title: Insulin-like growth factor 1 increases apical fibronectin in blastocysts to increase blastocyst attachment to endometrial epithelial cells in vitro.
abstract: OBJECTIVE
  Does insulin-like growth factor 1 (IGF1) increase adhesion competency of blastocysts to increase attachment to uterine epithelial cells in vitro?
  
  
  CONCLUSIONS
  IGF1 increases apical fibronectin on blastocysts to increase attachment and invasion in an in vitro model of implantation.
  
  
  BACKGROUND
  Fibronectin integrin interactions are important in attachment of blastocysts to uterine epithelial cells at implantation.
  
  
  METHODS
  Mouse blastocysts (hatched or near completion of hatching) were cultured in serum starved (SS) medium with varying treatments for 24, 48 or 72 h. Treatments included 10 ng/ml IGF1 in the presence or absence of the PI3 kinase inhibitor LY294002, an IGF1 receptor (IGF1R) neutralizing antibody or fibronectin. Effects of treatments on blastocysts were measured by attachment of blastocysts to Ishikawa cells, blastocyst outgrowth and fibronectin and focal adhesion kinase (FAK) localization and expression. Blastocysts were randomly allocated into control and treatment groups and experiments were repeated a minimum of three times with varying numbers of blastocysts used in each experiment. FAK and integrin protein expression on Ishikawa cells was quantified in the presence or absence of IGF1.
  
  
  METHODS
  Fibronectin expression and localization in blastocysts was studied using immunofluorescence and confocal microscopy. Global surface expression of integrin αvβ3, β3 and β1 was measured in Ishikawa cells using flow cytometry. Expression levels of phosphorylated FAK and total FAK were measured in Ishikawa cells and blastocysts by western blot and image J analysis. Blastocyst outgrowth was quantified using image J analysis.
  
  
  RESULTS
  The presence of IGF1 significantly increased mouse blastocyst attachment to Ishikawa cells compared with SS conditions (P < 0.01). IGF1 treatment resulted in distinct apical fibronectin staining on blastocysts, which was reduced by the PI3 kinase inhibitor LY294002. This coincided with a significant increase in blastocyst outgrowth in the presence of IGF1 (P < 0.01) or fibronectin (P < 0.001), which was abolished by LY294002 (P < 0.001). Apical expression of integrin αvβ3, β3 and β1 in Ishikawa cells was unaltered by IGF1. However, IGF1 increased phosphorylated FAK (P < 0.05) and total FAK expression in Ishikawa cells. FAK signalling is linked to integrin activation and can affect the integrins' ability to bind and recognize extracellular matrix proteins such as fibronectin. Treatment of blastocysts with IGF1 before co-culture with Ishikawa cells increased their attachment (P < 0.05). This effect was abolished in the presence of LY294002 (P < 0.001) or an IGF1R neutralizing antibody (P < 0.05).
  
  
  CONCLUSIONS
  This study uses an in vitro model of attachment that uses mouse blastocysts and human endometrial cells. This involves a species crossover and although this use has been well documented as a model for attachment (as human embryo numbers are limited) the results should be interpreted carefully.
  
  
  CONCLUSIONS
  This study presents mechanisms by which IGF1 improves attachment of blastocysts to Ishikawa cells and documents for the first time how IGF1 can increase adhesion competency in blastocysts. Failure of the blastocyst to implant is the major cause of human assisted reproductive technology (ART) failure. As growth factors are absent during embryo culture, their addition to embryo culture medium is a potential avenue to improve IVF success. In particular, IGF1 could prove to be a potential treatment for blastocysts before transfer to the uterus in an ART setting.
authors.affiliation: Discipline of Physiology, Bosch Institute, Sydney Medical School, University of Sydney, K25 - Medical Foundation Building, Sydney 2006, Australia.
authors.forename: Charmaine J
authors.initials: CJ
authors.lastname: Green
authors.affiliation: Discipline of Physiology, Bosch Institute, Sydney Medical School, University of Sydney, K25 - Medical Foundation Building, Sydney 2006, Australia Discipline of Anatomy and Histology, Sydney Medical School, University of Sydney, K25 - Medical Foundation Building, Sydney 2006, Australia.
authors.forename: Stuart T
authors.initials: ST
authors.lastname: Fraser
authors.affiliation: Discipline of Physiology, Bosch Institute, Sydney Medical School, University of Sydney, K25 - Medical Foundation Building, Sydney 2006, Australia margotd@physiol.usyd.edu.au.
authors.forename: Margot L
authors.initials: ML
authors.lastname: Day
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D018920:Coculture Techniques; D004477:Ectogenesis / Q000187:drug effects; D046149:Embryo Culture Techniques; D004717:Endometrium / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005300:Fertility Agents, Female / Q000378:metabolism / Q000494:pharmacology*; D005353:Fibronectins / Q000819:agonists* / Q000378:metabolism; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D008815:Mice, Inbred Strains; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D021381:Protein Transport / Q000187:drug effects; D017526:Receptor, IGF Type 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: D004791:Enzyme Inhibitors; D005300:Fertility Agents, Female; D005353:Fibronectins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011994:Recombinant Proteins; C519530:insulin-like growth factor-1, mouse; D007334:Insulin-Like Growth Factor I; D058539:Phosphatidylinositol 3-Kinase; D017526:Receptor, IGF Type 1; D051416:Focal Adhesion Protein-Tyrosine Kinases
keywords: PI3 kinase; fibronectin; implantation; insulin-like growth factor 1; integrin