Embryonic Development Research Articles

P-191 Pronuclear transfer overcomes embryonic developmental arrest in Padi6-deficient mice

Abstract Study question Can pronuclear transfer (PNT) overcome embryonic developmental arrest (EDA) observed in the created Padi6-/- mouse model? Summary answer Applying PNT significantly improved the blastocyst rate of Padi6-/- zygotes, indicating its potential in overcoming female-related infertility. What is known already Various components of the subcortical maternal complex (SCMC), including peptidyl arginine deiminase 6 (PADI6), play a crucial role in early embryonic development and female fertility. Recent studies have revealed that mutations in PADI6 can result in EDA and thus female infertility, which is hard to overcome by current available treatment methods. The PNT technique, designed to replace compromised cytoplasm with a healthy counterpart, has been proposed to overcome specific female-related infertility indications. The Padi6-/- mouse displaying EDA, offers a valuable model to investigate the efficacy of PNT to overcome female-related infertility. Study design, size, duration From April 2022 to December 2023, an experimental study was conducted with CRISPR/Cas9 created Padi6 knockout mice. For fertility evaluation, Padi6-/- and Padi6+/- females were paired with wildtype males. Next, 30 wildtype and 25 Padi6-/- oocytes were collected for parthenogenetic activation. A total of 81, 54 and 145 zygotes were collected from wildtype, Padi6+/- and Padi6-/- females, respectively, for evaluating embryonic development and the effectiveness of PNT in overcoming EDA. Participants/materials, setting, methods The C57BL/6J Padi6-/- mouse model was established by introducing Padi6 c.1577_1578insT with CRISPR/Cas9. Female mice aged 6-12 weeks underwent ovarian hyperstimulation and mated with wildtype males. Oocytes were parthenogenetically activated with SrCl2 in presence of Cytochalasin D, and checked for 2-cell and blastocyst rates. PNT treatment was applied by transferring the 2PN from Padi6-/- to wildtype enucleated zygotes using electrofusion, and the efficiency was evaluated in terms of embryonic developmental potential. Main results and the role of chance After mating Padi6-/- female mice with wild-type males, no offspring could be obtained. In contrast, Padi6+/- females exhibited normal fertility, yielding an average of 7.3 offspring per litter. In addition, Padi6-/- males are also fertile. Twenty-eight wildtype oocytes (n = 30) survived parthenogenetic activation, with 85.71% (24/28) reaching the 2-cell stage, and 75% (18/24) subsequently forming blastocysts. In contrast, all Padi6-/- oocytes (n = 25) survived after activation, but only 20% (5/25) reached the 2-cell stage (P < 0.0001), with none progressing to blastocyst (P = 0.0039). Regarding zygote development, 82.14% (46/56) of wildtype zygotes and 98.14% (53/54) of Padi6+/-embryos reached the 2-cell stage, showing blastocyst rates of 95.65% (44/46) and 100% (53/53) respectively. For zygotes obtained from Padi6-/- females, 72.50% (87/120) of them progressed to the 2-cell stage (P = 0.1908, vs wildtype), but only 4.60% (4/87) reached blastocysts (P< 0.0001, vs wildtype), exhibiting the EDA phenotype. Following PNT to wildtype cytoplasm (n = 25), 23 reconstructed PNT zygotes were obtained. Of these, 91.30% (21/23) reached the 2-cell stage (P = 0.4924, vs wildtype), and 85,71% (18/21) developed to the blastocyst stage (P = 0.3151, vs wildtype), which is equivalent to the wildtype zygotes. Limitations, reasons for caution This study is limited by the small sample size of the PNT, which will still be extended. Next to the blastocyst rate, additional assessment methods such as multi-omics analyses will be employed to assess the efficiency and safety of PNT treatment. Wider implications of the findings This study provides further insight into the potential of the nuclear transfer technology for female-related infertility issues due to cytoplasmic incompetence. For couples experiencing recurrent EDA after ICSI due to inferior oocyte quality, PNT still offers them the possibility of having genetically related children. Trial registration number Not applicable

P-548 An investigation into the mechanisms of active enhancers regulating the initial lineage differentiation in mammals

Abstract Study question What role do active enhancers play in determining the fate of early embryonic cells? Are there differences in active enhancers between mouse and human blastocysts? Summary answer Tissue-specific active enhancers in inner cell mass (ICM) and trophoblast (TE) are essential for blastocyst formation, regulating gene expression through interactive loops with promoters. What is known already At the two-cell stage of nuclear transfer embryos, the absence of interaction between Zscan4d promoter and distal active enhancer leads to reduced Zscan4d expression, resulting in lower developmental rates. Overexpressing the demethylase Kdm4d alleviates reprogramming-resistant H3K9me3 modification, restoring the structural loop between the Zscan4d promoter and active enhancer and enhancing developmental efficiency. These findings strongly suggest a close association between the formation of regulatory chromatin interactions in early embryonic development and histone modifications. Indicating active enhancers marked with H3K27ac may regulate differentiation gene expression through distal interactions in blastocyst. Study design, size, duration Analyze variations in histone H3K27ac modification, chromatin accessibility, higher-order structures, and transcriptomes during mouse embryo differentiation from 8-cell stage to ICM and TE formation. Identify tissue-specific active enhancers in ICM and TE, validate their gene expression regulation, explore their role in early differentiation-associated genes in mice. Compare and clarify similarities and differences in enhancer regulation mechanisms during early embryo development in humans and mice. Participants/materials, setting, methods The 8-cell, morula and blastocyst stage embryos were collected from the oviducts and uterus of super-ovulated female ICR mice mated with male mice. The clinical human blastocysts were collected and separated the ICM and TE. Low-input multi-omics sequencing, including Cut-Tag, ATAC-seq, Hi-C, and RNA-seq, was performed on the embryonic samples. After analyzing and identifying active enhancers, tissue-specific ones were selected and validated for their impact on gene expression via CRISPR-Cas9 knockout in embryos. Main results and the role of chance Inhibiting ep300 expression in mouse embryos through siRNA interference reduces blastocyst formation from 90% to approximately 40%. Introducing the small molecule inhibitor A485 at the 8-cell stage, blocking the active center of CBP/p300 enzymes, completely arrests embryos at the morula stage, preventing their development into blastocysts. Emphasizing the crucial role of correctly establishing active enhancers marked by H3K27ac modification in embryonic differentiation. Multi-omics data demonstrates high repeatability and quality. PCA clustering analysis reveals a clear differentiation path in the distribution of H3K27ac-modified histones, chromatin accessibility sites, and the transcriptome from the 8-cell stage to the blastocyst. Differences in H3K27ac histone modification distribution between the ICM and TE indicate the presence of tissue-specific active enhancers in blastocyst. The disparities in H3K27ac modification distribution between blastocyst ICM and TE primarily occur in distal gene regions rather than proximal ones. Moreover, these distal enhancers exhibit a positive correlation with the expression of lineage-specific genes, suggesting a probable regulatory role of distal enhancers in gene modulation during embryonic differentiation. Tissue-specific enhancer-promoter (EP) interaction loops emerge during embryonic differentiation, allowing direct interaction between tissue-specific active enhancers and promoters, thereby activating the expression of tissue-specific genes. Limitations, reasons for caution While DNA sequences with H3K27ac modification are currently considered markers for active enhancers, H3K4me modification is another histone marker for enhancers. However, enhancers associated with H3K4me modification may not necessarily be in an activated state. Future research can simultaneously incorporate appropriate detection methods for H3K4me modification. Wider implications of the findings After elucidating the mechanisms through which active enhancers regulate mouse differentiation, comparing the differences in active enhancers during the first lineage differentiation process between mice and humans can help identify factors that enhance human blastocyst quality. This can potentially improve clinical outcomes in assisted reproduction. Trial registration number not applicable

