Characterization of marker chromosomes by microdissection and fluorescence in situ hybridization

We characterized by microdissection and fluorescence in situ hybridization (FISH) two marker chromosomes: (1) a de novo, acrocentric marker chromosome detected in 88 per cent of the amniotic fluid cells of one of two physically and developmentally normal twins; and (2) a metacentric marker chromosome present in a phenotypically normal female. Analysis of FISH probes developed from the marker chromosomes indicated that the marker chromosomes in cases 1 and 2 were del(14)(q11) and a derivative chromosome from a Robertsonian translocation, respectively. Microdissection in combination with FISH may prove to be a valuable technique in determining the chromosomal origin of de novo marker chromosomes and unbalanced structural rearrangements detected during prenatal diagnosis.     

Cytogenetic analysis of chorionic villi for prenatal diagnosis: An ACC collaborative study of U.K. data

A prospective 3-year collaborative study was undertaken in 1987 to collect cytogenetic data from diagnostic chorionic villus samples (CVS) in the U.K. in order to determine the predictive value of the chromosome abnormalities encountered. Twenty-seven laboratories contributed a total number of 7595 cases, of which 97·6 per cent were successful. Excluding single cell anomalies, a total of 480 cytogenetic abnormalities were reported, of which 137 were familial structural rearrangements and 343 were de novo problems. Non-mosaic trisomies of chromosomes 13, 18, and 21 (n=157), non-mosaic sex chromosome abnormalities (n=33), and triploidy (n=6) were all confirmed in cells of fetal origin where follow-up information was available. Of the nine remaining non-mosaics including tetraploidy, trisomies of other autosomes, and extra markers, only a trisomy 16 and a case of a supernumerary marker proved genuine. Eighty-eight cases of mosaicism were reported to the study, of which only nine were confirmed as genuine: two cases involving chromosome 13, one trisomy 18, two examples of extra marker chromosomes, three 45,X, and one 47,XXX. There were no reports of false-negative findings. Presumptive maternal cell contamination was encountered in 39 cases, a detected incidence of 0·5 per cent. Four cases of presumptive ‘vanishing twin’ were recorded: in three of these, direct preparations showed a female karyotype, whereas cultures indicated a male (with male fetuses in two cases). The fourth case was of a female fetus with male and female cells in the CVS cultures. Subtle structural chromosome abnormalities were missed in three instances. Accurate prediction of the fetal karyotype was shown to require detailed knowledge of both the nature and the distribution of abnormal cells in the extra-embryonic tissues. In many cases, this could only be made where results from direct preparations and cultured cells were available. A number of conclusions were reached from these and similar data in the literature regarding the reliability of chromosome findings in CVS.     

Prenatal diagnosis of a de novo supernumerary marker derived from chromosome 16

Marker chromosomes are supernumerary chromosomes of unknown origin and are seldom found in prenatal diagnosis. Application of fluorescent in situ hybridization (FISH) allows the identification of the chromosomal origin of markers. Estimation of the risk of an abnormal phenotype outcome can be enabled by collecting data on phenotypes associated with markers of the same chromosomal origin. So far only very few cases of prenatal diagnosis of de novo supernumerary markers derived from chromosome 16 have been reported. Here the prenatal diagnosis of a de novo supernumerary marker chromosome 16 is described and the relevant literature discussed. Copyright © 2001 John Wiley & Sons, Ltd.     

A 10-year survey, 1980–1990, of prenatally diagnosed small supernumerary marker chromosomes,identified by fish analysis. Outcome and follow-up of 14 cases diagnosed in a series of 12 699 prenatal samples

A cytogenetic survey and follow-up studies were made of 14 cases with supernumerary marker chromosomes, identified among 12 699 prenatal samples, investigated at our institution over a 10-year period from 1980 to 1990. FISH (fluorescence in situ hybridization) techniques were employed to identify the chromosomal origin of the marker chromosomes. Five cases were familial, all derived from acrocentric chromosomes, and all without apparent phenotypic effects in the children. Nine cases represented de novo aberrations. In two cases (one with a marker from chromosome 14 or 22, the other with a ring-like marker derived from chromosome 17), the pregnancies continued and apparently normal babies were delivered at term, but the child with a marker derived from chromosome 17 showed slight psychomotor retardation at 2 years of age. All other pregnancies with de novo markers were terminated. In three cases, significant abnormalities were found at autopsy. One of these had an isochromosome 12p and the phenotype was consistent with Pallister-Killian syndrome. In conclusion, marker chromosome identification, as well as clinical follow-up, is essential for the purpose of improving genetic counselling.     

