Inner Cell Mass Quality Research Articles

The density of the inner cell mass is a new indicator of the quality of a human blastocyst: a valid supplement to the Gardner scoring system.

Can the density of the inner cell mass (ICM) be a new indicator of the quality of the human blastocyst? The densification index (DI) developed in this study can quantify ICM density and provide positive guidance for ploidy, pregnancy, and live birth. In evaluating the quality of ICM, reproductive care clinics still use size indicators without further evaluation. The main disadvantage of this current method is that the evaluation of blastocyst ICM is relatively rough and cannot meet the needs of clinical embryologists, especially when multiple blastocysts have the same ICM score, which makes them difficult to evaluate further. This observational study included data from 2272 blastocysts in 1991 frozen-thawed embryo transfer (FET) cycles between January 2018 to November 2021 and 1105 blastocysts in 430 preimplantation genetic testing cycles between January 2019 and February 2023. FET, ICSI, blastocyst culture, trophectoderm biopsy, time-lapse (TL) monitoring, and next-generation sequencing were performed. After preliminary sample size selection, the 11 focal plane images captured by the TL system were normalized and the spatial frequency was used to construct the DI of the ICM. This study successfully constructed a quantitative indicator DI that can reflect the degree of ICM density in terms of fusion and texture features. The higher the DI value, the better the density of the blastocyst ICM, and the higher the chances that the blastocyst was euploid (P < 0.001) and that pregnancy (P < 0.001) and live birth (P = 0.005) were reached. In blastocysts with ICM graded B and blastocysts graded 4BB, DI was also positively associated with ploidy, pregnancy, and live birth (P < 0.05). ROC analysis showed that combining the Gardner scoring system with DI can more effectively predict pregnancy and live births, when compared to using the Gardner scoring system alone. Accurate calculation of the DI value places high demands on image quality, requiring manual selection of the clearest focal plane and exposure control. Images with the ICM not completely within the field of view cannot be used. The association between the density of ICM and chromosomal mosaicism was not evaluated. The associations between the density of ICM and different assisted reproductive technologies and different culture conditions in embryo laboratories were also not evaluated. Prospective studies are needed to further investigate the impact of ICM density on clinical outcomes. ICM density assessment is a new direction in blastocyst assessment. This study explores new ways of assessing blastocyst ICM density and develops quantitative indicators and a corresponding qualitative evaluation scheme for ICM density. The DI of the blastocyst ICM developed in this study is easy to calculate and requires only TL equipment and image processing, providing positive guidance for clinical outcomes. The qualitative evaluation scheme of ICM density can assist embryologists without TL equipment to manually evaluate ICM density. ICM density is a simple indicator that can be used in practice and is a good complement to the blastocyst scoring systems currently used in most centers. This work was supported by the National Key Research & Development Program of China (2021YFC2700603). The authors report no financial or commercial conflicts of interest. N/A.

P-238 Evaluating the role of blastocyst quality in blighted ovum: a propensity score matching analysis

Abstract Study question What is the impact of blastocyst stage and inner cell mass (ICM) grade on the formation of blighted ovum in in vitro fertilization? Summary answer Analysis indicates blastocyst stage influences gestational sac formation, while ICM grade significantly predicts blighted ovum occurrence, highlighting potential predictive markers What is known already It is well known that the blastocyst grading, encompassing stage, ICM and trophectoderm (TE) is correlated with the pregnancy outcome. Although Gardner criteria is widely used for blastocyst grading, the significance and clinical implications of each component remain challenging to assess. A blight ovum, a presence of GSAC without advancement to FHT, commonly results from fetal aneuploidy. The quality of ICM, evolving into the fetus, is thought to reflect fetal euploidy, whereas TE develops into the placenta. However, the specific influence of these blastocyst grading components on blighted ovum incidence remains underexplored. Study design, size, duration This study analyzed 897 patient clinical data from 2011 to 2022, collected from three IVF clinics. It included maternal age, embryo transfer method (fresh or frozen), and outcomes (GSAC, FHT). Day 5 embryos were graded for blastocyst stage, ICM, and TE using Gardner criteria. Participants/materials, setting, methods Of 897 IVF cycles, 416 led to GSAC formation and 379 to FHT. Thirty-seven cycles resulted in blighted ovum. Logistic regression analyzed blastocyst stage, ICM, and TE grades’ effects on GSAC and FHT, accounting for confounding factors. Due to the small sample size, propensity score matching (PSM) was performed at 1:3 and 1:4 ratios matched blighted ovum cases with controls based on age and embryo status. Main results and the role of chance As anticipated, ANOVA and logistic regression analyses identified blastocyst stage, ICM, and TE as significant predictors of GSAC and FHT development, underscoring the relevance of Gardner criteria. However, the limited number of blighted ovum cases necessitated further analysis. Propensity score matching (PSM) was thus applied to 37 blighted ovum cases, compared against non-blighted ovum cases at ratios of 1:3 and 1:4, selected randomly from the dataset. The standardized mean differences pre- and post-PSM were 0.14 and 0, indicating well-balanced group characteristics. GSAC formation probability increased with advanced blastocyst stages (5,6) compared to earlier stages (3,4), with ICM and TE grades showing no significant impact. Conversely, FHT advancement from GSAC was unaffected by blastocyst stage, but the likelihood of FHT occurrence was 3.618 and 3.476 times higher for ICM grades A or B compared to C, at 1:3 and 1:4 ratios respectively. Limitations, reasons for caution The study accounted for age and cycle type (fresh or thawed) as confounding factors affecting pregnancy outcomes. To further minimize potential biases, controlling additional factors is crucial. Moreover, with data derived from three clinics, external validation is vital to substantiate the findings. Wider implications of the findings Blighted ovum, an early form of miscarriage, is deeply distressing for patients. This study highlights the potential role of ICM grades in predicting blighted ovum occurrences. Such insights could be invaluable for embryo selection and patient counseling. Trial registration number not applicable

