Department of Biomedical Sciences, University of South Carolina School of Medicine Greenville, 701 Grove Road, Greenville, SC, 29605, USA.
School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, 19104, USA.
J Assist Reprod Genet. 2022 Jul;39(7):1515-1522. doi: 10.1007/s10815-022-02510-3. Epub 2022 May 11.
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.
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).
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.
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.
在人类 IVF 胚胎中成功鉴定转录组生物标志物，可能会提高着床预测能力，并提供通过常规胚胎活检基因组分析无法获得的见解。我们通过检查凋亡基因的相对存在，证明了一种使用囊胚腔液条件培养基进行 qPCR 评估胚胎整体基因表达的方法的概念验证。
在 ICSI-IVF 囊胚第 5 天的滋养外胚层活检后，从 19 个胚胎（11 个整倍体）中收集囊胚腔液条件培养基。通过 qPCR 评估培养基中的凋亡基因表达。通过 Wilcoxon 符号秩检验（整体表达）、多元方差分析（功能基因组）和独立性卡方检验（基因水平）对基因表达进行统计分析。
观察到整倍体胚胎与非整倍体胚胎之间的整体凋亡基因表达显著更高（p=0.001）。在着床和未着床胚胎之间，促凋亡基因的表达显著更高（p=0.045）。所有整倍体胚胎的促凋亡与抗凋亡基因表达均接近显著（p=0.053）。基于倍性状态的说法在基因水平上得到进一步证实，与非整倍体胚胎相比，整倍体胚胎中 BBC3（p=0.012）和 BCL2L13（p=0.003）的表达显著更高。
在这项初步研究中，我们证明了：（1）囊胚腔液条件培养基基因表达的定性分析是可行的；（2）表达的总体趋势可能与临床结局有关；（3）存在基因水平的表达趋势，并且可能是比较样本之间表达的另一种可行指标。在这个样本量下进行的分析中存在统计学意义，这表明囊胚腔液条件培养基作为未来 IVF 案例的额外有益预测工具值得进一步研究。