A CRISPR/Cas9 screen in embryonic stem cells reveals that Mdm2 regulates totipotency exit

During early embryonic development, the transition from totipotency to pluripotency is a fundamental and critical process for proper development. However, the regulatory mechanisms governing this transition remain elusive. Here, we conducted a comprehensive genome-wide CRISPR/Cas9 screen to investigate the 2-cell-like cells (2CLCs) phenotype in mouse embryonic stem cells (mESCs). This effort led to the identification of ten regulators that play a pivotal role in determining cell fate during this transition. Notably, our study revealed Mdm2 as a significant negative regulator of 2CLCs, as perturbation of Mdm2 resulted in a higher proportion of 2CLCs. Mdm2 appears to influence cell fate through its impact on cell cycle progression and H3K27me3 epigenetic modifications. In summary, the results of our CRISPR/Cas9 screen have uncovered several genes with distinct functions in regulating totipotency and pluripotency at various levels, offering a valuable resource for potential targets in future molecular studies.

P-253 Development of embryos from aged mothers transferred to the uterus of young mice

Abstract Study question Is the development of embryos from advanced maternal age (AMA) mice, similar to those from young maternal age (YMA) mice when transferred to young uteri? Summary answer AMA mouse embryos are characterized by altered organ morphology, particularly of the brain, which may predispose to neurodevelopmental disorders. What is known already Human and animal studies have linked AMA pregnancy to elevated risk of neurodevelopmental disorders and behavioural alterations in offspring, primarily attributed to compromised uterine (maternal) conditions. In human, it was also suggested that postnatal care of older mother may influence offspring behaviour. Animal studies revealed that behavioural alterations of AMA offspring are programmed during pregnancy and so they persist in litters fostered by young mouse. It remains unclear whether an embryo of older mother will undergo normal development after embryo transfer to younger female’s uterus. Study design, size, duration Embryos for Advanced Maternal Age, AMA (n = 105) and control Young Maternal Age, YMA (n = 44) groups were collected from thirty females 8-14 months old and six females 3-5 months old, respectively and transferred to pseudo-pregnant mice for further development. At embryonic day 16 (E16) foetuses were collected, fixed for histology and evaluated using Theiler and Kaufman’s classification of mouse development, where E16 corresponds to Theiler stage 24 (TS24) Participants/materials, setting, methods CBA x C57BL6 hybrid mice were used throughout the experiment. At E16, embryo survival rate (number of foetuses collected / number of transferred embryos) was scored for both groups. Foetuses were assessed for visible abnormalities and underwent histological evaluation of organs accordingly to TS 24. Parameters such as size/diameter, signs of vacuolization, cell density and layers of cerebral cortex in brain of foetuses were analysed. Values were considered different when p < 0.05, Mean±SD; Mann-Whitney test. Main results and the role of chance At E16, the survival of AMA embryos did not differ significantly from YMA control (39/105; 37% vs. 20/44; 45%, respectively) however, 5% of AMA foetuses (2/39) displayed oedema or cerebral haemorrhage. Histological analysis revealed alterations in the head/brain of AMA in comparison to YMA foetuses, such as smaller head size (head area in mid-sagittal section: 25.25±4.87 vs. 31.39±3.79 mm2, p < 0.05), thinner cerebral cortex (435.78±12.95 vs. 515.22±17.78µm, p < 0.005), reduced ratio of cortical plate (CP) to cerebral cortex thickness (0.20±0.03 vs. 0.26±0.009, p < 0.05) and an increased ratio of ventricular zone (VZ) to cerebral cortex thickness (0.29±0.002 vs. 0.24±0.002, p < 0.05). Moreover, decreased cellular density in CP layer (10378.86 vs. 12066.40 cells/mm2, P < 0.05) was noted. AMA brains were characterised by high degree of cellular vacuolisation, particularly in medulla oblongata, suggestive for spongiosis (1550.27 vs. 571.92 vacuoles/mm2, p < 0.05). These brain alterations may cause neurodevelopmental disorders in offspring. AMA foetuses had: smaller bronchial diameter (31.52 vs. 36.62µm, p < 0.05), no ossification centres and reduced skin wrinkling, corresponding to TS23 (that is previous developmental stage). These observations may suggest a delayed development of AMA foetuses. Limitations, reasons for caution The results of controlled animal experiments play a key role in developing conceptual models and formulating hypothesis about target species. However, extrapolation of these results to humans requires caution because species-specific developmental features may differ. Wider implications of the findings Our study demonstrates that advanced maternal age (AMA) impacts oocyte/embryo programming in mice. Morphological changes in AMA-conceived foetuses suggest potential risks for neurodevelopmental and other health issues in offspring, even when carried in the uterus of a young female, emphasizing the far-reaching effects of AMA on progeny health. Trial registration number not applicable

