Since the circulating levels of E2 and progesterone are high during the third trimester, E2 was employed with MPA to mimic the gestational steroidal milieu. MPA was used instead of native progesterone, which is rapidly metabolized in vitro. The cultures were washed twice with PBS, to remove residual serum components, and switched to a serum-free defined medium (DM) that consisted of BM plus ITS+ premix (BD Biosciences, Bedford, MA), 5 im FeSO4, 50 im ZnSO4, 1 nm CuSO4, 20 nm Na2SeO3, trace elements (Invitrogen), 50 ig/ml ascorbic acid (Sigma), and 50 ng/ml epidermal growth factor (BD Biosciences).
The corresponding vehicle or steroid(s) with or without TNF and IL1B (R&D Systems) was added to DM.
After 24 h of incubation, the concentrations of immunoreactive CSF1 and MIF in conditioned DM were measured by specific ELISAs according to the instructions provided by the manufacturer (R&D Systems). The levels of CSF1 and MIF were normalized to the total cell culture protein content, as measured by Lowry protein assay (Bio-Rad Laboratories, Hercules, CA). The intra- and inter-assay coefficients of variation and assay sensitivity were 2.3%, 6.5% and 15.62 pg/ml and 5.0%, 6.9%, and 31.25 pg/ml, respectively, for CSF1 and MIF.