Specimens of spontaneous abortions of a pregnancy-period from 3 to 28 weeks (mean value: about 9 weeks) were obtained from 60 females, age 23 to 42 years (mean age: 31.45). Interphase FISH karyotyping of 63 spontane­ous abortion specimens, including three sets of twins, ob­tained from the hospital of the Moscow Setchenov’s Med­ical Academy, Moscow, Russia, was carried out (Table 1). Tissues of spontaneous abortions were processed for FISH as follows: the chorionic villi or embryonic tissue samples were washed three times in physiological solution. In order to clean the specimens of the rest of maternal decidua and blood, samples were washed in 70% ethanol several times, then rinsed with 60% acetic acid for 30 seconds and placed in a solution of 60% acetic acid for 15-20 min. at room temperature and periodically mixed by inversion. Dispersed single cell suspensions were fixed in 3:1 metha­nol: acetic fixative three times for 10, 20 and 30 min. The cells were placed onto wet slides and air-dried at room temperature. Two slides with two drops of cell suspension were prepared for each sample.

Chromosome-specific DNA probes for chromosomes 1, 9, 13/21, 14/22, 16, 18, X and Y were used in FISH experiments. Alphoid DNA probes were cloned in the Laboratory of Cytogenetics of National Research Center of Mental Health, Moscow, Russia, precisely as described previously [10-12].

The following combinations of DNA probes were used: 1) Cy3-labeled chromosome Y-specific probe, X-specific fluoresceine-labeled chromosome probe, and bio­tinylated chromosome 1-specific probe; 2) biotinylated chromosome 9-specific probe and Cy3-labeled chromo­some 13/21-specific probe; 3) fluoresceine-labeled chro­mosome 16-specific probe and Cy3-labeled chromosome 18-specific probe; 4) biotinylated chromosome 1-specific probe and Cy3-labeled chromosome 14/22-specific probe. Each DNA probe was taken at final concentration of 25-50 ng/10 mL of hybridizing solution (10% dextran sulfate and 2 x SSC in 60% formamide, pH 7). In situ hybridiza­tion and detection protocols were performed as described earlier [10,13]. Mixtures of DNA probes (5-10 mL) were placed onto the slides, covered by cover slips and dena­tured for 3-5 min. at 72°C. After 4 hours (or overnight) hybridization at 37°C, the slides were washed for 15 min. in 50% formamide, 2 x SSC (pH 7) at 42°C, followed by two washes for 5 min. each in 2 x SSC, 0.1% Tween 20 at 37°C. The slides were then incubated with 5mg/mL AMCA-avidin (Vector Laboratories, Burlingame, CA, USA) for detection of the chromosome 1-specific biotin-labeled DNA probe, co-hybridized with the Cy3-labeled chromosome Y-specific probe, and X-specific fluores- ceine-labeled chromosome probe. Finally, interphase nu­clei were counterstained with DAPI to allow simultaneous observation of interphase nuclei and hybridized probes. Hybridization signals were detected simultaneously using a Leitz-Orthoplan fluorescence microscope (Ernst Leitz, Wetzlar GmbH, Wetzlar, Germany), equipped with a com­bination of appropriate filters and objectives of 63X or 100X. From 100 to 400 cells were analyzed for each spe­cimen/probe.