BACKGROUND:Stem cell-based embryo models (SCBEMs) are clusters of pluripotent stem cells that can mimic morphological and functional aspects of early human embryos to different degrees. When cultured from human cells, SCBEMs offer technically scalable and amenable tools that can help refine, reduce, and, in the future, perhaps replace the use of animals and human embryos in fundamental and clinical research. These advantages propelled the development of SCBEMs, and several distinct types have been generated over the past decade, including gastruloids, axioloids, blastoids, and post-implantation-like embryoids. For purposes of governance, advisory reports distinguish between SCBEMs based on their presumed capacity to continuously undergo organized human development-referred to here as developmental potential. However, since functionally testing this potential by transferring human SCBEMs to a uterus would be unethical and is recommended to be prohibited, scientists lack clear or consistent ways to assess it. OBJECTIVE AND RATIONALE:This narrative review aims to tackle the question of how to assess developmental potential in SCBEMs by clarifying the different ways in which it can be and is being conceptualized. We achieve this by synthesizing insights from governance, science, and ethics. First, we examine how developmental potential is described in contemporary governance frameworks, and which aspects are emphasized. Next, we discuss biological markers for developmental potential and show how their scientific basis (in embryos, let alone SCBEMs) remains poorly understood. Then, we explore how the aspects considered relevant for assessments of developmental potential in governance and science may pre-emptively hinge on underlying conceptual interpretations and lead to differing normative implications. SEARCH METHODS:This narrative review combines insights from both the academic and grey literature on the (ethics of) embryo models. Original and review articles were selected from PubMed and Biorxiv with the main focus on articles published since 2015. Search terms included: embryo quality, in vitro fertilization, Gardner system, blastoid, gastruloid, embryo research, potentiality argument, developmental potential, transcriptomics, epigenetics, embryo metabolism, and related terms. Additional sources were identified through snowballing. This work focuses predominantly on human SCBEMs, but references to animal models are made. OUTCOMES:Comparison of the descriptions currently recommended for governance suggests at least three criteria that are used to assess developmental potential in SCBEMs: composition, organization, and interaction. Scientifically, developmental potential is multifaceted and only partly characterized, making it necessary to measure a broader range of aspects, using human embryos as benchmarks when possible. Since the range and significance of these aspects can be shaped by underlying accounts of developmental potential, contemporary advisory reports are examined to explore if and how they connote interpretations of developmental potential as possibility, probability, and predisposition. WIDER IMPLICATIONS:Categorization of the regulatory and scientific criteria currently used to assess developmental potential shows that they are underpinned by distinct interpretations of the concept, revealing tensions and questions for further inquiry. By synthesizing insights from governance, science, and ethics, this review thus aims to contribute to the responsible advancement of the SCBEM field and to support its coherent and transparent governance. REGISTRATION NUMBER:N/A.

Study question Can a multimodal deep learning approach integrating time-lapse imaging, clinical parameters, and genomic data predict embryo ploidy status in assisted reproductive technology (ART)? Summary answer The multimodal deep learning model can effectively predict ploidy status of embryos using merely embryo imaging. What is known already Current embryo selection relies on subjective morphological assessment or invasive preimplantation genetic testing for aneuploidy (PGT-A). Although PGT-A can diagnose embryo ploidy status and is claimed to enhance pregnancy rates, it presents several limitations including invasive biopsy, high cost, and potentially inaccuracies in detecting mosaicism. Recent artificial intelligence (AI) models show promise but primarily focus on viability prediction rather than genome-wide chromosomal analysis. Despite advances in time-lapse imaging and AI, no validated model currently integrates multimodal data for non-invasive ploidy prediction. Furthermore, existing methods lack the capability to distinguish between meiotic and mitotic chromosomal abnormalities, crucial for understanding developmental potential. Study design, size, duration We retrospectively analyzed 1,400 embryos from PGT cycles (2022-2024) using a stratified random sampling approach. We used ResNet152 as backbone to develop the model, using a 70/20/10 split for training, validation, and testing. Multiple logistic regression and bootstrapping (1000 iterations) were performed for statistical validation. Model performance was assessed using ROCAUC analysis. Participants/materials, setting, methods We included embryos from patients undergoing PGT at our center, cultured in EmbryoScope® time-lapse incubators. Imaging frames were extracted at 2-hour intervals. Morphokinetic parameters (from fertilization to blastocyst formation) and imaging texture features, e.g., density were analyzed. Chromosomal status was determined via whole-genome sequencing of trophectoderm biopsies using haplarithmisis. We then applied class activation mapping (CAM) heatmaps to visualize pixel regions predictive of ploidy outcomes for model interpretation. Main results and the role of chance The multimodal model achieved a ploidy prediction ROCAUC of 0.72, significantly outperforming morphology-only model (ROCAUC: 0.67, p < 0.001). Texture analysis revealed significant correlations between density patterns and ploidy status (Spearman’s ρ = 