores were not present in this dataset. Wider implications of the findings Although prediction capability of iDAScore remains perfectible, the AI score is still significantly associated with LB probability. Euploid or mosaic blastocysts with low AI scores (<8.0) processed a decreased LB rate, indicating the potential of annotation-free iDAScore system as a decision support tool for deselecting embryos with poor post-implantation development. Trial registration number not applicable

O-121 Temporal development of scores from a time-lapse based artificial intelligence provides no additional benefit compared to the latest score

Abstract Study question Does the temporal development of scores from an artificial intelligence (AI) based embryo selection model provide additional predictive power compared to the latest score? Summary answer Earlier predictions provide no additional predictive power given the latest prediction. Knowing the development of AI scores is thus not beneficial for embryo selection. What is known already Traditionally, embryo grading takes into consideration the final morphology score together with the history of embryo development events. Recent publications have shown that time-lapse based AI models trained on clinical outcomes can automatically rank embryos by the likelihood of implantation that equal or surpass traditional methods, without the necessity for manual evaluation. AI-based methods based on time-lapse data may account for the impact of the full temporal development on likelihood of implantation. In this study, we examine if the addition of previous scores adds to the performance of an embryo selection model. Study design, size, duration A retrospective multicenter study of transferred embryos (n = 2422) with known implantation data (KID) between 2012-2020 from 21 international clinics. Embryos were cultured in EmbryoScope time-lapse incubators for at least 5 days. Both single and multi-embryo transfers in fresh and warmed cycles were included. Embryos were classified as positive (KIDp) or negative (KIDn) by the presence of fetal heartbeat. Implantation likelihood was evaluated at cleavage- and blastocyst stages by a 3D CNN model; iDAScore v2.0. Participants/materials, setting, methods A kernel-based conditional independence test was used to evaluate if a score at x hours post insemination (hpi) provide extra information compared to a score at y hpi. The test estimates the likelihood that a prediction after x hpi does not provide any additional information with regards to predicting KIDp when a prediction after y hpi is available. These likelihoods (p-values) were Bonferroni corrected to adjust for multiple comparisons. Main results and the role of chance KIDp predictions were computed for all embryos at 40, 44, 64, 68, 112, and 116 hpi corresponding to early and median time of transfer for day 2, 3, and 5. For all time points, the conditional independence test showed that there was no added information from knowing predictions at an earlier time point (p > 0.21). Similarly, it was tested if a known later prediction will improve model performance for an earlier prediction. This was always the case (p < 0.01) except for predictions close in time (68 vs 64 hpi; p = 1.0) and (44 vs 40 hpi; p = 1.0). This may reflect that there was little additional embryo development information during a 4-hour period at this development stage. The above shows that knowledge of the temporal score development does not improve model performance compared to using latest available score. This means that an AI model based on time-lapse videos likely considers earlier embryo development events in the context of the entire development history. Limitations, reasons for caution This study is a retrospective study that only looks at AI based on time-lapse videos. There is a potential bias as the embryos were likely of higher quality having been chosen for transfer. It is possible that the results do not generalize to simpler non-3D CNNs. Wider implications of the findings Knowing the temporal score development can introduce subjectivity that might lower implantation rates as the earlier scores were shown to not provide any significant prediction improvement. Trial registration number not applicable

P-262 Comparative ability of iDAScore and embryo morphology grade to predict clinical pregnancy: Retrospective cohort study of 1510 fresh and frozen elective single embryo transfers

