Residual risk for cytogenetic abnormalities after prenatal diagnosis by interphase fluorescence in situ hybridization (FISH)

Results from conventional cytogenetic studies on 21 609 amniotic fluid specimens were analyzed retrospectively to determine the residual risk for a cytogenetic abnormality if interphase FISH, capable of only detecting aneuploidy for chromosomes 13, 18, 21, X and Y, was performed and did not reveal an abnormality. Detection rates (the probability of detecting a cytogenetic abnormality when an abnormality is present) and residual risks (the likelihood of a cytogenetic abnormality, in view of normal interphase FISH results) were calculated for the four major clinical indications for prenatal diagnosis (advanced maternal age, abnormal maternal serum screen indicating increased risk for trisomy 18 or trisomy 21, abnormal maternal serum screen indicating increased risk for neural tube defects and ultrasound abnormality). Differences in detection rates were observed to depend on clinical indication and presence or absence of ultrasound abnormalities. The detection rate ranged from 18.2 to 82.6% depending on the clinical indication. The detection rates of abnormalities significant to the pregnancy being evaluated (i.e. abnormalities excluding familial balanced rearrangements and familial markers) were between 28.6 and 86.4%. The presence of ultrasound abnormalities increased the detection rate from 72.2 to 92.5% for advanced maternal age and from 78.6 to 91.3% for abnormal maternal serum screen, indicating increased risk for trisomy 18 or trisomy 21. With regard to residual risk, the risk for a clinically significant abnormality decreased from 0.9–10.1%, prior to the interphase FISH assay, to a residual risk of 0.6–1.5% following a normal interphase FISH result in the 4 groups studied. Providing patients with detection rates and residual risks, most relevant to their situation (clinical indication and presence or absence of ultrasound abnormality) during counseling, could help them better understand the advantages and limitations of interphase FISH in their prenatal diagnostic evaluation. Copyright © 2003 John Wiley & Sons, Ltd.     

Uterine cavity lavage: Adding fish to conventional cytogenetics for embryonic sexing and diagnosing common chromosomal aberrations

This study was undertaken to examine the efficacy for early prenatal diagnosis of uterine cavity lavage at the level of the internal os and to assess the rate of maternal contamination. In phase I, uterine cavity lavage was performed in 38 women scheduled for pregnancy termination between 6 and 12 weeks. In addition to short- and long-term cultures, one-colour FISH (fluorescence in situ hybridization) with Y and X probes was used for fetal sexing. Two-colour FISH was used in all known male fetuses for the assessment of maternal contamination. In phase II, lavage was performed on 16 women. Fetal sex was diagnosed with direct labelled X and Y probes and common numerical chromosomal aberration was attempted with 18 and 21 direct labelled probes. Fetal sexing was successful in all cases in phases I and II. Out of 34 patients in which tissue was obtained, only FISH was done in six. Long-term cell cultures were successful in the other 28 cases, but complete karyotyping in 19 (56 per cent). No chromosomal aberration was found with the direct labelled probes 18 and 21 in FISH. Maternal contamination was assessed to be 5–10 per cent. This simple and easy-to-master technique is very effective in obtaining fetal cells early in pregnancy for genetic diagnosis, especially by FISH. However, the safety of the procedure must be tested in ongoing pregnancies.     

Prenatal aneuploidy detection in interphase cells by fluorescence in situ hybridization (Fish)

