Estrogen and Progesterone Differentiation of Endometrial Stromal Fibroblasts

eSF were treated with ovarian steroids as described [57 (link)]. This treatment protocol allows eSF to differentiate appropriately in response to estradiol and progesterone [15 , 57 (link)], including proper induction of decidualization, the progesterone-driven differentiation of eSF during the secretory phase in preparation for implantation. Briefly, cells were treated with 0.1% ethanol as vehicle control, 10 nM estradiol (Sigma-Aldrich), or 10 nM estradiol and 1 μM progesterone (Sigma-Aldrich) in SCM made with 2% instead of 10% FBS. Cells were treated for 14 d, with feeding every other day. Decidualization of cells in the presence of estradiol and progesterone was confirmed by a human IGF-binding protein 1 (IGFBP1) ELISA kit (Alpha Diagnostics International and Abcam). Samples treated with estradiol and progesterone effectively decidualized as assessed by secretion of IGFBP-1 (level > 500 ng/100K cells in 0.5 ml of culture) [15 ].