Diagnosis of miscarriages by molecular karyotyping: Benefits and pitfalls

Abstract

Purpose: About 50% of spontaneous abortions are caused by fetal chromosome abnormalities. Identification of these abnormalities helps to estimate recurrence risks in future pregnancies. However, due to culture failures or maternal contamination often no fetal karyotype can be obtained. Array comparative genomic hybridization can overcome some of these limitations. Methods: In this study, we analyzed 103 miscarriages by both T-banding and 1-Mb array comparative genomic hybridization. Results: We found an overall abnormality rate of 35% (34 of 96). In a comparison of 70 samples that were successfully analyzed by both techniques, 54 (77%) had identical karyotypes (42 normal, 12 abnormal) and 16 (23%) cases showed discrepancies. Most of these differences were due to maternal contamination during cell culture, which resulted erroneously in a normal female karyotype. Conclusion: These results demonstrate the improved diagnostic yield of array comparative genomic hybridization as compared with conventional karyotyping. Therefore, we implemented this technique in the diagnostic workup of miscarriages. Genet Med 2009:11(9):646 -654.Key Words: miscarriage, array comparative genomic hybridization, array CGH, chromosomal imbalance S pontaneous abortions are common, with 10% to 15% of all clinically recognized pregnancies ending in early pregnancy loss. Cytogenetic analysis has shown that about 50% of first trimester miscarriages are caused by fetal chromosome abnormalities, most of which consist of numerical abnormalities (86%), including trisomies, monosomies, and polyploidies. Structural abnormalities represent another 6% of anomalies found. 1-3 Identification of the cause of a spontaneous abortion helps to estimate recurrence risks in future pregnancies and, when an anomaly is found, comforts parents. Over the years, routine analysis of products of conception (POC) has been performed by karyotyping of metaphase spreads after tissue culture. However, due to failure of culture growth, suboptimal chromosome preparations, or possible maternal contamination 4,5 either no result or an erroneous result is obtained.New molecular cytogenetic methods avoid some of these pitfalls. Interphase fluorescence in situ hybridization (FISH), quantitative fluorescence polymerase chain reaction, and subtelomeric multiplex ligation-dependent probe amplification are rapid techniques that do not require cell culture and can be performed in 24 to 48 hours. 6 -9 Their main disadvantage, however, is the use of probes and primers that target only a selection of chromosomes or only specific subtelomeric loci, thereby missing information about the remaining genome.Comparative genomic hybridization (CGH) uses DNA isolated directly from the fetus or extra embryonic tissues and, after labeling with fluorescent dyes, hybridizes this DNA together with a labeled reference sample to normal metaphase spreads. 10,11 It has been applied successfully by several groups to detect fetal chromosome abnormalities in POC. 5,12-17 Array CGH takes this technique one st...

Reflex fluorescent in situ hybridization testing for unsuccessful product of conception cultures: A retrospective analysis of 5555 samples attempted by conventional cytogenetics and fluorescent in situ hybridization

“…Normal cutoffs, which were previously established in our laboratory (data not shown), were then used to determine whether the sample was abnormal. The normal cutoffs for trisomy of chromosomes 13,16,18,21,22, X, and Y were 3%, 8.5%, 6%, 4.5%, 7%, 10%, and 10%, respectively. Monosomy X and triploidy/tetraploidy had normal cutoffs of 20% and 5%, respectively.…”

“…Materials and Methods: All products of conception samples were simultaneously processed for both chromosome analysis and FISH analysis. If the chromosome analysis was unsuccessful, interphase FISH was performed for chromosomes 13,16,18,21,22, X, and Y. To assess the performance of the FISH assay, a 3-year retrospective comparative analysis of the FISH results versus chromosome results was performed.…”

“…All POC samples received in the laboratory were simultaneously processed for both chromosome analysis (cultured) and FISH analysis (uncultured). On an unsuccessful chromosome analysis, an interphase FISH assay consisting of probes for chromosomes 13,16,18,21,22, X, and Y was performed on the uncultured fixed-cell pellet. Using this algorithm during a 3-year period, our laboratory attempted 5555 POC chromosome analyses of which 943 were subsequently analyzed by our reflex FISH panel test.…”

Prenatal BoBsTM in the cytogenetic analysis of products of spontaneous miscarriage

“…[1] Chromosomal abnormalities are the most common cause, more than 50% of aborted fetuses in the first trimester showing numerical abnormalities including trisomies, X monosomies and tripoidies. Aneuploidies become less prevalent as pregnancy progresses (30% in the second trimester), because most abnormal fetuses have miscarried earlier.…”

“…However, while it is possible to investigate several regions with FISH, the number of regions that can be explored with QF-PCR is limited, and MLPA requires the use of two commercial kits to invesigate subtelomere regions. [8] An additional approach is array-comparative genomic hybridisation (CGH), which has a better diagnostic yield than conventional karyotyping in the screening of products of conception (POC) [1] but with the disadvantage of high cost.…”