P-250 Only euploidy does not ensure optimal chances for successful live birth through single blastocyst cryotransfer

Abstract Study question What does a euploid blastocyst require to provide the greatest probability of successful birth? Summary answer Morphokinetic and morphological features of the inner cell mass (ICM) and trophectoderm (TE) are crucial for optimizing live birth chances in cryo-transferred euploid blastocysts. What is known already Embryo selection through preimplantation genetic testing for aneuploidy (PGT-A) alongside frozen embryo transfer strategy enhances outcomes of single-embryo transfer cycles. While euploidy is a crucial indicator, investigations on the identification of additional criteria influencing implantation and live birth rates remain imperative. Studies suggest morphokinetic and morphological quality may supplement predictive factors for euploid embryo success, with varying correlations. The impact of factors like age or gamete origin on euploid blastocyst success remains uncertain, differentiating it from clear effects on non-diagnosed embryos. Optimizing selection criteria involves navigating the intricate interplay of these variables. Study design, size, duration This retrospective study analyses the pregnancy rate (PR) in 596 single embryo cryotransfers of full euploid blastocysts (without detected mosaicism) between February 2019 and September 2023. From these, 437 had a known outcome, and the live birth rate (LBR) was calculated. The study explored the potential impact of gamete origin, oocyte provider age, recipient age, morphokinetics, and morphological quality of ICM or TE (Gardner score) on the reproductive success of the transferred embryo. Participants/materials, setting, methods All embryos were biopsied at the blastocyst stage and successively vitrified. High-resolution next-generation sequencing was used for PGT-A. Only deferred transferences of single post-thawing intact embryos were included. Morphokinetic was assessed using the KIDscoreD5 3.1. Quantitative variables (oocyte provider age, recipient age, and morphokinetic score) were compared using the t-student test, while qualitative variables (gamete origin, morphological quality of ICM and TE) were assessed using the chi-square test. Main results and the role of chance Oocytes leading to pregnancies and live births were derived from women with ages comparable to those without success (Pregnancy: 30.0±7.1 vs. 30.8±6.8, p = 0.174; Live Birth: 30.1±7.1 vs. 30.5±6.9, p = 0.499). Similar age was also observed among recipients achieving pregnancy (41.5±5.4) and live births (41.1±5.1) compared to those who did not (Pregnancy: 41.8±5.1, p = 0.506; Live Birth: 41.5±5.3, p = 0.411). The origin of oocytes and semen (own or donor) did not significantly affect PR (Oocytes: 199/273 vs. 217/323, p = 0.130; Semen: 328/473 vs. 88/123, p = 0.224) or LBR (Oocytes: 113/205 vs. 116/232, p = 0.285; Semen: 183/347 vs. 46/90, p = 0.783). Morphokinetics played a crucial role, with euploid embryos resulting in pregnancy (7.5±1.7) and live births (7.6±1.6) exhibiting significantly higher scores than those without success (6.9±1.7; 7.1±1.7; p &amp;lt; 0.05).199 ICM quality did not significantly impact PR (A:211/296, B:190/275, C:15/25; p = 0.469). However, higher miscarriage rates in C-quality ICM blastocysts significantly reduced the LBR (A:116/211, B:107/205, C:6/21; p &amp;lt; 0.05). TE quality exhibited significant differences in PR (A:221/289, B:184/289, C:11/18; p &amp;lt; 0.05). Since the miscarriage rate was similar among groups (p = 0.787), LBR remained higher in A-quality TE blastocysts (A:124/209, B:98/213, C:7/17; p &amp;lt; 0.05). Limitations, reasons for caution As a retrospective analysis, this study is inherently limited. The influence of expansion grade on success rates remains unexplored, given the use of assisted hatching on day 3 of culture. Performing assisted hatching at the time of biopsy could potentially overcome this limitation. Wider implications of the findings The pursuit of a euploid embryo often becomes the priority in IVF laboratories. However, once the milestone is achieved, morphokinetics and embryonic morphological quality must continue to be considered. This study provides valuable insights for conveying a detailed message to patients with euploid embryos about their prospects for homebirth success. Trial registration number not applicable

P-446 Personalization of the euploid embryo transfer guided by the endometrial receptivity analysis in patients with &amp;gt;1 previous failed transfer