Abstract Study question Can iDAScore predict ongoing clinical pregnancy (CP) with a sensitivity and specificity equivalent to that associated with manual morphology assessment and grading? Summary answer The fully automated iDAScore was able to predict CP with equivalent performance to manual morphology assessment and grading in this retrospective cohort study. What is known already iDAScore is an artificial intelligence (AI) based algorithm developed by applying machine learning to morphokinetic time laps (TL) image data of embryos with a known treatment outcome. Embryos are automatically assessed on day 5 of culture and ranked according iDAScore, ranging from 1 to 9.9. Embryos may be prioritised for transfer based on highest sore. Along with other published AI algorithms, iDAScore has been proposed to optimise the chance of CP following ET by improving objectivity of embryo assessment compared with manual scoring systems. The fully autonomous assessment of embryos by AI algorithms has beneficial implications for laboratory workload. Study design, size, duration Retrospective audit of 787 fresh and 723 frozen single embryo transfer cycles which took place from April 2019 to September 2022. All recipient, surrogacy, warmed oocyte, embryo biopsy, cleavage stage and slow thaw frozen ET cycles were excluded. Participants/materials, setting, methods Selection for transfer was based on blastocyst morphology grade. iDAScores were obtained retrospectively. The area under the receiver operator characteristic (AUROC) curve and sensitivity and specificity for CP prediction of both iDAScore and blastocyst morphology grade was compared overall and in two stratified analyses. The first assessed fresh and frozen cycles separately, the second compared their performance in female age groups of ≤ 35 and >35 years. CP was defined by ultrasound detection of foetal heartbeat. Main results and the role of chance The mean grading of blastocysts by iDA was 8.34 ± 1.4, with a strong correlation to classic morphological scoring (r = 0.69, P < 0.001). The clinical pregnancy rate for fresh and frozen embryo transfer was 31.0% (95%CI 27.9-34.3) and 44.0% (95%CI 40.8-48.0) respectively. iDA score was positively associated with clinical pregnancy rates in both fresh (adjOR 1.69, 95%CI 1.44-1.99) and frozen embryo transfers (adjOR 1.45, 95%CI 1.26-1.67), independent of maternal age. There was no difference in the AUROC for iDA (AUC 0.64, 95%CI 0.62-0.67) versus conventional morphology (AUROC 0.63, 95CI 0.61-0.66) when all eSETs were considered, or when fresh eSETS (AUROC for fresh iDA 0.66 95%CI 0.62-0.70 vs morphology 0.65 95%CI 0.62-0.69) or frozen iDA 0.63 95%CI 0.59-0.67 vs morphology 0.61 95%CI 0.57-0.64) eSETS were considered separately. The iDA score exhibited slightly better performance in the age stratified analyses, with a higher AUROC in women >35 years; iDA 0.68 95%CI 0.64-0.71 vs morphology 0.64 95%CI 0.60-0.67, p = 0.021) but no difference was observed in younger women. This age difference in iDA performance was primarily driven by frozen embryo transfers (p = 0.002). For women >35years an iDA score of 8.75 was associated with an AUROC of 0.63 and 67% sensitivity and 60% specificity for prediction of clinical pregnancy. Limitations, reasons for caution This was a retrospective single centre study and the performance of iDA needs to be confirmed prospectively in a multi-centre trial. That selection of embryos for transfer was based on morphology, may have contributed to overestimation of model performance. Wider implications of the findings The application of AI based embryo ranking algorithms to embryo selection for transfer, has the potential to maintain clinical pregnancy rates while reducing the time burden associated with conventional morphology / morphokinetic assessments. Trial registration number Nil

Toxic Effects of Gemcitabine and Paclitaxel Combination: Chemotherapy Drugs Exposure in Zebrafish.

Pharmaceuticals are widely recognized as potentially hazardous to aquatic ecosystems. In the last two decades, the constant intake of biologically active chemicals used in human healthcare has been related to the growing release of these agents into natural environments. As reported by several studies, various pharmaceuticals have been detected, mainly in surface water (seas, lakes, and rivers), but also in groundwater and drinking water. Moreover, these contaminants and their metabolites can show biological activity even at very low concentrations. This study aimed to evaluate the developmental toxicity of exposure to the chemotherapy drugs gemcitabine and paclitaxel in aquatic environments. Zebrafish (Danio rerio) embryos were exposed to doses of gemcitabine 15 μM in combination with paclitaxel 1 μM from 0 to 96 h post-fertilization (hpf) using a fish embryo toxicity test (FET). This study highlights that both gemcitabine and paclitaxel exposure at single non-toxic concentrations affected survival and hatching rate, morphology score, and body length after exposure in combination. Additionally, exposure significantly disturbed the antioxidant defense system and increased ROS in zebrafish larvae. Gemcitabine and paclitaxel exposure caused changes in the expression of inflammation-related, endoplasmic reticulum stress-related (ERS), and autophagy-related genes. Taken together, our findings underline that gemcitabine and paclitaxel increase developmental toxicity in zebrafish embryos in a time-dependent manner.

