title: Effects of combined epidermal growth factor, brain-derived neurotrophic factor and insulin-like growth factor-1 on human oocyte maturation and early fertilized and cloned embryo development.
abstract: BACKGROUND
  Human cloned blastocysts generated from oocytes following in vitro maturation (IVM) are a potential resource for embryonic stem cells (ESC) with homologous immune systems. The purpose of this study was to evaluate the effects of multiple growth factors [epidermal growth factor (EGF), brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1)] on human oocyte maturation, early embryo development, blastocyst formation and ESC line generation.
  
  
  METHODS
  Patients (n= 344) undergoing IVF owing to male factor infertility were enrolled in this study. Metaphase II oocytes were separated into four grades based on their morphology. Spindle assembly from IVM oocytes with or without growth factor treatment was assessed by immunostaining. Piezo-assisted micromanipulation technology was used to produce fertilized (ICSI) and cloned [(somatic cell nuclear transfer (SCNT)] embryos. Embryos received four different growth factor treatments; embryo development rates from pronuclear to blastocyst stage and embryo grading (for quality) at the 8-cell stage were analyzed. The presence of receptors on human cumulus cells and IVM oocytes was assessed by immunofluorescence. The blastocysts generated from fertilized and cloned embryos were used for ESC derivation.
  
  
  RESULTS
  The combination of EGF, BDNF and IGF-1 can effectively increase oocyte maturation rate in vitro, and significantly improve the oocyte quality in terms of morphology and normal spindle levels (P< 0.05). Also, the developmental competence of fertilized oocytes to 8-cell and blastocyst stages was improved by the addition of growth factors (P< 0.05). However, there were no significant differences among the four groups in 8-cell grading. Blastocyst formation in cloned embryos cultured with the three growth factors was higher than the control group (23.1 versus 4.3%, P< 0.05). Receptors for the three growth factors were present in cumulus cells and IVM oocytes, and four human ESC lines were derived from fertilized blastocysts but none from cloned blastocysts.
  
  
  CONCLUSIONS
  This study demonstrated that EGF, BDNF and IGF-1 can improve oocyte maturation rate and quality in vitro, and consequently increase early embryo development and blastocyst formation, which is very beneficial in improving the reprogramming efficiency of SCNT. The present study has identified a valuable culture system for IVM and cloned human embryos, potentially using these embryos to derive human therapeutic ESC.
authors.affiliation: Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, No. 49 HuaYuan North Road, HaiDian District, 100191 Beijing, The People's Republic of China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu
authors.forename: Jie
authors.initials: J
authors.lastname: Yan
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.forename: Liying
authors.initials: L
authors.lastname: Yan
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhao
authors.forename: Ying
authors.initials: Y
authors.lastname: Lian
authors.forename: Rong
authors.initials: R
authors.lastname: Li
authors.forename: Ping
authors.initials: P
authors.lastname: Liu
authors.forename: Jie
authors.initials: J
authors.lastname: Qiao
mesh_terms: D001755:Blastocyst / Q000166:cytology; D019208:Brain-Derived Neurotrophic Factor / Q000378:metabolism*; D019976:Cloning, Organism; D054885:Cumulus Cells / Q000166:cytology; D047108:Embryonic Development / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000166:cytology; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005260:Female; D005306:Fertilization; D005307:Fertilization in Vitro; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D007248:Infertility, Male / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007621:Karyotyping; D008297:Male; D008677:Metaphase; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D009865:Oocytes / Q000166:cytology; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I