0.68, p < 0.001). Texture feature scores were significantly different across embryo development process, such as key indicators at fertilization, day3, and day5. CAM heatmap analysis identified inner cell mass region as the strongest morphological predictor. Limitations, reasons for caution Our study has limitations due to its retrospective nature and the single-center design, which may restrict generalizability. Selection bias may occur as we only included embryos progressing to biopsy. External validation across different laboratories and diverse datasets is needed to confirm these findings. Wider implications of the findings Our method demonstrated the potential to reduce reliance on invasive PGT-A, mitigating procedural risks and costs. By enabling non-invasive euploid embryo selection, we aim to enhance the accessibility to precision reproductive care and refine transfer strategies for chromosomal abnormal embryos. Trial registration number No

Study question Can non-invasive preimplantation genetic testing (niPGT) using spent culture medium (SCM) accurately detect monogenic disorders and the meiotic/mitotic origin of aneuploidies? Summary answer Our approach enables concurrent genome-wide haplotyping and copy number profiling, enabling the detection of monogenic disorders and aneuploidies with the ability to distinguish meiotic/mitotic aberrations. What is known already Preimplantation genetic testing (PGT) is typically performed through invasive embryo biopsy to detect aneuploidies and monogenic disorders. SCM contains cell-free DNA, offering a non-invasive alternative for embryo testing. However, existing methods mainly focus on aneuploidy detection and fail to determine the meiotic or mitotic origin of chromosomal abnormalities, limiting clinical applicability. Distinguishing between these errors is crucial, as meiotic errors occur during gametogenesis and affect the entire embryo, whereas mitotic errors arise post-fertilization and lead to mosaicism. A non-invasive method capable of accurately detecting genetic disorders and aneuploidies while identifying their segregational origin would advance reproductive care. Study design, size, duration In an exploratory retrospective study, 90 SCM samples were analyzed from 53 blastocysts affected by monogenic disorders from 14 families enrolled in diagnostic PGT. SCM was obtained after embryo transfer to a biopsy dish to avoid interference. The study aimed to identify and develop the optimal experimental protocol and computational method for niPGT, respectively. The resulting haplotypes and copy number profiles were compared to invasive biopsy results. Participants/materials, setting, methods SCM samples from 53 blastocyst were split into halves, yielding 90 samples. Five whole-genome amplification protocols were tested to determine the best method for niPGT. Whole-genome sequencing was performed, followed by haplarithmisis-based haplotyping to reconstruct genome-wide haplotypes. Quality metrics, including allele drop-in, drop-out, and parental contamination, were assessed. Copy number profiles were generated, and results were compared to biopsy outcomes to evaluate diagnostic concordance, haplotype accuracy, and the ability to distinguish meiotic from mitotic aberrations. Main results and the role of chance Parallel genome-wide haplotyping and copy number profiling of cfDNA derived from SCM, enabled the identification of monogenic aberrations and aneuploidies. In 74 samples, the sex identified from SCM matched the biopsy results, while 16 showed mismatches, suggesting (maternal) contamination. High-quality embryos assessed with the optimal amplification method had a median haplotype concordance of 92.5% ([89.83-95.27], n = 15). Notably, all meiotic aberrations (n = 3) detected via biopsy were also observed in SCM, demonstrating niPGT’s ability to detect inherited chromosomal abnormalities. In contrast, mitotic aberrations were inconsistently detected, likely due to embryo mosaicism or variable DNA shedding into SCM. These results indicate that niPGT can reliably detect monogenic disorders and meiotic aneuploidies but may be discordant for mitotic errors, which is inherent to mosaicism. Limitations, reasons for caution While niPGT successfully detects meiotic aberrations and monogenic disorders, contamination remains a major challenge. Mitotic errors are inconsistently detected due to mosaicism caused by chromosomal instability. Further validation in larger cohorts is necessary to refine methods and improve reliability before widespread clinical implementation. Wider implications of the findings By eliminating the need for invasive biopsy, our method could transform reproductive medicine, offering a new paradigm for embryo selection. Future improvements in contamination control will further advance the clinical applicability of niPGT. This approach enables non-invasive genetic testing and could be extended to other areas of personalized medicine. Trial registration number No

To investigate the clinical utility of preimplantation genetic testing for aneuploidy origin (PGT-AO), which determines the meiotic/mitotic origin of aneuploidies and their level of mosaicism. PGT-AO together with sequencing-based PGT-M avoids unnecessary transfer of embryos with meiotic trisomies and most monosomies. Determining meiotic/mitotic origin based on mosaicism level is incorrect. Rising global parental age increases aneuploidy risk in offspring, driving demand for advanced PGT. Preimplantation genetic testing for aneuploidy (PGT-A) is widely used in in vitro fertilization for embryo selection. However, large clinical trials have questioned its efficacy, as current PGT-A may discard viable embryos. PGT-A overlooks the mosaic nature of embryos, causing concern in the field, since it is now evident that mosaic embryos, i.e. embryos that contain both euploid and aneuploid cells, may lead to healthy live births. We evaluated the clinical utility of PGT-AO, which determines the meiotic/mitotic origin of aneuploidies. In a nationwide non-selection