Preclinical Validation Research Articles

OC 36.2 Machine-Learning Based Risk Prediction of Cancer Associated Thrombosis: Internal, External, and Pre-Clinical Validation Studies

OC 36.2 Machine-Learning Based Risk Prediction of Cancer Associated Thrombosis: Internal, External, and Pre-Clinical Validation Studies

Multi-camera optical tracking and fringe pattern analysis for eye surface profilometry in ocular proton therapy

Background and purposeAn optical tracking system for high-precision measurement of eye position and orientation during proton irradiation of intraocular tumors was designed. The system performed three-dimensional (3D) topography of the anterior eye segment using fringe pattern analysis based on Fourier Transform Method (FTM). Materials and methodsThe system consisted of four optical cameras and two projectors. The design and modifications to the FTM pipeline were optimized for the realization of a reliable measurement system. Of note, phase-to-physical coordinate mapping was achieved through the combination of stereo triangulation and fringe pattern analysis. A comprehensive pre-clinical validation was carried out. Then, the system was set to acquire the eye surface of patients undergoing proton therapy. Topographies of the eye were compared to manual contouring on MRI. ResultsPre-clinical results demonstrated that 3D topography could achieve sub-millimetric accuracy (median:0.58 mm) and precision (RMSE:0.61 mm) in the clinical setup. The absolute median discrepancy between MRI and FTM-based anterior eye segment surface reconstruction was 0.43 mm (IQR:0.65 mm) ConclusionsThe system complied with the requirement of precision and accuracy for image guidance in ocular proton therapy radiation and is expected to be clinically tested soon to evaluate its performance against the current standard.

Pre-Clinical Validation of Targeting Cholesterol Metabolism for Radiotherapy Sensitization in Head and Neck Cancer

This study examines the potential benefits of targeting cholesterol metabolism in combination with radiation therapy (RT) for treating Head and Neck Squamous Cell Carcinoma (HNSCC). Despite advancements in RT delivery, 60% of patients still experience a relapse within 2 years, highlighting the need for improved treatment strategies. Our hypothesis is that a specific, biological response to RT by a subset of tumor cells drives radio-resistance and recurrence, and that targeting cholesterol metabolism may be an effective way to improve RT efficacy. To identify radiation-induced biological responses, whole-genome RNA sequencing analysis was carried out on Cal27 (HPV-) and UMSCC47 (HPV+) cell lines after X-Ray (6MeV Linac) or proton (pencil beam scanning, center part of SOBP) radiations. Potential radio-resistance targets were identified by comparing RT-induced transcriptomic changes with publicly available GEO and TCGA transcriptomic data from human HNSCC samples. Cholesterol metabolism as a driver of HNSCC radio-resistance was validated in vitro by pharmacological and genetic approaches. Transcriptomic analysis revealed a significant increase in 11 genes related to sterol and cholesterol in both the Cal27 and UMSCC47 cell lines after radiation exposure (pathway functional enrichment analysis, p = 2.05e-05). High expression of these RT-induced cholesterol gene signatures correlated with decreased overall survival among HNSCC patients based on the TCGA database (log rank p = 0.022, HR = 1.4). Moreover, a Gene Set Enrichment Analysis of the GSE23036 dataset (pre-treatment HNSCC tumors: locoregional recurrence vs. nonrecurrence) also linked cholesterol metabolism and local recurrence (p = 0.002, FDR = 0.07, NES = 1.72). Single-cell RNA sequencing data from the GSE164690 dataset revealed high expression of cholesterol metabolism genes in human HNSCC epithelial and mesenchymal fractions. In vitro studies demonstrated that RT increased the nuclear translocation of the master transcription factors controlling cholesterol gene expression SREBP2 and increased the expression of the SREBP2 target gene SQLE at the protein level. In accordance with this result, RT increased cellular cholesterol content and uptake in human (p = 0.027) and murine (p = 0.015) HNSCC cell lines. Drug or genetic targeting of SREBP2 reduced cellular cholesterol levels in HNSCC cells and increased toxicity when combined with RT in both 2D and 3D cell cultures compared to RT alone. Targeting cholesterol metabolism in combination with RT could be beneficial for treating HNSCC. In vivo validation in immunocompetent mouse models and orthotopic patient-derived xenograft models is ongoing.

