Apoptotic qPCR gene expression array analysis demonstrates proof-of-concept for rapid blastocoel fluid-conditioned media molecular prediction

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.

Keywords: Aneuploidy; Apoptosis; Blastocoel fluid; Diagnostic; Embryo selection; IVF; Self-correction; qPCR.

Fig. 1

Relative apoptotic gene expression within the blastocoel fluid-conditioned media of all embryonic samples (in columns) with respect to many critical apoptotic genes demonstrates proof of capacity to assess quantitatively the relative concentration of gene expression within the cell-free fluid. A. Pro-apoptotic gene expression is shown. B. Anti-apoptotic and other classified gene expression is shown. Note that gray boxes represent gene expression for which qPCR analysis yielded no sustained amplification, likely implying the lack of any mRNA for the specific gene of interest within the specified blastocoel fluid-conditioned media. Sample expression is visualized upon a heat map that has color changes signifying exponential changes in expression. Hence, color differences represent the order of magnitude differences in expression. Comparative analysis (represented by colors) occurs between the comparison of gene expression for each gene (horizontal) (please refer to Supplemental Table 1 for citations used to classify genes as pro- or anti-apoptotic in this figure)