study, we retrospectively analyzed the genomes of 5,975 embryos sequenced for PGT for monogenic disorders (PGT-M) using either genotyping-by-sequencing (n = 2,493) or whole-genome sequencing (n = 3,482). Genome haplarithmisis determined the meiotic/mitotic origin of aneuploidies and their mosaicism level. The aberrations were correlated with recorded clinical outcomes, such as birth status, heart rate, HCG status, ovarian stimulation protocol and duration, maternal and paternal age, endometrial thickness, and pregnancy complications. Couples were counseled by clinical geneticists at University Medical Centers (UMCs) of the Netherlands, in Maastricht, Amsterdam, Utrecht, and Groningen, and enrolled in the diagnostic PGT procedure after signing an informed consent form. Embryos (n = 946) eligible for transfer based on PGT-M (i.e. chromosomal aneuploidies were not accounted for) are analyzed by haplarithmisis-based PGT-AO including parental information. This information allows for the detection of chromosomal abnormalities and their mosaicism, and parental and segregational origin. Among 946 transferred embryos, 253 (26.7%) resulted in a live birth, while 693 (73.3%) failed, including implantation failure (n = 546, 78.8%), early embryonic arrest (n = 104, 15.0%) and pregnancy loss (n = 43, 6.2%). All embryos with a meiotic trisomy failed, whereas 17.0% of embryos with a mosaic mitotic trisomy resulted in a healthy live birth. Furthermore, only 6.8% of monosomic embryos lead to a healthy live birth. Remarkably, different ovarian stimulation protocols affected transfer outcomes and aneuploidy rates. Maternal age at ovum pickup is positively correlated with meiotic trisomy rate in contrast to mitotic trisomy rate. Our results show embryos with meiotic trisomies and most monosomies should not be selected for embryo transfer. In contrast to embryos with meiotic trisomies, embryos with mitotic trisomies are often mosaic and the abnormality may not be uniformly distributed throughout the blastocyst and could potentially be considered for transfer. Haplarithmisis relies on the inclusion of parental DNA to phase the embryonic genome and thus determine the segregational origin of aberrations. Additionally, mitosis and meiosis II trisomies cannot be discerned when there is no crossover event in the chromosome of interest. Embryos with meiotic trisomies and most monosomies should be considered non-viable, while those with mitotic trisomies may result in live births. PGT-AO combined with sequencing-based PGT-M avoids unnecessary embryo transfer. If applied non-invasively, it may increase successful embryo transfers following IVF. We envision PGT-AO making a paradigm-shift in embryo selection. No

Infertility affects ≈17.5% of the adult population worldwide, posing significant clinical, emotional, and socioeconomic challenges. Recent advances at the intersection of reproductive medicine and bioengineering offer promising avenues to enhance assisted reproductive technologies (ART). This review synthesizes emerging microengineered and stem cell-based platforms designed to improve key ART stages from gamete handling and fertilization to embryo culture and implantation. State-of-the-art microfluidic systems that refine sperm selection by leveraging directional behavioral responses, enhancing motility, and preserving DNA integrity is discussed. Moreover, devices for oocyte denudation and cryopreservation have been developed to mitigate cellular stress associated with conventional processing techniques. While microengineered platforms demonstrate promise in sperm sorting and reducing stress on gametes, their broader application in ART, particularly for oocyte handling and embryo culture, requires further development. The review further addresses stem cell-based embryo models and bioengineered endometrial platforms, which aim to recapitulate the dynamic in vivo microenvironments needed for successful fertilization, embryo development, and implantation. Despite encouraging preliminary results, challenges such as scalability, reproducibility, clinical validation, and ethical considerations remain. By identifying these gaps and proposing future directions, this review considers integrating microengineering and bioengineering approaches to streamline ART procedures, ultimately enabling more personalized and effective reproductive therapies.

Research question: What is the level of understanding, and what are the attitudes and considerations regarding preconception carrier screening (PCS) among couples seeking IVF or intracytoplasmic sperm injection (ICSI)? Design: A mixed-methods design was used. Nine interviews were conducted with couples or individual partners (n =16) who had an initial consultation for IVF/ICSI in the 2 years preceding this study. A questionnaire was completed by 115 participants. No actual PCS was offered. Results: All interviewed couples expressed a positive attitude towards PCS, and over half of the respondents stated that they would pursue or seriously consider pursuing PCS if possible. Some couples falsely believed that PCS could identify a cause for their fertility problems and increase their chance of conceiving. The desire to make an informed reproductive decision was the most important argument in favour of PCS. The primary argument against PCS was the apprehension of being confronted with reproductive dilemmas. The longer the delay to IVF/ICSI treatment required to perform PCS, the more couples would be inclined to decline screening. Participants indicated that they would prefer to receive information about PCS from a medical specialist at an early stage in their IVF/ICSI treatment. Conclusion: Although attitudes towards PCS were generally positive, some concerns were raised about treatment delays and potential reproductive dilemmas, and some couples had misconceptions about the purpose of screening within the context of their IVF/ICSI treatment. These fi ndings highlight the importance of tailoring information and counselling to the specific needs of couples seeking IVF/ICSI.