Development of Engineered-U1 snRNA Therapies: Current Status.

Splicing of pre-mRNA is a crucial regulatory stage in the pathway of gene expression. The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. Targeting aberrant RNA(s) may thus provide an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. To that purpose, the use of engineered U1 snRNA (either modified U1 snRNAs or exon-specific U1s-ExSpeU1s) has been applied as a potentially therapeutic strategy to correct splicing mutations, particularly those affecting the 5' splice-site (5'ss). Here we review and summarize a vast panoply of studies that used either modified U1 snRNAs or ExSpeU1s to mediate gene therapeutic correction of splicing defects underlying a considerable number of genetic diseases. We also focus on the pre-clinical validation of these therapeutic approaches both in vitro and in vivo, and summarize the main obstacles that need to be overcome to allow for their successful translation to clinic practice in the future.

In vitro anticancer efficacy of Cyperus rotundus (L.) on breast adenocarcinoma cells via the induction of DNA fragmentation and apoptosis

Introduction and Aim: Cyperus rotundus (L.), commonly known as nutgrass, has been in use in Indian medicine traditional system against several ailments. This investigation was performed to assess the in vitro antineoplastic activity of extracts prepared from water and ethanol with C. rotundus rhizomes. Materials and Methods: Toxic effects induced by the C. rotundus extracts on MCF-7 breast adenocarcinoma cells was evaluated using Sulphorhodamine- B (SRB) Assay, Trypan blue assay, Hoechst nuclear staining assay, Acridine Orange-Ethidium bromide (AO-EB) differential fluorescence staining assay and DNA fragmentation assays. Results: The results of SRB assay indicated the IC50 value of aqueous extract as 510.887 µg/mL and that of ethanol extract as 122.98 µg/mL. Trypan blue assay also indicated the similar results, wherein ethanol extract exerted higher cytotoxicity compared to the aqueous extract. MCF-7 cells treated with aqueous and ethanol extracts of C. rotundus rhizomes were exposed to Hoechst staining and AO-EB nuclear staining assays, wherein and ethanol extract induced clear apoptotic bodies in the tested cells. Further, the cells were treated with the ethanol extract and DNA fragmentation was confirmed by DNA ladder assay. MCF-7 cells administered with ethanol extract were subjected to DNA fragmentation assay for assessing the apoptosis induction nature of the extract. Conclusion: Results from our study indicate a promising approach into the possibility of developing C. rotundus ethanol extract into an effective anticancer moiety after appropriate pre-clinical and clinical validations.

Computational nodal displacement analysis of acetabulum fossa for injection molded cemented polyethylene acetabular liner

The acetabular liner (AL) is one of the key components that determine the functionality and durability of the total hip joint replacement (THR) device. The performance of Ultra high molecular weight polyethylene (UHMWPE)-based AL depends critically on the manufacturing route and its properties, which are evaluated pre-clinically using a host of experimental and computational analyses. The conventional manufacturing of an AL involves multiple stages, including extrusion/compression molding followed by machining, which is time/cost intensive and leads to material loss. In such a scenario, injection molding is a promising alternative, yet its feasbility remains unexplored for the manufacturing of AL for THA applications. Against this backdrop, the two-fold objectives of this work are to report our recent efforts to establish the efficacy of the injection molding of new generation UHMWPE biomaterial; HU (60 wt% HDPE- 40 wt% UHMWPE blend) for manufacturing AL prototype and to present the key biomechanical response analysis of this prototype, in silico. A range of manufacturing relevant material properties, as well as customized mold design to manufacture HU-based AL with external design features, are discussed. Such guidelines are particularly relevant to mold polymeric parts with a higher thickness (>8 mm). As part of the pre-clinical validation of AL with new design features, a less explored in silico approach to assess biomechanical micro-strain in the acetabulum fossa is presented, and the results are analysed in accordance with the mechanostat theory. The outcomes revealed that for a 100 kg subject weight, average micro-strain in the remodelling region was 1132, while it was determined as 723 for a 55 kg subject weight. Such results highlight the influence of subject weight on micro-strain generation and distribution in the acetabulum fossa. The von Mises stress in AL also increased with subject weight from 17 MPa in a subject weight of 55 kg to 28 MPa in a subject weight of 100 kg. Taken together, this work demonstrates the feasibility and competence of this new generation biomaterial in terms of implant manufacturing via injection molding with a clinically desired biomechanical response.