Prenatal detection of trisomy 21 in uncultured amniocytes by fluorescence in situ hybridization: A prospective study

A prospective study was undertaken to evaluate the use of fluorescence in situ hybridization (FISH) for the detection of trisomy 21 in interphase nuclei of uncultured amniotic fluid cells. Five hundred cases were analysed in situ and classified as normal or abnormal; the results were subsequently checked against the cytogenetic findings. Four hundred and ninety-three were correctly identified as normal with an 86·6 per cent average frequency of scored nuclei exhibiting two signals; six cases were correctly identified as trisomic for chromosome 21 with 81·7 per cent of scored nuclei exhibiting three signals; and one abnormal case involving an unbalanced chromosome 21·21 translocation was falsely scored as normal due to poor hybridization/detection efficiency. The method has been substantially improved and simplified so that it is suitable for the rapid detection of trisomy 21. As aneuploidy detection in interphase does not identify structural chromosome aberrations, it is not a substitute for fetal chromosome analysis.     

Prenatal diagnosis of two rare de novo structural aberrations of the Y chromosome: cytogenetic and molecular analysis

Two rare de novo structural aberrations of the Y chromosome were detected during routine prenatal diagnosis: a satellited non-fluorescent Y chromosome (Yqs), the first de novo Yqs to be reported in a fetus, and a terminal deletion of the Y chromosome long arm del(Y)(q11). In both cases detailed cytogenetic and molecular analyses were undertaken. In the case of the Yqs it was demonstrated by fluorescence in situ hybridization (FISH) that the satellites were derived from chromosome 15. In the case of the del(Yq), it was shown with molecular analysis by polymerase chain reaction (PCR) amplification of sequence-tagged sites (STS-PCR) that the deleted portion of the long arm of chromosome Y included the azoospermia factor loci, AZFb and AZFc. The clinical significance of these findings is discussed. Copyright © 2001 John Wiley & Sons, Ltd.     

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 pregnancies are characterized by an increased frequency of cytogenetic abnormalities found at prenatal diagnosis.     

Molecular and cytogenetic characterization of extra-structurally abnormal chromosomes (ESACs) found prenatally: outcome and follow-up

A 40-year-old woman underwent amniocentesis at 15.3 weeks of gestation. Chromosome analysis performed using QFQ, DA-DAPI and CBG banding revealed two de novo extra-chromosomal markers (ESACs) in 11 of the 16 colonies analysed. Fluorescence in situ hybridization (FISH) showed that both chromosomes came from the Yq11.22.1 region of the Y chromosome. PCR analysis of genes and STS localized on the Y chromosome excluded the Yp presence specifically of the SRY gene, and most of the euchromatic region of Yq. After extensive genetic counselling and considering both laboratory and second-level ultrasound data, the couple decided to continue the pregnancy. At 37.4 weeks of gestational age, a girl weighing 2750 g was born with an Apgar score of 9/10. A blood sample taken from the umbilical cord showed three cellular lines:mos47,XX, +mar1 ish.der (Y)(wcpY+) [21%]/48,XX, +mar1 ish.der (Y)(wcpY+), +mar2 ish.der (Y)(wcpY+) [41%]/46,XX [38%]. One year after birth, the baby was developing normally and had normal psychomotorial activity. Copyright © 2003 John Wiley & Sons, Ltd.     

Complications of cordocentesis in high-risk pregnancies: Effects on fetal loss or preterm delivery

Between 1990 and 1993, 166 cases underwent cordocentesis and were followed for at least the following 4 weeks in the Prenatal Diagnosis and Therapy Centre of Vienna University. The indications for the procedure were structural malformations in 46·4 per cent of the cases, other high-risk diagnoses in 48·8 per cent, and maternal age over 35 years in only 4·8 per cent. We investigated retrospectively all cases of complications resulting in fetal loss or preterm labour. Abortion, intrauterine fetal death, chorioamnionitis, and preterm delivery occurred in 0·6, 5·4, 0·6 and 9·0 per cent of these cases, respectively, adding up to a total of 26 cases (15·7 per cent). Although this rate looks relatively high, 20 of the 26 cases had already displayed signs implying a complicated prognosis. Neither maternal age, gestational age, number of attempts, nor placental location correlated with fetal loss or preterm delivery. Significantly higher rates of fetal loss or preterm delivery were observed when cordocentesis was performed in cases diagnosed as duodenal/intestinal stenosis or hydrops–ascites–hydrothroax/hygroma colli (P=0·0488 and P=0·0005). The frequency of complications did not decrease as the experience of the operators increased.     

