OBJECTIVE: This study was carried out to investigate the effects of 3-dimensional co-culture of human endometrial cells decidualized with progesterone and TGF-beta1 on the development of 2-cell mouse embryos. METHODS: Stromal and epithelial cells isolated from human endometrial tissue were immunostained for cytokeratin and vimentin. Expression of TGF-beta1, its receptor-1,-2, integrin-beta3 and prolactin in mono or co-culture according to three different hormone conditions was investigated by RT-PCR. Differential staining was used to investigate the number of ICM and trophectoderm of hatched mouse blastocysts in different three conditions. RESULTS: The immunohistochemical study was positive for cytokeratin or vimentin and confirmed that epithelial and stromal cells were isolated from endometrial tissue successfully. In co-culture, TGF-beta1, its receptor-1, integrin-beta3 and prolactin except TGF-beta1-r2 were expressed in progesterone dominant condition. The hatching and attaching rate were higher in the co-culture with decidualized cells (p<0.05). However, we observed that lots of the incomplete hatched blactocysts attached on non-decidualized cells. The ICM number of hatched mouse blastocysts was higher in co-culture with decidualized and non decidualized cells than media only culture (p<0.05). The trophectoderm number of hatched blastocyst was higher in the co-culture with decidualized cells than non-decidualized cells or media only culture (p<0.05). CONCLUSION: The administration of progesterone, estrogen and TGF-beta could induce decidualization of stromal and epithelial cells isolated from human endometrial tissue using 3-dimensional co-culture, and the decidualization of human endometrial cells could increase the hatching and attaching rate of 2-cell mouse embryos.