Identification of a novel inhibitor of SARS-CoV-2 main protease: an in silico, biochemical, and cell-based approach.

The recurrent nature of coronavirus outbreaks, severity of the COVID-19 pandemic, rapid emergence of novel variants, and concerns over the effectiveness of existing vaccines against novel variants have highlighted the need to develop therapeutic interventions. Targeted efforts to identify inhibitors of crucial viral proteins are the preferred strategy. In this study, we screened FDA-approved and natural product libraries using in silico approach for potential hits against the SARS-CoV-2 main protease (Mpro) and experimentally validated their potency using in vitro biochemical and cell-based assays. Seven potential hits were identified through in silico screening and were subsequently evaluated in SARS-CoV-2-based cell-free assays, followed by testing in the HCoV-229E-based culture system. Of the tested compounds, 4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-1-isopropyl-1H-benzofuro[3,2-b]pyrazolo[4,3-e]pyridin-3(2H)-one (PubChem CID:71755304, hereafter referred to as STL522228) exhibited significant antiviral activity. Subsequently, its potential as a novel COVID therapeutic molecule was validated in the SARS-CoV-2-culture system, where STL522228 demonstrated superior antiviral activity (EC50 = 0.44 μm) compared to Remdesivir (EC50 = 0.62 μm). Based on these findings, we report the strong anti-coronavirus activity of STL522228, and propose that it as a promising pan-coronavirus Mpro inhibitor for further experimental and preclinical validation.

Pre-clinical validation of a pan-cancer CAR-T cell immunotherapy targeting nfP2X7

Chimeric antigen receptor (CAR)-T cell immunotherapy is a novel treatment that genetically modifies the patients’ own T cells to target and kill malignant cells. However, identification of tumour-specific antigens expressed on multiple solid cancer types, remains a major challenge. P2X purinoceptor 7 (P2X7) is a cell surface expressed ATP gated cation channel, and a dysfunctional version of P2X7, named nfP2X7, has been identified on cancer cells from multiple tissues, while being undetectable on healthy cells. We present a prototype -human CAR-T construct targeting nfP2X7 showing potential antigen-specific cytotoxicity against twelve solid cancer types (breast, prostate, lung, colorectal, brain and skin). In xenograft mouse models of breast and prostate cancer, CAR-T cells targeting nfP2X7 exhibit robust anti-tumour efficacy. These data indicate that nfP2X7 is a suitable immunotherapy target because of its broad expression on human tumours. CAR-T cells targeting nfP2X7 have potential as a wide-spectrum cancer immunotherapy for solid tumours in humans.

Nanoparticles that target the mitochondria of tumor cells to restore oxygen supply for photodynamic therapy: Design and preclinical validation against breast cancer

Photodynamic therapy, in which photosensitizers locally generate cytotoxic reactive oxygen species, can treat tumor tissue with minimal effects on surrounding normal tissue, but it can be ineffective because of the anoxic tumor microenvironment. Here we developed a strategy to inactivate the mitochondria of tumor cells in order to ensure adequate local oxygen concentrations for photodynamic therapy. We conjugated the photosensitizer 5-aminolevulinic acid to the lipophilic cation triphenylphosphine, which targets mitochondria. Then we packaged the conjugate into nanoparticles that were based on biocompatible bovine serum albumin and coated with folic acid in order to target the abundant folate receptors on the tumor surface. In studies in cell culture and BALB/c mice bearing MCF-7 xenografts, we found that the nanoparticles helped solubilize the cation-photosensitizer conjugate, prolong its circulation, and enhance its photodynamic antitumor effects. We confirmed the ability of the nanoparticles to target tumor cells and their mitochondria using confocal laser microscopy and in vivo assays of pharmacokinetics, pharmacodynamics, and tissue distribution. Our results not only identify a novel nanoparticle system for treating cancer, but they demonstrate the feasibility of enhancing photodynamic therapy by reducing oxygen consumption within tumors.

