Frequency of fetal karyotype abnormalities in women undergoing invasive testing in the absence of ultrasound and other high-risk indications.

OBJECTIVES: No previous studies have reported the frequencies of individual chromosomal anomalies in normal-appearing fetuses stratified by maternal age (MA) and gestational age (GA). We therefore sought to (1) characterize the frequency of all fetal karyotype anomalies in sonographically normal appearing fetuses without pretest risk factors, and (2) assess MA and GA impact on the proportion of anomalies targeted by screening and consequent impact on residual risk following a negative result. METHODS: Fetal karyotypes from samples without prior risk assessment or ultrasound anomalies were analyzed. We calculated, per single-year MA and in two GA intervals, the predicted frequency of each cytogenetic defect. RESULTS: A total of 129 263 karyotypes were analyzed. The risk for significant, cytogenetically visible chromosomal anomalies, at 15 to 20 weeks GA, varies between 1/301 at MA of 18 years, and 1/9 at MA of 48 years. The proportion of clinically significant anomalies not addressed by current screening methods is 47% at MA of 18 years and 5% at MA of 48 years. CONCLUSIONS: By determining frequencies for individual karyotype anomalies stratified by MA and GA, in the setting of normal-appearing fetuses, a more personalized risk assessment, including the residual risk after a normal fetal aneuploidy screening result, can be provided. © 2016 John Wiley & Sons, Ltd.

QF-PCR as a substitute for karyotyping of cytotrophoblast for the analysis of chorionic villi: advantages and limitations from a cytogenetic retrospective audit of 44,727 first-trimester prenatal diagnoses.

OBJECTIVES: Karyotyping on chorionic villous samples (CVS) includes the analysis of both cytotrophoblast (STC) and mesenchyme (LTC). This approach requires complex laboratory organization and trained technicians. The introduction of quantitative fluorescent polymerase chain reaction (QF-PCR) instead of conventional karyotyping in low-risk pregnancies opened its application in CVS analysis. Discordant QF-PCR and CVS cytogenetic results were reported, and strategies for CVS analysis were introduced to minimize this risk. The possibility to substitute the STC with QF-PCR was reported. The aim of this study is to evaluate benefits and limitations of the approach QF-PCR + LTC compared with the traditional method STC + LTC and to quantify the associated risks of false results. METHOD: This study is based on a retrospective cytogenetic audit of CVS results (n = 44 727) generated by the STC + LTC analytic approach. False-negative risks related to true fetal mosaicism type IV, imprinting syndromes and maternal contamination in LTC were calculated. RESULTS: Compared with STC + LTC, QF-PCR + LTC approach is associated with a cumulative false-negative risk of ~1/3100-1/4400. Costs and reporting time of STC in a high-throughput cytogenetic lab are similar to a CE-IVD marked QF-PCR analysis. CONCLUSIONS: These results should be clearly highlighted in the pre-test counseling and extensively discussed with the couple prior to testing for informed consent.

Chromosome abnormalities investigated by non-invasive prenatal testing account for approximately 50% of fetal unbalances associated with relevant clinical phenotypes.

During the past 20 years non-invasive screening tests have been increasingly utilized in prenatal diagnosis (PD) practice. Considerable effort has been exerted by multicenter consortia to evaluate the reliability of non-invasive screening tests in detecting those women with an increased risk of having a pregnancy affected by trisomies 21, 18, and 13, monosomy X, and triploidies. To what extent this group of abnormal karyotypes accounts for the total number of phenotypically relevant fetal chromosome abnormalities has, however, never been investigated. The present report is an attempt aimed to quantify this proportion. A retrospective analysis of a homogeneous survey of 115,128 consecutive invasive prenatal tests was undertaken. All cases were classified in accordance with the indication given for the invasive testing. Cytogenetic results regarding 96,416 karyotype analyses performed because of advanced maternal age (>or=35 years) or gestational anxiety (<35 years) were considered since these are the patients who usually undergo non-invasive screening tests. We calculated the number of cases (T21, T18, T13, 45,X, and triploidy) that would have been detected by prenatal screening on the basis of the published detection rate of the combined-2 test or the quadruple test. Our findings indicate that the chromosomal abnormalities investigated by screening tests represent <50% of the fetal chromosomal abnormalities associated with an abnormal outcome ranging from intermediate-to-severe in women <35 years (45.8% and 39.6% in the first and second trimesters, respectively), and sensitivity >50% in women >or=35 years (65.1% and 61.8%, respectively). To conclude, approximately 50% of the phenotypically relevant abnormal karyotypes cannot be detected by non-invasive prenatal screening tests.

Prenatal detection by subtelomeric FISH and MLPA of unbalanced meiotic recombinants resulting from parental pericentric inversions.

OBJECTIVE: Pericentric inversion carriers are predisposed to produce unbalanced gametes that result in conceptuses having either a partial trisomy for one distal segment and a partial monosomy for the other or vice versa. Larger inversions result in smaller unbalanced distal segments and a higher likelihood of a viable fetus. In these cases the structure of the recombinant chromosome is similar to the original balanced inverted or normal ones despite the (unbalanced) genetic content. Such cases may not be detected prenatally by conventional cytogenetic analysis. METHODS: In all prenatal samples from the pericentric inversion carriers we applied subtelomeric FISH probes specific for the chromosome involved in order to detect parental meiotic recombinants resulting from a single cross-over event. Confirmatory MLPA was also applied in unbalanced fetuses. RESULTS: The occurrence of a duplication deficiency unbalance from pericentric inversion carriers was successfully detected in all three fetuses by FISH. MLPA assays applied in two cases confirmed these results. CONCLUSIONS: The application of commercial FISH subtelomeric probes is a reliable method that could be routinely applied for the detection of single cross-over meiotic recombinants. MLPA is a sound alternative technique.

Confirmation of mosaicism and uniparental disomy in amniocytes, after detection of mosaic chromosome abnormalities in chorionic villi.

Chromosome mosaicism is detected in about 1-2% of chorionic villi samples (CVS), and may be due to a postzygotic nondisjunction event generating a trisomic cell line in an initially normal conceptus (mitotic origin) or the postzygotic loss of one chromosome in an initially trisomic conceptus (meiotic origin and trisomy rescue). Depending on the distribution of the abnormal cell line, the mosaic can be confined to the placenta (CPM) or generalised to the fetus (TFM, true fetal mosaicism). Trisomy rescue could theoretically be associated with a 33.3% probability of uniparental disomy (UPD) in the fetus. The aim of this study was to determine the risk of fetal involvement in a cohort of numerical and structural chromosome mosaics revealed in chorionic villi by means of combined direct and long-term culture analyses; we also determined the incidence of UPD associated with mosaic aneuploidies and supernumerary markers involving imprinted chromosomes. A total of 273 of a consecutive series of 15,109 CVS evaluated during a period of 5 years showed a mosaic condition in direct preparations and/or long-term cultures; confirmatory amniocentesis was performed in 203 cases. The abnormal cell line was extended to the fetus in 12.8% cases in terms of structural and numerical abnormalities involving autosomes and sex chromosomes; the risk of TFM varied and depended on the placental tissue distribution of the abnormal cell line. One of the 51 cases in which the mosaic involved an imprinted chromosome showed UPD, thus indicating a risk of 1.96%.