**1.3 Culture of placental explants with hydrogen peroxide**

The amount of villi used was 11 mg of placental tissue that was cultured in each well of 24-well plates (SPL Life Sciences, Korea) for 24 h for stabilization [63]. Cultures were performed in vitro in the absence of hydrogen peroxide or in the presence of 10, 100, and 1,000 μM of H2O2 for 4 h and 24 h in RPMI 1640 culture medium supplemented with 2 mM L-glutamine (Sigma-Aldrich, St Louis, MO, USA), 40 mg/ml antibiotic/antimycotic (Sigma-Aldrich), and 10% fetal bovine serum (Gibco BRL Life Technologies, The Netherlands) inactivated (complete RPMI medium).

After the culture periods, the explants were removed and submitted to RNA extraction for further analysis of the expression of genes related to inflammation (cytokines), autophagy, and inflammasome. Culture supernatants were obtained, centrifuged at 2,000 g for 10 min and stored at −80°C for determination of cytokines.