Getting to the Core: Exploring the Embryonic Development from Notochord to Nucleus Pulposus

The intervertebral disc (IVD) is the largest avascular organ of the human body and plays a fundamental role in providing the spine with its unique structural and biomechanical functions. The inner part of the IVD contains the nucleus pulposus (NP), a gel-like tissue characterized by a high content of type II collagen and proteoglycans, which is crucial for the disc’s load-bearing and shock-absorbing properties. With aging and IVD degeneration (IDD), the NP gradually loses its physiological characteristics, leading to low back pain and additional sequelae. In contrast to surrounding spinal tissues, the NP presents a distinctive embryonic development since it directly derives from the notochord. This review aims to explore the embryology of the NP, emphasizing the pivotal roles of key transcription factors, which guide the differentiation and maintenance of the NP cellular components from the notochord and surrounding sclerotome. Through an understanding of NP development, we sought to investigate the implications of the critical developmental aspects in IVD-related pathologies, such as IDD and the rare malignant chordomas. Moreover, this review discusses the therapeutic strategies targeting these pathways, including the novel regenerative approaches leveraging insights from NP development and embryology to potentially guide future treatments.

O-076 Novel method to enhance preantral follicle growth by granulocyte colony-stimulating factor administration improves embryos and pregnancy rate in poor ovarian reserve: a randomized controlled trial

Abstract Study question Whether priming with granulocyte colony-stimulating factor (G-CSF) before ART improves embryo and pregnancy rate while increasing anti-Müllerian hormone (AMH) in patients with poor ovarian reserve. Summary answer G-CSF priming improved embryonic development and pregnancy rate in ART with poor ovarian reserve, simultaneously increasing serum AMH. Enhanced preantral follicle growth likely was responsible. What is known already Eleven years ago, we treated repeated implantation failure in 10 women with diminished ovarian reserve by administering G-CSF with embryo transfers. No implantation succeeded directly, but serendipitously 3 of the 10 spontaneously became pregnant 2 months later. One of these pregnancies involved twins in a 45-year-old woman without ovulation induction. We hypothesized that G-CSF administration might have stimulated preantral follicle growth, with improved ovulation after 2 months. In animal models with diminished ovarian reserve, G-CSF administration consistently increased ovarian preantral follicles and AMH. Therefore, we designed this clinical trial. Study design, size, duration Patients were prospectively randomized to priming or control groups. All patients initially underwent conventional infertility treatment for 2 consecutive cycles in which the priming group but not controls received a subcutaneous G-CSF priming injection during the early luteal phase. Each group then underwent 1 cycle of IVF/ICSI and fresh embryo transfer (IVF/ICSI-fresh ET), followed by cryopreserved ET if needed until live birth or embryo depletion. AMH was measured before and after priming. Participants/materials, setting, methods Hundred ART patients 20 to 42 years old with AMH below 2 ng/mL were randomized to priming or control groups (50 patients each). None had over 1 ART failure, day-3 follicle-stimulating hormone (FSH) above 30 IU/L, uterine anomalies, or a partner with azoospermia. Main results and the role of chance Fertilization rate, embryonic development, and implantation rate by fresh ET were significantly improved by G-CSF priming. Clinical and ongoing pregnancy rates by IVF/ICSI-fresh ET were significantly higher with priming (30% and 26% in 47 ART patients; 3 delivered with conventional treatment) than in controls (12% and 10% in 49 ART patients; 1 dropped out). With priming, significantly more patients achieved cryopreservation of redundant blastocysts (53% with priming vs. 24% in controls). The cumulative live birth rate was 32% in 50 patients with priming, significantly higher than 14% in 49 controls (relative risk, 2.8; 95% confidence interval, 1.04-7.7). Infants derived from priming had no congenital anomalies, while infant weights, birth weeks, and Apgar scores were similar between groups. Among 4 variables (age, day-3 FSH, AMH, and priming), logistic regression significantly associated age and priming with cumulative live birth. Priming significantly increased serum AMH. The efficacy of G-CSF priming was not associated with genotypes for killer-cell immunoglobulin-like receptors. No adverse effects of priming were observed. Limitations, reasons for caution Enhancement of preantral follicle growth by G-CSF administration needs to be confirmed histologically as this clinical trial could not allow ovarian biopsies for its purpose of infertility treatments. Wider implications of the findings This study proposes a novel, simple, and safe treatment for poor ovarian reserve. In addition, enhancement of preantral follicle growth by G-CSF priming may also improve ovulatory dysfunctions in polycystic ovary syndrome with elevated AMH, reflecting pooling of retarded/arrested growing preantral follicles. Trial registration number UMIN000013956