Development and pre-clinical validation of next-generation sequencing gene panel to detect clinically relevant mutations in thyroid cytological and histological samples

Development and pre-clinical validation of next-generation sequencing gene panel to detect clinically relevant mutations in thyroid cytological and histological samples

Defining the scope of extended NIPS in Western China: evidence from a large cohort of fetuses with normal ultrasound scans

BackgroundStandard noninvasive prenatal screening(NIPS) is an accurate and reliable method to screen for common chromosome aneuploidies, such as trisomy 21, 18 and 13. Extended NIPS has been used in clinic for not only aneuploidies but also copy number variants(CNVs). Here we aim to define the range of chromosomal abnormalities that should be able to identify by NIPS in order to be an efficient extended screening test for chromosomal abnormalities.MethodsA prospective study was conducted, involving pregnant women without fetal sonographic structural abnormalities who underwent amniocentesis. Prenatal samples were analyzed using copy number variation sequencing(CNV-seq) to identify fetal chromosomal abnormalities.ResultsOf 28,469 pregnancies included 1,022 (3.59%) were identified with clinically significant fetal chromosome abnormalities, including 587 aneuploidies (2.06%) and 435 (1.53%) pathogenic (P) / likely pathogenic (LP) CNVs. P/LP CNVs were found in all chromosomes, but the distribution was not uniform. Among them, P/LP CNVs in chromosomes 16, 22, and X exhibited the highest frequencies. In addition, P/LP CNVs were most common on distal ends of the chromosomes and in low copy repeat regions. Recurrent microdeletion/microduplication syndromes (MMS) accounted for 40.69% of total P/LP CNVs. The size of most P/LP CNVs (77.47%) was < 3 Mb.ConclusionsIn addition to aneuploidies, the scope of extended NIPS should include the currently known P/LP CNVs, especially the regions with recurrent MMS loci, distal ends of the chromosomes, and low copy repeat regions. To be effective detection should include CNVs of < 3 Mb. Meanwhile, sufficient preclinical validation is still needed to ensure the clinical effect of extended NIPS.

BCL11A-targeted γ-globin gene induction by triterpenoid glycosides of Fagonia indica: A preclinical scientific validation of indigenous herb for the treatment of β-hemoglobinopathies

Pharmacological induction of fetal hemoglobin has proven to be a promising therapeutic intervention in β-hemoglobinopathies by reducing the globin chain imbalance and inhibiting sickle cell polymerization. Fagonia indica has shown therapeutic relevance to β-thalassemia. Therefore, we study the ethnopharmacological potential of Fagonia indica and its biomarker compounds for their HbF induction ability for the treatment of β-thalassemia. Here, we identify, compound 8 (triterpenoid glycosides) of F. indica. as a prominent HbF inducer in-vitro and in-vivo. Compound 8 showed potent erythroid differentiation, enhanced cellular proliferation, ample accumulation of total hemoglobin, and a strong notion of γ-globin gene expression in K562 cultures. Compound 8 treatment also revealed strong induction of erythroid differentiation and fetal hemoglobin mRNA and protein in adult erythroid precursor cells. This induction was associated with simultaneous downregulation of BCL11A and SOX6, and overexpression of the GATA-1 gene, suggesting a compound 8-mediated partial mechanism involved in the reactivation of fetal-like globin genes. The in vivo study with compound 8 (10 mg/kg) in β-YAC mice resulted in significant HbF synthesis demonstrated by the enhanced level of F-cells (84.14 %) and an 8.85-fold increase in the γ-globin gene. Overall, the study identifies compound 8 as a new HbF-inducing entity and provides an early “proof-of-concept” to enable the initiation of preclinical and clinical studies in the development of this HbF-inducing agent for β-thalassemia.

A system for monitoring the functional status of older adults in daily life

Functional decline in older adults can lead to an increased need of assistance or even moving to a nursing home. Utilising home automation, power and wearable sensors, our system continuously keeps track of the functional status of older adults through monitoring their daily life and allows health care professionals to create individualised rehabilitation programmes based on the changes in the older adult’s functional capacity and performance in daily life. The system uses the taxonomy of the International Classification of Functioning, Disability and Health (ICF) by the World Health Organization (WHO). It links sensor data to five ICF items from three ICF categories and measures their change over time. We collected data from 20 (pre-)frail older adults (aged ge 75 years) during a 10-month observational randomised pilot intervention study. The system successfully passed the first pre-clinical validation step on the real-world data of the OTAGO study. Furthermore, an initial test with a medical professional showed that the system is intuitive and can be used to design personalised rehabilitation measures. Since this research is in an early stage further clinical studies are needed to fully validate the system.

