In the current study we have complemented and extended our previous investigation by investigating the possibility that local production of TGFb1 Foretinib c-Met inhibitor within the endometrium plays a critical role in triggering the process of menstruation in cells from non-pregnant endometrium by inhibiting biosynthesis and/or secretion of PRL, IGFBP-1 and tissue factor via a SMAD-dependent pathway. We have also examined the effects of TGFb1 in cells obtained from early pregnancy to compare the TGFb1 response between stromal cells decidualized in vitro and in vivo. In the present study we have demonstrated that TGFb1 reduces the expression and secretion of PRL, IGFBP-1, and TF by human ESCs decidualized in vitro. Notably the latter appeared more refractory to the treatment. Targeted knockdown of SMAD 4, the protein which translocates phosphorylated SMAD members to the nucleus mediating the transcriptional downstream biological actions of TGFb1, revealed that the impact of TGFb1 on expression and release of IGFBP1was SMAD independent. In contrast inhibition of PRL protein release was SMAD-dependent demonstrating that TGFb1 can act via more than one signalling pathway in this cell type. Previous studies have reported that TGFb1 can alter expression of decidual proteins although impacts on endometrial decidualization have been inconsistent. To our knowledge the current study reports the first data directly comparing the response to TGFb1 in cells decidualized in vitro with primary cells recovered from decidua i.e. those exposed to the presence of a blastocyst. Primary ESCs, obtained from non-pregnant endometrium and decidualized in vitro, are considered a model for cells that decidualize during the non-pregnant menstrual cycle. In primary ESCs we demonstrated incubation of cells with TGFb1 reduced both the concentrations of IGFBP-1 and PRL mRNAs as well as the amounts of these proteins secreted into the culture media. The findings in the current study are in agreement with a number of studies reporting a marked inhibitory effect of TGFb1 on basal and stimulated PRL secretion, mRNA levels and de novo PRL synthesis in rat anterior pituitary cells, decidual cells from 1st trimester and term pregnancy. However in contrast to the current findings, it has been reported that TGFb1 can potentiate the decidualization process in ESCs with increased production of PRL independent of the presence of progesterone. With a further study reporting a TGFb1-dependent increase in expression of PRL in ESCs although these cells were not exposed to a decidualization stimulus. One limitation to our study is that all the decidual markers we examined are also regulated by progesterone. As we have cultured all our cells in the presence of MPA we are unable to reject the possibility that augmentation of the decidual markers is occurring as an indirect consequence of TGFb1 mediated suppression of PR expression. Interestingly, we detected a very rapid reduction in protein release for both IGFBP-1 and PRL in ESCs that preceded any reduction in total concentrations of the mRNAs.