Novel Algorithms for Improved Sensitivity in Non-Invasive Prenatal Testing.

Non-invasive prenatal testing (NIPT) of cell-free DNA in maternal plasma, which is a mixture of maternal DNA and a low percentage of fetal DNA, can detect fetal aneuploidies using massively parallel sequencing. Because of the low percentage of fetal DNA, methods with high sensitivity and precision are required. However, sequencing variation lowers sensitivity and hampers detection of trisomy samples. Therefore, we have developed three algorithms to improve sensitivity and specificity: the chi-squared-based variation reduction (χ2VR), the regression-based Z-score (RBZ) and the Match QC score. The χ2VR reduces variability in sequence read counts per chromosome between samples, the RBZ allows for more precise trisomy prediction, and the Match QC score shows if the control group used is representative for a specific sample. We compared the performance of χ2VR to that of existing variation reduction algorithms (peak and GC correction) and that of RBZ to trisomy prediction algorithms (standard Z-score, normalized chromosome value and median-absolute-deviation-based Z-score). χ2VR and the RBZ both reduce variability more than existing methods, and thereby increase the sensitivity of the NIPT analysis. We found the optimal combination of algorithms was to use both GC correction and χ2VR for pre-processing and to use RBZ as the trisomy prediction method.

Unexplained False Negative Results in Noninvasive Prenatal Testing: Two Cases Involving Trisomies 13 and 18.

Noninvasive prenatal testing (NIPT) validation studies show high sensitivity and specificity for detection of trisomies 13, 18, and 21. False negative cases have rarely been reported. We describe a false negative case of trisomy 13 and another of trisomy 18 in which NIPT was commercially marketed directly to the clinician. Both cases came to our attention because a fetal anatomy scan at 20 weeks of gestation revealed multiple anomalies. Karyotyping of cultured amniocytes showed nonmosaic trisomies 13 and 18, respectively. Cytogenetic investigation of cytotrophoblast cells from multiple placental biopsies showed a low proportion of nontrisomic cells in each case, but this was considered too small for explaining the false negative NIPT result. The discordant results also could not be explained by early gestational age, elevated maternal weight, a vanishing twin, or suboptimal storage or transport of samples. The root cause of the discrepancies could, therefore, not be identified. The couples involved experienced difficulties in accepting the unexpected and late-adverse outcome of their pregnancy. We recommend that all parties involved in caring for couples who choose NIPT should collaborate to clarify false negative results in order to unravel possible biological causes and to improve the process of patient care from initial counseling to communication of the result.

Detection of fetal chromosomal anomalies: does nuchal translucency measurement have added value in the era of non-invasive prenatal testing?

OBJECTIVES: The objective of this study is to determine what percentage of fetal chromosomal anomalies remains undetected when first trimester combined testing is replaced by non-invasive prenatal testing for trisomies 13, 18, and 21. We focused on the added clinical value of nuchal translucency (NT) measurement. METHODS: Data on fetal karyotype, ultrasound findings, and pregnancy outcome of all pregnancies with an NT measurement ≥3.5 mm were retrospectively collected from a cohort of 25,057 singleton pregnancies in which first trimester combined testing was performed. RESULTS: Two hundred twenty-five fetuses (0.9 %) had an NT ≥3.5 mm. In 24 of these pregnancies, a chromosomal anomaly other than trisomy 13, 18, or 21 was detected. Eleven resulted in fetal demise, and ten showed fetal ultrasound anomalies. In three fetuses with normal ultrasound findings, a chromosomal anomaly was detected, of which one was a triple X. CONCLUSIONS: In three out of 25,057 pregnancies (0.01%), non-invasive prenatal testing and fetal ultrasound would have missed a chromosomal anomaly that would have been identified by NT measurement. © 2015 John Wiley & Sons, Ltd.

From karyotyping to array-CGH in prenatal diagnosis.

Conventional karyotyping detects chromosomal anomalies in up to 35% of pregnancies with fetal ultrasound anomalies, depending on the number and type of these anomalies. Extensive experience gained in the past decades has shown that prenatal karyotyping is a robust technique which can detect the majority of germline chromosomal anomalies. For most of these anomalies the phenotype is known. In postnatal diagnosis of patients with congenital anomalies and intellectual disability, array-CGH/SNP array has become the first-tier investigation. The higher abnormality detection yield and its amenability to automation renders array-CGH also suitable for prenatal diagnosis. As both findings of unclear significance and unexpected findings may be detected, studies on the outcome of array-CGH in prenatal diagnosis were initially performed retrospectively. Recently, prospective application of array-CGH in pregnancies with ultrasound anomalies, and to a lesser extent in pregnancies referred for other reasons, was studied. Array-CGH showed an increased diagnostic yield compared to karyotyping, varying from 1-5%, depending on the reason for referral. Knowledge of the spectrum of array-CGH anomalies detected in the prenatal setting will increase rapidly in the years to come, thus facilitating pre- and posttest counseling. Meanwhile, new techniques like non-invasive prenatal diagnosis are emerging and will claim their place. In this review, we summarize the outcome of studies on prenatal array-CGH, the clinical relevance of differences in detection rate and range as compared to standard karyotyping, and reflect on the future integration of new molecular techniques in the workflow of prenatal diagnosis.