Characteristics of research-focused human islet preparations from organ donors with type 2 diabetes

ABSTRACT Type 2 diabetes mellitus (T2D) affects 463 million individuals worldwide. β-cell dysfunction and relatively inadequate β-cell mass has been implicated in the pathogenesis of T2D. Primary human islets from T2D patients can reveal the islet dysfunction and the underlying mechanisms and thus have become valued resources for diabetes research. Our center (Human Islet Resource Center, China) has prepared a number of batches of human islets from T2D organ donors. The present study aims to characterize islet isolation processes, islet yields, and qualities of T2D pancreases by comparing with non-diabetic (ND) ones. Overall, 24 T2D and 80 ND pancreases were obtained with informed research consents. The digestion time, islet purity, yield, size distribution, islet morphology score, viability, and function in each islet preparation were analyzed. We found that at digestion stage, T2D pancreases need significantly longer digestion duration and have worse digestion rates and lower gross islet yields. At purification stage, T2D pancreases have poorer purity, purification rate, morphology score, and islet yields after purification. Functional evaluation by GSI assay showed that the human T2D islets have significantly lower glucose stimulated insulin secretion ability. In conclusion, the features of longer digestion duration, lower yields and quality, and impaired insulin secretion in T2D group are consistent with the pathological condition of this disease. Both islet yields and islet function evaluation results did not support human T2D islets as clinical transplantation resources. However, they could serve as good research models for T2D disease studies and promote the advancement of diabetes research.

Structural changes in lung morphology detected by MRI after modulating therapy with elexacaftor/tezacaftor/ivacaftor in adolescent and adult patients with cystic fibrosis

IntroductionElexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) improves CFTR function in cystic fibrosis (CF) patients homozygous or heterozygous for F508del mutation. The aim of the study was to evaluate the response to ELX/TEZ/IVA treatment both clinically and morphologically in terms of bronchiectasis, bronchial wall thickening, mucus plugging, abscess and consolidations. MethodsWe retrospectively collected data from CF patients followed at Parma CF Centre (Italy) treated by ELX/TEZ/IVA between March and November 2021. Post-treatment changes in respiratory function, quality of life, sweat chloride concentration, body mass index, pulmonary exacerbations and lung structure by chest magnetic resonance imaging (MRI) were assessed. T2-and T1-weighted sequences were acquired with a 20 min-long scanning protocol on a 1.5T MRI scanner (Philips Ingenia) without administration of intravenous contrast media. Results19 patients (32.5 ± 10.2 years) were included in the study. After 6 months of treatment with ELX/TEZ/IVA, MRI showed significant improvements in the morphological score (p < 0.001), with a reduction in bronchial wall thickening (p < 0.001) and mucus plugging (p 0.01). Respiratory function showed significant improvement in predicted FEV1% (58.5 ± 17.5 vs 71.4 ± 20.1, p < 0.001), FVC% (79.0 ± 11.1 vs 88.3 ± 14.4, p < 0.001), FEV1/FVC (0.61 ± 0.16 vs 0.67 ± 0.15, <0.001) and LCI2.5% (17.8 ± 4.3 vs 15.8 ± 4.1 p < 0.005). Significant improvement was found in body mass index (20.6 ± 2.7 vs 21.9 ± 2.4, p < 0.001), pulmonary exacerbations (2.3 ± 1.3 vs 1.4 ± 1.3 p 0.018) and sweat chloride concentration (96.5 ± 36.6 vs 41.1 ± 16.9, p < 0.001). ConclusionsOur study confirms the efficacy of ELX/TEZ/IVA in CF patients not only from a clinical point of view but also in terms of morphological changes of the lungs.