FISH is a quick, inexpensive, accurate, sensitive and relatively specific method for aneuploidy detection in samples of uncultured chorionic villus cells and amniotic fluid cells. FISH allows detection of the autosomal trisomies 13, 18 and 21 and X and Y abnormalities and any other chromosome abnormality for which a specific probe is available. The detection rate of these abnormalities is high in informative samples which have a concordance of > 99.5% with cytogenetic results. A relatively high number of abnormal cases are found in uninformative samples. However, such samples should be regarded as samples to be investigated further. Clinical experience with the use of FISH for prenatal diagnosis is now beyond 10,000 cases; a number of clinical protocols and smaller trials have also been carried out, resulting in 90% of attempted analyses giving informative results with a high detection rate and extraordinarily low false-positive and false-negative rates Unsolved problems remain, such as occasional technical failures, admixtures of maternal blood and up to 20% uninformative scoring results, especially for abnormal specimens. FISH is at present used as an adjunct to classical cytogenetic analysis. However, this should not be interpreted as meaning that FISH could not be used as a methodology in its own right. If FISH were to be considered a Diagnostic test then this might be the case, due to the risk of false-negative and false-positive results and the fact that FISH does not allow a diagnosis of certain structural abnormalities. If, on the other hand, FISH is considered a screening test, which means that in all abnormal (or indeterminate) cases, classical cytogenetic analysis would follow the abnormal screening test, the accuracy which is potentially higher than for other screening methods, for example in cases of trisomy 21, justifies FISH as a prenatal screening test in its own right.     

Evaluation of X,Y, 18, and 13/21 alpha satellite DNA probes for interphase cytogenetic analysis of uncultured amniocytes by fluorescence in situ hybridization

The major aneuploidies diagnosed prenatally involve the autosomes 13, 18, and 21, and sex chromosomes. Fluorescence in situ hybridization (FISH) allows rapid analysis of chromosome copy number in interphase cells. This prospective study evaluated the use of four commercially available centromeric DNA probes (DXZ1, DYZ1, D18Z1, and D13Z1/D21Z1) for direct analysis of uncultured amniocytes. One hundred and sixteen amniotic fluid samples were analysed by FISH and standard cytogenetics. This evaluation demonstrated that FISH with, X, Y, and 18 alpha satellite DNA probes could accurately and rapidly detect aneuploidies involving these chromosomes and could be used in any prenatal clinical laboratory. In contrast, the 13/21 alpha satellite DNA probe hybridizing both chromosomes 13 and 21 was unreliable for prenatal diagnosis in uncultured amniocytes.     

Prenatal detection of structural abnormalities of chromosome 18: associations with interphase fluorescence in situ hybridization (FISH) and maternal serum screening

We describe two cases of prenatally ascertained isochromosome 18. Case 1 included both an isochromosome 18p and an isochromosome 18q, while Case 2 involved only an isochromosome 18q. Both of these cases were associated with a positive maternal serum triple screen trisomy 18 risk (greater than 1 in 100 risk). In addition, fluorescence in situ hybridization (FISH) was performed on uncultured amniotic fluid interphase cells in both cases looking for aneuploidy for chromosomes 13, 18, 21, X and Y. The results of the interphase analyses support the common knowledge that careful interpretation of interphase FISH analysis is necessary and that results should be followed by full cytogenetic analysis. To our knowledge these are the first reported cases of structurally abnormal chromosomes 18 being associated with a positive maternal serum triple screen for trisomy 18. Copyright © 2002 John Wiley & Sons, Ltd.     

Monosomy X rescue explains discordant NIPT results and leads to uniparental isodisomy

Noninvasive prenatal testing accurately detects trisomy for chromosomes 13, 21, and 18, but has a significantly lower positive predictive value for monosomy X. Discordant monosomy X results are often assumed to be due to maternal mosaicism, usually without maternal follow-up. We describe a case of monosomy X-positive noninvasive prenatal testing that was discordant with the 46,XX results from amniocentesis and postnatal testing. This monosomy X pregnancy doubled the single X chromosome, leading to 45,X/46,XX mosaicism in the placenta and uniparental isodisomy X in the amniotic fluid. Thus, at least some discordant monosomy X results are due to true mosaicism in the pregnancy, which has important implications for clinical outcome and patient counseling.     

