TY - JOUR
T1 - Comprehensive analysis of soluble RNAs in human embryo culture media and blastocoel fluid
AU - Kirkegaard, Kirstine
AU - Yan, Yan
AU - Sørensen, Boe S.
AU - Hardason, Thorir
AU - Hanson, Charles
AU - Ingerslev, Hans Jakob
AU - Knudsen, Ulla Breth
AU - Kjems, Jørgen
AU - Lundin, Kersti
AU - Ahlström, Aisling
PY - 2020/9/1
Y1 - 2020/9/1
N2 - 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.
AB - 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.
KW - ART
KW - Biomarker
KW - Culture medium
KW - miRNA
KW - tiRNA
UR - http://www.scopus.com/inward/record.url?scp=85088089172&partnerID=8YFLogxK
U2 - 10.1007/s10815-020-01891-7
DO - 10.1007/s10815-020-01891-7
M3 - Journal article
C2 - 32681282
AN - SCOPUS:85088089172
SN - 1058-0468
VL - 37
SP - 2199
EP - 2209
JO - Journal of Assisted Reproduction and Genetics
JF - Journal of Assisted Reproduction and Genetics
IS - 9
ER -