Benchmarking of early ontogeny parameters as predictors of the first-feeding larvae vitality in spotted rose snapper Lutjanus guttatus

A common problem in marine fish hatcheries is the lack of functional embryonic parameters reliably predicting favorable larval outcomes. Embryos characterized as high-quality by typical assessment methods frequently result in weak larvae with high mortality during the endo-exotrophic transition. Thus, because there is no universal consensus on what parameters are most associated with successful larval survivability, this study aims to determine the most reliable, practical, and real-time predictor of vitality at the earliest developmental stage for first-feeding larvae of L. guttatus. From 18 mass volitional spawnings, the reproductive performance and quality of embryos and larvae were assessed by following quality parameters: total fecundity per spawning, percentage of floating eggs, fertilization rate, blastomere morphology score (at 8–32 cell stages), egg and oil globule volumes, coefficient of variation of eggs and oil globule diameters, percentage of poly-globular eggs, oil globule-to-egg volume percentage, morphological appearance and survival rates at 24 (hatching), 48 and 72 h post-spawning, larvae transition rate, and survival activity index (SAI). SAI was used as a larval vitality benchmark to determine the predictive capacity of the abovementioned parameters. Our results show that blastomere morphology score was the only parameter positively correlated with SAI. However, when examining each component of blastomere morphology to SAI prediction, only the presence of cell inclusions, but not size, symmetry, margins, or adhesions of blastomeres, predict the SAI. This reveals that embryos with defective blastomeres can develop into normal-appearing larvae but are weak to starvation. Therefore, cell inclusions are the best real-time, early, reliable, and functional predictor of first-feeding larvae vitality in L. guttatus.

Spike count and morphology in the classification of epileptiform discharges.

The purpose of this study is to investigate the impact of Bergen Epileptiform Morphology Score (BEMS) and interictal epileptiform discharge (IED) candidate count in EEG classification. We included 400 consecutive patients from a clinical SCORE EEG database during 2013-2017 who had focal sharp discharges in their EEG, but no previous diagnosis of epilepsy. Three blinded EEG readers marked all IED candidates. BEMS and IED candidate counts were combined to classify EEGs as epileptiform or non-epileptiform. Diagnostic performance was assessed and then validated in an external dataset. Interictal epileptiform discharge (IED) candidate count and BEMS were moderately correlated. The optimal criteria to classify an EEG as epileptiform were either one spike at BEMS > = 58, two at > = 47, or seven at > = 36. These criteria had almost perfect inter-rater reliability (Gwet's AC1 0.96), reasonable sensitivity of 56-64%, and high specificity of 98-99%. The sensitivity was 27-37%, and the specificity was 93-97% for a follow-up diagnosis of epilepsy. In the external dataset, the sensitivity for an epileptiform EEG was 60-70%, and the specificity was 90-93%. Quantified EEG spike morphology (BEMS) and IED candidate count can be combined to classify an EEG as epileptiform with high reliability but with lower sensitivity than regular visual EEG review.

Novel interpretation of sperm stress test and morphology for maturity assessment of young Norwegian Red bulls

The use of genomic selection significantly reduces the age of dairy bulls entering semen production compared to progeny testing. The study aimed to identify early indicators that could be used for screening bulls during their performance testing period and could give us insight into their future semen production performance, acceptance for the AI station, and prediction of their future fertility. The study population consisted of 142 young Norwegian Red bulls enrolled at the performance test station, followed until we received semen production data, semen doses, and, subsequently, non-return rates (NR56) from the AI station. A range of semen quality parameters were measured with computer-assisted sperm analysis and flow cytometry from ejaculates collected from 65 bulls (9–13 months). The population morphometry of normal spermatozoa was examined, showing that Norwegian Red bulls at 10 months of age have homogenous sperm morphometry. Norwegian Red bulls could be separated into 3 clusters according to their sperm's reaction patterns to stress test and cryopreservation. Results of semi-automated morphology assessment of young Norwegian Red bulls showed that 42% of bulls rejected for the AI station and 18% of bulls accepted had ejaculates with abnormal morphology scores. For the youngest age group at 10 months, the mean (SD) proportion of spermatozoa with normal morphology was 77.5% (10.6). Using novel interpretation of sperm stress test combined with sperm morphology analysis and consecutive cryopreservation at a young age allowed identification of the candidate's sperm quality status. This could help breeding companies introduce young bulls earlier to the AI stations.

