Abstract
Improved embryo prioritization is crucial in optimizing the results in assisted reproduction, especially in light of increasing utilization of elective single embryo transfers. Embryo prioritization is currently based on morphological criteria and in some cases incorporates preimplantation genetic testing for aneuploidy (PGT-A). Recent technological advances have enabled parallel genomic and transcriptomic assessment of a single cell. Adding transcriptomic analysis to PGT-A holds promise for better understanding early embryonic development and implantation, and for enhancing available embryo prioritization tools. Our aim was to develop a platform for parallel genomic and transcriptomic sequencing of a single trophectoderm (TE) biopsy, that could later be correlated with clinical outcomes. Twenty-five embryos donated for research were utilized; eight for initial development and optimization of our method, and seventeen to demonstrate clinical safety and reproducibility of this method. Our method achieved 100% concordance for ploidy status with that achieved by the classic PGT-A. All sequencing data exceeded quality control metrics. Transcriptomic sequencing data was sufficient for performing differential expression (DE) analysis. All biopsies expressed specific TE markers, further validating the accuracy of our method. Using PCA, samples clustered in euploid and aneuploid aggregates, highlighting the importance of controlling for ploidy in every transcriptomic assessment.
Highlights
Www.nature.com/scientificreports non-viable embryos and understanding the process of implantation and early development is important for optimizing fertility treatment outcomes for the entire infertile population[14]
In this work we have shown that it is clinically feasible to explore the transcriptome of an embryo alongside its ploidy status from a single trophectoderm biopsy of 4–6 cells
The addition of RNAseq did not interfere with the workflow of preimplantation genetic testing for aneuploidy, as it is performed in clinics, nor did it significantly alter the clinical quality control metrics (Table 3)
Summary
Www.nature.com/scientificreports non-viable embryos and understanding the process of implantation and early development is important for optimizing fertility treatment outcomes for the entire infertile population[14]. Advances in bioinformatic tools which can better control for increased sequencing noise, batch effects, and entanglement of biological and technical variability, have made it more feasible to integrate multiple layers of data (RNAseq, DNA methylation, histone modifications etc.)[32,33,34,35,36,37] These advancements, in turn, have driven the development of several methods for parallel sequencing of both mRNA and gDNA from the same cell or cell population. We introduce PGT-AT (Preimplantation Genetic Testing for Aneuploidy and Transcriptome), in which we create a clinically applicable tool for simultaneous assessment of chromosomal copy number by low pass whole genome sequencing, and transcriptomic profile using whole transcriptome RNAseq For this proof of concept study, embryos donated for research with known ploidy were selected (either aneuploid or euploid embryos harboring a disease causing gene mutation, deeming them not desirable for transfer). Once both the safety and the feasibility of this method are established, the transcriptome of euploid embryos destined for transfer will be assessed and the gene expression correlated with clinical outcomes
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