Rapid prenatal diagnosis of 14 cases of triploidy using fish with multiple probes

Fluorescence in situ hybridization (FISH) of chromosome-specific probes to interphase nuclei can rapidly identify aneuploidies in uncultured amniotic fluid cells. Using DNA probe sets specific for chromosomes 13, 18, 21, X, and Y, we have identified 14 fetuses where the hybridization pattern was consistent with a triploid chromosome constitution. In each case, the identification of fetal abnormalities by ultrasound examination initiated a request for rapid determination of ploidy status via prenatal FISH analysis of uncultured amniocytes. FISH produced a three-signal pattern for the three autosomes in combination with signals indicating an XXX or XXY sex chromosome complement. This hybridization pattern was interpreted to be consistent with triploidy. Results were reported to the physician within 2 days of amniocentesis and subsequently confirmed by cytogenetics. These cases demonstrate the utility of FISH for rapid prenatal identification of triploidy, particularly when fetal abnormalities are seen with ultrasonographic examination.     

Prenatal investigation of a 45,X/46,X,r(?) karyotype in amniocytes using fluorescence in situ hybridization with an X-centromeric probe

The karyotype of cultured amniotic fluid cells obtained on the indication of advanced maternal age was shown to be a mosaic 45,X/46,X,r(?). The small size and banding pattern made it difficult to determine whether the ring was derived from and X or a Y chromosome, or even from an autosome. By using an X-centromeric probe and fluorescence in situ hybridization (FISH), we demonstrated the ring to have an X centromere. Thus, a more complete genetic counselling was possible. This confirms the usefulness of FISH in identifying and characterizing this and other chromosome rearrangements in prenatal diagnosis.     

Estimating the risk of a fetal autosomal trisomy at mid-trimester using maternal serum alpha fetoprotein and age: A retrospective study of 142 pregnancies

Risks appropriate for mid-trimester prenatal screening for autosomal trisomies have been estimated from a combination of maternal age and maternal serum (MS) alpha-fetoprotein (AFP) levels at 16–20 weeks gestation. Published data on the frequency of Down's syndrome births relative to maternal age were modified to include the additional age-related frequency of trisomy 18 and trisomy 13 cases to provide an overall risk for an autosomal trisomy at midtrimester. MSAFP results from a retrospective study of 142 affected (114 trisomy 21, 19 trisomy 18, and 9 trisomy 13)and 113 000 unaffected pregnancies were converted to multiples of the appropriate gestational median (MOM). The AFP levels in the autosomal trisomy pregnancies were found to be significantly reduced at 0.72 MOM of the unaffected pregnancies. Risks (likelihood ratios) were derived from the overlapping log Gaussian distributions for affected and unaffected pregnancies and combined with maternal age risks to give the overall odds of an affected pregnancy. A mid-trimester cut-off risk of 1:280 gave an estimated 37 per cent detection rate for autosomal trisomies in the west of Scotland population for a follow-up (false-positive) rate of 6.6 per cent. These figures compare with a 30 per cent detection and 6.7 per cent false-positive rate if age 35 years and over is used as the sole criterion for selection of at-risk pregnancies.     

A first trimester trisomy 13/trisomy 18 risk algorithm combining fetal nuchal translucency thickness,maternal serum free β-hCG and PAPP-A

This study examines 45 cases of trisomy 13 and 59 cases of trisomy 18 and reports an algorithm to identify pregnancies with a fetus affected by trisomy 13 or 18 by a combination of maternal age fetal nuchal translucency (NT) thickness, and maternal serum free β-hCG and PAPP-A at 11–14 weeks of gestation. In this mixed trisomy group the median MoM NT was increased at 2.819, whilst the median MoMs for free β-hCG and PAPP-A were reduced at 0.375 and 0.201 respectively. We predict that with the use of the combined trisomy 13 and 18 algorithm and a risk cut-off of 1 in 150 will for a 0.3% false positive rate allow 95% of these chromosomal defects to be identified at 11–14 weeks. Such algorithms will enhance existing first trimester screening algorithms for trisomy 21. Copyright © 2002 John Wiley & Sons, Ltd.     