O-039 Con - the faster the better

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

P-127 The RNA m6A landscape of mouse oocytes and preimplantation embryos

Abstract Study question Despite the significance of N6-methyladenosine (m6A) in gene regulation, the requirement for large amounts of RNA has hindered m6A profiling in mammalian early embryos. Summary answer We apply picoMeRIP-seq to map m6A in mouse oocytes and preimplantation embryos. We define the landscape of m6A during the maternal-to-zygotic transition. What is known already N6-methyladenosine (m6A), the most prevalent internal modification in eukaryotic messenger RNA (mRNA), plays key regulatory roles in many biological processes (for example, RNA stability, splicing, transport and translation) and is involved in a variety of physiological processes (for example, cell differentiation and reprogramming, embryonic development and stress responses). Study design, size, duration We recently developed an m6A mapping assay for low RNA input, low-input methyl RNA immunoprecipitation and sequencing (picoMeRIP-seq), which enables mapping of the m6A methylome using a low number of cells. In this article, we applied picoMeRIP-seq to multiple developmental stages of mouse oocytes and early embryos to profile their m6A landscapes. Participants/materials, setting, methods We use mouse as study materials.Using picoMeRIP-seq, we generated transcriptome-wide m6A maps for six stages (GV, MII, zygote, two-cell, eight-cell and blastocyst), with two biological replicates per stage and 49–110 oocytes/embryos per replicate. Main results and the role of chance We define the landscape of m6A during the maternal-to-zygotic transition, including stage-specifically expressed transcription factors essential for cell fate determination. Both the maternally inherited transcripts to be degraded post fertilization and the zygotically activated genes during zygotic genome activation are widely marked by m6A. In contrast to m6A-marked zygotic ally-activated genes, m6A-marked maternally inherited transcripts have a higher tendency to be targeted by microRNAs. Moreover, RNAs derived from retrotransposons, such as MTA that is maternally expressed and MERVL that is transcriptionally activated at the two-cell stage, are largely marked by m6A. Our results provide a foundation for future studies exploring the regulatory roles of m6A in mammalian early embryonic development. Limitations, reasons for caution The effect of m6A on the turnover of retrotransposon-derived transcripts abundant in oocytes and early embryos needs to be further addressed in future studies. Wider implications of the findings Our results provide a foundation for future studies exploring the regulatory roles of m6A in mammalian early embryonic development. Trial registration number not applicable

Effect of Calcium Ionophore on the Embryonic Outcome of the Intracytoplasmic Sperm Injection in Female Patients with Good & Bad Quality Oocytes: Histomorphometric Study

Abstract Introduction Fertilization failure is the most common problem in assisted reproductive technology . In incident of ovum and/or spermatozoa correlated fertilization difficulties after intracytoplasmic sperm injection, approaches of modified ICSI techniques, which can be done using Ca 2+ ionophores. The objective of the research Effectiveness of Ca 2+ ionophores was studied on the embryonic outcome in female patients with bad quality oocytes while her partner has normal semen analysis. Materials and Methods Thirty-eight couples have undergone ICSI, with female age ranging between 40 to 45 years old. The retrieved oocytes were subdivided into 4 groups; good quality oocytes, good quality ovums with adding Ca 2+ ionophore treatment, bad quality ovums in addition, bad quality ovums with adding Ca 2+ ionophore treatment, all were inserted by the corresponding sperms. Examination of the embryos was done on day two after fertilization, cleavage stage, and blastulation stage. Results Treated good quality oocytes showed fertilization of (92%) of the oocytes compared to (68%) of the untreated good quality oocytes. Treated bad quality oocytes showed fertilization of (85%) of the oocytes compared to (65%) of the untreated bad quality oocytes. The quality of embryos was enhanced on day two after fertilization, day three (cleavage stage), and day five (blastulation stage) in both treated groups than in the untreated ones. Conclusion The activated oocytes resulted in better fertilization and embryonic development. Therefore, ICSI combined with artificial oocyte activation using Ca 2+ ionophore is useful in mid-age female patients with good and bad quality oocytes regardless the cause.

P-637 Different diffusion of Bisphenol-A from serum to follicular fluid in women undergone IVF cycles. EcoFoodFertility for Women Project

Abstract Study question BisphenolA (BPA) is used as a raw material in the production of polycarbonate plastics and epoxy resins, today it is considered an endocrine disruptor. Summary answer BPA diffusion from serum to the follicular fluid is not homogenous in women both with regular menstruation or with pathologies of the reproductive system. What is known already It is well established that BPA is present in serum and urine of women who permanently live in areas with a high environmental impact. Many studies report different diffusion of BPA in biological fluids, in fact, it is not clear how BPA bioaccumulates in tissues. Despite the large number of animal experiments in this regard, there are few human studies on BPA levels in various body fluids, such as FF (Follicular fluid). Furthermore, there are few report in literature of BPA effects on the endocrine system, oocyte morphology and embryonic development. Study design, size, duration We enrolled 118 women underwent to a first IVF cycle to evaluate the diffusion of BPA between serum and Ff for a multicenter study. Group A, 49 women with regular menstruation and ovulation; Group B, 31 women with regular menstruation and ovulation but they took cortisone before the oocyte pick-up; Group C, 38 women of which 14 with ovarian endometriosis and 24 with PCOS. The age was homogeneous in the three groups, 28.7±4.1. Participants/materials, setting, methods We obtained serum and FF from women who underwent to IVF cycles, in the period between April 2017 and March 2019. BPA concentration was performed with ELISA enzyme immunoassay kit and the values are expressed in ng/mL. It was used 5 mL glass, BD Vacutainer® USP Type III, without silicon, red-capped, additive-free tubes and held horizontally. Follicular fluids were bloodless with negative albumin test. Main results and the role of chance In all samples examined (serum and FF) BPA was in different concentrations, the highest values were: 99,3 ng/mL in the serum and 85,2 ng/mL in the FF, while the lowest values in serum 42,5ng/mL and in FF 10,9 ng/mL.In group A, there is a significant diffusion of BPA from the serum to the FF (p < 0.0001), with concentrations on average lower in the FF: BPA serum (ng/mL) 68,5±14,1, BPA FF(ng/mL) 42,4±12,8. In group B, a higher BPA concentration emerged on average in the FF compared to the serum, maybe the diffusion is facilitated by the use of cortisone (p < 0.005):BPA serum (ng/mL) 65,8±13,4, BPA FF(ng/mL) 74,3±9,2. In Group C, it emerged that the concentrations of BPA in the FF were lower than in the serum, probably due to a greater resistance of the blood-follicular barrier to the passage of BPA (p < 0.0001): BPA serum (ng/mL) 83,4±14,1, BPA FF(ng/mL) 38,7±19,2. Limitations, reasons for caution This study highlights the different mechanism of BPA diffusion in tissues. Further studies are needed to understand the reason for a lower diffusion of BPA in the FF in the presence of pathologies of the reproductive system. More data are needed to support the mechanisms of BPA bioaccumulation in FF. Wider implications of the findings From our data emerges that the use of cortisone in the period preceding the oocyte pick-up seems to promote the diffusion of BPA in the FF. We can hypothesize that this mechanism is also valid for other tissues and it could be one of many mechanisms of BPA bioaccumulation. Trial registration number 001

