title: MicroRNAs in spent blastocyst culture medium are derived from trophectoderm cells and can be explored for human embryo reproductive competence assessment.
abstract: OBJECTIVE
  To assess whether extracellular microRNAs (miRNAs) can be accurately profiled from spent blastocyst culture media (SBM) and used as embryonic biomarkers.
  
  
  METHODS
  Prospective cohort study.
  
  
  METHODS
  Private and academic in vitro fertilization centers.
  
  
  METHODS
  Inner cell mass-free trophectoderm (TE) samples and their relative SBM from five good-quality human blastocysts.
  
  
  METHODS
  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.
  
  
  METHODS
  Analysis of miRNAs in culture media in relation with TE cells and comparison of miRNA profiles between implanted and unimplanted euploid blastocysts.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: GENERA, Centre for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy; GENETYX, Molecular Genetics Laboratory, Vicenza, Italy. Electronic address: capalbo@generaroma.it.
authors.forename: Antonio
authors.initials: A
authors.lastname: Capalbo
authors.affiliation: GENERA, Centre for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy; GENETYX, Molecular Genetics Laboratory, Vicenza, Italy.
authors.forename: Filippo Maria
authors.initials: FM
authors.lastname: Ubaldi
authors.affiliation: GENERA, Centre for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy; GENETYX, Molecular Genetics Laboratory, Vicenza, Italy.
authors.forename: Danilo
authors.initials: D
authors.lastname: Cimadomo
authors.affiliation: Division of Women's Health and Assisted Conception Unit, King's College of London, Guy's Hospital, London, United Kingdom.
authors.forename: Laila
authors.initials: L
authors.lastname: Noli
authors.affiliation: Division of Women's Health and Assisted Conception Unit, King's College of London, Guy's Hospital, London, United Kingdom.
authors.forename: Yakoub
authors.initials: Y
authors.lastname: Khalaf
authors.affiliation: Department of Public Health and Infectious Diseases, University of Rome La Sapienza, Rome, Italy.
authors.forename: Alessio
authors.initials: A
authors.lastname: Farcomeni
authors.affiliation: Division of Women's Health and Assisted Conception Unit, King's College of London, Guy's Hospital, London, United Kingdom.
authors.forename: Dusko
authors.initials: D
authors.lastname: Ilic
authors.affiliation: GENERA, Centre for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy; GENETYX, Molecular Genetics Laboratory, Vicenza, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Rienzi
mesh_terms: D001755:Blastocyst / Q000166:cytology / Q000378:metabolism*; D002460:Cell Line; D019295:Computational Biology; D003198:Computer Simulation; D017077:Culture Media, Conditioned / Q000378:metabolism*; D030541:Databases, Genetic; D046149:Embryo Culture Techniques; D010064:Embryo Implantation; D004624:Embryo Transfer; D053595:Embryonic Stem Cells / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro; D018507:Gene Expression Regulation, Developmental; D053263:Gene Regulatory Networks; D005819:Genetic Markers; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011446:Prospective Studies
chemical_list: D017077:Culture Media, Conditioned; D005819:Genetic Markers; D035683:MicroRNAs
keywords: Biomarkers of implantation; blastocyst evaluation; blastocyst-endometrial dialogue; embryo quality; microRNA