Arnav Lal1,2, Allison Kranyak1, Jonathan Blalock1, Deepti Athavale1, Alyssa Barré1, Addison Doran1, T Arthur Chang3, Randal D Robinson3, Shawn Zimmerman4, J David Wininger5, Lauren A Fowler1, William E Roudebush1, Renee J Chosed6. 1. Department of Biomedical Sciences, University of South Carolina School of Medicine Greenville, 701 Grove Road, Greenville, SC, 29605, USA. 2. School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, 19104, USA. 3. Department of Obstetrics and Gynecology, University of Texas Health Science Center, San Antonio, TX, 78229, USA. 4. VIOS Fertility Institute, Swansea, IL, 62226, USA. 5. Department of Obstetrics and Gynecology-Reproductive Medicine, Wake Forest School of Medicine, Winston-Salem, NC, 27101, USA. 6. Department of Biomedical Sciences, University of South Carolina School of Medicine Greenville, 701 Grove Road, Greenville, SC, 29605, USA. chosed@greenvillemed.sc.edu.
Abstract
PURPOSE: Successful identification of transcriptomic biomarkers within human IVF embryos may enhance implantation prediction and provide insights not available through conventional embryo biopsy genomic analysis. We demonstrate proof-of-concept for a methodology to assess overall embryo gene expression using qPCR with blastocoel fluid-conditioned media by examining the comparative presence of apoptotic genes. METHODS: Blastocoel fluid-conditioned media were collected from 19 embryos (11 euploid) following trophectoderm biopsy of day-5 ICSI-IVF blastocysts. Media were assessed for apoptotic gene expression via qPCR. Statistical analysis of gene expression was conducted via Wilcoxon Signed-Ranks test (overall expression), multivariate ANOVA (functional gene groups), and chi-square test of independence (gene level). RESULTS: A significantly higher overall apoptotic gene expression within euploid versus aneuploid embryos (p = 0.001) was observed. There was significantly (p = 0.045) higher expression of pro-apoptotic genes between implanted and not implanted embryos. Pro- vs. anti-apoptotic gene expression from all euploid embryos approached significance (p = 0.053). The ploidy status-based claim is further substantiated at the gene level with significantly higher expression of BBC3 (p = 0.012) and BCL2L13 (p = 0.003) in euploid embryos compared to aneuploid embryos. CONCLUSIONS: In this preliminary study, we demonstrate that (1) qualitative analysis of blastocoel fluid-conditioned media gene expression is possible, (2) global trends of expression are potentially related to clinical outcomes, and (3) gene-level expression trends exist and may be another viable metric for comparative expression between samples. The presence of statistical significance within analyses conducted with this sample size warrants a larger investigation of blastocoel fluid-conditioned media as an additional beneficial predictive tool for future IVF cases.
PURPOSE: Successful identification of transcriptomic biomarkers within human IVF embryos may enhance implantation prediction and provide insights not available through conventional embryo biopsy genomic analysis. We demonstrate proof-of-concept for a methodology to assess overall embryo gene expression using qPCR with blastocoel fluid-conditioned media by examining the comparative presence of apoptotic genes. METHODS: Blastocoel fluid-conditioned media were collected from 19 embryos (11 euploid) following trophectoderm biopsy of day-5 ICSI-IVF blastocysts. Media were assessed for apoptotic gene expression via qPCR. Statistical analysis of gene expression was conducted via Wilcoxon Signed-Ranks test (overall expression), multivariate ANOVA (functional gene groups), and chi-square test of independence (gene level). RESULTS: A significantly higher overall apoptotic gene expression within euploid versus aneuploid embryos (p = 0.001) was observed. There was significantly (p = 0.045) higher expression of pro-apoptotic genes between implanted and not implanted embryos. Pro- vs. anti-apoptotic gene expression from all euploid embryos approached significance (p = 0.053). The ploidy status-based claim is further substantiated at the gene level with significantly higher expression of BBC3 (p = 0.012) and BCL2L13 (p = 0.003) in euploid embryos compared to aneuploid embryos. CONCLUSIONS: In this preliminary study, we demonstrate that (1) qualitative analysis of blastocoel fluid-conditioned media gene expression is possible, (2) global trends of expression are potentially related to clinical outcomes, and (3) gene-level expression trends exist and may be another viable metric for comparative expression between samples. The presence of statistical significance within analyses conducted with this sample size warrants a larger investigation of blastocoel fluid-conditioned media as an additional beneficial predictive tool for future IVF cases.
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