title: Unconditioned commercial embryo culture media contain a large variety of non-declared proteins: a comprehensive proteomics analysis.
abstract: OBJECTIVE
  Which non-declared proteins (proteins not listed on the composition list of the product data sheet) are present in unconditioned commercial embryo culture media?
  
  
  CONCLUSIONS
  A total of 110 non-declared proteins were identified in unconditioned media and between 6 and 8 of these were quantifiable and therefore represent the majority of the total protein in the media samples.
  
  
  BACKGROUND
  There are no data in the literature on what non-declared proteins are present in unconditioned (fresh media in which no embryos have been cultured) commercial embryo media.
  
  
  METHODS
  The following eight commercial embryo culture media were included in this study: G-1 PLUS and G-2 PLUS G5 Series from Vitrolife, Sydney IVF Cleavage Medium and Sydney IVF Blastocyst Medium from Cook Medical and EmbryoAssist, BlastAssist, Sequential Cleav and Sequential Blast from ORIGIO. Two batches were analyzed from each of the Sydney IVF media and one batch from each of the other media. All embryo culture media are supplemented by the manufacturers with purified human serum albumin (HSA 5 mg/ml). The purified HSA (HSA-solution from Vitrolife) and the recombinant human albumin supplement (G-MM from Vitrolife) were also analyzed.
  
  
  METHODS
  For protein quantification, media samples were in-solution digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For in-depth protein identification, media were albumin depleted, dialyzed and concentrated before sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gel was cut into 14 slices followed by in-gel trypsin digestion, and analysis by LC-MS/MS. Proteins were further investigated using gene ontology (GO) terms analysis.
  
  
  RESULTS
  Using advanced mass spectrometry and high confidence criteria for accepting proteins (P < 0.01), a total of 110 proteins other than HSA were identified. The average HSA content was found to be 94% (92-97%) of total protein. Other individual proteins accounted for up to 4.7% of the total protein. Analysis of purified HSA strongly suggests that these non-declared proteins are introduced to the media when the albumin is added. GO analysis showed that many of these proteins have roles in defence pathways, for example 18 were associated with the innate immune response and 17 with inflammatory responses. Eight proteins have been reported previously as secreted embryo proteins.
  
  
  CONCLUSIONS
  For six of the commercial embryo culture media only one batch was analyzed. However, this does not affect the overall conclusions.
  
  
  CONCLUSIONS
  The results showed that the HSA added to IVF media contained many other proteins and that the amount varies from batch to batch. These variations in protein profiles are problematic when attempting to identify proteins derived from the embryos. Therefore, when studying the embryo secretome and analyzing conditioned media with the aim of finding potential biomarkers that can distinguish normal and abnormal embryo development, it is important that the medium used in the experimental and control groups is from the same batch. Furthermore, the proteins present in unconditioned media could potentially influence embryonic development, gestation age, birthweight and perhaps have subsequent effects on health of the offspring.
  
  
  BACKGROUND
  The study was supported by the Danish Agency for Science, Technology and Innovation. Research at the Fertility Clinic, Aarhus University Hospital is supported by an unrestricted grant from Merck Sharp & Dohme Corp and Ferring. The authors declare no conflicts of interest.
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus C., Denmark tfd@mb.au.dk.
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Dyrlund
authors.affiliation: The Fertility Clinic, Aarhus University Hospital, Aarhus N, Denmark Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Kirstine
authors.initials: K
authors.lastname: Kirkegaard
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus C., Denmark.
authors.forename: Ebbe Toftgaard
authors.initials: ET
authors.lastname: Poulsen
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus C., Denmark Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus C., Denmark.
authors.forename: Kristian W
authors.initials: KW
authors.lastname: Sanggaard
authors.affiliation: The Fertility Clinic, Aarhus University Hospital, Aarhus N, Denmark.
authors.forename: Johnny J
authors.initials: JJ
authors.lastname: Hindkjær
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus C., Denmark Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus C., Denmark.
authors.forename: Jørgen
authors.initials: J
authors.lastname: Kjems
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus C., Denmark Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus C., Denmark.
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Enghild
authors.affiliation: The Fertility Clinic, Aarhus University Hospital, Aarhus N, Denmark.
authors.forename: Hans Jakob
authors.initials: HJ
authors.lastname: Ingerslev
mesh_terms: D003470:Culture Media / Q000737:chemistry*; D046149:Embryo Culture Techniques / Q000379:methods*; D006801:Humans; D011506:Proteins / Q000032:analysis*; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D003470:Culture Media; D011506:Proteins
keywords: IVF; culture media; embryo; proteomics; secretome