Transabdominal chorionic villus sampling and amniocentesis for prenatal diagnosis: 5 years' experience at a University Centre

The fetal loss rates and fetal congenital birth defects in 821 transabdominal (TA) chorionic villus sampling (CVS) and 771 amniocentesis (AC) cases were evaluated from a 5-year period (1987–1991) at the University Central Hospital of Turku. The parents were given the option of choosing between the two sampling procedures. CVS was performed, in most cases, at 11 weeks of gestation; and AC, at 15 weeks. The rate of total post-procedure loss was 6·7 per cent in the CVS group and 4·4 per cent in the AC group (p=0·08). The rate of spontaneous abortions was 1·9 per cent in the CVS group and 1·0 per cent in the AC group (p=0·10). The number of birth defects was low in both study groups. No limb reduction cases were observed. Mosaicism was noted in 14 CVS cases and in five AC cases. We conclude that TA-CVS is a safe and practical alternative to AC in prenatal fetal karyotyping.     

Second-trimester maternal serum screening using alpha-fetoprotein,human chorionic gonadotrophin,and unconjugated oestriol: Experience of a regional programme

Over a 2-year period from January 1991 to December 1992, second-trimester maternal serum screening for Down's syndrome using alpha-fetoprotein (aFP), human chorionic gonadotrophin (hCG), and unconjugated oestriol (uE₃) was made available to five health districts in East Anglia, with a total population of 1·2 million. Amniocentesis was offered when the risk of Down's syndrome at term was 1:200 or greater. 25359 singleton pregnancies were screened, representing an uptake of 77 per cent. The recall rate for the 24 per cent of women who had not had a dating scan prior to the test was 9·4 per cent compared with 3·9 per cent for those who had been scanned (P<0·0005). Seventy-five per cent (36/48) of Down's syndrome pregnancies were detected for a false-positive rate of 4·0 per cent. Twenty-five out of 36 of detected Down's syndrome pregnancies were dated by scan prior to sampling, and in the 11 remaining cases, the dates were confirmed by scan after a high-risk result was obtained. The exclusion of uE₃ from the screening protocol would have reduced the detection rate to 52 per cent (25/48) for the same false-positive rate. Eighty-five per cent of women identified at high risk accepted the offer of an amniocentesis. Other fetal abnormalities detected were trisomy 18 (3), trisomy 13 (2), 45,X (6), 69,XXX (5), other chromosome abnormalities (9), open neural tube defects (26), hydrocephalus (7), abdominal wall defects (4), and steroid sulphatase deficiency (6).     

Mosaicism of autosomes and sex chromosomes in morphologically normal,monospermic preimplantation human embryos

We have previously detected chromosome abnormalities in human embryos whilst identifying the sex for preimplantation diagnosis of X-linked disease. In this study we assess the incidence of these abnormalities, both for sex chromosomes and autosomes 1 and 17, using dual fluorescent in situ hybridization (FISH). Sixty-nine normally fertilized embryos of good morphology at the 6–10 cell stage (day 3 post-insemination) were examined. The embryos were spread whole using HCl and Tween 20 to dissolve the cytoplasm. Thirty-four embryos were analysed for the sex chromosomes and 35 for autosomes 1 and 17. All probes were directly labelled with fluorochromes allowing analysis in 2 h. Control lymphocytes demonstrated that the probes were of high specificity. For the sex chromosomes, five embryos were mosaic (15 per cent) with the remaining 29 being uniformly XX or XY. In no case was an XX nucleus found in an otherwise XY embryo, indicating that even though mosaicism for the sex chromosomes is present, such abnormalities would not lead to a misdiagnosis of sex. For the autosomes, 16 embryos were abnormal (46 per cent); one embryo was triploid, one was monosomic for chromosome 1, and ten others were diploid mosaics (three diploid/aneuploid, three diploid/polyploid, and four diploid/haploid). A further four embryos had variable chromosome numbers in the majority of nuclei which appeared to be the result of uncontrolled mitotic division. The presence of haploidy or double monosomy, which occurred in 15 per cent of nuclei, has important implications for the diagnosis of trisomies and dominant disorders.     

