title: Factors affecting the gene expression of in vitro cultured human preimplantation embryos.
abstract: OBJECTIVE
  What is the relative effect of common environmental and biological factors on transcriptome changes during human preimplantation development?
  
  
  CONCLUSIONS
  Developmental stage and maternal age had a larger effect on the global gene expression profile of human preimplantation embryos than the culture medium or oxygen concentration used in in vitro culture.
  
  
  BACKGROUND
  Studies on mouse and bovine embryos have shown that different conditions in the in vitro culture of embryos can lead to changes in transcriptome profiles. For humans, an effect of developmental stage on the transcriptome profile of embryos has been demonstrated, but studies on the effect of maternal age or culture conditions are lacking.
  
  
  METHODS
  Donated, good quality, day 4 cryopreserved human preimplantation embryos (N = 89) were randomized to be cultured in one of two culture media (G5 medium or HTF medium) and one of two oxygen concentrations (5% or 20%), with stratification for maternal age. Next to these variables, developmental stage after culture was taken into account in the analysis.
  
  
  METHODS
  Embryos that developed to morula or blastocyst stage during these 2 days whose amplified mRNA passed our quality control criteria for microarray hybridization were individually examined for genome-wide gene expression (N = 37).
  
  
  RESULTS
  Based on the number of differentially expressed genes (DEGs), developmental stage (3519 DEGs) and maternal age (1258 DEGs) had a larger effect on the global gene expression profile of human preimplantation embryos than either tested culture medium (596 DEGs) or oxygen concentration (492 DEGs) used during in vitro culture. Interactions between the factors were found, indicating that culture conditions might have a different effect depending on the developmental stage or the maternal age of the embryos. Affected pathways included metabolism, cell cycle processes and oxidative phosphorylation.
  
  
  CONCLUSIONS
  Culture of embryos for only 2 days might have limited the effect on global gene expression by the investigated culture conditions. Earlier stages of development (Day 0 until Day 4) were not analyzed and these embryos might respond differently to the experimental conditions. The freezing and thawing procedures might have had an effect on gene expression. RT-PCR validation was not performed due to scarcity of the material.
  
  
  CONCLUSIONS
  Our results show that when studying gene expression in single human preimplantation embryos under various experimental conditions, one should take into account the confounding effect of biological variables, such as developmental stage and maternal age. This makes these experiments different from gene expression experiments where these variables can be tightly controlled, for example when using cell lines.
  
  
  BACKGROUND
  This study received no external funding and there were no competing interests.
authors.affiliation: Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands MicroArray Department and Integrative Bioinformatics Unit (MAD-IBU), Swammerdam Institute for Life Sciences, Faculty of Science (FNWI), University of Amsterdam, 1090 GE Amsterdam, The Netherlands.
authors.forename: E
authors.initials: E
authors.lastname: Mantikou
authors.affiliation: MicroArray Department and Integrative Bioinformatics Unit (MAD-IBU), Swammerdam Institute for Life Sciences, Faculty of Science (FNWI), University of Amsterdam, 1090 GE Amsterdam, The Netherlands Netherlands Bioinformatics Center (NBIC), 6525 GA Nijmegen, The Netherlands.
authors.forename: M J
authors.initials: MJ
authors.lastname: Jonker
authors.affiliation: Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.
authors.forename: K M
authors.initials: KM
authors.lastname: Wong
authors.affiliation: Department of Obstetrics and Gynaecology, GROW school for Oncology and Developmental Biology, Maastricht University Medical Center, 6202 AZ Maastricht, The Netherlands.
authors.forename: A P A
authors.initials: AP
authors.lastname: van Montfoort
authors.affiliation: MicroArray Department and Integrative Bioinformatics Unit (MAD-IBU), Swammerdam Institute for Life Sciences, Faculty of Science (FNWI), University of Amsterdam, 1090 GE Amsterdam, The Netherlands Present address: GenomeScan B.V., Plesmanlaan 1d, 2333BZ Leiden, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: de Jong
authors.affiliation: MicroArray Department and Integrative Bioinformatics Unit (MAD-IBU), Swammerdam Institute for Life Sciences, Faculty of Science (FNWI), University of Amsterdam, 1090 GE Amsterdam, The Netherlands Netherlands Bioinformatics Center (NBIC), 6525 GA Nijmegen, The Netherlands.
authors.forename: T M
authors.initials: TM
authors.lastname: Breit
authors.affiliation: Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.
authors.forename: S
authors.initials: S
authors.lastname: Repping
authors.affiliation: Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands s.mastenbroek@amc.uva.nl.
authors.forename: S
authors.initials: S
authors.lastname: Mastenbroek
mesh_terms: D001755:Blastocyst / Q000378:metabolism*; D003470:Culture Media; D046149:Embryo Culture Techniques; D047108:Embryonic Development; D015870:Gene Expression; D006801:Humans; D008423:Maternal Age; D010100:Oxygen / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D003470:Culture Media; D012333:RNA, Messenger; D010100:Oxygen
keywords: culture medium; developmental stage; gene expression; human preimplantation embryos; in vitro fertilization; maternal age; oxygen concentration