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Comprehensive analysis of soluble RNAs in human embryo culture media and blastocoel fluid

  • Embryo Biology
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

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.

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Data availability

All data will be made accessible on request.

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Acknowledgments

Anne Færch Nielsen is thanked for critical reading of the manuscript. Anette Gabrielsen is thanked for assistance in collecting spent media at Regional Hospital Horsens.

Funding

This work was supported by unrestricted grants from Vitrolife (Grant for the development of ART), the Hjalmar Svensson Foundation, VP legacy on recommendation from Novo Nordisk, and the Danish Council for Independent Research Medical Sciences.

Author information

Authors and Affiliations

  1. Department of Clinical Biochemistry, Aarhus University Hospital, Palle Juul Jensens Boulevard, 8200, Aarhus N, Denmark

    Kirstine Kirkegaard & Boe S. Sørensen

  2. Department of Gynecology and Obstetrics, Aarhus University Hospital, Palle Juul Jensens Boulevard, 8200, Aarhus N, Denmark

    Kirstine Kirkegaard

  3. Interdisciplinary Nanoscience Center (iNANO) and Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Yan Yan & Jørgen Kjems

  4. Livio Fertilitetscentrum Gothenburg, Carlandersparken 24, 402 29, Gothenburg, Sweden

    Thorir Hardarson

  5. Unit of Reproductive Medicine, Sahlgrenska University Hospital, 41345, Gothenburg, Sweden

    Charles Hanson, Kersti Lundin & Aisling Ahlström

  6. Fertility Unit and Centre for Preimplantation diagnosis, Aalborg University Hospital, Søndre Skovvej 3, 9000, Aalborg, Denmark

    Hans J Ingerslev

  7. Fertility Clinic, Regional Hospital Horsens, Sundvej 30, 8700, Horsens, Denmark

    Ulla Breth Knudsen

  8. Inst. Clin. Medicine, Aarhus University, Palle Juul-Jensens Boulevard 88, 8200, Aarhus N, Denmark

    Ulla Breth Knudsen

Authors
  1. Kirstine Kirkegaard
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  2. Yan Yan
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  3. Boe S. Sørensen
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  4. Thorir Hardarson
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  5. Charles Hanson
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  6. Hans J Ingerslev
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  7. Ulla Breth Knudsen
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  8. Jørgen Kjems
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  9. Kersti Lundin
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  10. Aisling Ahlström
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Contributions

KK, KL, and AA designed the study. KL and AA designed and acquired samples for the spent culture media analysis. AA performed the experiments on surplus embryos embryos. UBK took part in study design and data acquisition at the region Hospital Horsens. YY designed and performed the deep sequencing experiments. JK designed the deep sequencing experiments and performed the interpretation of the sequencing analysis. BS contributed substantially to the acquisition of data and interpretation of the miRNA analysis. JI, TH and CH contributed substantially to the interpretation of data. KK interpreted the miRNA data and wrote the first draft. All authors critically reviewed and approved the final version of the manuscript.

Corresponding author

Correspondence to Kirstine Kirkegaard.

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Conflict of interest

The authors declare that they have no conflict of interest.

Ethics approval

The study was approved by the Ethics committee in Gothenburg (Dnr: 066-15), by the Central Denmark Region Committees on Biomedical Research Ethics and the Danish Data Protection Agency.

Consent to participate

Informed consent was obtained from all individual participants included in the study.

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Kirkegaard, K., Yan, Y., Sørensen, B.S. et al. Comprehensive analysis of soluble RNAs in human embryo culture media and blastocoel fluid. J Assist Reprod Genet 37, 2199–2209 (2020). https://doi.org/10.1007/s10815-020-01891-7

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  • DOI: https://doi.org/10.1007/s10815-020-01891-7

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