Rapid detection of aneuploidy in uncultured chorionic villus cells using fluorescence in situ hybridization

Rapid detection of aneuploidy using chromosome-specific repetitive DNA probes and the potential diagnostic accuracy of fluorescence in situ hybridization (FISH) on interphase cells of chorionic villus samples (CVS) are presented. Analyses demonstrated the ability to correctly identify aneuploidy using FISH in uncultured CVS. Our preliminary investigation suggests that this technique offers a significant clinical potential to circumvent problems of culture, time, and cost in cytogenetic analysis.     

Tetrasomy 12p—unusual presentation in CVS

CVS direct preparations usually achieve limited resolution and are better at detecting numerical rather than structural abnormalities. A CVS direct preparation analyzed using G-banding revealed a 47,XY,+G karyotype in 5 of 11 cells and was reported as mosaic for trisomy 21. Subsequent analysis of the CVS culture found only normal male cells. Amniocentesis revealed both normal male cells and cells with an extra F-group chromosome. Fluorescence in situ hybridization (FISH) identified this chromosome to be an isochromosome from the short arm of chromosome 12 [i(12)(p10)]. The amniocyte karyotype was reported as 47,XY,+i(12)(p10)[12]/46,XY[8].ish i(12)(p10)(wcp12+), which is associated with Pallister–Killian syndrome. Reexamination of the CVS direct preparation by FISH with a chromosome 12 centromere probe confirmed the karyotype of this tissue to be 47,XY,+mar[5]/46,XY[6].nuc ish 12cen(D12Z3 × 3)/12cen(D12Z3 × 2). Thus, multiple studies, including amniocentesis and fluorescence in situ hybridization, may be required to fully and accurately evaluate abnormalities detected by CVS. This case also indicates that mosaicism for supernumerary isochromosomes may have a complex origin. Copyright © 2003 John Wiley & Sons, Ltd.     

Rapid sexing of human embryos by non-radioactive in situ hybridization: Potential for preimplantation diagnosis of X-linked disorders

Sixty spare human embryos at various stages of preimplantation development were prepared for cytogenetic analysis. Fluorescent staining of those with metaphases allowed scoring for the presence of a Y chromosome. In situ hybridization was then performed using a biotinylated Y-specific sequence, and the probe was detected by a standard streptavidinlinked alkaline phosphatase system. This enabled comparison of the chromosomal sex with that obtained after in situ hybridization in 28 embryos, and the sexing result obtained by the two methods was concordant in all cases. A further 21 embryos in which no metaphase chromosomes were obtained were sexed by biotinylated in situ hybridization only. Overall, 66 per cent of male interphase nuclei demonstrated a Y-specific hybridization signal. Results were obtained in under 24 h, which may permit the sexing of an embryo biopsied during cleavage and the transfer of sexed embryos at the blastocyst stage to the mother's uterus in the same cycle as oocytes are collected for in vitro fertilization.     

Molecular cytogenetic analysis of term placentae suspected of mosaicism using fluorescence in situ hybridization

In first-trimester chorionic villus sampling (CVS) for prenatal diagnosis, abnormal chromosomal findings, such as mosaicism, trisomies, or suspect abnormal karyotypes, are found more frequently than at amniocentesis. The fact that these chromosomal abnormalities do not always reflect the fetal karyotype but may be restricted to the placenta is a major problem in diagnosis and counselling. In this paper we present the results of fluorescence in situ hybridization (FISH) studies on interphase nuclei of three term placentae investigated because of false-positive findings at first-trimester CVS. The chorionic villi of the first case showed a mosaic chromosome pattern involving a trisomy 10 cell line and a normal cell line, those of the second case a total trisomy 8 cell line, while in the third case a complete monosomy X was found. Follow-up amniocentesis in each of these three cases revealed a normal karyotype. By using FISH, we were able to confirm the presence of the aberrant cell lines, which were all confined to one part of the placenta. FISH on interphase nuclei allows the investigation of large numbers of cells for the existence of numerical chromosome aberrations in a quick and reliable way.     

Maternal uniparental isodisomy 10 and mosaicism for an additional marker chromosome derived from the paternal chromosome 10 in a fetus

An Erratum has been published for this article in Prenatal Diagnosis 22(11) 2002: 1056. We report a case of maternal isodisomy 10 combined with mosaic partial trisomy 10 (p12.31-q11.1). Chromosome examinations from a CVS sample showed a karyotype 47,XY,+mar/46,XY. The additional marker chromosome which was present in 6/25 interphase nuclei was shown by fluorescence in situ hybridization (FISH) to have been derived from a pericentromeric segment of chromosome 10. DNA analysis was performed from umbilical cord blood from the fetus after termination of the pregnancy at 18 weeks. The results showed that the two structurally normal chromosomes 10 were both of maternal origin, whereas the marker chromosome derived from the father. Autopsy of the fetus revealed hypoplasia of heart, liver, kidneys and suprarenal glands, but, apart from a right bifid ureter, no structural organ abnormalities. This fetus represents the second reported instance of a maternal uniparental disomy (UPD) 10. Copyright © 2002 John Wiley & Sons, Ltd.     

