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Keywords:

  • De novo synthesis;
  • first trimester;
  • hCG;
  • hPL;
  • human;
  • third trimester;
  • trophoblast culture

PROBLEM: To compare the capacity of de novo hormone synthesis by cultured trophoblast cells isolated from early and term placenta as cytotrophoblast, and to determine the ability of these cells to proliferate in culture. METHOD OF STUDY: Cytotrophoblast cells were isolated from term (TP, 38–42 weeks) and early placentae (EP, 8–13 weeks) by enzymatic digestion and subsequent purification on a percoll gradient. The net synthesis of the hormones human placental lactogen (hPL) and human chorionic gonadotropin (hCG) was determined as the release during culture+cell content after culture−cell content before culture. Proliferation was determined using a dedicated colorimetric reagent (CellTiter 96TM). RESULTS: Using a percoll gradient we were able to isolate three cell bands with densities of 1.051, 1.058, and 1.063 g/mL, which were predominantly cytotrophoblast cells as shown by immunocytochemical analysis. The cytotrophoblast cells with the highest density (1.063 g/mL) were used because they were found to release the highest amount of hormones and have shown the lowest rate of cell death after 6 days in culture. Both hCG and hPL showed different patterns of release during the first 2–3 days of culture between TP and EP. While the release by EP cytotrophoblast cells continued during 6 days of culture (n=4), the concentrations for TP cytotrophoblast (n=4) reached a plateau between 4 and 6 days. Net de novo synthesis calculated for 3×104 TP trophoblast cells cultured for 6 days (mean±SD, n=4) was 8.65±9.05 mU for hCG and 0.95±0.45 ng for hPL. For EP, it was 395.5±265.5 mU for hCG and 148.8±84.2 ng for hPL. Net synthesis of hCG was>10-fold (TP) and>70-fold (EP) higher than the initial cell content. While at term, hPL synthesis was only a fraction of the initial cell content, production by EP cytotrophoblast was 106 times the initial cell content. The extent of cell death after 6 days in culture was significantly ( P<0.02) higher for term (30–40%) than for early trophoblast (10–20%). Using a proliferation detection agent during the first 3 days of culture with first trimester cytotrophoblast cells, we did not find any changes in the proliferative activity. CONCLUSIONS: There are differences in the functional activity between trophoblast cells obtained from first and third trimester. The in vitro findings are difficult to reconcile with the different patterns of plasma concentrations of the two hormones observed in vivo during the course of pregnancy.