Fifty-eight patients aged 28-38 years (mean 32.2) were undergoing IVF-ET procedure due to infertility of different etiology. The ovarian stimulation was applied according to previously described standard protocols [31]. The cells were obtained after standard IVF-ET procedure [33,34] at the Infertility Treatment Program “Technobios”, Medical University, Sofia, Bulgaria. A hundred and three cells were included in the present study: 54 unfertilized human oocytes, 34 arrested human zygotes and 15 polar bodies. Informed written consent was obtained from the patients before their surplus material was used for scientific studies. The study was approved by the Ethical Committee of the Medical University, Sofia, Bulgaria and complied with the ethical rules and laws of the European Union.

Oocytes and 2PN zygotes were considered to be unfertilized if they lacked pronuclei, and arrested if they contained two pronuclei but no sign of cleavage on the day of the embryo transfer, i.e., after 48-52 hours of in vitro incubation after their in vitro insemination. Cell-fixation procedure used the following steps: 1) zona pellucida removal by Tyrode’s solution (pH 2.5) treatment, observed with a stereomicroscope; polar bodies were fixed together with the unfertilized oocytes and arrested zygotes; 2) hypotonic incubation (1% sodium citrate with 5% BSA) for 20 min. at room temperature; 3) cells were placed onto acetone-precleaned slides and a cold fixative (1:3 acetic acid:methanol) was applied. Disappearance of the cytoplasm during fixation was controlled using phase contrast microscopy.

Oocyte metaphases were stained with Giemsa for visualization and evaluation of chromosomes. Only oocytes with good metaphase spread (40 cells) or with PCC of the sperm chromatin beside the female chromosomes (14 cells) were included in the study. The cells were then discolored and frozen for further analysis by FISH. Slides with zygotes and polar bodies were frozen without Giemsa staining.

The chromatin of oocytes, pronuclei and polar bodies was FISH-analyzed for chromosomes 18, 21, X and Y to investigate the ploidy status of the cells. The slides were prepared for FISH by dehydration in ethanol (70, 85 and 100%) and additional drying and fixation at 60C for 1 hour. Fluorescent in situ hybridization was performed in two cycles using a centromere- and a locus-specific probe at each step. At the first cycle of hybridization, probes for chromosomes 18 (centromere-specific, CEP 18 Spectrum Green; Vysis, Stuttgart, Germany) and 21 (locus-specific, LSI 21 Spectrum Orange; Vysis) were used. The probes and the chromatin were simultaneously denatured at 73C for 7 min. Hybridization was performed in a humid chamber for 5 hours at 37C. The chromatin was counterstained by DAPI (4',6-diamidino-2-phenylindole). Between the two FISH steps, the results were registered and the slides were then washed in dH₂O for 10 seconds at 71C, dehydrated in ethanol series and warmed to 60C again. At the second cycle of hybridization, probes for chromosomes X and Y (centromere-specific, FITC-labeled for chromosome X and classical satellite probe, Texas Red Labeled for chromosome Y; Q-Biogene, Heidelberg, Germany) were applied. The procedures of denaturation, hybridization and counterstaining were the same as at the first step. Results of FISH were observed by fluorescent microscopy (BX60; Olympus, Hamburg, Germany), digitally registered (VC45; Olympus) and analyzed using ISIS software (Metasystems, Altlussheim, Germany).