The association between alpha-fetoprotein and βhcg levels prior to and following chorionic villus sampling in cases that spontaneously miscarried

Maternal serum alpha-fetoprotein (AFP) and beta-human chorionic gonadotropin (βhCG) measurements taken prior to chorionic villus sampling (CVS) in 21 patients who subsequently miscarried were compared with measurements in a control group of 113 patients with uneventful pregnancies. Patients with AFP levels of 10 iu/ml or more prior to the CVS had a 4·3 times greater risk of miscarriage (95 per cent confidence interval 1·3–13·6). AFP levels obtained 1 week after the CVS in the 13 patients with late miscarriages were higher than in the control group (P = 0·06). Patients miscarrying had a greater rise in AFP (P = 0·06) and a greater fall in βhCG levels (P = 0·04) following the CVS procedure, compared with the control subjects. Each 10-unit change in the difference between AFP or βhCG levels prior to and 1 week following the CVS was associated with a significantly increased risk for late miscarriage. Elevated maternal serum AFP levels early in pregnancy and changes in AFP and βhCG levels following CVS may predict an increased risk for subsequent miscarriage.     

Magnetic-activated cell sorting (MACS) significantly decreases the hybridization efficiency of fluorescence in situ hybridization (FISH)

Fetal cells were enriched from maternal blood using density gradient centrifugation of Histopaque followed by magnetic-activated cell sorting (MACS) to select CD71-positive cells. For each specimen, cells partially purified by Histopaque were split into equal portions, and each portion was subjected to purification by MACS in parallel. Cells before and after MACS were subjected to dual-color fluorescence in situ hybridization (FISH) analysis with X- and Y-chromosome-specific probes. We found that the hybridization rates were decreased by approximately 10% after MACS based on duplicated analysis for each sample. Copyright © 2001 John Wiley & Sons, Ltd.     

Fluorescent in situ hybridization (FISH): A new application in the delineation of true vs. pseudomosaicism in prenatal diagnosis

Metaphase chromosomes and interphase nuclei from nine amniotic fluid cultures were studied with fluorescence in situ hybridization (FISH). The samples were initially analyzed with routine G-banding and were diagnosed as having true mosaicism (five patients) or pseudomosaicism (four patients). In our study, FISH analysis could provide additional information to distinguish pseudo– from true mosaicism by allowing interphase studies and analysis of an increased number of metaphase spreads. These results suggest a multilinear origin of ‘in situ’ colonies of cells.     

Mosaicism for trisomy 12: Four cases with varying outcomes

Trisomy 12 observed in chorionic villus sampling (CVS) may reflect generalized mosaicism or indicate mosaicism confined to only the placenta. In this report, four cases of trisomy 12 observed in CVS or cultured placental biopsies with varying outcomes are presented. Seven dinucleotide repeat polymorphisms for chromosome 12 were used to determine the chromosome 12 origins in the fetus or child and to delineate the mechanism(s) that gave rise to the trisomy. In two cases (cases A and C), the mosaicism was confined to the placenta, resulting in normal liveborns. Although, in one case, the molecular results suggested an apparent duplication of one paternal chromosome 12 in the placenta, normal biparental inheritance was found in the diploid fetal cell line in both cases. In two other cases (cases B and D), trisomy 12 was observed in both extraembryonic and fetal tissues. In one of these pregnancies, a child was born by Caesarean section at 37 weeks because of intrauterine growth retardation and oligohydramnios, and resulted in neonatal death. Molecular markers and fluorescence in situ hybridization (FISH) revealed low-level trisomy 12 mosaicism in the spleen. In the fourth case, fetal abnormalities were detected on ultrasound and low-level trisomy 12 mosaicism was observed in amniotic fluid cells using conventional cytogenetics and FISH. Molecular markers revealed a maternal meiosis I non-disjunction of chromosome 12 in DNA from a cultured placental biopsy. Although predicting the outcomes of pregnancies involving confined placental mosaicism remains difficult, molecular techniques are valuable tools for distinguishing uniparental from biparental disomy and mechanisms of mosaicism.     

