Features of chromosomal abnormalities in spontaneous abortion cell culture failures detected by interphase FISH analysis

Abstract

Cytogenetic analysis of reproductive wastage is an important stage in understanding the genetic background of early embryogenesis. The results of conventional cytogenetic studies of spontaneous abortions depend on tissue culturing and are associated with a significant cell culture failure rate. We performed interphase dual-colour FISH analysis to detect chromosomal abnormalities in noncultured cells from two different tissues -cytotrophoblast and extraembryonic mesoderm -of 60 first-trimester spontaneous abortions from which cells had failed to grow in culture. An original algorithm was proposed to optimize the interphase karyotype screening with a panel of centromere-specific DNA probes for all human chromosomes. The overall rate of numerical chromosomal abnormalities in these cells was 53%. Both typical and rare forms of karyotype imbalance were found. The observation of six cases (19%) of monosomy 7, 15, 21 and 22 in mosaic form, with a predominant normal cell line, was the most unexpected finding. Cell lines with monosomies 21 and 22 were found both in cytotrophoblast and mesoderm, while cells with monosomy 7 and 15 were confined to the cytotrophoblast. The tissue-specific compartmentalization of cell lines with autosomal monosomies provides evidence that the aneuploidy of different human chromosomes may arise during different stages of intrauterine development. The effect of aneuploidy on selection may differ, however, depending on the specific chromosome. The abortions also revealed a high frequency of intratissue chromosomal mosaicism (94%), in comparison with that detected by conventional cytogenetic analysis (29%; Po0.001). Confined placental mosaicism was found in 25% of the embryos. The results of molecular cytogenetic analysis of these cell culture failures illustrate that the diversity and phenotypic effects of chromosomal abnormalities during the early stages of human development are underestimated.

Array-CGH testing in spontaneous abortions with normal karyotypes

“…In an attempt to overcome these problems other techniques such as fluorescent polymerase chain reaction (Diego-Alvarez et al, 2005), interphase-FISH (Horiuchi et al, 1997;Lebedev et al, 2004, Vorsanova et al, 2005, chromosome-CGH (Daniely et al, 1998;Fritz et al, 2001), MLPA (Diego-Alvarez et al, 2006) and array-CGH have been introduced in the study of POCs.…”

“…In two cases in which a normal male karyotype (46,XY) had been found, array-CGH revealed the presence of an additional chromosome 7 in one of them and of an additional chromosome 13 in the other. Although the presence of an undetected mosaicism cannot be ruled out, considering that the presence of mosaicism is a rather frequent finding in abortion material (Lebedev et al 2004;Schaeffer et al, 2004;Vorsanova et al 2005), it is also possible that it went undetected by the conventional cytogenetic analysis because of the previously mentioned difficulties usually presented by abortion material. In the third case in which conventional analysis of cells from a long term culture revealed a 46,XX karyotype and the array-CGH analysis showed a male complement with an additional chromosome 18, very likely the analyzed cells were of maternal origin.…”

“…Thus, in addition to being a time-consuming test, karyotyping does not always lead to a result; furthermore, in about 4.4% to 29% of cases the result does not correspond to the actual fetal karyotype, mainly due to maternal cell contamination as shown by other techniques such as fluorescence in situ hybridization (FISH) and others (Bell et al, 1999;Lomax et al, 2000;Diego-Alvarez et al, 2005;Karaoguz et al, 2005;Nikitina et al, 2005) DNA-based technologies do not require dividing cells thus, overcoming one of the main limitations associated with conventional cytogenetic analysis of spontaneous abortion material. Several of these methods have been used as diagnostic tools, including fluorescent polymerase chain reaction (Diego-Alvarez et al, 2005), interphase-FISH (Horiuchi et al, 1997;Lebedev et al, 2004, Vorsanova et al, 2005, chromosome-CGH (Daniely et al, 1998, Fritz et al, 2001, and multiplex ligation probe amplification (MLPA) (Diego-Alvarez et al, 2006). More recently, array-CGH has been considered as a particularly useful alternative to conventional karyotyping in the field of diagnosis (Schaeffer et al, 2004;Benkhalifa et al, 2005;Shimokawa et al, 2006), as it allows screening gains and losses in thousands of targets simultaneously.…”

Incidence and spectrum of chromosome abnormalities in spontaneous abortions: New insights from a 12-year study

“…The utilization of comparative genomic hybridization (CGH) and interphase fluorescence in situ hybridization (iFISH) has allowed cytogenetic laboratories to evaluate POC samples that have failed to grow. 24,25 In order to determine the impact of these new techniques, an accurate representation of current abnormality rates, which reflect current reproductive trends, is needed to serve as a comparison.…”

Estudio cromosómico en abortos espontáneos

“…Ésta última se revela por una preponderancia de sexo femenino entre las muestras con cariotipo normal y por el fenómeno común de encontrar una línea celular 46,XX además de una con cariotipo anormal. Una gran ventaja de disponer de estudio de FISH para aquellos casos en que no prospera el cultivo celular, permite entregar resultados concretos sobre algunas anomalías más prevalentes (11,12). La utilización del examen de FISH con un panel de sondas para los cromosomas 13, 16, 18, 21, 22, X e Y, permite detectar aproximadamente el 70% de todas las alteraciones cromosómicas encontradas por cariotipo, aún cuando no provee resultados para todos los cromosomas.…”