We present the first study that investigates the effect of maternal body mass index (BMI) on the quantity of circulating fetal cells available to use in cell-based noninvasive prenatal test (cbNIPT). ...cbNIPT has been proposed as a superior alternative to noninvasive prenatal test from cell-free fetal DNA. Kølvraa et al. Prenat Diagn. 2016 Dec; 36(12): 1127–34 established that cbNIPT can be performed on as few as one fetal cell, and Vestergaard et al. Prenat Diagn. 2017 Nov; 37(11): 1120–4 demonstrated that these fetal trophoblast cells could be used successfully in cbNIPT to detect chromosomal and sub-chromosomal abnormalities. This study on 91 pregnant women with high-risk pregnancies suggests that cbNIPT should not be hampered by an increased BMI because every pregnancy, irrespective of the BMI, has rendered fetal cells for downstream genetic analysis. The mean number of fetal cells per sample was 12.6, with a range of 1–43 cells in one sample. ANOVA showed that increasing maternal BMI tends to decrease the number of fetal cells, but not significantly.
Introduction:
Circulating fetal cells isolated from maternal blood can be used for prenatal testing, representing a safe alternative to invasive testing. The present study investigated the potential ...of cell-based noninvasive prenatal testing (NIPT) for diagnosing monogenic disorders dependent on the mode of inheritance.
Methods:
Maternal blood samples were collected from women opting for prenatal diagnostics for specific monogenic disorders (
N
= 7). Fetal trophoblasts were enriched and stained using magnetic activated cell sorting and isolated by fluorescens activated single-cell sorting. Individual cells were subject to whole genome amplification, and cells of fetal origin were identified by DNA-profiling using short tandem repeat markers. The amplified fetal DNA was input for genetic testing for autosomal dominant-, autosomal recessive-, X-linked and repeat expansion disorders by direct variant analysis and haplotyping. The cell-based NIPT results were compared with those of invasive testing.
Results:
In two cases at risk of skeletal dysplasia, caused by variants in the
FGFR3
gene (autosomal dominant disorders), cell-based NIPT correctly stated an affected fetus, but allelic dropout of the normal alleles were observed in both cases. Cell-based NIPT gave an accurate result in two cases at risk of autosomal recessive disorders, where the parents carried either different diastrophic dysplasia causing variants in the
SLC26A2
gene or the same cystic fibrosis disease-causing variant in the
CFTR
gene. Cell-based NIPT accurately identified an affected male fetus in a pregnancy at risk of Duchenne muscular dystrophy (
DMD
gene, X-linked recessive disorders). In two cases at risk of the myotonic dystrophy type 1 (
DMPK
gene, repeat expansion disorder), cell-based NIPT correctly detected an affected and an unaffected fetus, respectively.
Discussion:
Circulating fetal cells can be used to detect both maternally- and paternally inherited monogenic disorders irrespective of the type of variant, however, the risk of allelic dropout must be considered. We conclude that the clinical interpretation of the cell-based NIPT result thus varies depending on the disorders’ mode of inheritance.
The existing risk of procedure-related miscarriage following invasive sampling for prenatal diagnosis is higher for twin pregnancies and some women are reluctant to test these typically difficultly ...obtained pregnancies invasively. Therefore, there is a need for noninvasive testing options that can test twin pregnancies at an early gestational age and ideally test the twins individually.
A pregnant woman opted for cell-based NIPT at GA 10 + 5. As cell-based NIPT is not established for use in twins, the test was provided in a research setting only, when an ultrasound scan showed that she carried dichorionic twins.
Fifty mL of peripheral blood was sampled, and circulating fetal cells were enriched and isolated. Individual cells were subject to whole-genome amplification and STR analysis. Three fetal cells were analyzed by chromosomal microarray (aCGH).
We identified 20 fetal cells all sharing the same genetic profile, which increased the likelihood of monozygotic twins. aCGH of three fetal cells showed the presence of two X chromosomes and a gain of chromosome Y. CVS from both placentae confirmed the sex chromosomal anomaly, 47,XXY and that both fetuses were affected.
NIPT options can provide valuable genetic information to twin pregnancies that help the couples in their decision-making on prenatal testing. Little has been published about the use of cell-based NIPT in twin pregnancies, but the method may offer the possibility to obtain individual cell-based NIPT results in dizygotic twins.
Purpose
Proof of concept of the use of cell-based non-invasive prenatal testing (cbNIPT) as an alternative to chorionic villus sampling (CVS) following preimplantation genetic testing for monogenic ...disorders (PGT-M).
Method
PGT-M was performed by combined testing of short tandem repeat (STR) markers and direct mutation detection, followed by transfer of an unaffected embryo. Patients who opted for follow-up of PGT-M by CVS had blood sampled, from which potential fetal extravillous throphoblast cells were isolated. The cell origin and mutational status were determined by combined testing of STR markers and direct mutation detection using the same setup as during PGT. The cbNIPT results with respect to the mutational status were compared to those of genetic testing of the CVS.