The results of aneuploidy screening in 276 couples undergoing assisted reproductive techniques

Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) using sequential in situ hybridization was applied for aneuploidy testing in 276 couples with 282 ART cycles. Patients with advanced maternal age (AMA, n = 147), recurrent implantation failure (RIF, n = 48), repeated early spontaneous abortion (RSA, n = 32) and abnormal gamete cell morphology (AGCM, n = 55) including macrocephal sperm forms or cytoplasmic granular oocytes were included. Embryo biopsy was performed on day 3 in a calcium–magnesium–free medium by using a noncontact diode laser system. After fixation and enzymatic treatment, fluorescent in situ hybridization (FISH) was carried out on 1147 blastomeres with specific probes for chromosomes 13, 16, 18, 21 and 22 for AMA group, 13, 18, 21, X and Y for AGCM group and 13, 16, 18, 21, 22, X and Y for RIF and RSA groups respectively. The overall chromosomal abnormality rate in analyzed embryos was 40.9%, with no significant difference between AMA, RIF and RSA groups (p > 0.05). However, AGCM group presented a higher rate of chromosomal aneuploidies (57.4%) than the other three groups (p < 0.01). A total of 84% biopsied embryos presented cleavage in 24 h and embryo transfer was realized in 278 cycles. In four cycles, no chromosomally normal embryo was found for embryo transfer. A total of 88 pregnancies (31.6%) were achieved, 19.3% resulted in abortion and 63 healthy births were obtained, with a total of 93 babies born. Aneuploidy testing in couples with poor prognosis undergoing ART cycles is a useful tool to increase the chance of ART success. Furthermore, abnormal gamete cell morphology should be considered one of the major indications for PGD in ART programs as high aneuploidy rates were observed in this group. Copyright © 2004 John Wiley & Sons, Ltd.     

Detection and isolation of fetal cells from maternal blood using the flourescence-activated cell sorter (FACS)

The presence of fetal cells in the maternal circulation during pregnancy has been suggested by repeated observations of small numbers of cells containing Y chromatin or a Y chromosome in the blood of pregnant women. With the fluorescence-activitated cell sorter (FACS), we have used antibodies to a paternal cell surface (HLA) antigen, not present in the mother, to select fetal cells from the lymphocyte fractions of a series of maternal blood samples, collected as early as 15 weeks of gestation. These sorted cells have been examined for a second paternal genetic marker, Y chromatin. Y chromatin-containing cells were found among the sorted cells from prenatal maternal blood specimens in 8 pregnancies subsequently producing male infants whose lymphocytes reacted with the same antibodies to paternal antigen used for sorting with the FACS. In each of 17 pregnancies resulting in male infants who failed to inherit the antigen detected by the antibodies used for cell sorting, Y chromatin-containing cells were not found prenatally. The use of two paternal genetic markers, a cell surface antigen and nuclear Y chromatin, to identify fetal cells in maternal blood permits us to conclude that these cells are present in the mother's circulation, as early as 15 weeks gestation. Further development of the techniques reported here could lead to widespread screening of maternal blood samples during pregnancy for detection of fetal genetic abnormalities.     

Prenatal diagnosis of partial monosomy 18p(18p11.2→pter) and trisomy 21q(21q22.3→qter) with alobar holoprosencephaly and premaxillary agenesis

A prenatal diagnosis of partial monosomy 18p(18p11.2→pter) and trisomy 21q(21q22.3→qter) in a fetus with alobar holoprosencephaly (HPE) and premaxillary agenesis (PMA) but without the classical Down syndrome phenotype is reported. A 27-year-old primigravida woman was referred for genetic counselling at 21 weeks' gestation due to sonographic findings of craniofacial abnormalities. Level II ultrasonograms manifested alobar HPE and median orofacial cleft. Cytogenetic analysis and fluorescence in situ hybridization (FISH) on cells obtained from amniocentesis revealed partial monosomy 18p and a cryptic duplication of 21q,46,XY,der(18)t(18;21)(p11.2;q22.3), resulting from a maternal t(18;21) reciprocal translocation. The breakpoints were ascertained by molecular genetic analysis. The pregnancy was terminated. Autopsy showed alobar HPE with PMA, pituitary dysplasia, clinodactyly and classical 18p deletion phenotype but without the presence of major typical phenotypic features of Down syndrome. The phenotype of this antenatally diagnosed case is compared with those observed in six previously reported cases with monosomy 18p due to 18;21 translocation. The present study is the first report of concomitant deletion of HPE critical region of chromosome 18p11.3 and cryptic duplication of a small segment of distal chromosome 21q22.3 outside Down syndrome critical region. The present study shows that cytogenetic analyses are important in detecting chromosomal aberrations in pregnancies with prenatally detected craniofacial abnormalities, and adjunctive molecular investigations are useful in elucidating the genetic pathogenesis of dysmorphism. Copyright © 2001 John Wiley & Sons, Ltd.     