In recent years, multiple efforts have been made to develop guidelines for research involving human embryo-like structures (ELS), also known as stem cell-based embryo models (SCBEMs). Policy recommendations have been proposed by advisory bodies in countries such as the Netherlands, Australia, France, Sweden, and the United Kingdom, as well as by expert committees of international professional organizations, including the International Society for Stem Cell Research (ISSCR) and the European Society of Human Reproduction and Embryology (ESHRE). A common ground across these guidelines is the distinction between ELS based on their foreseeable capacity for undergoing continuous human development—a normative recommendation that has given rise to (implicit or explicit) conceptual dichotomies like “integrated vs. non-integrated,” “complete vs. incomplete,” and “complex vs. simple”, among others. Some argue that this capacity confers upon ELS that possess it a moral status comparable to that of human embryos. Others suggest that, while it increases their moral value, it does not necessarily equate them to embryos. In short, there is consensus that ELS with a developmental potential akin to human embryos deserve some degree of moral consideration, but disagreement about the extent of that consideration. This underscores the need for accurate methods to assess and differentiate the developmental potential of specific types of ELS. From an experimental perspective, addressing this need is challenging. Developmental potential is difficult to quantify, and there is no “ideal” human embryo against which to benchmark, given the variability introduced by genetic, epigenetic, and environmental factors. Quantifiable standards are thus hard to establish, and the methods currently in use—such as visual assessments of blastocyst morphology—fail to provide a comprehensive picture of developmental potential. Philosophically, the challenge lies in qualifying developmental potential. Developmental potential may be considered morally valuable for two (non-mutually exclusive) reasons: (1) extrinsic—grounded in symbolic, relational, or instrumental value; and (2) intrinsic—based on properties or interests valued for their own sake. If the moral relevance of developmental potential stems from extrinsic factors, assessing it depends at least partly on empirical studies of how stakeholders—such as patients, scientists, and ethicists—perceive and assign meaning to ELS with such potential. This raises a further challenge: how can we ensure that these insights are interpreted and applied in a consistent, non-arbitrary way in ethical deliberation and policymaking? If alternatively (or additionally), developmental potential is considered morally relevant for intrinsic reasons, then identifying its biological markers becomes key. Yet interpretations of what constitutes developmental potential vary. An “active” view emphasizes internal parameters—such as organismic wholeness, self-organization, neural development, or organ formation—while a “passive” view focuses on external enabling factors, such as environmental cues or support systems necessary for further development. Different interpretations lead to the identification of different biological criteria, making it necessary to first clarify the specific interpretation(s) at stake. Clarifying the basis for considering some ELS morally more significant than others on account of their foreseeable developmental potential is also essential for responsibly translating the normative weight of this potential into national and international regulation. If developmental potential is regarded as intrinsically valuable, then ELS that possess it may warrant stronger protection than if their value were seen as merely extrinsic. The latter depends on context (i.e., is derivative), whereas the former does not (i.e., is non-derivative). Ultimately, how developmental potential is understood—and who determines its moral significance—will shape the governance of ELS research and its broader societal implications. Exploring the ethical bearing and biological markers of developmental potential is thus essential to determining how ELS can and should be used in (future) research and, therefore, the focus of this lecture.