Abstract Study question Does personalized embryo transfer (pET) guided by Endometrial Receptivity Analysis (ERA) increase reproductive outcome in patients with &amp;gt;1 previous failed transfer of an euploid embryo? Summary answer pET guided by ERA resulted in a significantly higher Pregnancy Rate (PR), Ongoing Pregnancy Rate (OGPR) and Live Birth Rate (LBR) versus standard embryo transfer. What is known already pET consists of synchronizing embryo transfer with the optimal endometrial receptivity status by the transcriptomics identification of the personalized window of implantation (WOI). Euploid transfers improve reproductive outcome controlling the embryo contribution in the implantation. Controversial results have been published regarding the clinical usefulness of pET in a range of clinical indications. Here we present data from patients with &amp;gt;1 previous failed embryo transfer (ET) in a subsequent euploid ET. Study design, size, duration Multicenter retrospective study involving 270 patients with at least one previous implantation failure between 2017 and 2021. These patients were divided into two groups according to their subsequent transfer: study group (pET guided by ERA) (n = 200), and control group (standard transfer) (n = 70). All the ET were done in Hormone Replacement Therapy cycles (HRT). PR, OGPR and LBR were compared between the two groups. Participants/materials, setting, methods Age, body mass index, previous failed attempts, inner cell mass (ICM) and trophectoderm (TE) quality of the transferred embryos were compared between the groups. Study group had an ERA result as standard WOI or displaced WOI and pET of an euploid embryo was carried out in a subsequent HRT cycle (following the same conditions and recommendations provided by ERA). Wilcoxon rank sum test was applied to quantitative comparations. Chi-square test was done for categorical variables. Main results and the role of chance Clinical results in terms of PR, OGPR and LBR were significantly higher when pET guided by ERA was performed when compared to the non-ERA group. PR was 65% (130/200) in pET versus 37.14% (26/70) in control group (P = 0.000088); OGPR was 49% (98/200) in pET versus 27.14% (19/70) in control group (P = 0.0024); and LBR was 48.20% (95/197) in pET versus 26.09% (18/69) in control group (P = 0.0022). Control and demographics variables did not show differences between the two groups: Age: 35.27 ± 3.86 versus 35.77 ± 3.35 (P = 0.31); BMI: 22.99 ± 3.02 versus 23.25 ± 3.72 (P = 0.52); Previous failed attempts: 2.42 ±1.78 versus 2.17 ±1.81 (P = 0.32); P values where 0.1 and 0.36 for comparisons of optimal quality of ICM and TE, respectively. Limitations, reasons for caution This is a retrospective study, having the limitations of its nature. Wider implications of the findings These results show that pET guided by the ERA test of an euploid embryo significantly increases the reproductive outcomes (PR, OGPR and LBR) in patients with ≥1 previous failed transfer compared to the standard frozen ET. Trial registration number Hospital Clínico de Valencia Ethic Committee approval nº. 2021/018.

P-175 Morphokinetic growth variance score as a tool to identify euploid embryos with the highest live birth potential

Abstract Study question Is the longitudinal growth pattern of euploid embryos associated with live birth rate? Summary answer Embryos with lower growth variance score (GVS), i.e., pointing towards a steadier growth during time-lapse monitoring (TLM), have higher live birth rates. What is known already Dynamic TLM imaging has been increasingly adopted by fertility clinics as an attempt to improve the ability of selecting embryos with the highest potential for implantation. Many authors have identified and incorporated markers of embryonic morphokinetics into decision algorithms for embryo (de)selection. However, longitudinal changes during this temporal process, and the impact of such changes on embryonic competence remains unknown. Aiming to model the reference ranges of euploid embryonic growth as a single growth trajectory might provide an additive value to blastocyst morphological grade to identify highly competent euploid embryos. Study design, size, duration This observational cohort study was performed in a tertiary referral IVF centre between October 2017 and June 2021, including a total of 340 single euploid autologous frozen embryo transfers. Pre-implantation genetic diagnosis for aneuploidy (PGT-A) performed by next generation sequency (NGS). Pregnancy outcomes were assessed as live birth, pregnancy loss or not pregnant. Participants/materials, setting, methods Standard morphokinetic parameters (tPB2 to tEB) of all euploid embryos cultured in TLM system were used to develop the reference ranges for the growth variance score (GVS) model. Nuclear errors at two- and four-cell stages, and blastocyst grade before trophectoderm (TE)-biopsy were also annotated. GVS of euploid embryos was evaluated for its association with pregnancy outcomes, and to its predictive ability to live birth potential over TE and inner cell mass (ICM) grade morphology. Main results and the role of chance A total of 189 (55.6%) euploid transfers resulted in a live birth, 48 (14.1%) in pregnancy loss and 103 (30. 3%) did not result in pregnancy. The median time for euploid embryo blastulation was 109.9 hours (95% CI:98.8-121.0 hours). Lower quality ICM (OR: 0.13, 95% CI: 0.05-0.33, P &amp;lt; 0.001) and TE (OR: 0.46, 95% CI:0.22-0.94, P = 0.034), multinucleation at 4-cell stage (OR: 0.042, 95% CI:0.21-0.84, P = 0.016) and higher GVS (OR: 0.59, 95% CI: 0.45-0.77, P &amp;lt; 0.001), significantly decreased the odds of live birth. In the multivariable analysis, only GVS (OR: 0.62, 95% CI: 0.46-0.84, P = 0.002) and lower ICM quality (OR: 0.21, 95% CI: 0.06-0.65, P = 0.009) had a significant effect on live birth. There was a significant trend towards higher live birth rates with lower GVS among embryos of similar ICM grades: 85% vs 65% live births in low vs. high GVS for embryos with ICM type A, 76% vs. 48% for ICM type B, 67% vs. 21% for ICM type C, respectively (P for trend &amp;lt;0.001). Addition of the GVS to embryo morphology model (ICM, TE, biopsy day) significantly improved the model’s ability to predict live birth (AUC: 0.67 vs. 0.62, P = 0.015). Limitations, reasons for caution The exclusion of IVF cases limits the utility of the model to ICSI-derived embryos. The utility of GVS should be replicated further in external cohorts to confirm its reproducibility in other clinical and laboratory settings. Wider implications of the findings By capturing the dynamic growth pattern of embryos, GVS may serve as a valuable tool in prioritizing PGT-A tested embryos for transfer. Its impact was even more prominent for selecting suboptimal blastocysts, which is a more challenging subset of embryos to choose due to their likelihood of reduced viability. Trial registration number Not Applicable

P-137 ICM and TE quality significantly impact the live birth in euploid frozen blastocyst transfer cycles