Fetal erythroblasts from maternal blood identified with 2,3-bisphosphoglycerate (BPG) and in situ hybridization (ISH) using Y-specific probes

Different types of fetal nucleated cells can be found in maternal blood, providing the possibility of non-invasive prenatal diagnosis. For this purpose, we have studied fetal erythroblasts. We discovered that haemoglobin-containing cells treated with 2,3-bisphosphoglycerate (BPG) can be visualized by a peroxidase reaction, which at the same time visualizes an in situ hybridization (ISH) signal, specific for the X, Y or 21 chromosome. In order to prove that the BPG-positive cells were erythroid, an anti-glycophorin A (GPA) antiserum combined with a staphylococcal rosette technique was used. To enrich for erythroblasts, leukocytes were depleted from maternal blood by treatment with anti-CD45 monoclonal antibody and passage over an anti-mouse IgG-coated glass bead column. To evaluate the potential of the method for clinical use, we studied maternal blood samples from 18 women referred to us for prenatal diagnosis between 6 and 19 weeks of gestation. Erythroblasts were found in 13 out of 14 normal pregnancies. Erythroblasts with a Y-signal were found as early as 9 weeks of gestation, but at 6 weeks the Y-signal was seen in BPG-negative cells only. These cells showed an epithelioid morphology indicating that they were cytotrophoblasts. The BPG-ISH method provides a simple technique for identifying erythroblasts and simultaneously visualizing a desired probe.     

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.     

Current concepts of Down syndrome screening tests in assisted reproduction twin pregnancies: another double trouble

Assisted reproductive technologies (ART) have increased both the number of pregnancies in women beyond the age of 35 and the incidence of multiple pregnancies. Various methods of screening for Down syndrome (DS) were introduced in clinical practice during the last two decades, and specific problems were encountered when they were applied for twin pregnancies. The current review aims to explore the problematic issue of prenatal DS screening in ART twins. Overall, more women with twin pregnancies (mainly those who conceived via assisted reproduction) are found to be false positive for DS. This is because mid-trimester maternal serum screening is associated with a higher false-positive rate secondary to changes in the feto-placental endocrinologic metabolism, reflected mainly in high human chorionic gonadotrophin (hCG) levels in the ART pregnancies. First-trimester nuchal translucency (NT) measurement in twin pregnancies is not affected by the problems encountered in serum screening. This sonographic screening approach enables a fetus-specific identification of those fetuses at high risk of DS and is associated with a lower false-positive rate than mid-trimester serum screening. DS screening in ART twins presents several challenges in determining the most appropriate screening test modality. Whether there is any significant benefit of adding first-trimester biochemistry or nasal bone scanning in screening ART-conceived twins awaits further investigation. Copyright © 2005 John Wiley & Sons, Ltd.     

Multicenter clinical experience with non-invasive cell-free DNA screening for monosomy X and related X-chromosome variants

Objective

We aimed to investigate how the presence of fetal anomalies and different X chromosome variants influences Cell-free DNA (cfDNA) screening results for monosomy X.

Methods

From a multicenter retrospective survey on 673 pregnancies with prenatally suspected or confirmed Turner syndrome, we analyzed the subgroup for which prenatal cfDNA screening and karyotype results were available. A cfDNA screening result was defined as true positive (TP) when confirmatory testing showed 45,X or an X-chromosome variant.