Validation of a platinum bioelectrode model for preclinical electrical and biological performance evaluation.

High throughput testing of clinically representative Pt electrodes requires an inexpensive, efficient method of production. The aim of this study was to develop a facile platinum (Pt) model electrode (PME) and assess its production process, stability, and reproducibility. In this study a new model electrode was developed using representative substrates and dimensions as state-of-the-art electrode arrays used for neural stimulation. It was found that the PME is a highly reproducible robust system with similar electrochemical performance but with lower variability than other neural prosthetic arrays.Clinical Relevance- As an estimate these novel model electrodes cost 300 times less than a cochlear implant, can be manufactured in a tenth of the time and with a less than 10% failure rate. It is expected that model electrodes with low variability of electrical properties will significantly improve preclinical validation testing of electrochemical stimulation, surface modifications, and coatings.

3,5,7-trihydroxyflavone restricts proliferation of androgen-independent human prostate adenocarcinoma cells by inducing ROS-mediated apoptosis and reducestumour growth.

Flavonoids are among the largest groups of secondary metabolites. Studies suggestthat dietary intake of flavonoids reducesthe risk of cancer. 3,5,7-trihydroxyflavone (THF) belongs to the flavone class of flavonoids and potentially inhibits the growth of many cancers; however, it is unexplored in prostate cancer. This study reports the antiproliferative potential of THF in prostate cancer cell line via reactive oxygen species (ROS)-mediated cascades and examines the tumour reduction potential in swiss albino mice. The potency of THF was evaluated by employing cytotoxicity assays and wound healing assays. Cell cycle, ROS, mitochondrial membrane potential (MMP), and Annexin-V-FITC assay were performed using a flow cytometer. In vivo, anticancer potential was achieved using the mice Ehrlich Ascites Carcinoma (EAC) model. THF inhibits cell growth with IC50 of 64.30 µM (MTT), 81.22 µM (NRU) and 25.81 µM (SRB), substantiated by cell migration assay. Cell-cycle analysis revealed that THF increases the subdiploid population. Furthermore, the Annexin-V-FITC assay evoked a significant induction of late apoptosis at a higher concentration of THF. THF also disrupts MMP, caused by an increased generation of ROS. In the EAC model, THF significantly inhibits tumour growth and increases the percent survival of mice and ROS levels in EAC cells. Hence, it may be concluded that THF might execute its antiproliferative effect via inducing ROS generation and could be a promising lead for preclinical and clinical validations.

Pharmacological characterisation of the effort for reward task as a measure of motivation for reward in male mice

RationaleMotivational deficits are a common symptom shared across multiple psychiatric and neurodegenerative disorders. Effort-based decision-making tasks are a translatable method for assessing motivational state. Much of the preclinical validation of the task derives from acute pharmacological manipulations in rats. However, mice currently offer a greater genetic toolkit to study risk genes and phenotypic models. Despite this, there is limited characterisation of their behaviour in this type of motivation task.ObjectivesHere, we investigate the effort for reward (EfR) task as a measure of motivational state in mice using drugs previously shown to modulate effort-based decision-making in rats and humans.MethodUsing male C57bl/6j mice, we test the effects of drugs which modulate DA transmission. We also test the effects of CP101-606 which does not act directly via DA modulation but has been shown to exert beneficial effects on motivational state. Finally, we test the sensitivity of the task to a chronic corticosterone (CORT) treatment.ResultsAmphetamine, methylphenidate, and CP101606 in mice increased high-effort responses for high-value reward, while administration of haloperidol decreased high-effort responses. Surprisingly, tetrabenazine had no effect at the doses tested. Chronic, low-dose CORT consumption did not alter task performance.ConclusionThese data suggest that the EfR task is sensitive to acute dopaminergic modulation and NR2B selective antagonism in mice. However, it may lack sensitivity to non-acute phenotypic models. Further work is required to demonstrate the utility of the task in this context.