Clinical and pathological factors in spontaneous abortion following chorionic villus sampling

Twenty-nine cases of spontaneous abortion following first-trimester chorionic villus sampling (CVS) were reviewed out of a series of 722 patients. Of the 29 cases, there were only four abnormal CVS results. Pathological examination was performed in 79 per cent of cases, and this did not identify any characteristic pathological feature associated with spontaneous abortion after CVS. There was no obvious difference in the pathological features following the transabdominal (TA) or the transcervical (TC) methods. The majority of miscarriages occurred within 4 weeks of the procedure, but 38 per cent of cases aborted between 7 and 14 weeks after CVS. The TC method was used in 22 patients; the TA in 6; and both methods in 1 patient. The TA method was associated with a significantly lower fetal loss rate than the TC method (TA 2 per cent, TC 9 per cent, p < 0.001).     

Risk factors associated with transcervical CVS losses

Factors found to be associated with pregnancy loss after transcervical CVS were race (higher for non-white), history of spontaneous abortion, unplanned pregnancy, history of spotting or bleeding during the pregnancy prior to CVS, and placental position (higher for fundal or lateral locations). Whether the increase in loss risk is due to the factor, per se, or the factor plus the CVS cannot be determined due to the lack of appropriate control data.     

Chorionic villus sampling utilization following reports of a possible association with fetal limb defects

Prenatal diagnosis choices were reviewed in 473 women who presented for genetic counselling prior to 11 weeks' gestation for the indication of advanced maternal age. Group A consisted of 336 patients who were unaware of a possible association between chorionic villus sampling (CVS) and limb defects. Group B consisted of 137 patients who were provided this information. Fifty-one per cent of patients in group A and 45 per cent of patients in group B chose CVS. This difference was not significant by χ² analysis (P = 0·7). Patterns of prenatal diagnosis procedure utilization from 1987 to 1992 revealed a significant reduction in CVS utilization accompanied by a corresponding increase in amniocentesis after the association between CVS and limb defects was publicized. Referrals for CVS counselling also significantly declined. However, acceptance rates did not change for those patients who received genetic counselling. First-trimester genetic counselling, including a discussion regarding a possible association between CVS and limb defects, helps patients make informed decisions concerning prenatal diagnosis options, and, in our population, resulted in no change in CVS acceptance rates.     

Transcervical (TC) and transabdominal (TA) CVS for prenatal diagnosis in rotterdam: Experience with 3611 cases

Data from 3611 consecutive CVS (TC, N= 1780; TA, N= 1831) were analysed with emphasis put on influence of maternal and gestational age at CVS on the fetal loss rate < 28 weeks. For TC-CVS the gestational age varied from 9.3–11.6 weeks, for TA-CVS from 9.3–20 weeks. Sampling efficacy at first attempt was 86.5 per cent and 95 per cent respectively. In 4.6 per cent an abnormal result was established. In older mothers (N=2362) the fetal loss rate was significantly higher (p = <0.05) when sampled before 12 weeks (TC-CVS 6.2 per cent, TA-CVS 5.8 per cent). When the CVS (TA) was performed after 12 weeks the fetal loss rate decreased to 2.4 per cent. In 1079 younger women the fetal loss rate remained low (TC 2.8 per cent; TA < 12 weeks 1.8 per cent; TA > 12 weeks 1.7 per cent) and was not influenced by gestational age at the time of sampling. We concluded both methods safe and reliable when the choice of application considers maternal age.     

Prenatal diagnosis of fragile x syndrome: (cgg)n expansion and methylation of chorionic villus samples

Fragile X syndrome is the most common form of inherited mental retardation, due to an expansion of the (CGG)_n trinucleotide repeat in the FMR-1 gene and hypermethylation of its 5′ upstream CpG island. Two major problems remain to be resolved for fragile X prenatal diagnosis: the abnormal methylation patterns of chorionic villus samples (CVS) and the inability to predict the mental status of females with the full mutation. We present here the results of ten prenatal diagnoses of fragile X syndrome using Southern blotting and polymerase chain reaction (PCR) amplification, and the analysis of 50 further CVS to test the methylation status of the CpG island of the FMR-1 gene. In the ten ‘at-risk’ CVS, eight normal (five males and three females) and two affected male fetuses were detected. Absence of methylation in the CVS was observed in two cases, which was not found upon subsequent examination of the newborn or of fetal tissues. In the 50 CVS not ‘at risk’ for fragile X syndrome, abnormal fragment patterns for probe StB12.3 were detected in 32 per cent for female and 24 per cent for male fetuses. This abnormal pattern could be due to absent or partial methylation of the CpG island of the FMR-1 gene in chorionic villus tissues.     

Does chorionic villus sampling compromise fetal umbilical blood flow?