Does confined placental mosaicism account for adverse perinatal outcomes in IVF pregnancies?

BACKGROUND: IVF singletons have poorer perinatal outcomes than singletons from spontaneous conceptions. This may be due to the influence of ovarian stimulation on the chromosomal constitution of the embryos which could be translated into localized chromosomal anomalies in the placenta. The aim of this study was to compare the incidence of confined placental mosaicism (CPM) in IVF/ICSI pregnancies and spontaneous conceptions. METHODS: We conducted a multi-centre retrospective analysis of karyotype results obtained by chorionic villus sampling (CVS), performed due to advanced maternal age (>or=36 years at 18 weeks of gestation), in the Netherlands between 1995 and 2005. RESULTS: From a total of 322 246 pregnancies, 20 885 CVS results were analysed: 235 in the IVF/ICSI group and 20 650 in the control group. The mean age of women in both groups was 38.4 years (mean difference -0.08, 95% CI -0.35 to 0.18). Data relating to the fetal karyotype were missing in 143 cases in the control group. When taking into account missing data, the incidence of CPM was lower in the IVF-ICSI group than in the control group, 1.3% versus 2.2% (odds ratio 0.59, 95% CI 0.19-1.85), whereas the incidence of fetal chromosomal anomalies was increased 4.3% versus 2.4% (odds ratio 1.81, 95% CI 0.95-3.42). Neither differences were statistically significant. CONCLUSIONS: The incidence of CPM is not increased in IVF/ICSI pregnancies compared with spontaneous conceptions. CPM probably does not account for the adverse perinatal outcomes following IVF/ICSI.

[Chromosomal mosaicism in the placenta]. / Chromosomaal mozaïcisme in de placenta.

Chromosomal mosaicism is when two (or more) cell lines with different chromosomal complements are found within one individual. Mosaicism can be found in all tissues but may also be confined to a specific tissue. During pregnancy the aberrant cell line, often with a trisomy, may be confined to the placenta. In prenatal diagnosis, more specifically in chorionic villus sampling, this can interfere with the results and may necessitate follow-up investigation with amniotic fluid cell culture. Confined placental mosaicism can have no visible effect whatsoever on foetal development, but can also lead to serious foetal problems, depending on the chromosome involved. Uniparental disomy in the diploid cell line of the mosaicism may also have a negative effect on the foetal phenotype. In the case of the reverse situation, where the normal diploid cell line is confined to the placenta and the foetus has trisomy 13 or 18, it has been suggested that the diploid cell line might play a role in intrauterine survival; a recent study by the present authors could not confirm this view.

FISH analysis of fetal nucleated red blood cells from CVS washings in cases of aneuploidy.

OBJECTIVE: In chorionic villus sampling (CVS) the chromosome analysis is inconclusive in 1-2% of the samples. In many cases follow-up amniocentesis is performed. Fetal nucleated red blood cells (FNRBCs) are present in washings of chorionic villus samples. We wanted to establish whether analysis of these true fetal cells, using fluorescence in situ hybridization (FISH), could support the CVS karyotype. METHODS: We analysed washings of first trimester chorionic villi from non-mosaic 45,X (n=6) and full trisomy 18 cases (n=7). FNRBCs were identified by immunostaining and FISH was performed with chromosome-specific probes for X, Y and 18. RESULTS: In all 13 samples FNRBCs were present (between 4 and 30 cells per sample). Five cases of monosomy X showed one X signal in 89-100% of the nuclei; in the other case 50% of the nuclei displayed one signal. In the trisomy 18 cases three spots were seen in 60-100% of the cells. CONCLUSION: The CVS aneuploidy was confirmed in FNRBCs in all samples, so FISH on FNRBCs can be used in cases of non-mosaic numerical chromosomal abnormalities. This test can confirm a CVS diagnosis of monosomy X or trisomy 18 and thus minimize the risk for false-positive diagnoses. An additional invasive test may be prevented.

A placental diploid cell line is not essential for ongoing trisomy 13 or 18 pregnancies.