Abstract Study question Is the live birth rate (LBR) in euploid frozen blastocyst transfer (FET) affected by the quality of ICM (Inner cell mass) and TE (Trophectoderm)? Summary answer ICM and TE significantly impacts the LBR with a decline of LB from 57.3% (ICM-A) to 48.5% (ICM-B) to 22.1% (ICM-C) (p &amp;lt; 0.001) What is known already The morphological blastocyst grading system proposed by Gardner-Schoolcraft remains the most accepted system to identify blastocysts with higher implantation potential. It relies on morphological features within the blastocyst, including ICM and TE. Several studies tried to identify the individual contribution of each. However, the conclusions remain contradictory and no clear consensus has yet been achieved. Due to heterogeneity of parameters evaluated between different publications, where embryos with unknown ploidy status were transferred in conjunction with a variability of stimulation protocols and in the number of transferred embryos, the real effect of the ICM and TE is difficult to infer. Study design, size, duration This two-center retrospective observational study includes a total of 977 euploid single FET cycles between March 2017 and March 2020 at ART Fertility Clinics Muscat, Oman and Abu Dhabi, UAE. Participants/materials, setting, methods Trophectoderm biopsies were analyzed with Next Generation Sequencing (NGS). All blastocysts available on D5 or D6 with a quality ≥ BL3CC were subjected to TE biopsy for PGT-A analysis and LBR was recorded. Vitrification/warming of blastocysts was performed using Cryotop method (Kitazato). Bivariate and multivariate analysis were performed between LB outcomes and ICM and TE grade while controlling for confounding factors. Main results and the role of chance A total of 977 single FET cycles were analyzed: 651 in hormone replacement therapy (HRT) and 326 in natural cycle regimen (NC) resulting in a 46.88% LBR. The mean patients’ age was 33.80 years with a mean Body Mass Index (BMI) of 26.80 kg/m2. Though all qualities of ICM and TE were associated with LB, blastocyst ICM-A LBR was statistically significantly higher (57.3%) than ICM-B (48.4%) and ICM-C (22.1%) (p &amp;lt; 0.001). Similarly, blastocyst TE-A LBR was statistically significantly higher (59.2%) than TE- B (48.6%) and TE- C (30.3%) (p &amp;lt; 0.001). Miscarriage rate was similar in all groups. The grade of ICM and TE were significantly associated with Anti-Mullerian-Hormone (AMH) and day of blastocyst biopsy. Mean AMH (ng/ml) was higher in ICM groups (A: 3.78, B: 3.24, p &amp;lt; 0.001) and TE group (A: 3.63, B: 3.38, p &amp;lt; 0.05) compared to lower grade (ICM-C: 2.86, TE-C: 2.82). In multivariate analysis, endometrial preparation for FET, BMI and AMH were the parameters influencing LBR: OR:1.45, [CI:1.07-1.96], p &amp;lt; 0.015) for NC; OR 0.96 [CI:0.93-0.99], p = 0.004 for BMI; OR 0.95 [CI:0.90-1.00], p = 0.033 for AMH; Both, ICM-C and TE-C, resulted in a significantly lower chance of LB [ICM: OR 0.32, CI:0.17-0.61, p &amp;lt; 0.001; TE: OR 0.44, CI:0.27-0.73, p = 0.002), compared to grade A. Limitations, reasons for caution The retrospective nature of the study and inter-observer variability in blastocyst scoring is a limitation. The physician/embryologist performing the embryo transfer could not been standardized due to the multicenter design. Randomized controlled studies are needed to determine whether ICM or TE should be prioritized in the selection of the blastocyst. Wider implications of the findings The ICM and TE scoring in FET may influence the LBR and should be considered as an important factor for the success of embryo transfer cycles. Whether these results can be extrapolated to fresh embryo transfer and to blastocysts with unknown ploidy status, needs further investigation. Trial registration number not applicable

P-235 An analysis of qualitative and quantitative morphokinetic parameters automatically annotated using CHLOE (Fairtility), an AI-based tool, finds AI score predictive of blastulation and ploidy

Abstract Study question What is the relationship between qualitative and quantitative morphokinetic parameters automatically annotated using CHLOE(Fairtility), an AI-based tool? Summary answer CHLOE score is associated with ploidy. DUC embryos have lower blastulation, form fewer good blastocysts, have increased fragmentation, slower development, lower implantation than non-DUCs. What is known already The introduction of time-lapse technologies in IVF has led to the discovery of quantitiative and qualitative morphokinetic parameters which are predictive of embryo viability (ESHRE Workshop group, 2020). The challenges of annotating videos manually remain: (i)operator variation, (ii)time-consuming; (iii)complexity of how to prioritise numerous features when determining which embryos to transfer, freeze or discard. CHLOE (Fairtility) is an AI-based tool designed to automatically capture these parameters from the time-lapse videos, removing the “black box” associated with AI, and, instead, bringing transparency and support to the embryologist responsible for the decision, thus, enhancing personalisation of care down to each individual embryo. Study design, size, duration Prospective cohort analysis on time-lapse data retrospectively collected at a single private fertility clinic in Spain between 2018-2020. 693 videos were automatically annotated (without training) using the CHLOE Artificial Intelligence (AI) tool for the following quantitative features: tPNa,tPNf,t2,t3,t4,t5,t6,t7,t8,t9,tM,tsB,tB,teB, size of ICM; and the following qualitative parameters: number of pronucleates, morphological quality of Inner Cell Mass and Trophectoderm (CHLOE Morphological scoring), identification of unusual embryo cleavages i.e. Direct Uneven Cleavage (DUCs), amongst other features. Participants/materials, setting, methods All embryos were cultured using the Embryoscope (Vitrolife) incubator. Using a range of algorithms, CHLOE generated a prediction of blastulation (at 30hpi) and implantation which were compared to outcome (blastocysts vs non-blastocysts; euploids vs Aneuploids&amp;Mosaics; Mosaics vs euploids&amp;aneuploids). Embryos identified as DUCS by CHLOE were compared with non-DUCs in terms of outcomes and in terms of endpoints generated by CHLOE (parametric continuous data assessed using 2-tail t-test, categorical data using chi-square). Main results and the role of chance Within all cleaved embryos analysed (n = 693), 29% were DUCs. DUC embryos were less likely to blastulate (DUCvsNonDUCs: 25vs50%,p&amp;lt;0.001), had a higher proportion of embryos with severe fragmentation (26% vs 3%,p&amp;lt;0.001), less likely to be suitable for biopsy (23vs87%, p &amp;lt; 0.001) lower blastulation prediction score (0.53vs0.76,p&amp;lt;0.001), lower implantation prediction score (0.21vs0.48,p&amp;lt;0.001) and slower embryo development across the all morphokinetic time-points assessed(p &amp;lt; 0.001), except for t5 (NS); than non-DUCs. DUCs and non-DUCs had similar proportion of 1,2,3PNs(5,83,5%vs 7,84,3%, NS). Within embryos that blastulated (n = 581), 25% were DUCs. DUC blastocysts were less likely to have a good quality ICM (7vs33%,p&amp;lt;0.001) or a good quality trophectoderm (9vs35%,p&amp;lt;0.05), lower implantation score (0.29vs0.52,p&amp;lt;0.05) and slower embryo development across the following morphokinetics time-points than non-DUC blastocysts. DUCs (n = 38) and non-DUC (n = 292) blastocysts had similar euploidy rate (50vs43%,NS), mosaicism rate (8vs11%,NS), and similar ratio of Euploids:Aneuploid:Mosaics (19:16:3vs126:133:33, NS). One DUC embryo was transferred, leading to an ongoing clinical pregnancy. Blastulation score was predictive of blastulation (AUC of 0.91, p &amp;lt; 0.001). Mosaic embryos had similar implantation score to non-mosaics (0.61vs0.67, NS). Euploid embryos had a higher implantation score than aneuploid blastocysts (0.71bs0.62, p &amp;lt; 0.02), so implantation score was predictive of ploidy. Limitations, reasons for caution This study involved the validation of (i) a specific AI based tool which may not be generalised across other AI tools; (ii) in a single centre. Results obtained did not involve training, suggestive of CHLOE’s ability to generalise across clinics. Presenting a framework for responsibly incorporating AI into clinical practice. Wider implications of the findings CHLOE can simplify the processing of time-lapse data to effectively, consistently, and efficiently quantify parameters that can help explain a comprehensive prediction of embryo viability. This provides a useful tool which will ultimately assist clinicians with selecting the most optimal embryos for transfer and avoid wastage from discarding viable embryos. Trial registration number not applicable