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

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

P-107 Sperm DNA fragmentation impacts embryonic developmental potential but is not associated with PGT-A outcomes

Abstract Study question Does sperm DNA Fragmentation Index (DFI) affect embryonic development and influence Preimplantation Genetic Testing for Aneuploidy (PGT-A) outcomes in IVF? Summary answer DFI adversely affects early embryonic development, diminishing blastocyst potential. Nonetheless, PGT-A outcomes are unaffected, underscoring the importance of selecting optimal sperm for enhanced IVF results. What is known already Several major factors are thought to cause DFI. The relationship between sperm DFI and IVF results remains debated. Study design, size, duration This study was conducted from January 2018 to March 2023, included 56 married couples undergoing IVF after performing a sperm DFI test. The DFI was assessed at the first semen examination and categorized into three groups: low (≤15%), moderate (15-30%), and high (≥30%). Participants/materials, setting, methods Intracytoplasmic sperm injection (ICSI) was performed on 1006 mature oocytes, followed by blastocyst culture. A total of 278 blastocysts meeting freezing criteria were vitrified. Trophectoderm biopsies from 216 blastocysts underwent Next-Generation Sequencing for aneuploidy analysis. Statistical analysis utilized the Cochran-Armitage test to compare outcomes among the DFI groups. Main results and the role of chance The average age of the husbands and wives who underwent IVF after the DFI examination was 42.3±4.2 and 41.4±2.4 years old. Normal fertility rates after ICSI in low, moderate, and high DFI were 85.5%, 84.5%, and 83.1%, with no significant difference. Similarly, the blastocyst freezing rates were 35.4%, 26.3%, and 29.6%, and tended to decrease as the DFI value increased (p < 0.05). However, the euploid rates for PGT-A were 22.4%, 16.3%, and 18.2%. There were no statistically significant differences between the DFI groups in terms of euploidy. Limitations, reasons for caution Cases declining ICSI due to patient preference or historical medical reasons were excluded from this study. Wider implications of the findings This suggests that sperm DNA imperfections have a negative impact on the developmental process during the early stages of division, but during late stages of division, the ability to repair DNA works to differentiate into blastocysts with normal developmental potential. Trial registration number not applicable

P-190 Impact of low lactate culture media in embryo development and IVF outcomes

Abstract Study question Can low lactate culture media improve embryo utilization rate in artificial reproductive technology (ART) cycles? Summary answer Embryos cultured in low lactate media showed a significant improved blastocyst utilization rate compared to embryos cultured in standard culture media. What is known already Culture media plays a key role in the embryonic development in the IVF lab. Currently, different culture medias are commercially available, yet no consensus on the most suitable media composition has been reached. Recently, minimizing metabolic stress for the embryo to enhance implantation and pregnancy rates has been suggested. Research on the metabolism of mammalian preimplantation embryos revealed that excess lactate in the culture medium, in addition to pyruvate and glucose, can burden metabolic efficiency, as embryos naturally produce lactate during energy production. Study design, size, duration The prospective, monocentric study was designed and conducted at the Kinderwunsch Institut Schenk GmbH (Dobl, Austria). A total of 60 women with an average age of 32.5 undergoing ICSI (intracytoplasmic sperm injection) treatment were included. All of them had at least 6 mature (MII) oocytes at time of insemination. Sibling oocytes were cultured in two different culture medias and outcomes were compared. Patients undergoing preimplantation genetic testing were excluded. Participants/materials, setting, methods Mature (MII) oocytes of each patient (total 621 MII oocytes) were inseminated by ICSI and then equally split into two culture medias: standard (313 oocytes) and low lactate (308 oocytes) covered with mineral-oil overlay and incubated in time-lapse under the same conditions. A total of 428 embryos were obtained. Embryos were evaluated using morphokinetic criteria. The fertilization rate, embryo utilization rate, pregnancy rate and time to Blastocyst were analyzed. Main results and the role of chance No differences were observed in the number of fertilized oocytes (n = 211 and 217 in the standard and low lactate media, respectively). The overall embryo utilization rate was significantly different (p < 0.008) resulting in 52% in standard media and 64.3% using low lactate media. Interestingly, age stratification (18-30; 31-35; 36-41) revealed overall higher embryo utilization rates when using low lactate media in all age groups. Blastocyst utilization rate was significantly different (p < 0.007) 43,1% and 58,05% in the standard and low lactate media, respectively. Regarding the time to blastocyst, it was observed that in the standard culture media 80% of the blastocysts reached blastocyst stage on D + 5 whereas in the low lactate media 92,07% reached blastocyst stage on D + 5, which was also significantly different (p < 0.001). No differences in pregnancy and live birth rates were observed. Limitations, reasons for caution The number of patients may be seen as a study limitation. Results should be confirmed with a bigger sample size. Wider implications of the findings Low lactate media could help improve IVF outcomes due to higher embryo utilization rate leading to better cumulative pregnancy rates. Excessive lactate concentration in culture media could negatively influence embryo metabolism. Trial registration number not applicable

P-103 Sperm selection for ICSI by rheotaxis and thermotaxis in In-Situ handmade microfluidics of fluidic walls: preliminary results of a Randomized Controlled Trial