Combination treatment of a novel CXCR3 antagonist ACT-777991 with an anti-CD3 antibody synergistically increases persistent remission in experimental models of type 1 diabetes.

Treatment of patients with recent-onset type 1 diabetes with an anti-CD3 antibody leads to the transient stabilization of C-peptide levels in responder patients. Partial efficacy may be explained by the entry of islet-reactive T-cells spared by and/or regenerated after the anti-CD3 therapy. The CXCR3/CXCL10 axis has been proposed as a key player in the infiltration of autoreactive T cells into the pancreatic islets followed by the destruction of β cells. Combining the blockade of this axis using ACT-777991, a novel small-molecule CXCR3 antagonist, with anti-CD3 treatment may prevent further infiltration and β-cell damage and thus, preserve insulin production. The effect of anti-CD3 treatment on circulating T-cell subsets, including CXCR3 expression, in mice was evaluated by flow cytometry. Anti-CD3/ACT-777991 combination treatment was assessed in the virally induced RIP-LCMV-GP and NOD diabetes mouse models. Treatments started at disease onset. The effects on remission rate, blood glucose concentrations, insulitis, and plasma C-peptide were evaluated for the combination treatment and the respective monotherapies. Anti-CD3 treatment induced transient lymphopenia but spared circulating CXCR3+ T cells. Combination therapy in both mouse models synergistically and persistently reduced blood glucose concentrations, resulting in increased disease remission rates compared to each monotherapy. At the study end, mice in disease remission demonstrated reduced insulitis and detectable plasma C-peptide levels. When treatments were initiated in non-severely hyperglycemic NOD mice at diabetes onset, the combination treatment led to persistent disease remission in all mice. These results provide preclinical validation and rationale to investigate the combination of ACT-777991 with anti-CD3 for the treatment of patients with recent-onset diabetes.

P-182 Validation of single-step warming for human blastocysts shows successful results are independent of the sucrose concentration used

Abstract Study question Can warming of blastocysts in a single step be successfully performed using different sucrose concentrations? Summary answer Blastocyst warming in a single step using sucrose concentrations between 0.25 M and 1.0 M shows similar rates of survival, re-expansion and development in vitro. What is known already Since the introduction of human blastocyst vitrification, excellent survival rates have been reported. Warming procedures usually involve three to five steps with exposure to solutions with decreasing concentrations of non-penetrating cryoprotectants. Successful blastocyst warming procedures are described using high as well as low starting levels of sucrose. Recent data show that a single step warming procedure can be equally effective in terms of survival and pregnancy rates. Moving towards simpler procedures can support further optimization of laboratory procedures. Study design, size, duration Blastocysts donated and consented by patients were used to evaluate a shorter warming procedure. Warming was performed in a single step using different sucrose concentrations. After warming, embryos were assessed for morphological survival and cultured in a time-lapse incubator to monitor re-expansion and development in vitro for 24 h. Three series of tests were performed. A control group using standard warming procedure was included in the first test Participants/materials, setting, methods For warming, carriers containing a single blastocyst were plunged into a warming solution at 37 °C, containing 0.25, 0.5 or 1.0 M sucrose and stayed in the solution for 1 or 2 minutes. Three series of experiments were performed, testing different sucrose concentrations or volume of warming solution. Warmed blastocysts were cultured following several rinsing steps and assessed for morphological survival. Re-expansion after 2 h, embryo characteristics and development were monitored until 24 h post warming. Main results and the role of chance Results of the different tests are summarized in the table, including information on sucrose levels, exposure times, volume of warming medium, numbers of blastocysts warmed, recovered, meeting criteria for embryo transfer, full re-expansion after 2 hours, viable after 24 h culture. No differences were observed between the different groups. Overall, 98 % of warmed blastocysts would be considered transferable after single step warming and 96 % were viable after 24 h culture. Results confirm blastocysts can be warmed at 37 °C in a single step using 0.25 M, 0.5 M or 1 M sucrose with no effect on survival or development in vitro. Limitations, reasons for caution This is a preclinical validation on use of a reduced warming time for vitrified blastocysts. Further evaluation and clinical validation of the findings is required to confirm safety of a simplified warming procedure. Working temperature rather than warming medium volume may be critical when aiming for shorter warming procedures. Wider implications of the findings Reducing the warming time and number of handling steps minimizes exposure of blastocysts to a suboptimal environment and operator or handling related stresses. It also allows further optimization of laboratory protocols and workflow. When confirmed clinically, these findings encourage investigating similar changes for other reproductive cells. Trial registration number not applicable

P-141 Independent external pre-clinical validation of iDAScore v1.0 in 1232 PGT-A cycles with 3604 biopsied blastocysts and 808 euploid transfers.