O-285 Artificial intelligence algorithms reach expert-level accuracy in automated grading of blastocyst morphology assessment based on static embryo images and Gardner criteria

Abstract Study question Can artificial intelligence (AI) algorithms reach expert-level accuracy in blastocyst morphology assessment according to Gardner criteria? Summary answer The prediction accuracy of the best performing AI algorithm (Deit), outperformed human-level mean accuracies compared to an embryologist majority vote for all Gardner morphological criteria. What is known already Routinely, morphological grading of blastocysts is performed visually according to Gardner criteria, which suggest expansion (EXP), quality of inner cell mass (ICM), and trophectoderm (TE) as key parameters to predict treatment outcome. Consequently, blastocyst scoring is prone to inter-and intra-observer variability, which may lead to inconsistencies in selecting blastocysts for transfer. AI-based algorithms may help to improve treatment outcome predictability, as it has been suggested recently. In those studies, parameters such as blastocyst quality or stage were annotated by experts from static or time-lapse-derived blastocyst images, to train AI algorithms, e.g. XCeption or YOLO, and compare them to human annotators. Study design, size, duration This retrospective study involves 2,270 images from 837 patients collected over a period of four years in a university IVF clinic. Participants/materials, setting, methods All images were annotated by one senior embryologist and divided into a training and a balanced test set. Subsequently, eight embryologists labeled 300 test set images such that every single image was seen by at least four embryologists. Annotators diverging from the ensemble vote for more than one standard deviation were excluded (n = 2) to set the ground truth labels. Finally, three AI architectures (XCeption, Swin, Deit) were trained and evaluated on that particular ground truth. Main results and the role of chance Out of nine annotators, labelling accuracy of two embryologists diverged from the consensus vote for more than one standard deviation for at least one of the three Gardner criteria. The consensus vote was built from the remaining seven annotators (mean accuracy EXP 0.81, ICM 0.70, TE 0.67). The Swin architecture outperformed the mean expert accuracy for all three criteria (EXP 0.82, ICM 0.76, TE 0.68), while the Deit and the XCeption architecture outperformed the mean expert accuracy in ICM accuracy (Deit 0.72, XCeption 0.73), and performed equal or worse in EXP and TE accuracy (Deit EXP 0.77, ICM 0.73; XCeption EXP 0.77, TE 0.66). When compared to a recent study conducted on time-lapse imaging data using AI algorithms, all our models outperform the ICM accuracy and achieve comparable TE accuracy. To minimize the role of chance in calculating the models' prediction accuracies, the SWA-Gaussian (SWAG) algorithm was used. SWAG is a method to reflect and calibrate uncertainty representation in Bayesian deep learning. It is based on modelling a Gaussian distribution for each networks' weight and applying it as a posterior over all neural network weights to perform Bayesian model averaging. Limitations, reasons for caution To reflect a real IVF lab scenario, embryologists of different origins and levels of experience were involved and no scoring training was offered to the participants. These facts could have potentially negatively affected the degree of consensus, although we excluded two annotators diverging from the mean labeling accuracy. Wider implications of the findings In the past, AI algorithms proved to reliably differentiate between good and bad prognosis blastocysts but not necessarily between blastocysts of similar quality. Further AI-supported differentiation on the basis of expansion and cell lineages will facilitate the ranking of blastocysts and would bring automated scoring closer to clinical application. 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

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

O-008 Low grade blastocysts result in healthy live births and should not be discarded

