title: Ammonium accumulation in commercially available embryo culture media and protein supplements during storage at 2-8°C and during incubation at 37°C.
abstract: Does ammonium accumulate in commercially available culture media and protein supplements used for in vitro development of human pre-implantation embryos during storage and incubation?
  
  
  
  Ammonium accumulates in ready-to-use in vitro fertilization (IVF) culture media during storage at 2-8°C and in ready-to-use IVF culture media and protein supplements during incubation at 37°C.
  
  
  
  Both animal and human studies have shown that the presence of ammonium in culture medium has detrimental effects on embryonic development and pregnancy rate. It is, therefore, important to assess the amount of ammonium accumulation in ready-to-use IVF culture media under conditions that are common in daily practice.
  
  
  
  Ammonium accumulation was investigated in 15 ready-to-use media, 11 protein-free media and 8 protein supplements.
  
  
  
  Ammonium was measured by the use of an enzymatic method with glutamate dehydrogenase. To simulate the storage and incubation conditions during IVF treatments, ammonium concentrations were measured at different time-points during storage at 2-8°C for 6 weeks and during incubation at 37°C for 4 days.
  
  
  
  All ready-to-use, i.e. protein supplemented, culture media showed ammonium accumulation during storage for 6 weeks (ranging from 9.2 to 99.8 µM) and during incubation for 4 days (ranging from 8.4 to 138.6 µM), resulting in levels that might affect embryo development. The protein supplements also showed ammonium accumulation, while the culture media without protein supplementation did not. The main sources of ammonium buildup in ready-to-use culture media were unstable glutamine and the protein supplements. No additional ammonium buildup was found during incubation when using an oil overlay or with the presence of an embryo in the culture droplet.
  
  
  
  In addition to the unstable glutamine and the protein supplements, other free amino acids might contribute to the ammonium buildup. We did not investigate the deterioration of other components in the media.
  
  
  
  Break-down of components into ammonium is more pronounced during incubation at 37°C, however, it is not negligible during storage at 2-8°C. This results in increasing ammonium levels in culture media over time that may affect embryo development. Therefore, it is important that the use of free l-glutamine in human embryo culture media is stopped and that the use of protein supplements is thoroughly evaluated.
  
  
  
  No funding or no competing interests declared.
  
  
  
  Not applicable.
authors.affiliation: Department of Obstetrics and Gynaecology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands.
authors.forename: Sander H M
authors.initials: SH
authors.lastname: Kleijkers
authors.affiliation: Department of Obstetrics and Gynaecology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands.
authors.forename: Aafke P A
authors.initials: AP
authors.lastname: van Montfoort
authors.affiliation: Central Diagnostic Laboratory, Maastricht University Medical Center, Maastricht, The Netherlands.
authors.forename: Otto
authors.initials: O
authors.lastname: Bekers
authors.affiliation: Department of Obstetrics and Gynaecology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands.
authors.forename: Edith
authors.initials: E
authors.lastname: Coonen
authors.affiliation: Department of Obstetrics and Gynaecology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands.
authors.forename: Josien G
authors.initials: JG
authors.lastname: Derhaag
authors.affiliation: Department of Obstetrics and Gynaecology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands.
authors.forename: Johannes L H
authors.initials: JL
authors.lastname: Evers
authors.affiliation: Department of Obstetrics and Gynaecology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands john.dumoulin@mumc.nl.
authors.forename: John C M
authors.initials: JC
authors.lastname: Dumoulin
mesh_terms: D064751:Ammonium Compounds / Q000032:analysis*; D001755:Blastocyst; D003080:Cold Temperature; D003470:Culture Media / Q000737:chemistry*; D046149:Embryo Culture Techniques; D006801:Humans; D013997:Time Factors
chemical_list: D064751:Ammonium Compounds; D003470:Culture Media
keywords: IVF/ICSI; ammonium; culture medium; glutamine; protein