Deep Infiltrating Endometriosis: Diagnostic Accuracy of Preoperative Magnetic Resonance Imaging with Respect to Morphological Criteria.

Several current guidelines recommend imaging in the diagnostic work-up of deep infiltrating endometriosis (DIE). The purpose of this retrospective diagnostic test study was to evaluate the diagnostic accuracy of MRI compared to laparoscopy for the identification of pelvic DIE, considering lesion morphology using MRI. In all, 160 consecutive patients were included who received pelvic MRI for evaluation of endometriosis between October 2018 and December 2020 and underwent subsequent laparoscopy within 12 months of the MRI examination. MRI findings were categorized for suspected DIE using the Enzian classification and were additionally graded using a newly suggested deep infiltrating endometriosis morphology score (DEMS). Endometriosis was diagnosed in 108 patients (all types, i.e., purely superficial and DIE), of which 88 cases were diagnosed with DIE and 20 with solely superficial peritoneal endometriosis (i.e., not deep infiltrating endometriosis/DIE). The overall positive and negative predictive values of MRI for the diagnosis of DIE, including lesions with assumed low and medium certainty of DIE on MRI (DEMS 1-3), were 84.3% (95% CI: 75.3-90.4) and 67.8% (95% CI: 60.6-74.2), respectively, and 100.0% and 59.0% (95% CI: 54.6-63.3) when strict MRI diagnostic criteria were applied (DEMS 3). Overall sensitivity of MRI was 67.0% (95% CI: 56.2-76.7), specificity was 84.7% (95% CI: 74.3-92.1), accuracy was 75.0% (95% CI: 67.6-81.5), positive likelihood ratio (LR+) was 4.39 (95% CI: 2.50-7.71), negative likelihood ratio (LR-) was 0.39 (95% CI: 0.28-0.53), and Cohen's kappa was 0.51 (95% CI: 0.38-0.64). When strict reporting criteria are applied, MRI can serve as a method to confirm clinically suspected DIE.

Expert consensus on the morphological evaluation of human cleavage-stage embryos and blastocysts.

Expert consensus on the morphological evaluation of human cleavage-stage embryos and blastocysts.

Human papillomavirus infection in women undergoing in-vitro fertilization: effects on embryo development kinetics and live birth rate

BackgroudSeveral studies showed that human papillomavirus (HPV) affects male fertility, but its impact on female fertility and in vitro fertilization (IVF) outcome is not yet clear.MethodsObjective of this observational, prospective, cohort study was to evaluate the prevalence of HPV infection in women candidate to IVF, and the effects of HPV infection on the kinetic of embryonic development and on IVF outcome. A total number of 457 women candidate to IVF were submitted to HR-HPV test; among them, 326 underwent their first IVF cycle and were included in the analysis on IVF results.Results8.9% of women candidate to IVF were HPV-positive, HPV16 being the most prevalent genotype. Among the infertility causes, endometriosis was significantly more frequent in HPV-positive than in negative women (31.6% vs. 10.1%; p < 0.01). Granulosa and endometrial cells resulted HPV-positive in 61% and 48% of the women having HPV-positive cervical swab, respectively. Comparing HPV-positive and negative women at their first IVF cycle, no significant difference was observed in the responsiveness to controlled ovarian stimulation (COS) in terms of number and maturity of retrieved oocytes, and of fertilization rate. The mean morphological embryo score was comparable in the two groups; embryos of HPV-positive women showed a quicker development in the early stages, with a significantly shorter interval between the appearance of pronuclei and their fusion. In the following days, embryo kinetic was comparable in the two groups until the early blastocyst stage, when embryos of HPV-positive women became significantly slower than those of HPV-negative women. Overall, these differences did not affect live birth rate/started cycle, that was comparable in HPV-positive and negative women (22.2 and 28.1%, respectively).Conclusions(a) the prevalence of HPV infection in women candidate to IVF is similar to that observed in the general female population of the same age range; (b) HPV infection migrates along the female genital apparatus, involving also the endometrium and the ovary, and perhaps participates in the genesis of pelvic endometriosis; (c) HPV slightly affects the developmental kinetic of in vitro-produced embryos, but does not exert an effect on live birth rate.