Abstract Study question Does transfer of low grade blastocysts results in acceptable live birth rates the birth of healthy babies? Summary answer While BC/CB/CC blastocysts have a reduced chance of live birth compared with AA/AB/BA/BB blastocysts, the absolute chances are still reasonable. What is known already Transfer of poorer quality embryos and blastocysts result in lower live birth rates, though to what extent is unclear, nor if there is an absolute threshold below which live births are very rare or even do not occur. Further, the developmental competence of the inner cell mass (ICM) or trophectoderm (TE) could at least theoretically impact the pregnancy and/or the health of the baby. Many clinics do not transfer or freeze poor quality embryos and blastocysts, and prefer to submit the patient to a further stimulation cycle. Study design, size, duration We performed a retrospective analysis of 10,978 couples undergoing singleton blastocyst transfers between 2009 and March 2020. We included all single blastocyst transfers for which there was complete data on blastocyst quality, singleton or twin births, birthweight and gestation at delivery, irrespective of blastocyst grading, female age, cause of infertility, ovarian response or endometrial thickness. We recorded live birth rates, birth weight and gestational age. Participants/materials, setting, methods Data from 14 clinics in 3 countries, 8 from China, 5 from New Zealand, and 1 from Australia were included in the final dataset. We compared the impact of blastocyst grading using multiple logistic regression. Blastocyst grading was based on the Gardner classification, in which the first letter denotes the grade of the inner cell mass (A is best), and the second letter the grade of the trophectoderm. Main results and the role of chance Overall, 10,978 single blastocyst cycles resulted in 4,261 live births (38.8%) (4195 singletons and 132 twins). Live birth rates were 47% after transfer of AA blastocysts (n = 2306); 42% after AB/BA (n = 2088); 33% after BC (n = 1973); 25% after CB (n = 715) and 14% after CC (n = 117). There were too few AC (n = 27) or CA (n = 12) blastocysts to include in the analysis. The odds of live birth for BC/CB/CC blastocysts compared with AA/AB/BA blastocysts, vary between 0.8 and 0.9. The live birth rate appears to be more dependent on ICM quality (C grade, n = 844, 23.2%) rather than TE quality (C grade, n = 2117, 32.1%), with the odds of live birth 0.43 and 0.57 respectively compared to A grade ICM or TE. The average birth weight (singleton only) was 3336.9+/-570.3 g (range 3323 to 3386 g), and the average gestation at delivery (singleton only) was 38+6+/-2.0 weeks (range 38+2 to 39+1). There was no significant difference for birth weight or gestational age at delivery between blastocysts of different grades. Limitations, reasons for caution This was a retrospective study. Grading was based on inner cell mass and trophectoderm and not on degree of expansion, or on day of transfer. It is likely that higher quality blastocysts were transferred first, in a fresh cycle, and poorer quality blastocysts frozen for later transfer. Wider implications of the findings The most important finding is that reasonable live birth rates are obtained in CC-blastocysts. We therefore advocate that CC-blastocysts should be replaced or frozen for later transfer. It is reassuring that there was no impact of blastocyst quality on birth weights or gestational age at the time of delivery. Trial registration number Not applicable

P-551 High concordance of the embryonic cell-free DNA with the inner cell mass: impact of blastocyst quality, patient age and mode of fertilization

Abstract Study question Does the embryonic cell-free DNA (cfDNA) in the culture medium represent the chromosomal content of the inner cell mass (ICM)? Which factors impact concordance rates? Summary answer There is high ploidy concordance between ICM biopsies and embryonic cfDNA. This value is independent of female age, insemination technique and embryo quality. What is known already The existence of embryonic cfDNA in spent blastocyst medium (SBM) has been confirmed in recent studies, opening a new era of possibilities for non-invasive preimplantation genetic testing for aneuploidy (niPGT-A). High concordance rates of cfDNA with trophectoderm (TE) biopsies and with whole blastocysts have been reported. However, the compartment(s) from where this DNA originates remain unclear. Both TE and ICM are potential sources, but, at the moment, the origin of this cfDNA is unknown as well as the mechanisms underlying its secretion into the medium. Study design, size, duration We carried out a prospective study to investigate the concordance of cfDNA with the corresponding TE and ICM biopsies. 141 day-6/7 blastocysts were donated for research after written informed consent signature for the project approved by the Ethics Committee. Embryos underwent TE biopsy and SBM collection in the same PGT-A cycle. ICM biopsy in thawed blastocysts was performed after TE biopsy diagnosis. cfDNA, TE and ICM biopsies were analyzed from January 2019 to November 2021. Participants/materials, setting, methods Embryos were cultured in routine conditions up to day 4. They were then washed and transferred to a new 10μl culture medium droplet. On day 6, SBM was collected and frozen at -20 °C; and blastocyst biopsy and vitrification were performed. Subsequently, blastocysts were thawed and ICM biopsy was conducted. All samples were analyzed by NGS (Ion ReproSeq PGS kit, ThermoFisher Scientific) and the results were analyzed with customized algorithms for TE, ICM and cfDNA. Main results and the role of chance In combination, the three sample types (cfDNA, ICM and TE) were informative in 81.6% of the blastocysts (115/141). Considering the ICM as the reference, ploidy concordance (i.e. being both euploid or aneuploid) for cfDNA was 86.1% (99/115) and for TE was 89.6% (103/115), without statistical difference. False positive rates were similar for cfDNA and for TE biopsies (6.1% and 9.6%, respectively), and false negative rates were not significantly different, but higher in cfDNA (7.8%) than in TE (0.9%), due to potential contamination with maternal DNA. Ploidy concordance between embryo cfDNA and TE biopsies was 89.6% (103/115). When the results were stratified by female age (≤37 or &amp;gt; 37 years), insemination technique (ICSI or IVF), blastocyst expansion degree (expanded, hatching or fully hatched), and ICM/TE quality (A or B), the informativity of the cfDNA was very similar between the different groups and ranged from 83.7% to 100%. Nevertheless, there were subtle differences for ICM-cfDNA ploidy concordance. It was slightly increased for the older female age group (88.3% vs 83.6% female age ≤37) as well as for ICSI (89.7% vs 82.5% in IVF) and for ICM quality B (88.4% vs 80.0% for ICM A). None of those differences reached statistical significance. Limitations, reasons for caution When stratifying according to the different criteria, the sample size analyzed was too small to draw strong conclusions. Therefore, more studies, with bigger sample size, are needed to replicate the results. Wider implications of the findings The embryonic cfDNA released to the culture medium provides information of the overall blastocyst chromosomal constitution, as suggested by the high ploidy concordance rates reported between ICM and cfDNA. This supports the use of niPGT-A as an alternative to other invasive aneuploidy detection methods that require a biopsy. Trial registration number ClinicalTrials.gov. ID NCT03520933

