Search

ObjectivesWe 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 MethodsStudy 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.ResultsStudy 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.ConclusionCirculating trophoblasts in the maternal circulation provide the potential of screening for aneuploidies and pathogenic CNVs covering the entire fetal genome.

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.

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

Background: 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.Case presentation: 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.Materials and Methods: 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).Results: 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.Conclusion: 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.

Background: Cell-free NIPT and cell-based NIPT are risk-free testing options using maternal blood samples to screen for fetal aneuploidies, but the methods differ. For cell-free NIPT, the fetal fraction of cell-free DNA in plasma is analyzed with a high background of maternal DNA. In contrast, for cell-based NIPT, a limited number of the rare, intact fetal cells are isolated for the genetic analysis. This case demonstrates the differences regarding testing for fetal sex-chromosomes anomalies (SCAs) between these two tests.Materials and Methods: A pregnant woman with mosaicism for Turner syndrome opted for NIPT in first trimester. For the cell-free NIPT analysis, DNA extraction, genome-wide massive parallel sequencing, and data analysis were carried out as described by the kit manufacturer (Illumina©, San Diego, CA, USA). For cell-based NIPT, the first sample gave no result, but the woman consented to repeat cell-based NIPT. After whole genome amplification and STR analysis, fetal DNA from three individual fetal cells was subjected to chromosomal microarray (aCGH, Agilent oligoarray, 180 kb).Results: Fetal fraction was 7%, and cell-free NIPT showed 2 copies of chromosomes 13, 18, and 21 and a decreased proportion of chromosome X, suggestive of fetal Turner syndrome. In contrast, the cell-based NIPT result showed no aneuploidy and two X-chromosomes in the fetus.Conclusion: cell-based NIPT may provide a non-invasive testing option to screen for SCAs in women with mosaicism for monosomy-X in blood, where cell-free NIPT cannot discriminate whether the X-loss is maternal or fetal.

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

OBJECTIVE: We aimed to develop cell-based NIPT for cystic fibrosis (CF) and test a pregnancy at risk of two common pathogenic variants.METHOD: A pregnant woman carrying monozygotic twins opted for prenatal testing as she and her partner were heterozygote carriers of F508del (c.1521:1523del). The partner was also positive for the CFTR-related variant R117H (c.350G>A). Fetal trophoblasts from maternal blood were enriched and isolated using antibodies and a capillary-based cell-picking instrument. Multiplex PCR-based fragment length analysis was performed on the extracted fetal DNA for STR-genotyping, fetal gender and F508del variant status. The R117H variant status was tested using SNaPshot analysis.RESULTS: The fetal origin of the isolated cells was verified by detection of two paternally inherited STR alleles and an Y chromosome marker, while no maternal DNA contamination was detected. The direct variant analysis detected F508del heterozygosity and the SNaPshot analysis for R117H detected only the normal allele. Thus, the results showed that the fetuses were healthy carriers of F508del, concordant with the findings of conventional prenatal testing.CONCLUSION: Cell-based NIPT could accurately state the fetal variant status and distinguish fetal trophoblasts from maternal cells. In the future, cell-based NIPT may provide an accurate less invasive alternative to chorionic villous sampling.

Objective Non-invasive prenatal testing (NIPT) based on fetal cells in maternal blood has the advantage over NIPT based on circulating cell-free fetal DNA in that there is no contamination with maternal DNA. This will most likely result in better detection of chromosomal aberrations including subchromosomal defects. The objective of this study was to test whether fetal cells enriched from maternal blood can be used for cell-based NIPT. Methods We present a method for enriching fetal cells from maternal blood, subsequent amplification of the fetal genome and detection of chromosomal and subchromosomal variations in the genome. Results An average of 12.8 fetal cells from 30 mL of maternal blood were recovered using our method. Subsequently, whole genome amplification on fetal cells resulted in amplified fetal DNA in amounts and quality high enough to generate array comparative genomic hybridization as well as next-generation sequencing profiles. From one to two fetal cells, we were able to demonstrate copy number differences of whole chromosomes (21, X−, and Y) as well as subchromosomal aberrations (ring X). Conclusion Intact fetal cells can be isolated from every maternal blood sample. Amplified DNA from isolated fetal cells enabled genetic analysis by array comparative genomic hybridization and next-generation sequencing. © 2016 John Wiley & Sons, Ltd.

