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.     

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.     

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.     

Transabdominal fine needle biopsy from chorionic villi in the first trimester

A technique for sampling first trimester chorionic villi for prenatal diagnosis by transabdominal fine needle biopsy is described. Specimens of chorionic villi were obtained from 49 out of 58 women, a success rate of 84.5 per cent. No fetal or maternal complications were demonstrated in the period before abortion. The procedure is useful for obtaining fetal tissue for culturing, DNA analysis and direct chromosome analysis.     

Enrichment of fetal nucleated cells from maternal blood: Model test system using cord blood

The presence of small numbers of fetal nucleated red cells in the maternal circulation has been a stimulus for the development of technologies for non-invasive prenatal genetic analysis. Our laboratory has been assessing the feasibility of density gradient centrifugation followed by magnetic activated cell sorting (MACS) of cells expressing CD32 and CD45, to deplete maternal nucleated blood cells. We have examined the efficiency of each of the steps of this procedure using cord blood from term pregnancies as a source of nucleated red blood cells. Cord blood was shown to contain highly variable numbers of nucleated red cells. Three different density gradients were examined. There was no major difference in the performances of the double and triple gradients. Density gradient centrifugation resulted in enrichments of nucleated red blood cells of about 1000-fold relative to the total cell count. However, it was apparent that the selection of the cell layers which were most enriched for these cells would result in significant losses of nucleated red cells in other layers. MACS sorting of cells using CD45 resulted in white cell depletions ranging from 7 to 34-fold. These data provide a foundation for comparison with other methods and for optimization of the MACS technique.     

Estimation of the weight of chorionic villus samples obtained from first trimester pregnancies by transcervical aspiration

Eight obstetricians experienced in chorionic villus sampling were asked to estimate the weight of samples of villi with and without a reference photograph. Referral to the photograph significantly improved the accuracy of their estimations.     

Chromosome analysis of first trimester chorionic villus biopsies Prepared by a maceration technique

A technique for the culture of chorionic villi for cytogenetic studies is described which is simple, reliable, rapid, and suitable for routine laboratory use. This method eliminates the need for multi-stage and time-consuming procedures inherent in previous techniques which have been developed for this tissue. Cultures suitable for analysis were obtained in an average of sixteen days and the risk of maternal cell contamination was evaluated. Results of cultures initiated from 50 chorion biopsies obtained by transcervical blind aspiration at a weekly termination clinic are presented, and the potential use of this technique as a routine procedure is discussed.     

Ultrastructure of first trimester chorionic villi with regard to the prenatal diagnosis of genodermatoses

Hopes are held out for chorion villus sampling, a technique which is gaining more and more importance for the first trimester prenatal diagnosis of chromosomal aberrations and metabolic abnormalities. A variety of inherited skin diseases can be diagnosed postnatally and prenatally (in the second trimester) by ultrastructural diagnostic markers. For evaluation of prenatal diagnosis in the first trimester, we investigated chorionic villi derived from the trophoblast layer of the early pregnancy by light microscopy and conventional electron microscopy. The ultrastructure of the cellular layers covering the villi, i.e., the inner cytotrophoblast and the outer syncytiotrophoblast, as well as that of the connective tissue of the inner extraembryonic mesoderm, are thoroughly described in relation to the ultra-structural changes in certain genodermatoses including epidermolyses and keratinization disorders. We found that chorionic villi have only a few of the characteristics differentiated in skin, and none of the structures which are relevant to the diagnosis of genodermatoses. In our view, the ultrastructural approach is not suitable for first trimester prenatal diagnosis of genodermatoses in chorionic villi.     

Pregnancy outcome after transcervical cvs with a flexible biopsy forceps: Evaluation of risk factors

The pregnancy outcome of 1936 women who had transcervical chorionic villus sampling (CVS) with a flexible biopsy forceps was evaluated. Follow-up until 4 weeks after delivery was 99.4 per cent. Various patient- and procedure-related risk factors for spontaneous loss (fetal or neonatal death) were analysed using stepwise logistic regression analysis. The overall spontaneous loss rate was 4.5 per cent. Factors found to be significantly associated with spontaneous loss were quantity of villi ≤ 15 mg (relative risk (RR) 2.13), a history of first-trimester miscarriage (RR 1.87) or delivery between 16 and 27 weeks (RR 3.87), cervical culture positive for anaerobes (RR 4.52) or group B streptococcus (RR 3.62), post-procedural bleeding >3 days (RR 1.99), and multiple insertions (RR 2.64). Significant differences in loss rates between individual operators were found. A learning effect was not present. There were no infants born with terminal transversal limb anomalies in our series.     

First trimester diagnosis of β-thalassaemia in a twin pregnancy

This paper reports the results of first trimester prenatal diagnosis in a twin pregnancy at risk for homozygous β°−thalassaemia (β°−39 mutant). Trophoblast samples from both twins were obtained at 10 weeks gestation with a forceps guided by ultrasound. Trophoblast DNA analysis, carried out with the oligonucleotide technique, revealed that one fetus was homozygous and the other heterozygous for the β−39 mutant. This diagnosis was confirmed at 17 weeks gestation by amniocyte DNA analysis. DNA polymorphism analysis within the α-globin gene provided useful genetic markers for twin differentiation.