Abstract Study question Is iDAScore v1.0 associated with euploidy and live-births (LBs) after euploid transfers? How often would it have affected embryologists’ clinical choices in a blinded analysis? Summary answer iDAScore v1.0 was associated with both euploidy (AUC:0.60) and LBs after euploid transfers (AUC:0.66). iDAScore v1.0 and embryologists would have frequently disagreed on embryo ranking. What is known already Embryo assessment/ranking to improve IVF efficiency (LB/transfer) is still challenging. The widely used static morphological evaluation suffers from subjectivity and intra-/inter-operator variability. Preimplantation genetic testing for aneuploidies (PGT-A) discriminates euploid/aneuploid embryos, improving IVF efficiency. Nevertheless, it requires manipulation and expertise and ∼50% euploid blastocysts still fail to implant. Time-lapse (TL) technology allows continuous undisturbed monitoring of embryo morphokinetics. The software iDAScore v1.0 is a deep learning algorithm trained on TL videos from implanted/non-implanted blastocysts to generate a score that should predict their implantation potential (score:1.0-9.9, from the lowest to the highest, respectively). Study design, size, duration Retrospective independent external pre-clinical validation of iDAScore v1.0 in PGT-A cycles (N = 1232) with fresh own oocytes and ≥1 biopsied blastocyst (N = 3604) (April-2013 to August-2022). The AI-based tool was investigated for associations with embryologists’ assessment, blastocysts’ karyotype, and LBs. Two simulations were then conducted to estimate how often iDAScore v1.0 would have ranked (i) euploid blastocysts first in presence of aneuploid as well, (ii) reproductively-competent blastocysts before incompetent in presence of ≥ 2 euploid and ≥1 LB. Participants/materials, setting, methods Embryos were cultured in EmbryoScope (Vitrolife). Only the first PGT-A cycles were included (maternal age 38.7 ± 3.4 years). The day of biopsy was defined based on the hours-post-insemination (hpi) and blastocyst morphology based on Gardner. ROC curve analyses were conducted to calculate the AUC for euploidy and LB discrimination based on embryologists’ assessment or iDAScore v1.0. Main results and the role of chance iDAScore v1.0 was associated with ICM-quality (A-grade, N = 2107, score: 7.5 ± 1.8; B-grade, N = 833, score: 5.6 ± 1.9; C-grade, N = 664, score: 4.4 ± 1.7; p &amp;lt; 0.01), trophectoderm-quality (A-grade, N = 1988, score: 7.5 ± 1.8; B-grade, N = 951, score: 5.9 ± 1.9; C-grade, N = 664, score: 4.3 ± 1.6; p &amp;lt; 0.01), and day ( ≤ 120hpi, N = 1462, score: 8.2 ± 1.5; 121-144hpi, N = 1874, score: 5.6 ± 1.7; &amp;gt; 144hpi, N = 268, score: 3.9 ± 1.4;p&amp;lt;0.01). Euploid blastocysts showed the highest score (N = 1443, 7.0 ± 2.1) versus both single-aneuploid (N = 1194, score: 6.5 ± 2.2, p &amp;lt; 0.01) and complex-aneuploid (N = 967, score: 5.8 ± 2.1, p &amp;lt; 0.01). AUC of 0.60 (95%CI 0.59−0.62) and 0.66 (95%CI 0.64−0.68) were reported for iDAScore v1.0 and embryologists’ assessment association with euploidy, respectively. Blastocysts resulting in a LB showed higher score (N = 361, 7.6 ± 1.8) than non-implanted/miscarried (N = 447, score: 6.5 ± 2.2; OR: 1.3, 95%CI 1.2-1.4, p &amp;lt; 0.01). AUC of 0.66 (95%CI 0.62-0-69) and 0.64 (95%CI 0.60-0-67) were reported for iDAScore v1.0 and embryologists’ assessment association with LBs, respectively. The simulations revealed that (i) across 587 cycles with both diagnoses, iDAScore v1.0 would have ranked euploid blastocysts as “top-quality” in 63% of the cases; (ii) across 202 cycles with sibling euploid blastocysts and a LB, it would have been equally, less, and more effective than embryologists, in 52%, 3% and 15% of the cases, respectively. Nonetheless, in 29% of the cases, this comparison was not doable because top-ranked euploid blastocysts were not transferred but a LB was achieved with worse-ranked blastocyst. Limitations, reasons for caution iDAScore v1.0 performance might be suboptimal on a dataset of mostly advanced-maternal-age women and on day-7/poor-quality blastocysts, due to their poor representation in the algorithm training. Although euploidy is a proxy of LB, iDAScore v1.0 was trained only to predict the latter. A prospective randomized study is now warranted Wider implications of the findings iDAScore v1.0 was significantly associated with euploidy and LB with results comparable to embryologists’ performance. Nevertheless, iDAScore v1.0 is more objective and reproducible than embryologists’ assessment. Embryo selection workflows, scientific debate, and patient counselling practice might all benefit from AI-based standardization of blastocyst evaluation. Trial registration number not available