Objective Trophoblastic fetal cells harvested from maternal blood have the capacity to be used for copy number analyses in a cell-based non-invasive prenatal test (cbNIPT). Potentially, this will result in increased resolution for detection of subchromosomal aberrations due to high quality DNA not intermixed with maternal DNA. We present 5 selected clinical cases from first trimester pregnancies where cbNIPT was used to demonstrate a wide range of clinically relevant aberrations. Method Blood samples were collected from high risk pregnancies in gestational week 12 + 1 to 12 + 5. Fetal trophoblast cells were enriched and stained using fetal cell specific antibodies. The enriched cell fraction was scanned, and fetal cells were picked using a capillary-based cell picking instrument. Subsequently, whole genome amplification (WGA) was performed on fetal cells, and the DNA was analyzed blindly by array comparative genomic hybridization (aCGH). Results We present 5 cases where non-invasive cell-based prenatal test results are compared with aCGH results on chorionic villus samples (CVS), demonstrating aneuploidies including mosaicism, unbalanced translocations, subchromosomal deletions, or duplications. Conclusion Aneuploidy and subchromosomal aberrations can be detected using fetal cells harvested from maternal blood. The method has the future potential of being offered as a cell-based NIPT with large high genomic resolution.

Objective Fetal cells from the maternal circulation (FCMBs) have the potential to replace cells from amniotic fluid or chorionic villi in a diagnosis of common chromosomal aneuploidies. Good markers for enrichment and identification are lacking. Method Blood samples from 78 normal pregnancies were used for testing the marker-set CD105 and CD141 for fetal cell enrichment. Fetal cell candidates were subsequently stained by a cocktail of cytokeratin antibodies, and the gender of the fetal cells was explored by fluorescence in situ hybridization (FISH) of the X and Y chromosomes. Results Fetal cell candidates could be detected in 91% of the samples, and in 85% of the samples, it was possible to obtain X and Y chromosomal FISH results for gender determination. The concordance between gender determined by FISH on fetal cells in maternal blood and gender found at birth reached 100% if three or more fetal cells with FISH signals could be found in a sample. Conclusion The marker set identifies fetal cells with specificity high enough to make cell-based noninvasive prenatal diagnosis realistic. © 2014 John Wiley & Sons, Ltd. Funding sources: The experiments described in this article were funded by the private company FCMB ApS. The ultimate goal of FCMB ApS was to develop a new method for noninvasive prenatal diagnostics based on identification of fetal cells in the maternal circulation. Conflicts of interest: The experiments described in this article were performed in the private company FCMB ApS in which the authors MB, RS, LH, KM, RHL, and BC were employed. SK and BC are founders of this company. The authors MB, RS, LH, BC, and SK have filed patent applications on the isolation and identification of fetal cells in maternal blood for noninvasive prenatal diagnostics.

Objective If noninvasive prenatal testing using next generation sequencing is to be effective for pregnant women, a cell-free fetal DNA (cffDNA) fraction above 4% is essential unless the depth of sequencing is increased. This study's objective is to determine whether physical activity has an effect on the proportion of cell-free DNA (cfDNA) arising from the fetus (fetal fraction). Methods Nine pregnant women carrying male fetuses at gestational age 12+0 weeks to 14+6 weeks were included. Plasma from nine pregnant women was drawn prior to, immediately after, and 30 min after 30 min of cycling with a pulse-rate of 150 beats per minute. The concentrations of cffDNA (DYS14) and cfDNA (RASSF1A) were assessed using quantitative real-time polymerase chain reaction. Results The fetal fraction decreased significantly in all participants after physical activity (p

Objective Different fetal cell types have been found in the maternal blood during pregnancy in the past, but fetal cells are scarce, and the proportions of the different cell types are unclear. The objective of the present study was to identify specific fetal cell markers from fetal cells found in the maternal blood circulation at the end of the first trimester. Method Twenty-three fetal cells were isolated from maternal blood by removing the red blood cells by lysis or combining this with removal of large proportions of maternal white blood cells by magnetic-activated cell sorting. Fetal cells identified by XY fluorescence in situ hybridization and confirmed by reverse-color fluorescence in situ hybridization were shot off microscope slides by laser capture microdissection. The expression pattern of a subset of expressed genes was compared between fetal cells and maternal blood cells using stem cell microarray analysis. Results Twenty-eight genes were identified as fetal cell marker candidates. Conclusion Of the 28 fetal marker candidate genes, five coded for proteins, which are located on the outer surface of the cell membrane and not expressed in blood. The protein product of these five genes, MMP14, MCAM, KCNQ4, CLDN6, and F3, may be used as markers for fetal cell enrichment. © 2012 John Wiley & Sons, Ltd.