Following 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 embryos following 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 
, 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 3-day-old human embryo enters the uterus at the morula stage, further 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 the human 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 
. Taking advantage of the availability of discarded human tri-pronuclear zygotes in IVF, 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 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. Selective single blastocyst transfer in patients with good prognosis has been shown to be effective in reducing multiple pregnancies without compromising the pregnancy rate 
. 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 normally-fertilized 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.
Successful implantation of the blastocyst is essential for reproduction. Implantation of blastocysts is a well-organized process regulated by multiple growth factors and cytokines 
. We demonstrated the facilitatory effects of key growth factors to promote blastocyst outgrowth. The trophectoderm cells of blastocysts differentiate during embryonic development to form the invasive trophoblasts that mediate implantation of embryos into the uterine wall. The outgrowth of trophoblast cells from cultured blastocysts is believed to reflect the proper differentiation of the embryo, important for trophoblast invasion of the endometrial stroma during implantation in utero [38,39]. Although blastocyst transfer is effective to select the best quality embryos with high implantation potential, overall implantation rate is ~30% 
, suggesting human embryo transfer might be improved. Due to the low amount of liquid in the uterine cavity, factors included in the transfer media could be retained in high concentrations. Indeed, embryo transfer in medium containing hyaluronan is effective in improving implantation rates in patients with recurrent implantation failure 
.Hyaluronan is the major glycosaminoglycan present in follicular, oviductal and uterine fluids and presumably promotes embryo–endometrial interactions during the initial phases of implantation. Because key growth factors promoted blastocyst outgrowth in vitro
, future supplementation of embryo transfer media with key growth factors could also promote implantation during embryo transfer.
Generating an autologous patient-specific embryonic stem cell line from SCNT embryos holds great promise for the treatment of degenerative human diseases. Successful derivation of embryonic stem cell lines following SCNT has been reported in mouse , rabbit , and non-human primates . However, the efficiency for the production of embryonic stem cell lines following SCNT is still low (<2%), particularly when adult somatic cells were used as the donor karyoplasts. Although many embryonic stem cell lines have been derived from surplus human blastocysts [47,48], no human cell-lines have been generated following SCNT. Among the many compounding factors, suboptimal in vitro culture condition contributes to the poor embryonic development of reconstructed embryos following SCNT. The present study represents an attempt to optimize the culture conditions for the development of human SCNT embryos. Although no blastocyst was obtained following fibroblast nuclear transfer, there was a trend to an augmented development of reconstructed embryos cultured with media containing autocrine/paracrine growth factors. Results from the present study provide the basis for future use of autocrine/paracrine factors to facilitate the derivation of patient-specific embryonic stem cells.
In conclusion, the present study demonstrated the utility of growth factor supplementation for optimal human early embryo development and blastocyst outgrowth. The findings may allow the design of better conditions for individual human embryo cultures, for estimating their developmental potentials using secretory products, and for the inclusion of growth factors in embryo transfer media to promote implantation. Although the present experimental design is based on the supplementation of endogenous growth factors diluted during assisted reproductive procedures, future studies on the potential side effects of these paracrine/autocrine factors on chromosomal numbers, genomic integrity, proteomic changes, and epigenetic modifications are essential before clinical use.