#5615 VALIDITY AND ADEQUACY OF ANIMAL MODELS IN HAEMODIALYSIS RESEARCH: A SYSTEMATIC REVIEW

Abstract Background and Aims with the worldwide dialysis population growing rapidly, improving haemodialysis (HD) outcomes is crucial and HD innovations are urgently needed. Prior to clinical application, novel HD technologies must undergo extensive preclinical testing. However, to date there is no consensus on the induction method of kidney failure in animal models nor which animal species is most suitable for research on HD innovations. Moreover, there is no consensus on which parameters should be used to validate adequate induction of kidney failure and functioning of novel HD devices. Using a systematic review approach, we summarized available literature of HD in kidney failure animal models. Method we performed a systematic search on PubMed and Embase for relevant studies up to February 4th 2022. After removal of duplicates, 5723 abstracts were screened for eligibility by three independent reviewers (JdV, KW and MK). Inclusion was based on publication of a HD intervention in an animal model with adequate (acute or chronic) kidney failure (independent of induction method). Detailed in- and exclusion criteria were registered in PROSPERO 2022 CRD42022307144. Data from individual reports, including animal species and sex, kidney injury parameters and HD details, were extracted in a pre-set data extraction form. Future efforts will focus on risk of bias and key quality indicators. Results of the 5723 abstracts screened, 195 records were included as full text and 41 full text articles were included for data-extraction (Figure 1). Current data extraction is completed for 31 articles (75%). Publication years range from 1973 to 2021 with over half (16/31) published after 2000. HD experiments were most frequently performed on dogs (45%), followed by rats (29%), goats (13%), pigs (10%), and sheep (3%) respectively. Parameters such as weight (81% reported), sex (58%) and age (29%) were not systematically reported. Most studies (67% of records reporting sex) used male animals only. Dog models were found in older studies, with no records after 2009. Other large animal studies were almost exclusively (88%) performed from the year 2007 onwards. Independent of animal species, studies mainly used acute kidney injury (AKI) models (84%), induced by bilateral nephrectomy (48%) or bilateral ureteral ligation (29%). The majority confirmed kidney failure by an increase in BUN and/or plasma creatinine. In AKI models, dialysis was on average initiated on AKI day 2 ± 0.8, and generally either HD (61%) or veno-venous hemofiltration as a single session (35%) was applied. Except for one clearance study, dialysis efficiency was reported as reduction ratios of BUN and/or plasma creatinine. Conclusion both large and small animal models have been used in HD research and preclinical validation of dialysis innovations. The use of dogs to test HD innovations has seen a sharp decline over the past decades, possibly resulting from a shift in public ethical perception. However, large animal models such as goat, sheep and pigs remain popular. A pressing issue appears the overall lack of adequate ARRIVE guideline adherence for reporting of animal parameters. We found no apparent improvement in reporting over time, emphasizing the need for clear guidelines to drive innovative HD research. Further (quantitative) analysis and meta-analysis are ongoing.