BACKGROUND The genetic composition of embryos generated by in vitro fertilization (IVF) can be examined with preimplantation genetic testing (PGT). Until recently, PGT was limited to detecting single-gene, high-risk pathogenic variants, large structural variants, and aneuploidy. Recent advances have made genome-wide genotyping of IVF embryos feasible and affordable, raising the possibility of screening embryos for their risk of polygenic diseases such as breast cancer, hypertension, diabetes, or schizophrenia. Despite a heated debate around this new technology, called polygenic embryo screening (PES; also PGT-P), it is already available to IVF patients in some countries. Several articles have studied epidemiological, clinical, and ethical perspectives on PES; however, a comprehensive, principled review of this emerging field is missing.OBJECTIVE AND RATIONALE This review has four main goals. First, given the interdisciplinary nature of PES studies, we aim to provide a self-contained educational background about PES to reproductive specialists interested in the subject. Second, we provide a comprehensive and critical review of arguments for and against the introduction of PES, crystallizing and prioritizing the key issues. We also cover the attitudes of IVF patients, clinicians, and the public towards PES. Third, we distinguish between possible future groups of PES patients, highlighting the benefits and harms pertaining to each group. Finally, our review, which is supported by ESHRE, is intended to aid healthcare professionals and policymakers in decision-making regarding whether to introduce PES in the clinic, and if so, how, and to whom.SEARCH METHODS We searched for PubMed-indexed articles published between 1/1/2003 and 1/3/2024 using the terms 'polygenic embryo screening', 'polygenic preimplantation', and 'PGT-P'. We limited the review to primary research papers in English whose main focus was PES for medical conditions. We also included papers that did not appear in the search but were deemed relevant.OUTCOMES The main theoretical benefit of PES is a reduction in lifetime polygenic disease risk for children born after screening. The magnitude of the risk reduction has been predicted based on statistical modelling, simulations, and sibling pair analyses. Results based on all methods suggest that under the best-case scenario, large relative risk reductions are possible for one or more diseases. However, as these models abstract several practical limitations, the realized benefits may be smaller, particularly due to a limited number of embryos and unclear future accuracy of the risk estimates. PES may negatively impact patients and their future children, as well as society. The main personal harms are an unindicated IVF treatment, a possible reduction in IVF success rates, and patient confusion, incomplete counselling, and choice overload. The main possible societal harms include discarded embryos, an increasing demand for 'designer babies', overemphasis of the genetic determinants of disease, unequal access, and lower utility in people of non-European ancestries. Benefits and harms will vary across the main potential patient groups, comprising patients already requiring IVF, fertile people with a history of a severe polygenic disease, and fertile healthy people. In the United States, the attitudes of IVF patients and the public towards PES seem positive, while healthcare professionals are cautious, sceptical about clinical utility, and concerned about patient counselling. WIDER IMPLICATIONS The theoretical potential of PES to reduce risk across multiple polygenic diseases requires further research into its benefits and harms. Given the large number of practical limitations and possible harms, particularly unnecessary IVF treatments and discarded viable embryos, PES should be offered only within a research context before further clarity is achieved regarding its balance of benefits and harms. The gap in attitudes between healthcare professionals and the public needs to be narrowed by expanding public and patient education and providing resources for informative and unbiased genetic counselling. Graphical Abstract The principles, estimated benefits, personal and societal harms, and clinical considerations of prioritizing IVF embryos based on their risk for late-onset, polygenic conditions.

BACKGROUND:Preimplantation genetic testing (PGT) is a reproductive technology that selects embryos without (familial) genetic variants. PGT has been applied in inherited cardiac disease and is included in the latest American Heart Association/American College of Cardiology guidelines. However, guidelines selecting eligible couples who will have the strongest risk reduction most from PGT are lacking. We developed an objective decision model to select eligibility for PGT and compared its results with those from a multidisciplinary team.METHODS:All couples with an inherited cardiac disease referred to the national PGT center were included. A multidisciplinary team approved or rejected the indication based on clinical and genetic information. We developed a decision model based on published risk prediction models and literature, to evaluate the severity of the cardiac phenotype and the penetrance of the familial variant in referred patients. The outcomes of the model and the multidisciplinary team were compared in a blinded fashion.RESULTS:Eighty-three couples were referred for PGT (1997-2022), comprising 19 different genes for 8 different inherited cardiac diseases (cardiomyopathies and arrhythmias). Using our model and proposed cutoff values, a definitive decision was reached for 76 (92%) couples, aligning with 95% of the multidisciplinary team decisions. In a prospective cohort of 11 couples, we showed the clinical applicability of the model to select couples most eligible for PGT.CONCLUSIONS:The number of PGT requests for inherited cardiac diseases increases rapidly, without the availability of specific guidelines. We propose a 2-step decision model that helps select couples with the highest risk reduction for cardiac disease in their offspring after PGT.

High-throughput sequencing technologies have increasingly led to discovery of disease-causing genetic variants, primarily in postnatal multi-cell DNA samples. However, applying these technologies to preimplantation genetic testing (PGT) in nuclear or mitochondrial DNA from single or few-cells biopsied from in vitro fertilised (IVF) embryos is challenging. PGT aims to select IVF embryos without genetic abnormalities. Although genotyping-by-sequencing (GBS)-based haplotyping methods enabled PGT for monogenic disorders (PGT-M), structural rearrangements (PGT-SR), and aneuploidies (PGT-A), they are labour intensive, only partially cover the genome and are troublesome for difficult loci and consanguineous couples. Here, we devise a simple, scalable and universal whole genome sequencing haplarithmisis-based approach enabling all forms of PGT in a single assay. In a comparison to state-of-the-art GBS-based PGT for nuclear DNA, shallow sequencing-based PGT, and PCR-based PGT for mitochondrial DNA, our approach alleviates technical limitations by decreasing whole genome amplification artifacts by 68.4%, increasing breadth of coverage by at least 4-fold, and reducing wet-lab turn-around-time by ~2.5-fold. Importantly, this method enables trio-based PGT-A for aneuploidy origin, an approach we coin PGT-AO, detects translocation breakpoints, and nuclear and mitochondrial single nucleotide variants and indels in base-resolution. This work demonstrates a novel preimplantation genetic testing (PGT) method that detects aberrations in both the nuclear and mitochondrial genomes of IVF embryos. The technology traces the origin of chromosomal aberrations to before or after fertilisation.

