To 144 h. Morphological changes were monitored for blastocyst formation at early, expanded, and hatched stages. Numbers of embryos developed to specific stages vs. total number of embryos studied are listed on top of each column. *, P,0.05 vs. control. GFs: growth factors. doi:10.1371/journal.pone.0049328.gHuman Embryo CultureFigure 4. Treatment with autocrine/paracrine growth factors promoted the development of day 3 human embryos to the blastocyst stage. Cryopreserved human day 3 embryos were thawed and evaluated based on their morphology. After discarding poor-quality embryos, goodquality embryos were divided into optimal (.6-cell-stage, grade 1 or 2) and suboptimal groups (.6-cell-stage, grade 3; 3- to 5-cell-stage, grade 1 to 3). Embryos were then cultured with or without key growth factors for 72 h in micro-drops of medium. At the end of culture, the proportion of blastocyst formation was evaluated. Numbers inside parentheses indicate blastocysts/total embryos for each group. *, P,0.05; N.S., no significant differences. A) Development of all blastocysts. B) Development of high quality blastocysts scored as 3AA to 5 AA. doi:10.1371/journal.pone.0049328.gin high quality blastocyst formation was found when all embryos were evaluated.Supplementation of Culture Media with Key Growth Factors Promoted Blastocyst OutgrowthTo evaluate the roles of Title Loaded From File ligand-receptor pairs of key growth factors in blastocyst implantation, their expressions in thawed cryopreserved day 5 embryos were determined by real-time RTqPCR. As shown in Fig. 5, all ligand-receptor pairs were expressed in blastocysts. Among the ligands, the levels of EGF and BDNF were high (Fig. 5, upper panel), suggesting possible dominant Title Loaded From File autocrine actions, whereas transcript levels of the receptors were comparable (Fig. 5, lower panel). Thawed cryopreserved day 5 embryos were cultured for 48 h to obtain hatching embryos before analyses of blastocyst adhesion and outgrowth in vitro. Embryos were cultured in a well coated with Matrigel with or without different growth factors (EGF, IGF-I, GM-CSF, BDNF, CSF-1,artemin, and GDNF) for 72 h before evaluation of their implantation potential. As shown in Fig. 6, the addition of growth factors to the culture media increased blastocyst outgrowth by 2.5fold. In contrast, treatment with growth factors did not affect blastocyst adhesion.Supplementation of Culture Media with Key Growth Factors Promoted the Development of SCNT EmbryosAfter warming, vitrified failed-to-be-fertilized oocytes served as recipients for SCNT using serum-starved (48 h) fibroblasts as the donor karyoplast. To confirm that the pronuclear formed after artificial activation was from injected fibroblast, we observed mitotic spindle 2 h following fibroblast injection with the assistance of OoSight. As shown in Fig. S1, only one birefringent spindle was seen in the center of each reconstructed oocyte. Among the 18 activated oocytes, 17 showed only one pronuclear in the cytoplasm. Because the development of reconstructed embryosHuman Embryo CultureFigure 5. Expresssion of key growth factors and receptors in human blastocysts. Cryopreserved human day 5 embryos were used for quantitative RT-PCR analyses of transcript levels for different ligand-receptor pairs (EGF/EGFR, IGF-I/IGF-IR, GM-CSF/GM-CSFR, BDNF/TrkB, CSF-1/CSF1R, artemin/GFRA3, and GDNF/GFRA1). Levels of all mRNAs were normalized based on those for b-actin in the same sample (mean 6 SEM, n = 3). doi:10.1371/journal.pone.00493.To 144 h. Morphological changes were monitored for blastocyst formation at early, expanded, and hatched stages. Numbers of embryos developed to specific stages vs. total number of embryos studied are listed on top of each column. *, P,0.05 vs. control. GFs: growth factors. doi:10.1371/journal.pone.0049328.gHuman Embryo CultureFigure 4. Treatment with autocrine/paracrine growth factors promoted the development of day 3 human embryos to the blastocyst stage. Cryopreserved human day 3 embryos were thawed and evaluated based on their morphology. After discarding poor-quality embryos, goodquality embryos were divided into optimal (.6-cell-stage, grade 1 or 2) and suboptimal groups (.6-cell-stage, grade 3; 3- to 5-cell-stage, grade 1 to 3). Embryos were then cultured with or without key growth factors for 72 h in micro-drops of medium. At the end of culture, the proportion of blastocyst formation was evaluated. Numbers inside parentheses indicate blastocysts/total embryos for each group. *, P,0.05; N.S., no significant differences. A) Development of all blastocysts. B) Development of high quality blastocysts scored as 3AA to 5 AA. doi:10.1371/journal.pone.0049328.gin high quality blastocyst formation was found when all embryos were evaluated.Supplementation of Culture Media with Key Growth Factors Promoted Blastocyst OutgrowthTo evaluate the roles of ligand-receptor pairs of key growth factors in blastocyst implantation, their expressions in thawed cryopreserved day 5 embryos were determined by real-time RTqPCR. As shown in Fig. 5, all ligand-receptor pairs were expressed in blastocysts. Among the ligands, the levels of EGF and BDNF were high (Fig. 5, upper panel), suggesting possible dominant autocrine actions, whereas transcript levels of the receptors were comparable (Fig. 5, lower panel). Thawed cryopreserved day 5 embryos were cultured for 48 h to obtain hatching embryos before analyses of blastocyst adhesion and outgrowth in vitro. Embryos were cultured in a well coated with Matrigel with or without different growth factors (EGF, IGF-I, GM-CSF, BDNF, CSF-1,artemin, and GDNF) for 72 h before evaluation of their implantation potential. As shown in Fig. 6, the addition of growth factors to the culture media increased blastocyst outgrowth by 2.5fold. In contrast, treatment with growth factors did not affect blastocyst adhesion.Supplementation of Culture Media with Key Growth Factors Promoted the Development of SCNT EmbryosAfter warming, vitrified failed-to-be-fertilized oocytes served as recipients for SCNT using serum-starved (48 h) fibroblasts as the donor karyoplast. To confirm that the pronuclear formed after artificial activation was from injected fibroblast, we observed mitotic spindle 2 h following fibroblast injection with the assistance of OoSight. As shown in Fig. S1, only one birefringent spindle was seen in the center of each reconstructed oocyte. Among the 18 activated oocytes, 17 showed only one pronuclear in the cytoplasm. Because the development of reconstructed embryosHuman Embryo CultureFigure 5. Expresssion of key growth factors and receptors in human blastocysts. Cryopreserved human day 5 embryos were used for quantitative RT-PCR analyses of transcript levels for different ligand-receptor pairs (EGF/EGFR, IGF-I/IGF-IR, GM-CSF/GM-CSFR, BDNF/TrkB, CSF-1/CSF1R, artemin/GFRA3, and GDNF/GFRA1). Levels of all mRNAs were normalized based on those for b-actin in the same sample (mean 6 SEM, n = 3). doi:10.1371/journal.pone.00493.