Abstract Study question Does sperm selection by rheotaxis and thermotaxis in In-Situ handmade microfluidics of fluidic walls optimize ICSI outcomes and workflow? Summary answer In-Situ handmade rheo/thermotaxis microfluidics of fluidic walls is as effective and more time-efficient than centrifugation density gradients to support pre-implantation embryonic development to blastocyst. What is known already Microfluidics technologies stand as the latest sperm selection methodology for ICSI. To outline their application in ART we conceived a lab-on-a-chip approach for sperm selection by In-Situ handmade microfluidics of fluidic walls (isM) in the same ICSI plate which integrates two operative zones: one to allow seminal plasma dispersion and sperm thermotaxis and another one to allow sperm rheotaxis. Our microfluidics system can be prepared with any media suitable for ICSI, disregarding centrifugation, washing, plasticware and other cells. The isM protocol proved non-inferior to centrifugation density gradients (CDG) to support embryonic development and implantation in a previous sibling-oocyte study. Study design, size, duration The present RCT was approved by our ethical committee for research with medical products (CEIm approval number 2023/394). This preliminary analysis aims to detect deviations in the pre-defined primary outcomes: Normal Fertilization Rate, Day 5/6 usable blastocyst rate per MII, and per zygote. This analysis includes data from 21 couples undergoing an ICSI treatment in our center during December 2023-January 2024. Only patients without clinical or analytical suspicion of sperm DNA fragmentation were included. Participants/materials, setting, methods Patients were randomly allocated to isM (n 9) or to CGD (n 12) groups. All participants signed an informed consent. Sperm samples were processed directly after liquefaction. For CGD, samples were prepared in 80-40% PureSperm® layers by isopycnic centrifugation followed by washing and dilution steps. The isM system was prepared by displaying three microdroplets of different volumes following a L-shape interconnection and allowing a temperature gradient of 2ºC between the first and the second microdroplet. Main results and the role of chance Sperm characteristics showed the following total mean values for the isM and CGD groups, respectively: concentration (58,2±38,0 × 106/ml and 52,7±29,1 × 106/ml) and progressive sperm concentration (16,6±17,0 × 106/ml and 16,8±13,4 × 106/ml). Women average age was 36,7±2,9 isM and 35,8±2,5 CDG. This analysis includes a total of 168 MII oocytes (n = 69 isM and n = 99 CDG), with a mean number of MII oocytes per patient of 7,4±2,1 isM and 8,3±1,9 CDG. Comparisons between the two groups showed that isM protocol optimizes ICSI outcomes by means of Normal Fertilization Rate: 88,4% vs 76,8%; p < 0,05, Day 5 A/B grade blastocyst rate: 36,1 vs 31,6%; ns, Day 5/6 usable blastocyst rate per MII: 58,0% vs 44,4%; ns and Day 5/6 usable blastocyst rate per diploid zygote: 65,6% vs 57,9%; ns. The isM protocol also optimizes the IVF workflow compared to CDG, as the time to obtain sperm for ICSI after liquefaction was 26 vs 77 minutes, respectively. Limitations, reasons for caution The study aims for a total size of 50 ICSI cycles to validate statistically our data at a power of 80%. The isM protocol depends on a free hand preparation. A ready-to-use surface would benefit the operability, albeit the use of templates and the training reduces inter and intra-operator variability. Wider implications of the findings The potential benefits of the isM protocol on fertilization could be significant for patients with a limited number of available oocytes for ICSI. According to the literature, microfluidics reduce the DNA fragmentation index in the selected sperm fraction. The isM method could be especially beneficial in patients with this pathology. Trial registration number NCT number assignment pending

P-203 Training of an artificial intelligence (AI) model to automatically detect nuclear errors during first cleavages

Abstract Study question Can an artificial intelligence (AI) algorithm be developed to automatically detect nuclear errors through dynamic images of first embryonic cleavages? Summary answer The AI model showed an accuracy and precision of above 60% in automatically detecting multinucleation in dynamic images. What is known already Implementation of dynamic monitoring by time-lapse (TL) incubators intends to enhance embryo selection, potentially leveraging algorithmic assistance. During embryonic development, certain morphological nuances, often overlooked in intermittent evaluations, become more apparent through TL monitoring. Some of these features are related to faulty karyokinesis in blastomeres, recognized as nuclear errors, which are linked to aneuploidy and reduced live-birth potential. Despite their significance in embryo selection, existing computer imaging AI models lack the ability to automatically detect these features. This research aims to develop the YOLO AI model’s ability to autonomously identify nuclear errors in dynamic embryonic cleavage imaging. Study design, size, duration Videos of 364 embryos recorded with four different TL incubators (Geri-Genea BiomedX, MIRI-ESCO, EmbryoScope/EmbryoScopePlus-Vitrolife) were randomly sampled from retrospective data. Videos started around the time of pronuclei fading (tPNf) and ceased at t5 (from 20hpi-50hpi). Each frame of each video was annotated by a senior embryologist. A total of 13246 frames were gathered with a resolution of 512x512 pixels, from which 9411 were used to train a YOLO model, 1977 for validation and test phases. Participants/materials, setting, methods For each frame, the embryologist outlined a box around the single nucleus observed in a blastomere and labelled it as “Normal”, and as “Multinucleated” if more than one nucleus or micronuclei were seen. 8% of the training dataset was composed of frames with no visible nucleus and 19% contained at least one multinucleated cell. The model thus further detected the presence of nuclei in a frame and classified it as “Normal” or “Multinucleated” when found. Main results and the role of chance Inferences and metrics were computed at the level of the embryo. No images of test videos were used in training. An embryo was considered “Normal” if no multinucleated cells were detected anywhere during the video. If at least one nuclear error was labelled, the embryo was considered as “Multinucleated”. To get a prediction at the embryo level, we considered the class predicted with the highest confidence during the video by the model. Another rule was to consider the embryo as “Multinucleated” if at least one cell was predicted as “Multinucleated” with a confidence above 0.5. When evaluated on the test dataset, the AI model reached 67% accuracy at detecting multinucleated embryos, with a precision of 63%, correctly identifying 61% of the multinucleated embryos. Conversely, when the model predicted that the embryo did not have any nuclear errors, it was right 61% of the time. Limitations, reasons for caution To reach the current set of results, the present model underwent previous multiple interfaces using several hundreds of embryos, all only trained by a single experienced embryologist. The model must undergo a cross-validation involving embryologists with varying levels of experience to assess its performance with a high degree of precision. Wider implications of the findings Continuous training and refinement of algorithms, informed by the accumulated knowledge of impacting features, play a crucial role in the evolution of automated systems. As these methods evolve, they will further refine embryo selection, improve outcomes, and contribute to the ongoing advancement of IVF. Trial registration number not applicable

P-022 Klinefelter syndrome couples are expected higher live birth rates than unexplained NOA couples after ICSI with testicular spermatozoa