Cross-hybridization of the chromosome 13/21 alpha satellite DNA probe to chromosome 22 in the prenatal screening of common chromosomal aneuploidies by fish

In a routine application of commercially available centromeric DNA probes for the prenatal screening of common trisomies involving the autosomes 13, 18, and 21, and sex chromosomes, four cases of discrepancy between fluorescence in situ hybridization (FISH) results and follow-up cytogenetic analysis were observed from a total of 516 cases of amniocentesis. In three of these cases, the results were false negative, and in one false positive. In this case, amniocentesis was performed because of a positive triple test in a 34-year-old woman with previous infertility treatment. The alpha satellite DNA probe for chromosomes 13/21 revealed five signals in 50 per cent of uncultured amniocytes, while standard cytogenetic analysis showed a normal karyotype. FISH analysis on metaphase chromosomes demonstrated the location of the additional signal in the centromeric region of chromosome 22. This additional signal was also present in the centromeric region of chromosome 22 of the mother, providing evidence for a possible inherited polymorphism in chromosome 22 responsible for unspecific hybridization with the alpha satellite probe for chromosomes 13/21 in this case. The observed polymorphism in centromeric regions may contribute to unreliability of the use of the 13/21 alpha satellite probe for prenatal screening by FISH.     

Prenatal identification of small mosaic markers of different chromosomal origins

In situ hybridization using a series of alphoid DNA probes has demonstrated the origin of two small accessory mosaic marker chromosomes ascertained from 1079 amniocenteses. These markers appeared to be de novo, derived from acrocentric chromosomes, and identical by traditional cytogenetic staining (G, Q, C, AgNOR, Hoechst-distamycin). Molecular characterization showed that one marker had originated from chromosome 14, the other from chromosome 22. Clinical outcome in both cases was normal.     

A comparison of amniotic fluid alpha-fetoprotein and acetylcholinesterase in the prenatal diagnosis of open neural tube defects and anterior abdominal wall defects

Amniotic fluid samples received for routine prenatal diagnosis of open neural tube defects were used for a study to compare amniotic fluid acetylcholinesterase (AChE) determination using a monoclonal antibody (4F19) enzyme antigen immunoassay and amniotic fluid alpha-fetoprotein (AFP) measurement as diagnostic tests for open neural tube defects. The study was based on 9964 women with singleton pregnancies and known outcome (including 6 with anencephaly and 18 with open spina bifida) having an amniocentesis at 14–23 weeks of gestation. The AChE immunoassay yielded detection rates for anencephaly of 100 per cent (95 per cent confidence interval (CI) 54·07–100 per cent), for open spina bifida of 100 per cent (95 per cent CI 81·47–100 per cent), for anterior abdominal wall defects of 20 per cent (95 per cent CI 0-51-71-64 per cent), and a false-positive rate of 0·22 percent (95 per cent CI 0·14–0·34 per cent) excluding anencephaly, open spina bifida, and anterior abdominal wall defects. For similar detection rates the false-positive rate of the AFP test was significantly higher, 0·74 per cent (95 per cent CI 0·58–0·94 per cent). On the basis of these findings, it is recommended that the technically simple AChE immunoassay should be used on all samples; the AFP test should only be used on the 0·5 per cent of the samples with concentrations of AChE activity ⩾ 8·5 nkat/1 for clear samples and blood-stained samples becoming clear after centrifugation, and ⩾ 25·0 nkat/1 for blood-stained samples that are discoloured after centrifugation; an AFP cut-off level of 2·0 MOM is recommended for this policy. Thereby, the detection rates for anencephaly, open spina bifida, and anterior abdominal wall defects would be 100, 100, and 20 per cent, respectively (95 per cent CIs 54·07–100, 81·47–100, and 0·51–71·64 per cent, respectively), and the false-positive rate would be 0·08 per cent (95 per cent CI 0·03–0·16 per cent) (excluding anencephaly, open spina bifida, and anterior abdominal wall defects).     

Hyperechoic fetal bowel: The perinatal consequences

A number of publications have reported an association between the finding of hyperechoic fetal bowel on prenatal sonogram and disorders such as aneuploidy and cystic fibrosis. To define more precisely the significance of this finding, we systematically reviewed the published material on the subject. Based on a total of 357 reported cases, we documented a high prevalence of cystic fibrosis (25·6 per cent) and chromosome abnormality (12·4 per cent) associated with increased bowel echogenicity in the fetus. High rates of intrauterine growth retardation (14·9 per cent), fetal demise (9·0 per cent), and prematurity (15·3 per cent) were also found. The data were obtained from a population at high a priori risk for aneuploidy and included fetuses at 1 in 4 risk for cystic fibrosis reported in two studies. This increased the bias towards an adverse outcome. The rate of complications when a hyperechoic abdomen is noted in a low-risk fetal population has so far not been delineated. Although the high frequency of complications found is of concern and warrants investigation, extrapolation of these risk figures to a fetal population at low a priori risk may not be appropriate.     

