Kirstine Kirkegaard1,2, Yan Yan3, Boe S Sørensen4, Thorir Hardarson5, Charles Hanson6, Hans J Ingerslev7, Ulla Breth Knudsen8,9, Jørgen Kjems3, Kersti Lundin6, Aisling Ahlström6. 1. Department of Clinical Biochemistry, Aarhus University Hospital, Palle Juul Jensens Boulevard, 8200, Aarhus N, Denmark. Kirstine.kirkegaard@clin.au.dk. 2. Department of Gynecology and Obstetrics, Aarhus University Hospital, Palle Juul Jensens Boulevard, 8200, Aarhus N, Denmark. Kirstine.kirkegaard@clin.au.dk. 3. Interdisciplinary Nanoscience Center (iNANO) and Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark. 4. Department of Clinical Biochemistry, Aarhus University Hospital, Palle Juul Jensens Boulevard, 8200, Aarhus N, Denmark. 5. Livio Fertilitetscentrum Gothenburg, Carlandersparken 24, 402 29, Gothenburg, Sweden. 6. Unit of Reproductive Medicine, Sahlgrenska University Hospital, 41345, Gothenburg, Sweden. 7. Fertility Unit and Centre for Preimplantation diagnosis, Aalborg University Hospital, Søndre Skovvej 3, 9000, Aalborg, Denmark. 8. Fertility Clinic, Regional Hospital Horsens, Sundvej 30, 8700, Horsens, Denmark. 9. Inst. Clin. Medicine, Aarhus University, Palle Juul-Jensens Boulevard 88, 8200, Aarhus N, Denmark.
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.
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 humanblastocysts were analyzed with array-based q-PCR analysis. Subsequently, deep sequencing of total and small RNA in day 5 spent culture medium from 5 humanblastocysts 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.
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