Quality and ReHability of Prenatal Cytogenetics

In the early fifties, prenatal investigation of amniotic fluid started with the evaluation of Rhesus sensitization (Bevis, 1950, 1952). It was followed by the discovery that fetal gender could be determined by the presence or absence of a sex chromatin body in the nuclei of cells in the amniotic fluid (Fuchs and Riis, 1956). In 1966, Steele and Breg demonstrated the possibility of culturing and karyotyping viabie amniotic fluid ceUs, rnainly of epithelial origin. Further progress was made with the refinernent of the technique and timing of arnniocentesis (Thiede et al., 1966; Jacobson and Barter, 1967), and the first prenatal diagnosis of Down syndrome (Valenti et al., 1968). Other important developments, in the same period, were the use of amniocentesis for the prenatal detection of biochemical abnormalities (Nadier and Gerbie, 1968), the development of ultramicrochemical techniques for rapid prenatal biochemical diagnosis (Galjaard et al., 1972, 1977, 1980; Niermeijer et al., 1975), and the finding of an association between a raised concentration of alpha-fetoprotein in arnniotic fluid and an open neural tube defect of the fetus (Brock and Sutcliffe, 1972). Cytogenetic investigations were improved by the discovery of the G-banding technique (Seabright, 1971). For almost fifteen years alnniocentesis was the only procedure for prenatal investigations. At the end of the eighties, more than a quarter of a million amniocenteses had been performed and the nurnber to date is probably in the rniilions. Nowadays, the safety of second trimester arnniocentesis together with the reliability, accuracy and efficiency is weil recognized; it is generally considered as the " gold standard"

Retrospective study of trisomy 18 in chorionic villi with fluorescent in situ hybridization on archival direct preparations

Trisomy 18 in direct chorionic villus preparations needs further investigation since the chromosome abnormality may be confined to the placenta and may not represent the actual fetal karyotype. We performed, retrospectively, fluorescent in situ hybridization (FISH) with the chromosome 18 centromere probe (L1.84) on interphase nuclei of destained slides of all cases of full trisomy 18 (n=22) and mosaic trisomy 18 (n=8) detected among 7600 first-trimester chorionic villus samples during an 8-year period (1985–1992). More nuclei displaying three signals were encountered in cases of full and mosaic trisomy 18 confirmed in fetal tissue than in non-confirmed cases. FISH can be useful for the verification of trisomy 18 in direct chorionic villus preparations

A chromosome 21-specific cosmid cocktail for the detection of chromosome 21 aberrations in interphase nuclei

Fluorescent in situ hybridization (FISH) with a 21q11-specific probe (CB21c1) consisting of three non-overlapping cosmids has been applied to interphase amniocytes of pregnancies at increased risk for fetal aneuploidy (N = 78) and to interphase lymphocytes, cultured and uncultured, of patients referred for Down syndrome (N = 19 and 28, respectively). In the uncultured amniocytes, six chromosome aberrations were detected: three cases of trisomy 21, a triploidy, a de novo 46,XX,t(21q21q), and a mosaic 46,XY/47,XY,+dic(21)(q11)/48,XY,+dic(21)(q11), +del(21)(q11). In 15 cultured and 20 uncultured blood samples, FISH correctly diagnosed trisomy 21 (full or mosaic) at the interphase level, which was confirmed in all cases by subsequent karyotyping. Because of specific and strong signals in interphase nuclei, CB21c1 appears to be a useful tool for the rapid detection of chromosome 21 abnormalities

Increased incidence of cytogenetic abnormalities in chorionic villus samples from pregnancies established by in vitro fertilization and embryo transfer (IVF-ET)

We studied 201 pregnancies that were established by in vitro fertilization and embryo transfer (IVF–ET) and compared the frequency of cytogenetic abnormalities with that found in a large control population matched for indication group (advanced maternal age) and time of sampling. A total of 252 IVF–ET fetuses were cytogenetically analysed by either chorionic villus sampling (CVS; n = 80) or amniocentesis (n = 172). Eleven chromosome abnormalities were found in the CVS group (13·8 per cent); among them, a 45, X/46, X, dic(q11)/46, X, del(Y)(q11) mosaic that was found in an IVF pregnancy established by intracytoplasmic sperm injection (ICSI), four cases of trisomy 21, and three cases of trisomy 7 confined to the placenta. The results indicate a statistically significant three‐to five‐fold increase in both confined placental abnormalities (P<0·008) and true fetal chromosome anomalies (P<0·04). In the amniocentesis group, identical rates (1·7 per cent) of chromosome abnormalities were found in the IVF–ET and control groups. It is concluded that late first trimester, but not early second trimester, IVF–ET pregnancie

False negative NIPT results: Risk figures for chromosomes 13,18 and 21 based on chorionic villi results in 5967 cases and literature review

Non-invasive prenatal testing (NIPT) demonstrated a small chance for a false negative result. Since the "fetal" DNA in maternal blood originates from the cytotrophoblast of chorionic villi (CV), some false negative results will have a biological origin. Based on our experience with cytogenetic studies of CV, we tried to estimate this risk. 5967 CV samples of pregnancies at high risk for common aneuplodies were cytogenetically investigated in our centre between January 2000 and December 2011. All cases of fetal trisomy 13,18 and 21 were retrospectively studied for the presence of a normal karyotype or mosaicism < 30% in short-term cultured (STC-) villi. 404 cases of trisomies 13,18 and 21 were found amongst 5967 samples (6,8%). Of these 404 cases, 14 (3,7%) had a normal or low mosaic karyotype in STC-villi and therefore would potentially be missed with NIPT. It involved 2% (5/242) of all trisomy 21 cases and 7.3% (9/123) of all trisomy 18 cases. In 1:426 (14/5967) NIPT samples of patients at high risk for common aneuploidies, a trisomy 18 or 21 will potentially be missed due to the biological phenomenon of absence of the chromosome aberration in the cytotrophoblast

Early embryonic development from zygote to blastocyst.

<p>The cytotrophoblast which is studied in short-term cultured villi (STC-villi) and with NIPT is derived from the trophoblast of the blastocyst, whereas the mesenchymal core, investigated in long-term cultured villi (LTC-villi) originates from the extra-embryonic mesoderm (EEM). Both EEM and fetus are derived from the inner cell mass (ICM) of the blastocyst.</p

Rapid aneuploidy detection with multiplex ligation-dependent probe amplification: A prospective study of 4000 amniotic fluid samples

The introduction of prenatal screening requires rapid high-throughput diagnosis of common aneuploidies. Multiplex ligation-dependent probe amplification (MLPA) allows for quick, easily automated multiplex testing of these aneuploidies in one polymerase chain reaction. We performed a large prospective study using MLPA on 4000 amniotic fluid (AF) samples including all indications and compared its value to karyotyping and fluorescence in situ hybridization (FISH). MLPA can reliably determine common aneuploidies with 100% sensitivity and 100% specificity. Moreover, some mosaic cases and structural chromosome aberrations were detected as well. In cases of a male fetus, triploidies can be detected by an aberrant pattern of probe signals, which mimics maternal cell contamination (MCC). Macroscopic blood contamination was encountered in 3.2% of the AF samples. In 20% of these samples, an MLPA pattern was found consistent with MCC, although there were no false negatives of the most common aneuploidies. As the vast majority of inconclusive results (1.7%) is due to potential MCC, we designed a protocol in which we determine whether MLPA can be performed on blood-contaminated AF samples by testing if blood is of fetal origi