Supplementary Material for: Methotrexate Induces Apoptosis of Postpartum Placental Cytotrophoblasts

<p><b><i>Background:</i></b> Though methotrexate (MTX) is known to inhibit proliferation of trophoblasts derived from ectopic and intrauterine pregnancies, its action on trophoblasts derived from postpartum placenta remains questionable. This study was designed to ascertain the efficacy of MTX in inducing cell death of postpartum placental cytotrophoblasts (PPTC). <b><i>Methodology:</i></b> Primary human cytotrophoblasts were isolated from placentae of 1st and 2nd trimester intrauterine pregnancies and from postpartum placentae. The isolated trophoblasts were identified based on the expression of cytokeratin 7. MTX-induced inhibition of proliferation of cytotrophoblasts was detected by flow cytometry combined with the WST-1 assay. Secretion of HCG-β and invasiveness were evaluated to assess the effect of MTX on blocking the differentiated cellular function of cytotrophoblasts in relation to the gestational age. The efficacy of MTX in inducing apoptosis of cytotrophoblasts was estimated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. <b><i>Results:</i></b> MTX significantly inhibited the proliferation and differentiation of cytotrophoblasts. MTX-induced cell apoptosis of PPTC was confirmed by increased expression of Fas, FasL, Bax, cleaved caspases 3, 7, 8, and 9, and decreased expression of Bcl-2. <b><i>Conclusion:</i></b> MTX inhibits replication and differentiation of cytotrophoblasts and appears to be an efficient inducer of PPTC apoptosis.</p>