Differentiation of human endometrial stromal cells into specialized decidual 
cells is critical for embryo implantation and survival of the conceptus. 
Initiation of this differentiation process is strictly dependent on elevated 
cAMP levels, but the signal intermediates that control the expression of 
decidual marker genes, such as prolactin (PRL) and IGFBP1, remain poorly 
characterized. Here we show that cAMP-dependent decidualization can be 
attenuated or enhanced upon treatment of primary cultures with a nicotinamide 
adenine dinucleotide phosphate (NADPH) oxidase inhibitor (diphenylen iodonium) 
or activator (apocynin), respectively. Time-course analysis demonstrated that 
cAMP enhances endogenous reactive oxygen species production, apparent after 12 h 
of stimulation, which coincides with a dramatic increase in decidual PRL and 
IGFBP1 expression. Knockdown of the Rho GTPase RAC1, which disables activation 
of the NADPH oxidase homologs NADPH oxidase (NOX)-1, NOX-2, and NOX-3, had no 
effect on PRL or IGFBP1 expression. In contrast, silencing of NOX-4, or its 
cofactor p22(PHOX), inhibited the expression of both decidual markers. Finally, 
we show that the NOX-4/p22(PHOX) complex regulates the DNA-binding activity of 
CCAAT/enhancer binding protein-β, a key regulator of human endometrial stromal 
cell differentiation. Thus, NOX-4 activation and reactive oxygen species 
signaling play an integral role in initiating the endometrial decidual response 
in preparation of pregnancy.