Blood transferrin receptor expression in chromosomally abnormal fetuses

In a cross-sectional study of 13 chromosomally abnormal fetuses, umbilical venous blood was obtained by cordocentesis at 17–32 weeks' gestation. Fetal blood transferrin receptor (CD71) expression (mean=79·8 per cent, range=60–98 per cent) and nucleated red cell count (mean=10·4 × 10⁹ per 1, range=1·0–25·0 × 10⁹ per 1) were significantly higher than the appropriate normal mean for gestation (z=3·92, P<0·0001 and z=3·69, P<0·001, respectively). These haematological changes in chromosomally abnormal fetuses would facilitate their prenatal diagnosis by analysis of fetal nucleated red blood cells isolated from the maternal circulation on the basis of CD71 expression.     

Serum PAPP-A and free β-hCG are first-trimester screening markers for down syndrome

Serum measurements of pregnancy-associated plasma protein A (PAPP-A) and the free β-human chorionic gonadotrophin (hCG) subunit were made in 13 women with Down syndrome (DS) pregnancies and six other women with fetal aneuploidy ascertained at chorionic villus sampling (CVS), as well as 89 women with contemporaneous normal control pregnancies. Median serum PAPP-A measurements (0·31 MOM, 95 per cent confidence interval (CI) 0·22–0·65 vs. normal 1·06, 95 per cent CI 0·89–1·20) were lower and free β-hCG subunit measurements (1·13 MOM, 95 per cent CI 0·93–2·63 vs. normal 0·91, 95 per cent CI 0·79–1·03) were higher at statistically significant levels. Receiver operator characteristic (ROC) curves showed that the highest sensitivity for detection, 71·2 per cent (95 per cent CI 54·7–87·6 per cent), was for depressed PAPP-A levels; the combination of low serum PAPP-A levels, maternal age, and elevated free β-hCG levels yielded a detection rate of 78·9 per cent (95 per cent CI 64·9–92·8 per cent) of the affected pregnancies at 8–12 weeks' gestation.     

Maternal serum free beta hCG screening: Results of studies including 480 cases of down syndrome

The median maternal serum free beta human chorionic gonadotropin (hCG) multiple of the median (MOM) of 480 Down syndrome cases in the second trimester was 2·64, significantly greater than the reported median MOM of intact hCG (p<0·0001). In 234 of these cases from retrospective and prospective studies, the effectiveness of maternal serum free beta hCG was evaluated in combination with alpha-fetoprotein (AFP) and maternal age in second-trimester Down syndrome screening. Down syndrome detection in the gestational age range of 14–16 weeks was 82 per cent. In all gestational weeks (14–22), a 77·7 per cent Down syndrome detection rate was achieved. In prospective screening of 44 272 patients under the age of 35 years, 69 per cent of Down syndrome cases were detected (73 per cent in gestational weeks 14–16). The false-positive rate for the prospective study was 3·8 per cent. The use of free beta hCG combined with maternal serum AFP and maternal age-related risk for Down syndrome in a screening population (i.e., women under 35 years) yields an improved detection efficiency over other protocols.     

First-trimester maternal serum schwangerschafts protein 1 (SP1) in pregnancies associated with chromosomal anomalies

The relationship between first-trimester maternal serum Schwangerschafts protein 1 (SP1) and the karyotype of the pregnancy was examined in 692 women who underwent chorionic villus biopsy at 6–12 weeks. There were 30 pregnancies with abnormal karyotypes, consisting of 14 Down's syndrome (DS), eight trisomy 18, and eight other anomalies, two of which were mosaics. The normal ranges and medians for gestation were defined from the 662 cases in which the karyotype was normal. The median SP1 (0·5 MOM) of the abnormal group was significantly lower than that of the normal group (10 MOM). This relationship was maintained for the DS pregnancies (0·4 MOM) and for anomalies other than trisomy 18 (0·43 MOM) but not trisomy 18 (1·1 MOM). It is possible that the use of SP1 as a screening test for chromosome anomalies in the first trimester could have a 43 per cent detection rate for a 5 per cent false-positive rate.