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.

Association between the quality of inner cell mass and first trimester miscarriage after single blastocyst transfer

BackgroundThe blastocyst morphology provided valuable roles for predicting pregnancy and live birth, but was still not fully understood for evaluating miscarriage. The aim of this study was to explore the association between blastocyst morphologic evaluation and first trimester miscarriage combined with karyotype of miscarried conceptus.MethodsThis retrospective cohort study included a total of 2873 clinical pregnancy cycles with single blastocyst transfer performed from January 2013 to April 2019. Chromosome karyotype of miscarried conceptus was analyzed via single nucleotide polymorphism array analysis. Miscarriage and karyotype of miscarried conceptus associated with blastocyst morphology were analyzed by chi-square and logistic regression analysis.ResultsA total of 354 (12.3%) cycles resulted in first trimester miscarriage. Miscarriage rates increased with trophectoderm (TE) grade from A to C (P = 0.012), while three morphologic parameters (blastocoele expansion degree, inner cell mass (ICM) and TE) showed no statistical significance with miscarriage after multivariable analysis. The rate of aneuploidy was 47.7% (83 of 174) in total miscarried conceptuses. For euploid miscarriages, the grade B of ICM occupied a higher proportion compared with aneuploidy, with OR of 2.474, (95% CI, 1.311–4.699), P = 0.005.ConclusionsChromosomal aberration of embryo is an important genetic factor for first trimester miscarriage, and the quality of ICM is a potential indicator for euploid miscarriage. Blastocysts with grade A of ICM should be given priority during single blastocyst transfer to reduce potential miscarriage.

Association between blastocyst morphology and pregnancy and perinatal outcomes following fresh and cryopreserved embryo transfer.

To assess the importance of each blastocyst morphological criteria with pregnancy and perinatal outcomes. This single-center retrospective cohort study included blastocyst single embryo transfers (SET) performed between 1/2012-2/2018. Poisson regression was used to evaluate pregnancy outcomes following fresh and cryopreserved embryo transfer (CET) for association with blastocyst expansion, inner cell mass (ICM) quality, and trophectoderm (TE) quality. Among cycles resulting in live birth, associations with preterm birth, small for gestational age (SGA) and large for gestational age (LGA), were evaluated using logistic regression. A total of 1023 fresh and 1222 CET cycles were included, of which 465 (45.1%) fresh and 600 (48.5%) CET cycles resulted in singleton live birth. Clinical pregnancy rates increased with increasing expansion among fresh transfers (p for trend = 0.001) but not CET (p = 0.221), and with TE quality for both fresh and CET cycles (p = 0.005 and < 0.0001, respectively). Live birth rates increased with increasing expansion (fresh p = 0.005, CET p = 0.018) and TE quality (fresh p = 0.028, CET p = 0.023). ICM grade was not associated with pregnancy outcomes; however, higher ICM quality among CET cycles was associated with increased chance of preterm birth (p =0.005). In blastocyst SET, blastocyst expansion and TE quality were each associated with clinical pregnancy and live birth. While higher ICM quality was associated with increased chance of preterm birth among CET, no other associations with perinatal outcomes were identified. Clinicians can be reassured that pregnancies from blastocysts with lower expansion, ICM, or TE qualities are not more likely to result in adverse perinatal outcomes.

Attempts for Generation of Embryonic Stem Cells from Human Embryos Following In Vitro Embryo Twinning.

In vitro embryo twinning can be used to increase the number of the human embryos available for production of human embryonic stem cell (hESC) lines. The aim of this study was to generate hESCs following the production of the twin embryos by in vitro embryo splitting procedures. In total 21 chromosomally abnormal (three pronuclei) embryos underwent in vitro embryo twinning and were allowed to develop to the blastocyst stage. As a result, 42 twin embryos were obtained, of which 24 developed to blastocyst stage. Using micromanipulation technique, the zona-free blastocysts were recovered and plated onto mitotically inactivated Yazd human foreskin fibroblast (Batch18; YhFF#18) feeder layers in microdrops. After 3 to 5 days of blastocyst culture onto human foreskin fibroblast feeder layers, the hESC-like outgrowths were passaged onto new feeders in microdrops. The initial outgrowths of hESC-like cells were generated, and cells were proliferated, passaged, and some of them expressed hESC and trophoblastic markers; however, no cell lines were established. This might be due to the low cell number and poor quality of inner cell mass within these twin blastocysts. In vitro embryo twinning by increasing the number of the human embryos could be useful in the future for the generation of new pluripotent stem cell lines. However, the challenge remains to optimize the methods.

Morphological and Kinetic Embryological Criteria and Correlation with Aneuploidy Rates: How Might they Be Used to Choose the Best IVF Embryo for Transfer?

