CASE REPORT: RNASEQ ANALYSIS OF BLASTOCOEL FLUID FROM EUPLOID EMBRYOS ORIGINATING FROM THE SAME PATIENT RESULTING IN DIFFERENT IMPLANTATION OUTCOMES REVEAL ALTERED EXPRESSION OF GENES ASSOCIATED WITH DEUBIQUITINATION PATHWAY

Abstract

Is the gene expression profile different between two 46, XX blastocysts, with identical morphology from the same patient? While Preimplantation Genetic Testing for Aneuploidies (PGT-A) allows for identification of ploidy status and morphology scoring provides additional information when selecting an embryo for transfer, euploid embryos with the same morphology do not always result in the same implantation outcome. This study aimed to evaluate global gene expression using RNASeq in blastocoel fluid-conditioned media from two 46, XX euploid embryos from the same 36-year old infertility patient from the same egg retrieval cycle. Retrospective analysis of day-5 euploid blastocoel fluid gene expression in two 46, XX embryos with different implantation outcomes from a 36-year old patient. Day-5 ICSI-generated blastocysts were scored via the Gardner and Schoolcraft grading system. Blastocoel fluid-conditioned media was obtained following biopsy of the day-5 blastocysts of same morphology grade (5AA). RNA was extracted and libraries prepared using a SMART-Seq Stranded kit followed by Illumina NextSeq500 sequencing performed at the University of South Carolina Functional Genomics Core. Sequences were aligned to the human genome, reads counted and gene expression determined. The resulting data set was then analyzed with the iDEP 9.1 web application to run a PGSEA pathway analysis utilizing Gene Ontology Biological Processes Geneset (Ge et al. BMC Bioinformatics 2018;19:534). Gene expression associated with various cellular pathways were compared between the blastocoel fluid sample from each blastocyst which revealed a decreased level of gene expression associated with the protein deubiquitination pathway in the positive implantation embryo, and no other analyzed pathway yielded significant difference in gene expression between the two blastocoel fluid samples. Specifically, genes encoding SHMT2, KAT2A, ASXL1 were all downregulated in blastocoel fluid from the embryo associated with successful implantation. RNASeq analysis of media samples from two 46, XX embryos with the same morphology from the same patient but associated with different implantation outcomes yielded differences in gene expression. Knowledge of chromosomal status and morphology of a preimplantation blastocyst stage embryo did not provide a complete picture of the underlying biological processes taking place within the embryo, nor could these measures fully predict an embryo’s implantation outcome. This case study provides evidence of a detectable difference in gene expression in blastocoel fluid obtained from embryos that would be considered indistinguishable by current methods employed by reproductive specialists. Moreover, this alteration in gene expression is associated with a difference in implantation outcome. While this case study identified the deubiquitination pathway as altered, further study is needed to determine if this difference in these euploid blastocysts is also found in embryos from other patients and/or with the same chromosomal status.