Abstract Study question What is PGT-AO’s impact on implantation and pregnancy rate per embryo transfer, by determining the prevalence and effect of (mosaic) aneuploidies and their segregational origin? Summary answer PGT-AO potentially enhances the efficiency of PGT and increases the pregnancy rate per embryo transfer by minimizing transfers with poor outcomes and improving implantation rates. What is known already Demographic factors such as advanced parental age increases risk of aneuploid offspring, driving demand for advanced PGT. Randomized controlled trials revealed that PGT-A does not increase live birth rates after IVF. PGT-A, the most frequent form of PGT, overlooks the mosaic nature and segregational origin of aneuploidies in IVF embryos. This possibly leads to avoiding the transfer of mosaic embryos and reduces the pregnancy rate per cycle. Prompting this study, five embryos transferred based on PGT-M, led to trisomic pregnancies terminated following invasive prenatal diagnosis. Subsequent genome haplarithmisis revealed that these embryos carried (meiotic) trisomies not previously detected by PGT-M. Study design, size, duration In this retrospective study, we included 1944 embryos (816 PGT-M embryos, 323 couples, 224 indications). Currently, 284 have been processed by genome haplarithmisis-based PGT-AO and analyzed to assess if (mosaic) aneuploidies and segregational origin would have altered transfer decisions and subsequent clinical outcomes. We evaluated our novel PGT- A approach, termed PGT-AO, providing a nuanced understanding of early embryonic development in clinical practice and more responsible PGT-A, potentially leading to viable mosaic embryo transfer. Participants/materials, setting, methods The couples were counseled by clinical geneticists at Dutch University Medical Centers (UMCs), in Maastricht, Amsterdam, Utrecht, and Groningen, and enrolled in the diagnostic PGT-M procedure after signing an informed consent form. Embryos (n = 816) eligible for transfer based on absence of the monogenic aberration of interest by PGT-M were analyzed by haplarithmisis-based PGT-AO including parental information. This information allows for the detection of (mosaic) chromosomal abnormalities and their parental and segregational origin. Main results and the role of chance Of 816 included embryos (323 couples, 224 indications), 284 were found to be suitable for transfer by PGT-M and were analyzed by PGT-AO. Of these, 183 were transferred. Of the transferred embryos, 65 (35.5%) had a positive HCG and 48 (26.3%) resulted in a liveborn. 14.6% (7/48) carried abnormalities following our PGT-AO approach. These abnormalities consisted of mitotic mosaic trisomies, mosaic monosomies, and mosaic duplications and a deletion. Of the transferred embryos, 132 (72.1%) did not result in a liveborn and 115 (62.8%) had negative HCG tests. Of the transferred embryos with negative HCG, 38.3% (44/115) contained a total of 90 abnormalities, consisting of 37 monosomies, 35 trisomies, 12 deletions, 2 duplications, 2 UPD and 1 ring chromosome. The 35 trisomies occurred in 20 embryos, of which 9 were of meiotic origin and 5 of mitotic origin, 1 of both meiotic and mitotic, and 5 could not be determined. Based on these preliminary results, PGT-AO can avoid the transfer of embryos with aneuploidy of meiotic origin and non-mosaic status. In contrast, embryos with aneuploidy of mitotic origin are often mosaic and the abnormality may not be uniformly distributed throughout the blastocyst and could potentially be considered for transfer. Limitations, reasons for caution Haplarithmisis relies on the inclusion of parental DNA to phase the embryonic genome and thus determine the segregational origin of aberrations. Additionally, mitosis and meiosis II trisomies cannot be discerned where a crossover event is absent for a chromosome with trisomy. Wider implications of the findings Implementation of PGT-AO will re-define the current embryo selection system. This enhances PGT by increasing pregnancy rate per embryo transfer and minimizing transfers with poor outcomes, disposal of potentially viable embryos, spontaneous pregnancy losses, and termination of aneuploid pregnancies. This reduces the emotional and financial burdens on families and healthcare. Trial registration number Not applicable

