Comprehensive profiling of Small RNAs in human embryo-conditioned culture media by improved sequencing and quantitative PCR methods. (3rd March 2020)

• Record Type: Journal Article
• Authors: Russell, Stewart J.; Menezes, Karen; Balakier, Hanna; Librach, Clifford
• Is Part Of: Systems biology in reproductive medicine. Volume 66:Number 2(2020:Apr.)
• Page Start: 129
• Page End: 139
• Publication Date: 2020-03-03
• Subjects: Small RNA -- miRNA -- embryo culture media -- non-invasive -- embryo -- blastocyst; Systems biology -- Periodicals; Andrology -- Periodicals; Generative organs, Male -- Diseases -- Periodicals; Biological systems -- Periodicals; Reproductive health -- Periodicals; Human reproduction -- Periodicals; 612.61
• DOI: 10.1080/19396368.2020.1716108
• Languages: English
• ISSNs: 1939-6368

Abstract

ABSTRACT: Embryo implantation depends on two primary factors: the quality of the embryo and endometrial receptivity. Small RNAs have been shown to be potent epigenetic regulators influencing cell proliferation, differentiation, and communication even in the context of early embryonic development. However, previous reports are limited to miRNAs and lack sensitivity. Here, we describe a platform for non-invasive small RNA biomarker discovery and validation from embryo-conditioned culture media (ECCM). We hypothesize that small non-coding RNAs (sncRNAs) are secreted by the embryo into the ECCM and test the limit of detection for profiling sncRNA by deep sequencing and quantitative PCR. In the first set of experiments, we evaluated sequencing sensitivity by comparing sncRNA profiles from pools of 10, 5, 3, and single ECCM drops. Next, we performed a similar test for TaqMan qPCR sensitivity by measuring select sncRNAs in 5, 3 and single drop ECCM pools. Finally, we compared the expression of an sncRNA panel by qPCR in single ECCM vs no-embryo control media . We report the first comprehensive sequencing of sncRNAs in ECCM with a sequencing sensitivity of 3 single embryo drops, capturing ~150 miRNAs and an abundance of tRNA-derived small RNAs (tsRNAs). We then profiled 15 sncRNAs by qPCR and determined that the assay maintains sensitivity in single ECCM drops. Finally, we found significant differences in these sncRNA expression between control and ECCM drops. Improving embryo selection is crucial for reducing time to pregnancy. Here we describe a sensitive technique for biomarker discovery by sequencing and qPCR validation in ECCM, demonstrating that the majority of sncRNAs are embryo derived. We also report an abundance of tsRNAs which suggests these sncRNAs may have functions in endometrial-maternal communication beyond the microRNAs which have been described previously. Abbreviations: PGT-A: Preimplantation genetic testing for aneuploidies; ECCM: Embryo-conditioned culture media; sncRNAs: Small non-coding RNAs; miRNAs: microRNAs; EVs: Extracellular vesicles; PCA: Principal component analysis.