Relationship between female sexual function, vaginal volume, vaginal resting tone, and pelvic floor muscle strength in women with stress urinary incontinence.

Vaginal morphology and pelvic floor muscle (PFM) strength may influence sexual stimulation, sensation, and orgasmic response. This study aimed to determine the relationship between female sexual function and PFM strength and vaginal morphology (represented by vaginal resting tone and vaginal volume) in women with stress urinary incontinence (SUI). Forty-two subjects with SUI were recruited for the study. Female sexual function was measured using the female sexual function index (FSFI) questionnaire. PFM strength was measured by digital palpation. Vaginal resting tone (mmHg) and vaginal volume (mL) were measured using a perineometer. The significance of the correlations between female sexual function and PFM function and hip muscle strength was assessed using Pearson's correlation coefficients. If a significant correlation between vaginal morphology and FSFI score was confirmed using Pearson's correlation, the cutoff value was confirmed through a decision tree. PFM strength was significantly correlated with desire (r=0.397), arousal (r=0.388), satisfaction (r=0.326), and total (r=0.315) FSFI scores. Vaginal resting tone (r=-0.432) and vaginal volume (r=0.332) were significantly correlated with the FSFI pain score. The cutoff point of vaginal resting tone for the presence of pain-related sexual dysfunction was >15.2 mmHg. PFM strength training should be the first strategy to improve female sexual function. Additionally, because of the relationship between vaginal morphology and pain-related sexual dysfunction, surgical procedures to achieve vaginal rejuvenation should be carefully considered.

Monotropein attenuates apoptosis and pyroptosis in chondrocytes and alleviates osteoarthritis progression in mice

BackgroundOsteoarthritis (OA) is a chronic degenerative joint disease characterized by loss of joint function, which seriously reduces the quality of life of the elderly and imposes a heavy socioeconomic burden worldwide. Monotropein (MON), the main active ingredient of Morinda officinalis F.C. How, has exhibited therapeutic effects in different disease models. However, its potential effects on chondrocytes in an arthritic model remain unclear. This study aimed to evaluate the effects of MON in chondrocytes and a mouse model of OA, and explore the potential mechanisms. Materials and methodsMurine primary chondrocytes were pretreated with 10 ng/ml interleukin (IL)-1β for 24 h to establish an in vitro model of OA, and then treated with different concentrations of MON (0, 25, 50 and 100 μM) for 24 h. The proliferation of the chondrocytes was assayed using ethynyl-deoxyuridine (EdU) staining. Immunofluorescence staining, western blotting and TUNEL staining were performed to assess the effects of MON on cartilage matrix degradation, apoptosis and pyroptosis. The mouse model of OA was constructed by surgical destabilization of the medial meniscus (DMM), and the animals were randomly divided into the sham-operated, OA and OA + MON groups. Following OA induction, the mice were given intraarticular injection of 100 μM MON or equal volume of normal saline twice a week for 8 weeks. The effects of MON on cartilage matrix degradation, apoptosis and pyroptosis were assessed as indicated. ResultsMON significantly accelerated the proliferation of chondrocytes, and inhibited cartilage matrix degradation, apoptosis and pyroptosis in the IL-1β-stimulated cells by blocking the nuclear factor-kappa B (NF-κB) signaling pathway. In the mouse model as well, MON treatment alleviated OA progression and promoted cartilage repair by inhibiting cartilage matrix degradation, and chondrocyte apoptosis and pyroptosis through the inactivation of the NF-κB signaling pathway. Furthermore, the MON-treated arthritic mice exhibited better articular tissue morphology and lower OARSI scores. ConclusionsMON alleviated OA progression by inhibiting cartilage matrix degradation, and the apoptosis and pyroptosis of chondrocytes via NF-κB pathway inactivation, and is a promising alternative for the treatment of OA.