STUDY QUESTION Can generative artificial intelligence (AI) models produce high-fidelity images of human blastocysts?SUMMARY ANSWER Generative AI models exhibit the capability to generate high-fidelity human blastocyst images, thereby providing substantial training datasets crucial for the development of robust AI models.WHAT IS KNOWN ALREADY The integration of AI into IVF procedures holds the potential to enhance objectivity and automate embryo selection for transfer. However, the effectiveness of AI is limited by data scarcity and ethical concerns related to patient data privacy. Generative adversarial networks (GAN) have emerged as a promising approach to alleviate data limitations by generating synthetic data that closely approximate real images.STUDY DESIGN, SIZE, DURATION Blastocyst images were included as training data from a public dataset of time-lapse microscopy (TLM) videos (n = 136). A style-based GAN was fine-tuned as the generative model.PARTICIPANTS/MATERIALS, SETTING, METHODS We curated a total of 972 blastocyst images as training data, where frames were captured within the time window of 110-120 h post-insemination at 1-h intervals from TLM videos. We configured the style-based GAN model with data augmentation (AUG) and pretrained weights (Pretrained-T: with translation equivariance; Pretrained-R: with translation and rotation equivariance) to compare their optimization on image synthesis. We then applied quantitative metrics including Frechet Inception Distance (FID) and Kernel Inception Distance (KID) to assess the quality and fidelity of the generated images. Subsequently, we evaluated qualitative performance by measuring the intelligence behavior of the model through the visual Turing test. To this end, 60 individuals with diverse backgrounds and expertise in clinical embryology and IVF evaluated the quality of synthetic embryo images.MAIN RESULTS AND THE ROLE OF CHANCE During the training process, we observed consistent improvement of image quality that was measured by FID and KID scores. Pretrained and AUG + Pretrained initiated with remarkably lower FID and KID values compared to both Baseline and AUG + Baseline models. Following 5000 training iterations, the AUG + Pretrained-R model showed the highest performance of the evaluated five configurations with FID and KID scores of 15.2 and 0.004, respectively. Subsequently, we carried out the visual Turing test, such that IVF embryologists, IVF laboratory technicians, and non-experts evaluated the synthetic blastocyst-stage embryo images and obtained similar performance in specificity with marginal differences in accuracy and sensitivity.LIMITATIONS, REASONS FOR CAUTION In this study, we primarily focused the training data on blastocyst images as IVF embryos are primarily assessed in blastocyst stage. However, generation of an array of images in different preimplantation stages offers further insights into the development of preimplantation embryos and IVF success. In addition, we resized training images to a resolution of 256 x 256 pixels to moderate the computational costs of training the style-based GAN models. Further research is needed to involve a more extensive and diverse dataset from the formation of the zygote to the blastocyst stage, e.g. video generation, and the use of improved image resolution to facilitate the development of comprehensive AI algorithms and to produce higher-quality images. WIDER IMPLICATIONS OF THE FINDINGS Generative AI models hold promising potential in generating high-fidelity human blastocyst images, which allows the development of robust AI models as it can provide sufficient training datasets while safeguarding patient data privacy. Additionally, this may help to produce sufficient embryo imaging training data with different (rare) abnormal features, such as embryonic arrest, tripolar cell division to avoid class imbalances and reach to even datasets. Thus, generative models may offer a compelling opportunity to transform embryo selection procedures and substantially enhance IVF outcomes.STUDY FUNDING/COMPETING INTEREST(S) This study was supported by a Horizon 2020 innovation grant (ERIN, grant no. EU952516) and a Horizon Europe grant (NESTOR, grant no. 101120075) of the European Commission to A.S. and M.Z.E., the Estonian Research Council (grant no. PRG1076) to A.S., and the EVA (Erfelijkheid Voortplanting & Aanleg) specialty program (grant no. KP111513) of Maastricht University Medical Centre (MUMC+) to M.Z.E.TRIAL REGISTRATION NUMBER Not applicable.

