Study question Is long-read nanopore sequencing feasible and reproducible as routine technique for aneuploidy detection, potentially allowing fresh embryo transfer PGT-A cycles? Summary answer Pre-clinical analysis of euploid and aneuploid DNA and single cells using long-read nanopore sequencing resulted in high concordance and reproducible results for aneuploidy detection. What is known already PGT-A is mostly performed using short-read next-generation sequencing, which requires high initial investment costs and high running expenses. Third-generation sequencing is a novel sequencing technology with the potential of fast, easy, and cost-effective sequencing analysis, possible even for small and less well-financed clinics. Long-read nanopore sequencing for PGT was mainly shown for structural variants and monogenetic disease, which comes with high costs and is far from clinical routine. PGT-A from trophectoderm biopsy samples using nanopore sequencing was so far demonstrated in a small pilot study and further pre-clinical and clinical studies are needed to transfer the technology into clinical routine. Study design, size, duration In this pre-clinical study, euploid and aneuploid DNA and single cells, as well as three to 20 cells were analyzed for aneuploidy using two different whole genome amplification (WGA) kits and long-read nanopore sequencing. In total, 44 different samples were analyzed after multiple displacement amplification (MDA) using REPLIg WGA kit (QIAGEN) and so far, 15 samples were analyzed using PicoPlex WGA kit (Takara) from April 2021. To confirm reproducibility, certain WGA samples were sequenced repetitively. Participants/materials, setting, methods Different euploid and aneuploid gDNA samples were diluted to 4.5 pg DNA per sample. Four different human fibroblast cell lines were diluted to approx. 20 cells, or single cells/three cells were picked using micromanipulation technique. DNA and cells were amplified, prepared for sequencing, and sequenced on MinION sequencer from Oxford Nanopore Technology. Data were analyzed using a custom pipeline consisting of pre-processing, alignment and copy-number calling. QC values and whole chromosome aneuploidies were determined automatically. Main results and the role of chance From 44 different single cells (n = 14), few cells (n = 9) or diluted gDNA (n = 21) samples amplified using MDA, 42 samples showed good quality sequencing results. Two samples result in QC failure, yielding a sample-success-rate (SSR) of 95.5%. Overall, sample sensitivity was 100%, specificity 95.2% with positive predictive value (PPV) of 95.5% and negative predictive value (NPV) of 100%. Per chromosome sensitivity was 100%, specificity 99.8% with PPV of 93.3% and NPV of 100%. Using PicoPlex WGA, 15 samples were analyzed so far: 7 gDNA and 8 single cell samples. The study is ongoing. All samples analyzed resulted in high quality sequencing data with the correct karyotype, leading to 100% SSR, 100% sensitivity, specificity, PPV, NPV per samples as well as per chromosome. Repetitive sequencing (n = 3) of four MDA amplified single cells showed identical sequencing results, indicating high reproducibility of library preparation, and sequencing. A 5p deletion in one cell line was correctly identified in all single cell analyses from either MDA or PicoPlex amplification, indicating sufficient resolution even for segmental aneuploidies. The whole workflow is feasible in under 24 hours. These results indicate high accuracy and high reproducibility of nanopore sequencing technology for single cell aneuploidy detection, possibly transferable for PGT-A. Limitations, reasons for caution The results of this pre-clinical study look very promising and workflow for sample preparation, sequencing and data analysis is fast, cost-efficient, and feasible for PGT-A applications. Nevertheless, no polar body, blastomere or trophectoderm biopsy sample was used and a clinical trial using real clinical samples is needed to confirm applicability. Wider implications of the findings Implementing novel technologies into clinical routine requires extensive systematic pre-clinical and clinical testing. To our knowledge, this is the first study that systematically analyzes sensitivity, specificity, PPV and NPV for aneuploidy detection using long-read sequencing technology. The fast workflow principally allows day 3/5 fresh embryo transfer after polar body biopsy. Trial registration number Not applicable

STUDY QUESTION: Is nanopore sequencing for PGT-A analysis of pooled polar bodies a reliable, fast, and cost-effective method and applicable for routine diagnostics in human reproductive care? SUMMARY ANSWER: Nanopore sequencing of pooled polar bodies (PB) revealed high concordance rate with traditional array comparative genomic hybridization (aCGH) analysis and the nanopore sequencing workflow was fast (feasible in under 5 hours) and cost-effective (100-150 Euro per sample), allowing fresh embryo transfer. WHAT IS KNOWN ALREADY: PGT-A using PB biopsy derives a clinical benefit by reducing number of embryo transfers and miscarriage rates but is currently not cost-efficient. Results are often unclear and require expert review. Nanopore sequencing technology opens possibilities by providing cost-efficient, fast sequencing results with uncomplicated sample preparation workflows. Interrogating the polar bodies avoids harming the embryo itself and is the only option for PGT-A in some jurisdictions. STUDY DESIGN, SIZE, DURATION: In this prospective clinical trial, 102 pooled PB samples from 20 patients treated for infertility between March and December 2022 were analyzed for aneuploidy using nanopore sequencing technology and compared with aCGH results generated as part of the clinical routine. All patients participating in this trial were treated for infertility at Wunschbaby Institut Feichtinger (WIF) in Vienna and chose aneuploidy screening of their polar bodies. All patients provided written informed consent. PARTICIPANTS/MATERIALS, SETTING, METHODS: PB samples were analyzed by aCGH for routine PGT-A. Aliquots of whole-genome amplified DNA were anonymized and prepared for sequencing by end-prepping, barcoding and adapter ligation. Samples were pooled equimolar for sequencing on a Nanopore MinION machine. Samples were sequenced for up to 9 hours for 6 pooled PB samples. Whole-chromosome copy-numbers were called by a custom bioinformatic analysis software after alignment and pre-processing. Automatically called results were compared to aCGH results. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 99 pooled polar body samples were compared, three samples were excluded because of failed or uninterpretable results. Overall, the Nanopore sequencing workflow showed high concordance rates with aCGH: 96 of 99 samples were consistently detected as euploid or aneuploid (concordance=97%, specificity = 0.957, sensitivity = 1.0, PPV = 0.906, NPV = 1.0) and 91 samples showed a fully concordant karyotype (92%). Chromosomal aneuploidies analyzed in this trial covered all 23 chromosomes with 98 trisomies, 97 monosomies in 70 aCGH samples. Detailed calculation of time and cost for the nanopore sequencing workflow was performed for different scenarios. Time calculation revealed that the whole nanopore workflow is feasible in under 5 hours (for one sample) with maximum time of 16 hours (for 12 samples in parallel). This enables fresh PB euploid embryo transfer. Material cost for the whole workflow range between 100Euro and 150Euro, including sequencing cost of only 40Euro per sample, resulting in cost-efficient aneuploidy screening. LIMITATIONS, REASONS FOR CAUTION: For some samples, the reference result remained unclear, due to increased noise and limited resolution of the aCGH method. In particular, the small chromosomes show higher variability in both platforms and manual review is often required. A larger study with follow-up data or a clinical non- selection trial would be beneficial for increased confidence. WIDER IMPLICATIONS OF THE FINDINGS: This is the first clinical study, systematically comparing nanopore sequencing for aneuploidy of pooled polar bodies with standard detection methods. High concordance rates confirmed feasibility of nanopore technology for this application. Additionally, the fast and cost-efficient sequencing workflow reveals clinical utility of this technology, making polar body PGT-A clinically attractive. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Wunschbaby Institut Feichtinger, Dr. Wilfried Feichtinger GmbH. The authors declare no competing conflict of interest. TRIAL REGISTRATION NUMBER: Not available

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