Human blastocysts diagnosed using Next-generation Sequencing with mosaicism demonstrate distinct transcriptomic profiles compared to euploid or aneuploid embryos

Objective: To determine if embryos diagnosed as mosaic using Next-generation sequencing (NGS) have distinct gene expression profiles compared to embryos diagnosed as euploid or aneuploid. Materials and methods: Fifteen blastocysts were diagnosed as mosaic with NGS including 7 full mosaic monosomies, 5 partial mosaic monosomies, 2 full mosaic trisomies, and 1 partial mosaic trisomy. Three NGS diagnosed euploid embryos, and 25 aneuploid embryos (9 NGS, 14 aCGH, 2 Single Nucleotide Polymorphism array) were used as comparisons. Replicate samples were available for all euploid and full aneuploid embryos. Aneuploid embryos were chosen if their aneuploid chromosome corresponded with one of the mosaic embryos. Complementary DNA from the samples was created using the SMATer v4 Ultra Low Kit. RNA-seq library preparation followed by sequencing on the Illumina HiSeq 2500 with 50 nucleotide length paired end reads was performed. The STAR/2.5 aligner was used to align the reads against the hg19 ensemble reference transcriptome. Differentially expressed genes (DEGs) were calculated using DES-seq2/3.5, and p values <0.05 were considered significantly differentially expressed. Pathway analysis was performed on the top DEGs among all of the mosaic embryos with p<0.05 considered significant. Main results: The 15 mosaic embryos had transcriptomic profiles which were distinct from full aneuploid embryos involving the same chromosome. Mosaic embryos had fewer DEGs compared to aneuploid embryos of the same chromosome when using euploid embryos as controls. Mosaic embryos were grouped on principal component analysis (PCA) and distinguishable from euploid and aneuploid embryos. Pathways involving cell proliferation, differentiation, and apoptosis were the most disrupted from the presence of a mosaic cell line. Overall design: Descriptive study of 43 blastocysts that underwent preimplantation genetic testing for aneuploidy (PGT-A) with either NGS or array Comparative Genomic Hybridization (aCGH) which were donated for IRB approved research.