ABSTRACT STUDY QUESTION What are the trends and developments in preimplantation genetic testing (PGT) in 2018 as compared to previous years? SUMMARY ANSWER The main trends observed in this 21st dataset on PGT are that the implementation of trophectoderm biopsy with comprehensive whole-genome testing is most often applied for PGT-A and concurrent PGT-M/SR/A, while for PGT-M and PGT-SR, single-cell testing with PCR and FISH still prevail. WHAT IS KNOWN ALREADY Since it was established in 1997, the ESHRE PGT Consortium has been collecting and analysing data from mainly European PGT centres. To date, 20 datasets and an overview of the first 10 years of data collections have been published. STUDY DESIGN, SIZE, DURATION The data for PGT analyses performed between 1 January 2018 and 31 December 2018 with a 2-year follow-up after analysis were provided by participating centres on a voluntary basis. Data were collected using an online platform, which is based on genetic analysis and has been in use since 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS Data on biopsy method, diagnostic technology, and clinical outcome were submitted by 44 centres. Records with analyses for more than one PGT for monogenic disorders (PGT-M) and/or PGT for chromosomal structural rearrangements (PGT-SR), or with inconsistent data regarding the PGT modality, were excluded. All transfers performed within 2 years after the analysis were included, enabling the calculation of cumulative pregnancy rates. Data analysis, calculations, and preparation of figures and tables were carried out by expert co-authors. MAIN RESULTS AND THE ROLE OF CHANCE The current data collection from 2018 covers a total of 1388 analyses for PGT-M, 462 analyses for PGT-SR, 3003 analyses for PGT for aneuploidies (PGT-A), and 338 analyses for concurrent PGT-M/SR with PGT-A. The application of blastocyst biopsy is gradually rising for PGT-M (from 19% in 2016–2017 to 33% in 2018), is status quo for PGT-SR (from 30% in 2016–2017 to 33% in 2018) and has become the most used biopsy stage for PGT-A (from 87% in 2016–2017 to 98% in 2018) and for concurrent PGT-M/SR with PGT-A (96%). The use of comprehensive, whole-genome amplification (WGA)-based diagnostic technology showed a small decrease for PGT-M (from 15% in 2016–2017 to 12% in 2018) and for PGT-SR (from 50% in 2016–2017 to 44% in 2018). Comprehensive testing was, however, the main technology for PGT-A (from 93% in 2016–2017 to 98% in 2018). WGA-based testing was also widely used for concurrent PGT-M/SR with PGT-A, as a standalone technique (74%) or in combination with PCR or FISH (24%). Trophectoderm biopsy and comprehensive testing strategies are linked with higher diagnostic efficiencies and improved clinical outcomes per embryo transfer. LIMITATIONS, REASONS FOR CAUTION The findings apply to the data submitted by 44 participating centres and do not represent worldwide trends in PGT. Details on the health of babies born were not provided in this manuscript. WIDER IMPLICATIONS OF THE FINDINGS The Consortium datasets provide a valuable resource for following trends in PGT practice. STUDY FUNDING/COMPETING INTEREST(S) The study has no external funding, and all costs are covered by ESHRE. There are no competing interests declared. TRIAL REGISTRATION NUMBER N/A.

Abstract Study question Which are the trends shown in data collection XXIII of the European Society of Human Reproduction and Embryology (ESHRE) PGT Consortium compared with previous years? Summary answer Data collection XXIV, the year 2021, represents valuable data on PGT activity in (mainly) Europe and reports on the main trends observed, being the further expansion of blastocyst biopsy and NGS-based comprehensive testing technologies. What is known already The ESHRE PGT Consortium was set up in 1997 and from that time has been collecting data on PGT-M, PGT-SR and PGT-A. Since 1999, the PGT consortium has collected 105 000 embryo analyses and published 21 previous data collection sets in 17 reports. Since the year 2016 a prospective cycle-by-cycle data collection is in place. Study design, size, duration IVF cycle management and genetic analysis techniques are getting more complex and require more details to be reported. Therefore, ESHRE uses an online data collection system in which data are collected prospectively from oocyte retrieval to analysis, embryo transfer and pregnancy/live birth. Data are collected cycle by cycle on a voluntary basis. Participants/materials, settings, method For the 2021 data, individual centres (36) from 19 countries directly entered the analysis data (n = 3067) into the PGT database through software developed by ESHRE. Data were analysed at ESHRE Central Office and include all aspects of the PGT/PGT-A cycles. Main results and the role of chance The indications for PGT included inherited chromosomal abnormalities (n = 386 analyses), monogenic disorders (n = 1329 analyses), aneuploidy testing (n = 1147 analyses), HLA typing (alone or in combination with PGT-M (n = 4 analyses), mitochondrial disorders (5 analyses) or combinations of the above (n = 111 analyses). 85 analyses were reported without indication. In addition, 894 clinical pregnancies and 496 deliveries have been analysed in detail. The methods used for biopsy were polar body (1%), cleavage stage biopsy (16%) and blastocyst biopsy (83%), showing a continuous increase in blastocyst biopsy compared to 2020 and earlier years. The methodology used for diagnosis is what evolved most over the last years but seems to stay comparable with 2020. Data set XXIV (2021) shows around 4% of FISH, 31% of PCR and 62% of WGA. Within WGA 84% of the analyses were done using NGS, in 8% of the cases SNP arrays were used, PCR accounted for 2.5% of the analyses and in 1% array-CGH was used. In the remaining cases, combinations of the above were used. The overall clinical pregnancy rate of 29% per analysis shows an increase compared to 2020. The baby data show that it is difficult for most centres to have a detailed follow-up. Limitations, reasons for caution The findings apply to the 36 participating centres and may not represent worldwide trends in PGT. Data were collected prospectively, but details of the follow-up on PGT pregnancies and babies born were limited. Wider implications of the findings The ESHRE PGT Consortium continues its activities as an important forum for PGT practitioners to share data and exchange experiences. The information extracted from the data collection helps to monitor quality issues in PGT and survey the introduction and effectiveness of new PGT technologies and methods. Trial registration number XXXX