Abstract Study question What are sperm retrieval rates (SRR) by microdissection TESE (micro TESE), fertilization rates, and pre- and post- implantation development in Klinefelter syndrome (KS) couples? Summary answer In KS couples, SRR, clinical pregnancy rates (CPR) per embryo transfer (ET), and live birth rates (LBR) per patient were higher than unexplained NOA couples. What is known already Micro TESE, in combination with ICSI, is presently used to treat infertility in cases of NOA including KS, which is the most common sex-chromosome disorder among infertile males, with a prevalence of 1 in 660 men and is a frequent cause of hypogonadism and infertility. There were few reports regarding ICSI outcomes in the couples of KS. The aim of this study is to assess the prevalence and the significance including SRR by micro TESE of ICSI outcomes with embryonic development in KS couples. Study design, size, duration A retrospective study was conducted in 212 patients with non-mosaic KS and 963 unexplained NOA patients with 46, XY without past history (unexplained NOA; not including after orchidopexy, KS, cryptozoospermia, post chemotherapy, mumps orchitis, etc) who underwent micro TESE and ICSI with fresh or cryopreserved testicular spermatozoa for their wives between September 2013 to November 2023. Participants/materials, setting, methods A total of 2204 azoospermic patients were examined for chromosomal analysis. We evaluated SRR of microdissection TESE, two pronuclei (2PN) rates, blastocyst development rates, good-quality blastocyst (Grade 3BB and above on day 5 by the Gardner scoring) rates, CPR per ET, and LBR per couple patient who underwent micro TESE. We did not undergo preoperative hormonal therapy for KS patients. Statistical analysis was performed using unpaired t-tests and chi-squared tests. Main results and the role of chance We identified 212 KS out of 2204 azoospermic patients (9.6%). SRR of the first attempt micro TESE in KS (91/175=52.0%) was significantly higher than unexplained NOA (166/768=21.6%) (p < 0.001). 2PN, blastocysts development, and good-quality blastocysts rates were 49.2%, 42.9%, 16.9% in KS and 50.8, 45.8%, 21.0% in unexplained NOA respectively. Only good-quality blastocysts rate of KS was significantly lower than that of unexplained NOA (p < 0.01). Taking into account deflection of wives age, each result was calculated at their age 38 or under, but they were the same. CPR and LBR per ET in KS (42.6%, 30.4%) were significantly higher than unexplained NOA (33.1%, 23.7%) (p < 0.01, p < 0.05). The couples were 26.4% (56/212) in KS and 12.0% (116/963) in unexplained NOA (p < 0.001) who have given birth or ongoing pregnancy up to now after underwent micro TESE. That is, KS couples were significantly easier to give birth than unexplained NOA. Rates of congenital disorders had no significant difference between KS and unexplained NOA. Limitations, reasons for caution We did not show the data using ejaculated sperm with KS. The natal outcomes and development of these children has not been fully investigated, long-term follow-up of babies of the KS couples is needed. Wider implications of the findings Micro TESE is particularly helpful for KS cases, but embryonic development depend on the quality of spermatozoa. However, if a blastocyst can be obtained, the expectation of having a child is considered to be high. These results were shown to be useful in informing azoospermic couples undergoing micro TESE. Trial registration number not applicable

Variation and variability in skeletal ossification of the gray short-tailed opossum, Monodelphis domestica

Abstract By reconstructing and comparing the sequence of ontogenetic (embryonic development and post-natal growth) events across species, developmental biologists have gained unique insights into the key processes underlying the evolution of modern lineages and their extinct relatives. However, despite the importance of intraspecific variation to evolutionary transformation and lineage divergence, variation in the sequence of developmental events is seldom acknowledged. Thus, how much variation or variability should be expected during ontogeny remains poorly understood and it is an open question to what extent it impacts interspecific comparisons of developmental patterns. To address this crucial question, we studied the skeletal development of the important biomedical and developmental model organism, Monodelphis domestica. We investigated cranial, forelimb, and hindlimb elements using Ontogenetic Sequence Analysis (OSA) to quantify and assess the full range of variation and variability in the sequence of ossification. Our study documented that previously unrecognized variation exists during M. domestica ontogeny — with over 5000 sequences for the full 92 event analysis. Further, OSA revealed unexpectedly high variability (i.e., the propensity to express variation) in the sequence of ossification for the skull and across the entire skeleton. Reconstructed modal sequences were generally in agreement with previously recognized patterns, including earlier ossification of the facial skeleton and a slight offset between forelimb and hindlimb development. However, the full range of variation shows that the majority of specimens in our analysis followed developmental trajectories distinct from those recovered by prior studies. This level of variation is quite remarkable and demonstrates the importance of assessing intraspecific ontogenetic variation. By quantifying sequence polymorphism and studying how developmental variation and variability differ among species, we can clarify more precisely how developmental patterns differ among species and gain insights into how ontogeny itself evolves.

From promoter motif to cardiac function: A single DPE motif affects transcription regulation and organ function in vivo.

Transcription initiates at the core promoter, which contains distinct core promoter elements. Here, we highlight the complexity of transcriptional regulation by outlining the effect of core promoter-dependent regulation on embryonic development and the proper function of an organism. We demonstrate in vivo the importance of the downstream core promoter element (DPE) in complex heart formation in Drosophila. Pioneering a novel approach utilizing both CRISPR and nascent transcriptomics, we show the effects of mutating a single core promoter element within the natural context. Specifically, we targeted the downstream core promoter element (DPE) of the endogenous tin gene, encoding the Tinman transcription factor, a homologue of human NKX2-5 associated with congenital heart diseases. The 7bp substitution mutation results in massive perturbation of the Tinman regulatory network orchestrating dorsal musculature, manifested as physiological and anatomical changes in the cardiac system, impaired specific activity features and significantly compromised viability of adult flies. Thus, a single motif can have a critical impact on embryogenesis and, in the case of DPE, functional heart formation.

