2.1.  Biological samples
Placental tissues from the first trimester legally induced abortions (8–12 weeks of gestation) were obtained from the Department of Obstetrics and Gynecology at the Broussais, Saint Vincent de Paul and Cochin Hospitals. Second trimester placental tissues were collected at the time of termination of pregnancy at 15 and 25 weeks of gestation (in weeks of amenorrhea) in trisomy 21-affected pregnancies. Fetal Down syndrome was diagnosed by karyotyping of amniotic fluid cells. The indication of amniocentesis was the maternal age. Term placentas were obtained after elective cesarean section from healthy mothers near term with uncomplicated pregnancies. With written informed consent of the pregnant woman, the following samples were collected at term: a fragment of the placenta, umbilical cord fetal blood and maternal blood. The placenta biopsy sample was collected at a depth of 1 cm and at a distance of 8 cm from the edge of the placenta, with the maternal side facing upward. The use of these biological samples was approved by local ethical committee. Villous cytotrophoblastic cells were isolated by sequential enzymatic digestion of chorionic villi from the first trimester, second trimester and term placenta and purified on a discontinuous percoll gradient, as described previously.47–49 These cells were positively stained for cytokeratin 7 at 95%, indicating the cytotrophoblastic origin of the cells. Placental fibroblasts were isolated by prolonged enzymatic digestion from first trimester chorionic villi as in Malassine et al.50 Fibroblastic cells (1.25 × 105 cells/mL) were plated on 35-mm plastic dishes (TPP, Switzerland) and cultured for 48 h, and the culture medium was changed daily. These cultured fibroblasts were characterized using immunocytochemistry. It was found that 98% of the cells were vimentin positive and also positive for a monoclonal antibody against specific fibroblast antigen (clone ASO2, Dianova, Hamburg, Germany). Owing to the limited amount of cells, only DNA was extracted.