MicroRNAs in spent blastocyst culture medium are derived from trophectoderm cells and can be explored for human embryo reproductive competence assessment

MicroRNAs can be reproducibly detected in blastocyst-spent media and most likely belong to trophectoderm cells. Primary clinical data suggest testing these noninvasive biomarkers for embryo selection in IVF cycles.

Antonio Capalbo, Ph.D., Maria Ubaldi Filippo, M.D., M.Sc., Danilo Cimadomo, M.Sc., Laila Noli, M.Sc., Yakoub Khalaf, M.D., M.Sc., Alessio Farcomeni, Ph.D., Dusko Ilic, M.D., Ph.D., Laura Rienzi, M.Sc.

Volume 105, Issue 1, Pages 224-235


To assess whether extracellular microRNAs (miRNAs) can be accurately profiled from spent blastocyst culture media (SBM) and used as embryonic biomarkers.

Prospective cohort study.

Private and academic in vitro fertilization centers.

Inner cell mass-free trophectoderm (TE) samples and their relative SBM from five good-quality human blastocysts.

Protocol for miRNA purification and analysis based on quantitative polymerase chain reaction set and validated on human embryonic stem cells (hESCs) and on SBM with and without biological variability.

Main Outcomes Measure(s):
Analysis of miRNAs in culture media in relation with TE cells and comparison of miRNA profiles between implanted and unimplanted euploid blastocysts.

Culture media from embryos in the cleavage, morula, and blastocyst stages were collected to investigate the presence of miRNAs. The SBM were prospectively collected from euploid implanted (n = 25) and unimplanted blastocysts (n = 28) for comparison. We hypothesized that human embryos secrete miRNAs in culture media that can be used as biomarkers. The comparative analysis of TE and SBM samples revealed that 96.6% (57 of 59; 95 CI, 88.3–99.6) of the miRNAs detected in the SBM were expressed from TE cells, suggesting a TE origin. The culture media collected from cleavage and morula stage embryos showed a pattern similar to blanks, suggesting that miRNAs profiling from spent culture media applies only for blastocysts. MicroRNAs analysis of SBM from euploid implanted and unimplanted blastocysts highlighted two miRNAs (miR-20a, miR-30c) that showed increased concentrations in the former and were predicted in silico to be involved in 23 implantation-related pathways.

MicroRNAs secreted from human blastocysts in culture media can be profiled with high reproducibility, and this approach can be further explored for noninvasive embryo selection.

  • Jorge De los Santos

    Dear Dr. Capalbo and colleagues,

    Congratulations! I read your paper and it looks that do not have a key citation. miRNA in media culture were described for first time by the Rosenbluth and Khatib lab.

    Dr. Khatib laboratory made a big contribution working in human and cattle, they were the first to find and published miRNA in IVF culture media.

    This is one of the articles that I am talking about; Kropp, J., S. M. Salih, and H. Khatib. 2014. Expression of microRNAs in bovine and human preimplantation embryo culture media. Front Genet 5:91.

    Thanks very much for your comments.

  • Daniel J. Kaser, MD

    Dear Dr. Capalbo and colleagues,

    Congratulations on this contribution, in which the miRNA profile of spent culture media from blastocyst is characterized. The differential expression of miRNA 20 and 30 among euploid embryos that implanted and those that failed to implant is intriguing, along with the description of your ongoing prospective study evaluating the efficacy of these non-invasive markers. Could you kindly elaborate on the design of your study for the readers–is it a non-selection study to allow you to calculate the performance characteristics of the assay?

    Thanks very much for your comments.

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