Results
Eight patients had blood collected between gestational weeks 10 and 13, from which 33 potential fetal cell samples were isolated. Twenty-seven out of 33 isolated cell samples were successfully tested (82%), of which 24 were of fetal origin (89%). This corresponds to a median of 2.5 successfully tested fetal cell samples per case (range 1–6). All fetal cell samples had a genetic profile identical to that of the transferred embryo confirming a pregnancy with an unaffected fetus, in accordance with the CVS results.
Conclusion
These findings show that although measures are needed to enhance the test success rate and the number of cells identified, cbNIPT is a promising alternative to CVS.
Trial registration number
N-20180001
In two cases, cell‐based noninvasive prenatal testing (cbNIPT) detected pathogenic copy number variations (CNVs) in the fetal genome. cbNIPT may potentially be an improved noninvasive alternative for ...the detection of smaller CNVs.
In two cases, cell‐based noninvasive prenatal testing (cbNIPT) detected pathogenic copy number variations (CNVs) in the fetal genome. cbNIPT may potentially be an improved noninvasive alternative for the detection of smaller CNVs
Background
In gestational trophoblastic disease, the prognosis is related to the genetic constitution. In some cases, taking a biopsy is contraindicated.
Methods
In a pregnant woman, ultrasound ...scanning suggested hydatidiform mole. To explore if the genetic constitution can be established without taking a biopsy (or terminating the pregnancy), cell‐free DNA and circulating gestational trophoblasts were isolated from maternal blood before evacuation of the uterus. The evacuated tissue showed the morphology of a complete hydatidiform mole. Without prior whole‐genome amplification, short tandem repeat analysis of 24 DNA markers was performed on the samples, and on DNA isolated from evacuated tissue, and from the blood of the patient and her partner.
Results
Identical genetic results were obtained in each of three circulating gestational trophoblasts and the evacuated tissue, showing that this conceptus had a diploid androgenetic nuclear genome. In contrast, analysis of cell‐free DNA was less informative and less specific due to the inherent presence of cell‐free DNA from the patient.
Conclusion
Our results show that it is possible to isolate and analyze circulating gestational trophoblasts originating in a pregnancy without maternal nuclear genome. For diagnosing gestational trophoblastic diseases, genotyping circulating gestational trophoblasts appears to be superior to analysis of cell‐free DNA.
The prognosis in gestational trophoblastic diseases is related to the genetic constitution. Performing biopsy may not be feasible due to the risk of miscarriage or bleeding. Analyzing circulating gestational trophoblasts is a feasible and an attractive alternative
Cell-based non-invasive prenatal testing (cbNIPT) based on circulating fetal extravillous trophoblasts (fEVTs) has shown to be possible in gestational week (GW) 10–13. Prenatal testing is relevant ...for a wider time period than GW 10–13, but it is unclear if fEVTs are present in sufficient numbers for cbNIPT at other time points during pregnancy. We present the first longitudinal study where the number of circulating fEVTs was determined from the mid first trimester to the mid second, specifically GW 6–8, 12–13, and 19–20. Blood samples from 13 women opting for assisted reproduction were collected at GW 6–8, 12–13, and 19–20. fEVTs were enriched using a magnetic-activated cell sorting system, stained with anti-cytokeratin antibodies, and fEVTs were identified with the use of a MetaSystem fluorescence microscope scanner. Blood samples drawn at GW 6–8 yielded an average of 5.5 fEVTs per 30 mL of blood. This increased significantly to an average of 11.8 in GW 12–13 (
P
value: 0.0070, Mann-Whitney test), and decreased significantly to an average of 5.3 in GW 19–20 (P value: 0.0063, Mann-Whitney test). In 9 out of 13 cases, the number of fEVTs peaked in GW 12–13 compared to GW 6–8 and GW 19–20. For the majority of cases, fEVTs can be identified at GW 6–8 and GW 19–20, but the highest number of fEVTs is observed at GW 12–13 indicating this is the optimal time point for cbNIPT.
Objectives
Cystic fibrosis (CF) is one of the most common severe autosomal recessive disorders. Prenatal or preconception CF screening is offered in some countries. A maternal blood sample in early ...pregnancy can provide circulating trophoblasts and offers a DNA source for genetic analysis of both the mother and the fetus. This study aimed to develop a cell‐based noninvasive prenatal test (NIPT) to screen for the 50 most common CF variants.
Methods
Blood samples were collected from 30 pregnancies undergoing invasive diagnostics and circulating trophoblasts were harvested in 27. Cystic fibrosis testing was conducted using two different methods: by fragment length analysis and by our newly developed NGS‐based CF analysis.
Results
In all 27 cases, cell‐based NIPT provided a result using both methods in agreement with the invasive test result.
Conclusion
This study shows that cell‐based NIPT for CF screening provides a reliable result without the need for partner‐ and proband samples.
Key points
What's already known about this topic?
Many pregnant women are positive towards prenatal screening for Cystic fibrosis (CF).
The only current pregnancy marker is echogenic bowel in the second trimester, but this test has low sensitivity and specificity.
What does this study add?
Prenatal screening for CF can be done using circulating trophoblasts isolated from maternal blood early in pregnancy
Circulating trophoblasts can provide screening for CF as a simple single‐visit setup without the need for a paternal sample.
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