Abstract Objective: To test the hypothesis that embryos reaching a certain developmental stage at a fixed time point (indicative of growth rate) and those of better morphology are more likely to be euploid than those reaching an earlier stage at the same point and those with poorer morphology. To test the hypothesis that quality of the inner cell mass (ICM) and trophectoderm (TE) independently predict ploidy status. 5 specific age groups were tested Design: Observational research study, retrospective analysis Setting: Clinical IVF laboratory, comprehensive chromosome screening (CCS) outsourced to specialist laboratory Patients: Those undergoing IVF with Comprehensive Chromosome Testing (CCS) by array Comparative Genomic Hybridization (aCGH). Interventions: All embryos grown to day 5 and 6. Those that formed advanced blastocysts were biopsied and cells were analyzed using aCGH Main Outcome Measures: developmental stage reached by a fixed time point and morphology of whole embryos and quality of ICM and TE; correlation to rates of aneuploidy for all chromosomes. Results: In addition to confirming a maternal age effect, evidence suggested that embryos reaching a more advanced stage of development on day 5 were more likely to be chromosomally normal than those not reaching the same stage until day 6. The poorer quality embryos were more likely to be aneuploid in the most age groups but this effect was seen more markedly in the trophectoderm (TE) than the inner cell mass. Conclusions: When comparing aneuploidy to growth rate, a complex pattern emerges in that, in the whole data set, it is the slower growing embryos that appear to be more likely to be aneuploid (but only in the younger and older age groups). By isolating the slower growing cohort (those not ready to biopsy until day 6) however, it seems that the relatively faster ones that continue to grow to day 6 are more likely to be aneuploid. Moreover, TE quality appears to be an important consideration for choosing embryos in all age groups.

Implantation ability of blastocyst from preimplantation genetic diagnosis (PGD) does not depend on the dedree of expansion after the initation of cavitation

Embryos biopsied on D3 undergo delayed compaction and alteration of hatching mechanism due to artificial hole in the zona pellucida. Therefore morphological grading system based on expansion degree and quality of inner cell mass (ICM) and trophoectoderm (TE) normally used for blastocyst selection might be not valid. The aim of this study was to establish each morphological parameter's contribution to the implantation potential of a chromosomally normal blastocyst from D3 biopsied embryos, attending to degree of blastocoel expansion and hatching status, size and compactness of ICM and cohesiveness and number of TE cells.

Influence of oocyte dysmorphisms on blastocyst formation and quality

To identify the association between oocyte dysmorphisms and blastocyst developmental competence. Case-control study. Private assisted reproduction center. This study included 5,516 embryos, which were obtained from 605 patients undergoing intracytoplasmic sperm injection cycles in which ET was performed on day 5 of embryo development. Intracytoplasmic sperm injection and ET. The morphologic characteristics of the blastocysts, namely the degree of blastocoel expansion and hatching status, the size and compactness of the inner cell mass (ICM), and the cohesiveness and number of trophectoderm cells, were correlated with oocyte dysmorphisms. Of the 5,516 embryos evaluated, 2,920 (52.9%) reached the blastocyst stage. The blastocysts' degree of expansion and hatching status were decreased by the presence of aggregates of smooth endoplasmic reticulum clusters, large perivitelline space, and shape abnormalities. The presence of a large perivitelline space also decreased the ICM and trophectoderm cell quality. Aggregates of smooth endoplasmic reticulum clusters also decreased the ICM quality, pregnancy rate, and the miscarriage rate. The results presented here suggest that the individual identification of oocyte dymorphisms may be a prognostic tool for blastocyst development and quality. However, the results do not diminish the importance of cleavage-stage embryo morphology assessment to predict blastocyst development competence.

The influence of patient and cohort parameters on the incidence and developmental potential of embryos with poor quality traits for use in human embryonic stem cell derivation

Human embryonic stem cells (hESCs) are most commonly derived from the inner cell mass (ICM) of blastocyst stage embryos. While the majority of hESC lines originate from good-quality embryos donated after cryogenic storage, poor-quality embryos (PQEs) not suitable for clinical use have also been shown to generate hESC. This provides a newfound function for embryos that would otherwise be discarded following IVF or ICSI. Owing to their lack of clinical importance, however, data on the poorest embryos in a cohort go largely unreported in the literature. It is therefore of interest to better understand the availability of PQEs from IVF/ICSI cycles and to determine their ability to develop into blastocysts with good-quality ICMs for use in hESC derivation. In this study, we investigate the influence of patient parameters and embryo cohort on PQE incidence, blastocyst development, ICM quality and successful hESC derivation from donated PQEs. PQEs from 736 patient cycles that did not meet our clinical criteria for transfer or cryopreservation were cultured until Day 6 of development and assessed for blastocyst formation and ICM quality. A subset of blastocysts with good-quality ICMs were then used for hESC derivation attempts. Anonymous patient data such as maternal age, embryo history and cohort parameters were then retrospectively compiled and analysed. PQEs made up 46.8% of two pronucleate embryos created from IVF/ICSI. Including embryos with abnormal fertilization, a mean of 3.6 ± 2.8 embryos were donated per cycle with 32.6% developing to the blastocyst stage. Good-quality ICM were produced in 13.9% of PQEs cultured. Of good-quality ICM, 15.4% of those used in hESC derivation attempts resulted in a novel line. The PQEs that originated from older patients (>37 year) or from cycles that did not result in pregnancy had significantly diminished blastocyst development and ICM quality. Maternal age was also shown to further influence the ability of good-quality ICMs to generate hESC. PQEs are an abundant source of embryos capable of developing to blastocysts with good-quality ICMs and subsequently generating novel hESC. We have shown that prognostic variables used to predict IVF/ICSI outcome can also help predict which PQEs have the best hESC developmental potential. Owing to the diversity of PQE origin, experiments designed to compare hESC derivation techniques or efficiency using PQEs should consider clinical IVF/ICSI parameters to establish groups with equal developmental competence. Additional investigation is needed to determine if these results are applicable to hESC derivation using good-quality embryos.