Comprehensive analysis of soluble RNAs in human embryo culture media and blastocoel fluid
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EMBRYO BIOLOGY
Comprehensive analysis of soluble RNAs in human embryo culture media and blastocoel fluid Kirstine Kirkegaard 1,2 & Yan Yan 3 & Boe S. Sørensen 1 & Thorir Hardarson 4 & Charles Hanson 5 & Hans J Ingerslev 6 & Ulla Breth Knudsen 7,8 & Jørgen Kjems 3 & Kersti Lundin 5 & Aisling Ahlström 5 Received: 30 April 2020 / Accepted: 13 July 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Purpose miRNAs have been suggested as biomarkers of embryo viability; however, findings from preliminary studies are divergent. Furthermore, the presence of other types of small RNA molecules remains to be investigated. The purpose of this study was to perform a comprehensive analysis of small non-coding RNA levels in spent and unconditioned embryo culture media, along with miRNA levels in blastocoelic fluid samples from human embryos. Methods miRNAs in unconditioned culture medium from 3 different manufacturers, along with miRNA from day 5 conditioned culture medium, control medium, and corresponding blastocoel fluid from 10 human blastocysts were analyzed with array-based q-PCR analysis. Subsequently, deep sequencing of total and small RNA in day 5 spent culture medium from 5 human blastocysts and corresponding controls was performed. Results In spite of using state-of-the-art sensitive detection methods, no miRNAs were found to be reliably present in the spent culture medium or the blastocoel fluid. Ct values were above the recommended limit for detection in the array-based analysis, a finding that was confirmed by deep sequencing. The majority of miRNAs identified by deep sequencing were expressed in all samples including control media and seem to originate from sources other than conditioned IVF media. Conclusions Our findings question the use of miRNAs as a reliable biomarker and highlight the need for a critical methodological approach in miRNA studies. Interestingly, tiRNA fragments appear to be overexpressed in conditioned IVF media samples and could potentially be a novel biomarker worthy of investigation. Keywords miRNA . tiRNA . Biomarker . Culture medium . ART
Introduction miRNAs are small, non-coding RNA molecules that have attracted attention in several areas of medicine, including assisted reproduction. miRNAs act by binding mRNA targets to repress their expression and one miRNA can target several different mRNAs [1]. In addition to regulating intracellular * Kirstine Kirkegaard [email protected] 1
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Department of Clinical Biochemistry, Aarhus University Hospital, Palle Juul Jensens Boulevard 8200 Aarhus N Denmark Department of Gynecology and Obstetrics, Aarhus University Hospital, Palle Juul Jensens Boulevard 8200 Aarhus N Denmark Interdisciplinary Nanoscience Center (iNANO) and Department of Molecular Biology and Genetics, Aarhus University, Aarhus Denmark
gene expression, miRNAs can be packaged into exosomes and released into the extracellular environment. In there, they can be taken up by recipient cells and modulate their gene expression [2]. As such,
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