Among which ,50 of them survived subsequent enucleation and fibroblast injection procedures. Following successful nuclear transfer, early embryonic development was improved with growth factor supplementation of culture media as reflected by the development of .5-fold increases in the proportion (40 , 6/15) of cultured SCNT oocytes to embryos with more than 4-cell stage embryos as compared those without growth factor supplementation (7 , 1/14). Although with low efficiency, the most advanced SCNT embryo reached the 16-cell stage when cultured in media supplemented with growth factors.DiscussionFollowing culturing individual human embryos in chemically defined serum-free media, we demonstrated the ability of key autocrine/paracrine growth Asiaticoside A site factors to promote early embryonic development and implantation. Treatment with key growth factors enhanced the development of abnormal tri-pronuclear zygotes, normally fertilized human embryos, and reconstructed embryosfollowing SCNT. The key growth factors not only stimulated embryo growth but also increased the proportion of morphologically good blastocysts, suggesting improvement of embryo quality. Although the facilitatory effects of individual growth factors on human embryo development have been reported [16,17,19,20,31,32], the present use of multiple growth factors likely exerts overlapping and redundant actions to allow optimal early embryo development. Culturing of individual embryos further minimizes cross-interaction of embryos and provides the basis to monitor the functions of single high quality embryos for transfer. Demonstration of the facilitatory effects of key growth 
factors to promote blastocyst outgrowth further provides future opportunity to include them in embryo transfer media to improve implantation success. Our immunofluorescence staining and real-time RT-qPCR analyses confirmed the expression of key ligand-receptor pairs in human early embryos, underscoring their importance as autocrine/paracrine factors. For each ligand-receptor pairs, ligand antigens were found in the cytoplasm of blastomeres whereas the receptor antigens were found in the plasma membrane, suggesting 
the secretion of these paracrine/autocrine ligands to act on membrane receptors in an autocrine/paracrine manner. As a 3day-old human embryo enters the uterus at the morula stage,Human Embryo CultureFigure 6. Treatment with autocrine/paracrine growth factors promoted blastocyst outgrowth. Cryopreserved human day 5 embryos were thawed and cultured for 48 h until hatching. High quality hatching embryos were then cultured with or without growth factors for 72 h in a well coated with Matrigel. At the end of culture, the proportion of blastocyst adhesion and outgrowth was evaluated. Numbers inside parentheses indicate blastocyst adhesion or outgrowth/total embryos for each group. *, P,0.05. doi:10.1371/journal.pone.0049328.gfurther development could also be regulated by paracrine factors secreted by the endometrium. Our RT-PCR and immunostaining studies confirmed the expression of these key growth factors in 223488-57-1 thehuman endometrium, suggesting their paracrine roles in support of embryo growth after the morula stage. Earlier studies indicated that tri-pronuclear zygotes are capable of developing into blastocysts albeit with lower efficiency [24]. Taking advantage of the availability of discarded human tripronuclear zygotes in IVF, we developed them into cleavage-stage embryos for analyzing the expression of different lig.Among which ,50 of them survived subsequent enucleation and fibroblast injection procedures. Following successful nuclear transfer, early embryonic development was improved with growth factor supplementation of culture media as reflected by the development of .5-fold increases in the proportion (40 , 6/15) of cultured SCNT oocytes to embryos with more than 4-cell stage embryos as compared those without growth factor supplementation (7 , 1/14). Although with low efficiency, the most advanced SCNT embryo reached the 16-cell stage when cultured in media supplemented with growth factors.DiscussionFollowing culturing individual human embryos in chemically defined serum-free media, we demonstrated the ability of key autocrine/paracrine growth factors to promote early embryonic development and implantation. Treatment with key growth factors enhanced the development of abnormal tri-pronuclear zygotes, normally fertilized human embryos, and reconstructed embryosfollowing SCNT. The key growth factors not only stimulated embryo growth but also increased the proportion of morphologically good blastocysts, suggesting improvement of embryo quality. Although the facilitatory effects of individual growth factors on human embryo development have been reported [16,17,19,20,31,32], the present use of multiple growth factors likely exerts overlapping and redundant actions to allow optimal early embryo development. Culturing of individual embryos further minimizes cross-interaction of embryos and provides the basis to monitor the functions of single high quality embryos for transfer. Demonstration of the facilitatory effects of key growth factors to promote blastocyst outgrowth further provides future opportunity to include them in embryo transfer media to improve implantation success. Our immunofluorescence staining and real-time RT-qPCR analyses confirmed the expression of key ligand-receptor pairs in human early embryos, underscoring their importance as autocrine/paracrine factors. For each ligand-receptor pairs, ligand antigens were found in the cytoplasm of blastomeres whereas the receptor antigens were found in the plasma membrane, suggesting the secretion of these paracrine/autocrine ligands to act on membrane receptors in an autocrine/paracrine manner. As a 3day-old human embryo enters the uterus at the morula stage,Human Embryo CultureFigure 6. Treatment with autocrine/paracrine growth factors promoted blastocyst outgrowth. Cryopreserved human day 5 embryos were thawed and cultured for 48 h until hatching. High quality hatching embryos were then cultured with or without growth factors for 72 h in a well coated with Matrigel. At the end of culture, the proportion of blastocyst adhesion and outgrowth was evaluated. Numbers inside parentheses indicate blastocyst adhesion or outgrowth/total embryos for each group. *, P,0.05. doi:10.1371/journal.pone.0049328.gfurther development could also be regulated by paracrine factors secreted by the endometrium. Our RT-PCR and immunostaining studies confirmed the expression of these key growth factors in thehuman endometrium, suggesting their paracrine roles in support of embryo growth after the morula stage. Earlier studies indicated that tri-pronuclear zygotes are capable of developing into blastocysts albeit with lower efficiency [24]. Taking advantage of the availability of discarded human tripronuclear zygotes in IVF, we developed them into cleavage-stage embryos for analyzing the expression of different lig.