To check for mutagenic effects of gestagens in vivo, the CB (Cytokinesis block method) MN test was applied to samples from 29 patients with a diagnosis of threatening spontaneous abortions, who had received gestagen therapy in the first 3 months of gestation. These patients have been cured with one of the models for hormonal substitution therapy: Gestormone (17-a-allylestr-4-en-17b-ol) 2 x 5 mg/day; Gravibinone (Hydroxyprogesteronum capronicum) 250 mg/third day IM; Progesterone (20-adihydroprogesterone) 250 mg/third day IM. For our cytogenetic study a classic method for cultivation of human peripheral blood lymphocytes was used [9,10] with some modifications that are necessary for applying the CB MN test [1].

For the cell cultures, 77% Parker 199 (Institute Torlak, Belgrade, Yugoslavia), 20% fetal calf serum (Sigma Chemical Company, St. Louis, MO, USA) and 3% PHA (INEP, Belgrade, Yugoslavia) were used. Under sterile conditions, 20 drops of whole heparinized blood were put into sterile bottles with 5 mL of medium. Cell cultures were incubated at 37°C, for a total of 72 hours. Cytochalasin B, at a final concentration of 4 mg/mL, was added after 48 hours from the beginning of incubation. The cell cultures were centrifuged, then the cell suspension was washed in 0.9% saline and treated with hypotonic solution (0.56% KCl). The cell suspension was then fixed for 2 x 15 minutes in a 3:1 fixation solution (methanol:acetic acid). The obtained material was spread onto specially prepared, dry, cold, and lamp-dried slides. After standing for 5 to 7 days, they were stained with 2% Giemsa stain (Alfapanon, Novi Sad, Yugoslavia). The MN frequency was determined by analysis of 1,000 binucleated cells per person. We have performed comparisons of average values by application of the Student-t test, and the variability of MN frequency was determined by application of quantitative genetic analysis (ANOVA).