Fast multicolor primed in situ protocol for chromosome identification in isolated cells may be used for human oocytes and polar bodies.

OBJECTIVE: To present and evaluate the use of a new ultra-fast multicolor primed in situ (PRINS) procedure for karyotyping human oocytes and first polar bodies. DESIGN: In situ chromosomal identification on isolated cells, using combinations of specific primers for chromosomes 1, 7, 9, 16, and 18 and fluorescent nucleotides. SETTING: Sixteen unfertilized oocytes were obtained from women participating in an IVF program. PATIENT(S): Five patients undergoing an IVF-ET. INTERVENTION(S): In vitro unfertilized oocytes were fixed on slides, and sequential PRINS reactions were performed on each preparation. MAIN OUTCOME MEASURE(S): Ultrarapid in situ identification of three or four chromosomes on oocyte and polar body chromosome spreads. RESULT(S): On the basis of the direct in situ mixing of the colors of fluorochromes (FITC, TRITC, Cascade Blue) that were incorporated in sequential PRINS reactions, this method allows rapid and efficient labeling of three or four individual chromosomes. Each PRINS reaction consists of a unique 4- to 6-minute step for both in situ annealing and elongation. The procedure can be combined with fluorescence in situ hybridization (FISH) reactions. CONCLUSION(S): By simplifying the multicolor PRINS procedure, this new protocol should facilitate the use and adaptation of PRINS to chromosome screening. This approach could be used in parallel or in combination with FISH for efficient aneuploidy assessment on isolated cells.

Fluorescence in situ hybridization analysis of human oocytes: advantages of a double-labeling procedure.

OBJECTIVE: To improve the fluorescence in situ hybridization (FISH) chromosomal analysis of human oocytes and first polar bodies. DESIGN: In situ chromosomal identification on isolated cells, with combinations of centromeric (or locus-specific) probes and whole-chromosome painting probes for chromosomes 9, 13, 16, 18, 21, and X. SETTING: Montpellier University Hospital. PATIENT(S): Women participating in an IVF program. INTERVENTION(S): Fifty-four in vitro unfertilized oocytes were fixed on slides, and simple or double FISH labeling procedures were performed on preparations. MAIN OUTCOME MEASURE(S): Simultaneous in situ visualization of specific domains and chromosome arms of each targeted chromosome. RESULT(S): Eight chromosomal abnormalities were identified, including two hyperhaploidies, three cases of extra single chromatid, and three cases of balanced separation of sister chromatids. Also, the double-labeling procedure allowed the avoidance of five interpretation errors, owing to additional artefactual signals. CONCLUSION(S): By ensuring precise identification of both chromosomes and single chromatids, the FISH double-labeling procedure limits the risk of erroneous interpretation and allows a more accurate cytogenetic analysis of human oocytes.