P-113 Comparison of mitochondrial function between expanded and hatching human blastocysts

Abstract Study question Is there a difference in mitochondrial function between expanded and hatching fresh and vitrified blastocysts? Summary answer Freezing and thawing may lead to mitochondrial dysfunction. What is known already Mitochondria, as crucial cellular organelles, play role in energy production through the electron transport chain, cell motility and organelle movement. They also have a significant impact on embryonic development. Furthermore, during the process of embryonic hatching, the energy generated by these organelles can affect ion pump activity, blastocyst expansion, and collapse. Therefore, factors that stress embryonic mitochondria may contribute to embryogenesis and hatching. Cooling and warming, as stress factors, maybe result in organelle damage, protein denaturation, membrane disruption, and DNA fragmentation. Additionally, they can cause changes in mitochondrial function, leading to decreased ATP levels and increased ROS levels. Study design, size, duration In this study, vitrified and fresh human 8-cell embryos were collected from April 18, 2021, to July 19, 2023. The embryos were cultured until the blastocyst stage, and then four samples were assessed in four groups. These groups included vitrified expanded blastocysts, vitrified hatching blastocysts, fresh expanded blastocysts, and fresh hatching blastocysts. Participants/materials, setting, methods Vitrified embryos were sourced from the embryo bank of the Royan Institute, while fresh embryos were obtained from good-quality 3PN embryos or excess embryos from couples who did not wish to freeze them. A stock solution containing lipophilic cationic JC-1 dye in dimethyl sulfoxide was prepared. Blastocysts were then exposed to a concentration of 1 μg/ml of JC-1 diluted in HamsF10-HSA. After washing with HF-HSA, imaging was performed using a 2-photon confocal microscope. Main results and the role of chance Our results showed that mitochondrial membrane potential in expanded blastocysts of both fresh and vitrified eight-cell embryos was significantly higher than in hatching blastocysts of fresh and vitrified embryos (P < 0.05). However, there was no significant difference in mitochondrial function between the vitrified and fresh groups, neither in expanded nor hatching blastocysts. Limitations, reasons for caution Due to the human origin of the samples, the minimum necessary replicates were considered for each experimental group. Wider implications of the findings In this study, it appears that plump zona breakers can be detected in hatching blastocysts using confocal microscopy. Previous studies, such as the one conducted by Sathananthan, identified these cells using a TEM microscope. Trial registration number -

O-273 A transcriptomic approach for embryo selection after preimplantation genetic diagnosis for aneuploidy

Abstract Study question Is it possible to predict an euploid chromosomal constitution from RNA-Seq data and identify a transcriptomic profile compatible with extended embryonic development? Summary answer It has been possible to obtain a karyotype comparable to PGT-A, in addition to acquiring a transcriptomic signature of embryos suggestive of improved implantation capacity What is known already Conventional assessment of embryo competence, based on morphology and morphokinetic, lacks knowledge of molecular aspects and faces controversy in predicting ploidy status. Understanding the embryonic transcriptome is crucial, as gene expression influences development and implantation. Preimplantation genetic testing (PGT) has improved pregnancy rates, but problems persist when high-quality euploid embryos do not reach term. In fact, around 50 to 60% implant, of which 10% miscarry. Comprehensive approaches, including RNA-Seq, offer potential to discover molecular markers of reproductive competence. Extended-embryo culture platforms up to day 14 can be utilized as a proxy to study embryo development at post-implantation stages Study design, size, duration Prospective pilot cohort study conducted from March 2023 to August 2023. A total of 30 vitrified human blastocysts with previous PGT-A diagnosis on day 5 (D5) or day 6 (D6) of development were analysed: n = 15 euploid and n = 15 aneuploid embryos. Finally, 21 embryo samples were included in the study (n = 10 euploid and n = 11 aneuploid), the rest (n = 9) were excluded due to poor quality after sequencing. All embryos were donated for research purposes Participants/materials, setting, methods Following warming and re-expansion, embryos underwent a second trophectoderm biopsy. The remaining embryo was cultured until day 11 to assess its development. After biopsy analysis by RNA-Seq, karyotypes were studied, as well as differential expression gene (DEG) of the comparison: unattached embryos (n = 12) vs embryos attached (n = 9) to the plate (padj<0.05 and FC > 1). In addition, we obtained a transcriptomic signature specific to embryos with a “theoretical” capacity for sustained implantation (raw p-value<0.05 and shrunk LFC>1.1) Main results and the role of chance Digital karyotype concordance was obtained with a sensitivity of 81%, a specificity of 83%, a Kappa index of 65.5% and an area under the ROC curve of 90%. At the gene level, 76 DEGs (56 upregulated and 20 downregulated) were found in the comparison of embryos unattached to the plate in extended culture versus those that were attached. To address the functional implications of these differences, significantly deregulated pathways according to GO and KEGG categories were identified. The mural trophectoderm of the analysed blastocysts showed 63 significantly deregulated terms (30 GO-Biological Process, 13 GO-Cellular Component, 12 GO-Molecular Function and 8 KEGG pathways), displaying upregulation of autophagy, apoptosis, protein kinase and ubiquitin-like protein ligase activity, and downregulation of ribosome, spliceosome, kinetochore, segregation, and chromosome condensation processes. The first described transcriptomic signature specific to embryos with theoretical enhanced implantation capacity consists of 501 genes, including: EMP2, AURKB, FOLR1, NOTCH3, LRP2, FZD5, MDH1, APOD, GPX8, COLEC12, HSPA1A, CMTM7, BEX3, which are related to implantation and embryonic development. These findings indicate that it would be possible to identify euploid embryos with a greater capacity for implantation and development, excluding those embryos that, although they could be implanted and viable, present chromosomal alterations Limitations, reasons for caution This study included a small sample size and a remarkable variability between samples. Structural chromosomal abnormalities were not included. It is not possible to diagnose mosaic embryos. Trophectoderm biopsy does not assure the chromosomal status of the whole embryo. Maximum number of days for in vitro development was 11 Wider implications of the findings Gene expression profiles of embryos with theoretical increased implantation capacity have been obtained. Clinical implementation of this technique, which assesses chromosomal content and transcriptomic profile, could be used to significantly increase clinical outcomes in assisted reproduction, allowing informed decisions to be made about future treatments and the prospects of success Trial registration number Not applicable