Results

We had cfDNA results, karyotype, and phenotype data for 55 pregnancies. cfDNA results were high risk for monosomy X in 48/55, of which 23 were TP and 25 were false positive (FP). 32/48 high-risk cfDNA cases did not show fetal anomalies. Of these, 7 were TP. All were X-chromosome variants. All 16 fetuses with high-risk cfDNA result and ultrasound anomalies were TP. Of fetuses with abnormalities, those with 45,X more often had fetal hydrops/cystic hygroma, whereas those with “variant” karyotypes had different anomalies.

Conclusion

Both, 45,X or X-chromosome variants can be detected after a high-risk cfDNA result for monosomy X. When there are fetal anomalies, the result is more likely a TP. In the absence of fetal anomalies, it is most often an FP or X-chromosome variant.     

Demonstration of spontaneously dividing male fetal cells in maternal blood by negative magnetic cell sorting and fish

We investigated a case of massive feto-maternal bleeding by using negative magnetic cell sorting (MACS) and fluorescent in situ hybridization (FISH). A 37-year-old pregnant woman had an uncomplicated amniocentesis for advanced maternal age at 16 weeks' gestation. The fetal karyotype was 46, XY. At 19 weeks' gestation, she had a minor car accident and slight vaginal bleeding. A subsequent Kleihauer-Betke test showed a 140 ml feto-maternal haemorrhage. Serial sonographic examinations indicated a normal fetus and placenta. We performed FISH analysis on maternal peripheral blood at 25 weeks. Anti-CD45 and MACS were used to deplete maternal leucocytes, enriching the proportion of fetal nucleated erythrocytes present. The isolated cells were analysed by using dual-colour FISH with X and Y specific probes. Approximately 65 800 nucleated cells were obtained after MACS depletion. A total of 234 cells were analysed by FISH. The results revealed that 70 of the nucleated cells (30 per cent) were male with one X and one Y signal. Among these cells, six male metaphases were observed in spontaneously dividing cells.     

Abnormal maternal serum chorionic gonadotropin levels in pregnancies with fetal chromosome abnormalities

The alpha subunit of human chorionic gonadotropin (alpha-hCG), human chorionic gonadotropin (hCG) and alpha fetoprotein (AFP) were measured in the serum of 25 women with chromosomally abnormal fetuses between 18 and 25 weeks of gestation and in 74 normal pregnancies. AFP levels less than 0.5 multiples of the median (MoM) or greater than 2.5 MoM were observed in 24 per cent of the abnormal pregnancies and in 6.76 per cent of the normal pregnancies. A low concentration of hCG (< 0.25 MoM) was observed in 8 per cent of abnormals and in 2.7 per cent of normals while an elevated concentration of hCG (>2.5 MoM) was observed in 56 per cent of abnormals and in 1.35 per cent of normals. Elevated hCG-alpha (>2.5 MoM) was observed in 28 per cent of abnormals and in none of the normals. Determination of elevated levels of hCG-alpha or hCG resulted in detection of 68 per cent of pregnancies with chromosomally abnormal fetuses with a false positive rate of 1.35 per cent. Determination of both elevated and depressed gonadotropin levels resulted in detection of 76 per cent of abnormal pregnancies with a false positive rate of 4.05 per cent. Measurement of hCG and hCG-alpha in maternal serum samples can be used as a screening procedure for detecting pregnancies at risk for fetal chromosome abnormalities.     

A single chorionic gonadotropin assay for maternal serum screening for Down's syndrome

A simple enzyme immunoassay measuring human chorionic gonadotropin in undiluted maternal serum has been developed in order to be used as a prenatal screening test for Down' s syndrome. A retrospective study of maternal serum sampled during pregnancies associated with trisomy 21 shows that with a 5% amniocentesis rate determined on a single test, the detection rate of trisomy 21 would be around two-thirds of the affected pregnancies. A prospective study of 9040 pregnant women under 38 years has confirmed the usefulness of the assay.