A possible association of limb reduction defects with chorionic villus sampling (CVS) may be related to compromised umbilical blood flow from the trauma of the procedure. We hypothesized that because CVS may disrupt or compromise umbilical blood flow to the fetus, either by vasoconstriction, bradycardia, or emboli, we would detect these changes using Doppler velocimetry. A cohort of 21 consecutive consenting patients undergoing first-trimester elective CVS for prenatal diagnosis were entered into a prospective longitudinal study. Colour flow Doppler velocimetry was performed on fetal umbilical arterial blood flow immediately before and after CVS to measure the pulsatility index, fetal heart rate, per cent flow time, and maximum flow velocity. Measurements were obtained from three consecutive cardiac cycles in three different umbilical segments and averaged. Potentially confounding variables also recorded included gestational age, method of CVS, number of passes, number of aspirations, placental location, tissue sample size, and operator. Umbilical velocimetry values before and after CVS were compared using the paired t-test and showed no statistically significant differences. No differences were found when data were analysed by gestational age, sample size, method, number of aspirations, placental location, or operator. We were unable to detect any significant change in fetal umbilical arterial blood flow velocimetry or heart rate after performing CVS. Umbilical blood flow does not appear to be routinely compromised by CVS.     

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.     

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.     

Cytogenetic analysis of 2928 CVS samples and 1075 amniocenteses from randomized studies

We report cytogenetic results from a randomized Danish chorionic villus sampling (CVS) and amniocentesis (AC) study including 2928 placental and 1075 amniotic fluid specimens processed in the same laboratory. The results are presented in groups comparing CVS with amniocentesis and transabdominal (TA) CVS with transcervical (TC) CVS as randomized. More abnormalities and more ambiguous diagnostic problems were found in placental tissues than in amniotic cells. There were no diagnostic errors and no incorrect sex predictions. Mosaicism was detected in 1 per cent of all cases of CVS (discordancies included). When confirmation studies were done, 90 per cent were found to be confined to the placenta. Eight cases (0.7 per cent) of mosaicism/pseudomosaicism were seen in amniotic fluid specimens, and two cases of five with confirmation studies were confirmed in the fetus. The rate of mosaicism/pseudomosaicism in CVS and AC specimens differed (P <0.05). The rate of pseudomosaicism in cultures of villi and amniotic fluid cells was 0.5 and 0.6 per cent, respectively. Single-cell aneuploidy was observed in 1.8 per cent of villi and 1.4 per cent of amniotic fluid cell specimens. Maternal cell contamination (MCC) was seen more often after TC sampling (4.5 per cent) compared with TA sampling (1.5 per cent), but posed no problems in interpretation. Compared with the processing of cultured specimens, the short-term method of preparation of villi in our laboratory doubled the technicians' workload. For practical and economic reasons we have ceased the routine use of short-term preparations.     

Rapid prenatal diagnosis of aneuploidy from uncultured amniotic fluid cells using five-colour fluorescence in situ hybridization

In this paper we describe the use of five-colour fluorescence in situ hybridization for prenatal diagnosis of aneuploidy using uncultured amniotic fluid cells. The analysis is based on ratio mixing of dual-labelled probes and digital imaging for the detection and visualization of five different probes specific for the five target chromosomes, 13, 18, 21, X, and Y. A retrospective blind analysis of 30 coded uncultured amniotic fluid samples correctly detected fetal sex and five trisomy 21 cases. Multicolour fluorescence in situ hybridization used in this way allows rapid and simultaneous detection of the most frequent aneuploidies.     

How does cell-based non-invasive prenatal test (NIPT) perform against chorionic villus sampling and cell-free NIPT in detecting trisomies and copy number variations? A clinical study from Denmark

Objectives

We aimed to compare cell-based NIPT (cbNIPT) to chorionic villus sampling (CVS) and to examine the test characteristics of cbNIPT in the first clinical validation study of cbNIPT compared to cell-free NIPT (cfNIPT).

Material and Methods

Study 1: Women (N = 92) who accepted CVS were recruited for cbNIPT (53 normal and 39 abnormal). Samples were analyzed with chromosomal microarray (CMA). Study 2: Women (N = 282) who accepted cfNIPT were recruited for cbNIPT. cfNIPT was analyzed using sequencing and cbNIPT by CMA.

Results

Study 1: cbNIPT detected all aberrations (32/32) found in CVS: trisomies 13, 18 and 21 (23/23), pathogenic copy number variations (CNVs) (6/6) and sex chromosome aberrations (3/3). cbNIPT detected 3/8 cases of mosaicism in the placenta. Study 2: cbNIPT detected all trisomies found with cfNIPT (6/6) and had no false positive (0/246). One of the three CNVs called by cbNIPT was confirmed by CVS but was undetected by cfNIPT, two were false positives. cbNIPT detected mosaicism in five samples, of which two were not detected by cfNIPT. cbNIPT failed in 7.8% compared to 2.8% in cfNIPT.

Conclusion

Circulating trophoblasts in the maternal circulation provide the potential of screening for aneuploidies and pathogenic CNVs covering the entire fetal genome.