Nd putative paracrine factors secreted by the reproductive tract were shown to enhance early embryonic development [11]. Recent progress in sequential culture media allowed extended culture of human embryos to the blastocyst-stage and blastocystHuman Embryo Culturetransfer is effective in selecting high-quality embryos for successful pregnancy [12], leading to the birth of several million IVF babies [14,15]. However, the efficiency of human blastocyst development in vitro remains to be improved. Several studies using surplus human material suggested the promotion of blastocyst development in vitro when culture media were supplemented with AKT inhibitor 2 web growth factors, including EGF [13], IGF-I [14], BDNF [18], and granulocyte macrophage colony-stimulating factor (GM-CSF) [19,20]. Because most routine human embryo cultures do not contain growth factors, we hypothesized that inclusion of autocrine/paracrine growth factors in the culture media could improve early embryonic development. We investigated the expression of key ligand-receptor pairs in cleavage-stage human embryos derived from tri-pronuclear zygotes and specific ligands in human uterine endometrium. We individually cultured abnormally fertilized zygotes and normally fertilized day 3 embryos from IVF programs in micro-drops using routine serum-free culture media supplemented with a group of key growth factors to improve embryo development. We examined if these growth factors could improve blastocyst outgrowth in vitro using normally fertilized hatching 
blastocysts. Because the development of reconstructed embryos after SCNT is sub-optimal, we also tested if supplementation of culture media with key growth factors could increase the development of SCNT-derived embryos.overnight at 4uC. The antibodies used include: anti-EGF (Abcam, Cambridge, MA), anti-IGF-I (Santa Cruz Biotech, Santa Cruz, CA), anti-GM-CSF (Abcam), anti-BDNF (Santa Cruz Biotech), anti-CSF-1 (Abcam), anti-artemin (Abcam), anti-GDNF (Abcam), anti-EGF receptor (Abcam), anti-IGF-I receptor (Abcam), antiGM-CSF receptor (Santa Cruz Biotech), anti-TrkB (R D systems, Minneapolis, MN), anti-CSF-1 receptor (Santa Cruz Biotech), Anti-GFR alpha 3 antibody (Abcam), After three washes in PBS containing 0.1 Tween 20, embryos were incubated with fluorescein isothiocyanate-conjugated secondary antibodies (Jackson Labs, West Grove, PA) for 1 h at 23uC. Nuclear status of embryos was evaluated by staining with 10 mg/ml propidium iodide for 10 min and examined with a confocal laser-scanning microscope (Zeiss LSM 510, Jena, Germany). For negative controls, the primary antibody was replaced with non-immune IgG.RT-PCR Analyses and Immunostaining of Growth Factors Expression in the Human EndometriumHuman endometrial samples at secretory phase were obtained from five patients, aged 36?2 years, who underwent gynecological surgery for uterine leiomyoma and exhibited normal menstrual cycles. The menstrual stage was confirmed based on (��)-Hexaconazole price urinary LH levels and ovarian ultrasonography. The expression of key growth factors in secretory phase endometrium was determined by conventional RT-PCR and immunostaining. For conventional RT-PCR, specific primers for growth factors and b-actin were used (Table S1). PCR 
reactions comprised 30 cycles of amplification with denaturation at 94uC for 30 sec, annealing at 60uC for 30 sec, and elongation at 72uC for 30 sec. For negative controls, no mRNA was included. To confirm identity, the PCR products were sequenced as describ.Nd putative paracrine factors secreted by the reproductive tract were shown to enhance early embryonic development [11]. Recent progress in sequential culture media allowed extended culture of human embryos to the blastocyst-stage and blastocystHuman Embryo Culturetransfer is effective in selecting high-quality embryos for successful pregnancy [12], leading to the birth of several million IVF babies [14,15]. However, the efficiency of human blastocyst development in vitro remains to be improved. Several studies using surplus human material suggested the promotion of blastocyst development in vitro when culture media were supplemented with growth factors, including EGF [13], IGF-I [14], BDNF [18], and granulocyte macrophage colony-stimulating factor (GM-CSF) [19,20]. Because most routine human embryo cultures do not contain growth factors, we hypothesized that inclusion of autocrine/paracrine growth factors in the culture media could improve early embryonic development. We investigated the expression of key ligand-receptor pairs in cleavage-stage human embryos derived from tri-pronuclear zygotes and specific ligands in human uterine endometrium. We individually cultured abnormally fertilized zygotes and normally fertilized day 3 embryos from IVF programs in micro-drops using routine serum-free culture media supplemented with a group of key growth factors to improve embryo development. We examined if these growth factors could improve blastocyst outgrowth in vitro using normally fertilized hatching blastocysts. Because the development of reconstructed embryos after SCNT is sub-optimal, we also tested if supplementation of culture media with key growth factors could increase the development of SCNT-derived embryos.overnight at 4uC. The antibodies used include: anti-EGF (Abcam, Cambridge, MA), anti-IGF-I (Santa Cruz Biotech, Santa Cruz, CA), anti-GM-CSF (Abcam), anti-BDNF (Santa Cruz Biotech), anti-CSF-1 (Abcam), anti-artemin (Abcam), anti-GDNF (Abcam), anti-EGF receptor (Abcam), anti-IGF-I receptor (Abcam), antiGM-CSF receptor (Santa Cruz Biotech), anti-TrkB (R D systems, Minneapolis, MN), anti-CSF-1 receptor (Santa Cruz Biotech), Anti-GFR alpha 3 antibody (Abcam), After three washes in PBS containing 0.1 Tween 20, embryos were incubated with fluorescein isothiocyanate-conjugated secondary antibodies (Jackson Labs, West Grove, PA) for 1 h at 23uC. Nuclear status of embryos was evaluated by staining with 10 mg/ml propidium iodide for 10 min and examined with a confocal laser-scanning microscope (Zeiss LSM 510, Jena, Germany). For negative controls, the primary antibody was replaced with non-immune IgG.RT-PCR Analyses and Immunostaining of Growth Factors Expression in the Human EndometriumHuman endometrial samples at secretory phase were obtained from five patients, aged 36?2 years, who underwent gynecological surgery for uterine leiomyoma and exhibited normal menstrual cycles. The menstrual stage was confirmed based on urinary LH levels and ovarian ultrasonography. The expression of key growth factors in secretory phase endometrium was determined by conventional RT-PCR and immunostaining. For conventional RT-PCR, specific primers for growth factors and b-actin were used (Table S1). PCR reactions comprised 30 cycles of amplification with denaturation at 94uC for 30 sec, annealing at 60uC for 30 sec, and elongation at 72uC for 30 sec. For negative controls, no mRNA was included. To confirm identity, the PCR products were sequenced as describ.