Viable trisomy 13 or 18 pregnancies may be supported by the presence of a diploid cell line, confined to the outer layer of the placenta (cytotrophoblast). To establish the presence of diploid cells we investigated five random biopsies from placentas of trisomy 13 (n = 8) and trisomy 18 cases (n = 6) of newborn infants and terminated pregnancies by means of fluorescence in situ hybridisation on interphase nuclei (n = 100). In 12 of these 14 placentas (including all five liveborns) 80% or more of the analysed nuclei showed three spots, suggestive of the presence of a full trisomy. In the other two placentas (both cases of trisomy 18) mosaicism was detected at most investigated sites. Thus, in contrast with earlier studies, these results show that a significant diploid cell line present in the placenta, confined to the trophoblast, is not a pre-requisite for intrauterine survival in the investigated cases.

Maternal uniparental disomy for chromosome 22 in a child with generalized mosaicism for trisomy 22.

We report on a case of generalized mosaicism for trisomy 22. At chorionic villus sampling (CVS) in the 37th week of pregnancy, a 47,XX,+22 karyotype was detected in all cells. The indication for CVS was severe unexplained symmetrical intrauterine growth retardation (IUGR) and a ventricular septal defect (VSD) was noted. In cultured cells from amniotic fluid taken simultaneously, only two out of ten clones were trisomic. At term, a growth-retarded girl with mild dysmorphic features was born. Lymphocytes showed a normal 46,XX[50] karyotype; both chromosomes 22 were maternal in origin (maternal uniparental disomy). Investigation of the placenta post-delivery using fluorescence in situ hybridization showed a low presence of trisomy 22 cells in only one out of 14 biopsies. In cultured fibroblasts of skin tissue, a mosaic 47,XX,+22[7]/46,XX[25] was observed. Clinical follow-up is given up to 19 months.

Cytogenetic characteristics of ectopic pregnancy.

During a 12 month period, tissue was collected from 30 surgically managed patients presenting with vital ectopic pregnancies. Chorionic villi of the removed tissue were successfully karyotyped by (semi-) direct chromosome technique in 22 cases. Only one abnormal chromosomal complement, a triploidy (69,XXX) was found. As controls, 10 cases of intrauterine pregnancies were investigated, all showing a normal karyotype. These findings do not suggest an important role for chromosome abnormalities in the aetiology of vital ectopic pregnancies.

The outcome of pregnancies with confined placental chromosome mosaicism in cytotrophoblast cells.

Cytogenetic findings and outcome of pregnancy are reported in 108 cases in which confined placental mosaicism (CPM, n = 101) or generalized mosaicism (n = 7) was found at or after first-trimester chorionic villus sampling. In all samples, a (semi)direct cytogenetic analysis of cytotrophoblast cells was performed. Two pregnancies with CPM ended in a spontaneous abortion before 28 weeks (1.9 per cent). In 15 cases the pregnancy was terminated: eight cases were shown to be examples of CPM; seven cases can be considered as examples of generalized mosaicism. A normal cytogenetic result was obtained after follow-up amniocentesis in 88 of the remaining 91 cases. In three cases, no amniocentesis was performed but confirmation of a normal karyotype was obtained in other cells. One of the 91 pregnancies was nevertheless terminated for psychosocial reasons. One child died perinatally and another on the seventh day after birth. The birth weight is known for 89 children; the curve shows a normal distribution. In 11 of these children (12.3 per cent), the birth weight was found to be below the tenth centile. The outcome in a subgroup of eight pregnancies with CPM and involvement of chromosome 13, 16, or 22, however, revealed two fetal losses and four children with a birth weight below the tenth centile (75 per cent).

The preserving of chorionic villi before establishing long-term cell cultures for cytogenetic analysis.

The possibility of preserving intact chorionic villi in culture medium for up to 7 days before establishing long-term cultures for prenatal chromosome analysis is demonstrated. The preserving itself had no negative effect on the growth capacity of the mesenchymal cells.

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.

Cytogenetic findings in 1250 chorionic villus samples obtained in the first trimester with clinical follow-up of the first 1000 pregnancies.

First-trimester chorionic villus sampling (CVS) was performed in a series of 1250 pregnancies. The direct method of karyotyping was successful in 1205 (96.4%). Abnormal laboratory findings resulted in 60 terminations of pregnancy (4.8%). In addition, six unexpected balanced chromosome rearrangements were detected. False-positive cytogenetic findings occurred in 2.3%, comprising 22 with mosaicism confined to the trophoblast, and a further six non-mosaic false-positive discrepancies were detected, four after termination of pregnancy. The outcome of the first 1000 pregnancies is known in all but one. There were no false-negative findings. Of 947 pregnancies meant to be continued, 34 (3.6%) ended in pregnancy loss before 28 weeks gestation. However, obstetricians with an experience of over 150 procedures had a pregnancy loss of 1.3%.