title: Endometrial signals improve embryo outcome: functional role of vascular endothelial growth factor isoforms on embryo development and implantation in mice.
abstract: OBJECTIVE
  Does vascular endothelial growth factor (VEGF) have important roles during early embryo development and implantation?
  
  
  CONCLUSIONS
  VEGF plays key roles during mouse preimplantation embryo development, with beneficial effects on time to cavitation, blastocyst cell number and outgrowth, as well as implantation rate and fetal limb development.
  
  
  BACKGROUND
  Embryo implantation requires synchronized dialog between maternal cells and those of the conceptus. Following ovulation, secretions from endometrial glands increase and accumulate in the uterine lumen. These secretions contain important mediators that support the conceptus during the peri-implantation phase. Previously, we demonstrated a significant reduction of VEGFA in the uterine cavity of women with unexplained infertility. Functional studies demonstrated that VEGF significantly enhanced endometrial epithelial cell adhesive properties and embryo outgrowth.
  
  
  METHODS
  Human endometrial lavages (n = 6) were obtained from women of proven fertility. Four-week old Swiss mice were superovulated and mated with Swiss males to obtain embryos for treatment with VEGF in vitro. Preimplantation embryo development was assessed prior to embryo transfer (n = 19-30/treatment group/output). Recipient F1 female mice (8-12 weeks of age) were mated with vasectomized males to induce pseudopregnancy and embryos were transferred. On Day 14.5 of pregnancy, uterine horns were collected for analysis of implantation rates as well as placental and fetal development (n = 14-19/treatment).
  
  
  METHODS
  Lavage fluid was assessed by western immunoblot analysis to determine the VEGF isoforms present. Mouse embryos were treated with either recombinant human (rh)VEGF, or VEGF isoforms 121 and 165. Preimplantation embryo development was quantified using time-lapse microscopy. Blastocysts were (i) stained for cell number, (ii) transferred to wells coated with fibronectin to examine trophoblast outgrowth or (iii) transferred to pseudo pregnant recipients to analyze implantation rates, placental and fetal development.
  
  
  RESULTS
  Western blot analysis revealed the presence of VEGF121 and 165 isoforms in human uterine fluid. Time-lapse microscopy analysis revealed that VEGF (n = 22) and VEGF121 (n = 23) treatment significantly reduced the preimplantation mouse embryo time to cavitation (P < 0.05). VEGF and VEGF165 increased both blastocyst cell number (VEGF n = 27; VEGF165 n = 24: P < 0.001) and outgrowth (n = 15/treatment: 66 h, P < 0.001; 74, 90, 98 and 114 h, P < 0.01) on fibronectin compared with control. Furthermore, rhVEGF improved implantation rates and enhanced fetal limb development (P < 0.05).
  
  
  CONCLUSIONS
  Due to the nature of this work, embryo development and implantation was only examined in the mouse.
  
  
  CONCLUSIONS
  The absence or reduction in levels of VEGF during the preimplantation period likely affects key events during embryo development, implantation and placentation. The potential for improvement of clinical IVF outcomes by the addition of VEGF to human embryo culture media needs further investigation.
  
  
  BACKGROUND
  This study was supported by a University of Melbourne Early Career Researcher Grant #601040, the NHMRC (L.A.S., Program grant #494802; Fellowship #1002028; N.J.H., Fellowship # 628927; J.E.; project grant #1047756) and L.A.S., Monash IVF Research and Education Foundation. N.K.B. was supported by an Australian Postgraduate Award. Work at PHI-MIMR Institute was also supported by the Victorian Government's Operational Infrastructure Support Program. There are no conflicts of interest to declare.
authors.affiliation: Department of Zoology, University of Melbourne, Parkville 3010, Australia Translational Obstetrics Group, Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Heidelberg, VIC 3084, Australia.
authors.forename: N K
authors.initials: NK
authors.lastname: Binder
authors.affiliation: MIMR-PHI Institute for Medical Research, 27-31 Wright St, Clayton 3168, Australia Department of Physiology, Monash University, Clayton, Australia.
authors.forename: J
authors.initials: J
authors.lastname: Evans
authors.affiliation: Department of Zoology, University of Melbourne, Parkville 3010, Australia.
authors.forename: D K
authors.initials: DK
authors.lastname: Gardner
authors.affiliation: MIMR-PHI Institute for Medical Research, 27-31 Wright St, Clayton 3168, Australia Department of Obstetrics and Gynecology, Monash University, Clayton, Australia.
authors.forename: L A
authors.initials: LA
authors.lastname: Salamonsen
authors.affiliation: Department of Zoology, University of Melbourne, Parkville 3010, Australia MIMR-PHI Institute for Medical Research, 27-31 Wright St, Clayton 3168, Australia Translational Obstetrics Group, Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Heidelberg, VIC 3084, Australia nhannan@unimelb.edu.au.
authors.forename: N J
authors.initials: NJ
authors.lastname: Hannan
mesh_terms: D000818:Animals; D003470:Culture Media; D046149:Embryo Culture Techniques; D010064:Embryo Implantation / Q000187:drug effects* / Q000502:physiology; D047108:Embryonic Development / Q000187:drug effects* / Q000502:physiology; D004717:Endometrium / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology* / Q000502:physiology
chemical_list: D003470:Culture Media; D042461:Vascular Endothelial Growth Factor A
keywords: assisted reproduction; embryo; endometrium; implantation; vascular endothelial growth factor