Cytogenetic and Fluorescence in situ Hybridisation (FISH) analysis of miscarriages.

Abstract

Spontaneous miscarriages account for 15-20% of recognised pregnancies. Approximately 50% of early pregnancy losses are due to chromosomal abnormalities, mainly autosomal trisomies and sex chromosome aneuploidies. The objective of this study was to establish and evaluate laboratory protocols for cytogenetic analysis of spontaneously aborted tissues in culture, and chromosome-specific FISH analysis in interphase cells. While conventional cytogenetics remains the gold standard, interphase FISH analysis is an essential adjunct to detect the common aneuploidies in cases of culture failure. Used alone, interphase FISH has the disadvantage of detecting only the common aneuploidies for which probe combinations are available. The concomitant use of both techniques enables a cytogenetic result to be given providing useful information for genetic counselling and reproductive decisions. 30 cases of spontaneous abortions between the 6th and 28th weeks of gestation were analysed cytogenetically on foetal skin or chorionic villus samples. Interphase FISH for the detection of aneuploidies of chromosomes 13, 16, 18, 21, X and Y was used in the cases of culture failure, and in six cases with an established karyotype. The optimal method for cell cultures used fine tissue fragments spread on culture flasks that were allowed to stand vertically for 30 minutes. Subsequent culture was carried out in Ham F-12 medium until cellular growth of the explants was observed. The optimal method for preparation of cells for interphase FISH analysis involved the use of acetic acid for isolating cell clumps and cytospin slide preparation. The success rate of conventional cytogenetics was 70%. The overall frequency of chromosomal abnormality was 23.3%, which was less than that reported in most studies, and attributable to the small sample size, the high proportion of second trimester abortuses, and the recurrent miscarriage patients included in the study. 17 (56.7%) of 30 cases had a 46,XX karyotype. Thus, maternal cell contamination could have been another reason for the low frequency of chromosomal abnormalities. Karyotype results showed 1 case of triploidy, 1 case of tetraploidy and 1 case of trisomy 16. Interphase FISH results showed 1 case of monosomy X, 1 case of trisomy 16, 1 case with combined trisomy 18, XXXY and monosomy 16, together with normal cell lines and 1 case of mosaic trisomy 16 (missed by karyotype). Interphase FISH should not be used in cases of culture failures only but (in certain cases) in conjunction with conventional cytogenetics, so the sensitivity of the test could be enhanced. Since a large number of nuclei (> 1 00) are analysed by interphase FISH a more accurate diagnosis is obtained leading to better patient management.