We developed them into cleavage-stage embryos for analyzing the expression of different ligand-receptor pairs known to play autocrine/paracrine functions in animal embryos. We also demonstrated that culturing these abnormally fertilized embryos in serum-free culture media supplemented with GDC-0941 PI3K inhibitor growth factors substantially promoted their development by more than 2-fold. The improvement of sequential culture systems for human IVF during the last decades has allowed extended culture of human early embryos to the blastocyst stage. Blastocyst transfer facilitates the selection of the best embryos with high implantation potential and therefore reduces the number of transferred embryos to avoid multiple pregnancies. However, the current human embryo culture system is still suboptimal and many embryos cannot develop to the blastocyst stage. Our results using normally fertilized day 3 embryos suggest that key autocrine/paracrine growth factors are beneficial to human embryonic development in vitro. These growth factors not only increase the rate of blastocyst formation, but also the quality of blastocysts. Indeed, culturing good-quality day 3 embryos in culture medium supplemented with these growth factors resulted in a 3.3-fold increase in the blastocyst formation rate and a 7.6-fold increase in the proportion of high quality blastocysts as compared to controls. These findings are consistent with the hypothesis that autocrine/paracrine factors secreted by early embryos are diluted during culture and growth factor supplementation is necessary to promote optimal blastocyst formation. Because most of the commercially available, chemically-defined media for human embryo cultures in IVF-ET do not contain growth factors, the present supplementation of widely used culture media with autocrine/paracrine growth factors has practical value in future IVF-ET procedures. Different from previously published reports showing small stimulatory effects of individual growth factors on human embryo development, our combined treatment with several autocrine/ paracrine factors showed a robust stimulation of normally fertilized day 3 embryos likely due to additive effects of different growth factors in the promotion of early embryonic development. Inclusion of IGF-I or GM-CSF increased the proportion of embryos developing to the blastocyst stage by 1.51-fold and 2.53-fold, respectively. In our study, treatment embryos with the growth factor cocktail showed a 3.3-fold increase in the proportion of blastocyst-stage-embryos. The ability of these paracrine/autocrine factors to promote development of early human embryos is consistent with findings showing zygote genome activation in human embryos at 4- to 8-cell stages on day 3 after fertilization when the expression of these growth factors begun to increase. In the present combination treatment protocol, several distinct signaling pathways could be activated by the autocrine/paracrine factors used: EGF, IGF-I and BDNF bind to respective receptor tyrosine kinases to activate downstream phophotidyinositol-3-kinase-Akt signaling, CSF1 and GM-CSF interact with type I cytokine receptors to activate the downstream JAK/STAT pathway, whereas GDNF and artemin interact with glycosylphosphatidyl- inositol-anchored receptors to activate downstream cRET and Src kinase pathways. Although the fresh tri-pronuclear zygotes used here were treated with five growth factors due to reagent availability, thawed normallyfertilized and SCNT embryos were treated with seven growth factors. It is likely that these divergent pathways exert overlapping and redundant actions on early embryo development and not all growth factors are needed for optimal embryo growth.