Isolation of fetal cells from transcervical samples by micromanipulation: Molecular confirmation of their fetal origin and diagnosis of fetal aneuploidy

Transcervical cell (TCC) samples have been shown to contain fetal cells amenable to molecular analysis. However, the presence of ‘contaminating’ maternal cells limits their use for prenatal diagnoses. In this report we show that clumps of fetal cells can be isolated from transcervical samples by micromanipulation and tested by fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR). Out of 129 clumps, isolated from mucus aspirates and transcervical lavages from 29 patients, 29 clumps from 11 patients were found to be exclusively of fetal origin as judged by the detection of chromosome 21-specific polymorphic DNA markers and Y-derived DNA sequences by PCR and FISH. One case of a male triploid fetus, diagnosed by the analysis of TCC samples obtained by mucus aspiration and lavage, was confirmed by testing clumps of cells isolated by micromanipulation.     

Detection of fetal cells in transcervical samples and prenatal diagnosis of chromosomal abnormalities

Transcervical samples collected by lavage, aspiration, and cytobrush from women between 6 and 13 weeks of gestation were tested for the presence of fetal cells using fluorescence in situ hybridization (FISH) with probes for chromosomes X, Y, 1, and 21, and by polymerase chain reaction (PCR) amplification of DNA sequences derived from chromosomes X, Y, and 21. With a few exceptions, a good correlation was observed between the results of sexing the fetuses using FISH or PCR on transcervical cell (TCC) samples retrieved by lavage and those obtained by testing fetal (placental) tissue. In a comparative study between TCC samples collected by lavage or cytobrush, the sex of the fetus was correctly diagnosed by PCR amplification of a Y-derived DNA sequence. Variable results were observed with samples obtained by aspiration, mainly because this procedure was found to be more prone to failure to remove thick mucus without previous injection of physiological saline. Chromosome 21-derived small tandem repeats (STRs) of fetal origin were successfully detected in about 40 per cent of TCC samples recovered by lavage. Two cases of chromosomal abnormalities, one of trisomy 21 and one of triploidy, were detected in TCC samples in the course of our investigations.     

Prenatal diagnosis of triple X using fetal cells obtained by endocervical lavage

Prenatal diagnosis of aneuploidies currently relies on invasive procedures such as chorionic villous sampling and amniocentesis. These methods can provide an accurate diagnosis of the fetal karyotype but are relatively invasive. Here, we report a case of the prenatal detection of triple X using fetal cells obtained by lavage of the endocervical canal prior to amniocentesis, following ultrasound diagnosis of hydrops fetalis. Copyright © 2003 John Wiley & Sons, Ltd.     

Ultrasound screening for chromosomal anomalies in the first trimester of pregnancy

For the last 6 years, sonographic signs for excessive fluid accumulation in the backs of 10- to 12-week-old fetuses have been looked for prior to transabdominal chorionic biopsy. In 1400 pregnancies, subsequent karyotype analyses revealed 28 cases of Down syndrome. In 15 ( = 54 per cent), a large fluid cushion over most of the back had been documented at the time of biopsy. Only a few chromosomally normal fetuses with the same peculiarity were observed. The cushion was also present in fetuses with trisomies 18 and 13 , and in Turner syndrome. Systematic first-trimester screening for nuchal fluid accumulation seems to be a recommended method for the detection of Down syndrome and other chromosome anomalies in young pregnant women at low risk. It compares favourably with current methods of maternal serum screening performed at 16–18 weeks which require a higher number of invasive procedures.     

Fetal nucleated red blood cells from CVS washings: an aid to development of first trimester non-invasive prenatal diagnosis

Fetal nucleated red blood cells (n-rbc) occur in the maternal circulation from 7 weeks of pregnancy. The enrichment of these cells from maternal blood will depend upon their stage of differentiation, which changes during development. We characterised the fetal n-rbc from chorionic villus sample (CVS) washings and used them to model first trimester non-invasive prenatal diagnosis. The ratio of ε- to γ-globin-producing cells declined rapidly from 10 to 13 weeks, as did the ratio of nucleated to non-nucleated rbc. By 13 weeks the great majority of cells containing γ- or ε-globin are anucleate. The fetal n-rbc were highly variable in size and density and sedimented over a wide density range with a high proportion (>80%) at a density overlapping with that of maternal rbc. We have devised an enrichment procedure using Orskoff lysis to differentially lyse the maternal cells followed by density centrifugation and separation using magnetic beads. This simple protocol allowed recovery of 70% (69±22%) of fetal cells when added at approximately 10 fetal cells/ml maternal blood. When 1 fetal cell/ml millilitre maternal blood was added (total volume 10 ml) the recovery was more variable but remained at approximately 70% (72±47%), with at least one fetal cell recovered in all cases. Copyright © 2001 John Wiley & Sons, Ltd.     

Significance of septations in isolated fetal cystic hygroma detected in the first trimester

Recent reports have indicated an increased risk for fetal chromosome abnormalities, especially autosomal trisomy, in fetuses with isolated cystic hygroma, or prominent nuchal membranes, detected by ultrasonography during the first trimester. However, these reports present contradictory information regarding the prognostic significance of septations within the cystic hygroma. We evaluated, in blind fashion, 55 consecutive cases of isolatd fetal cystic hygroma detected at or before 13·9 weeks' gestation to determine the association between septations and fetal chromosome complement. Septations were associated (P<0·05) with an increased risk for fetal chromosome abnormalities. However, the incidence of chromosome abnormalities was also increased (12·5 per cent) among cases not characterized by septations. Thus, we believe it prudent to offer invasive prenatal testing to all women found to be carrying fetuses with cystic hygroma, irrespective of the presence or absence of septations.