Whole-Chromosome Karyotyping of Fetal Nucleated Red Blood Cells Using the Ion Proton Sequencing Platform.

The current gold standard for the definitive diagnosis of fetal aneuploidy uses either chorionic villus sampling (CVS) or amniocentesis, both of which are which are invasive procedures carrying a procedure-related risk of miscarriage of up to 0.1-0.2%. Non-invasive prenatal diagnosis using fetal nucleated red blood cells (FNRBCs) isolated from maternal peripheral venous blood would remove this risk of miscarriage since these cells can be isolated from the mother's blood. We aimed to detect whole-chromosome aneuploidies from single nucleated fetal red blood cells using whole-genome amplification followed by massively parallel sequencing performed on a semiconductor sequencing platform. Twenty-six single cells were picked from the placental villi of twelve patients thought to have a normal fetal genotype and who were undergoing elective first-trimester surgical termination of pregnancy. Following karyotyping, it was subsequently found that two of these cases were also abnormal (one trisomy 15 and one mosaic genotype). One single cell from chorionic villus samples for two patients carrying a fetus with trisomy 21 and two single cells from women carrying fetuses with T18 were also picked. Pooled libraries were sequenced on the Ion Proton and data were analysed using Ion Reporter software. We correctly classified fetal genotype in all 24 normal cells, as well as the 2 T21 cells, the 2 T18 cells, and the two T15 cells. The two cells picked from the fetus with a mosaic result by CVS were classified as unaffected, suggesting that this was a case of confined placental mosaicism. Fetal sex was correctly assigned in all cases. We demonstrated that semiconductor sequencing using commercially available software for data analysis can be achieved for the non-invasive prenatal diagnosis of whole-chromosome aneuploidy with 100% accuracy.

Biology of human primitive erythroblasts for application in noninvasive prenatal diagnosis.

OBJECTIVE: Human primitive erythroblasts produced during early embryogenesis have been found in maternal circulation at early gestation and are considered good target cells for noninvasive prenatal diagnosis. We aimed to gain a better understanding of the biology of primitive erythroblasts and maximize their potential utility for noninvasive prenatal diagnosis. METHODS: Cells were obtained from first trimester human placental tissues. Biological properties including surface antigen composition, differentiation, proliferation, enucleation, and degeneration were studied as gestation progressed. A microdroplet culture system was developed to observe the behavior of these cells in vitro. RESULTS: Histology showed that primitive erythroblasts undergo maturation from polychromatic to orthochromatic erythroblasts and can differentiate spontaneously in vitro. Cell surface markers and nuclear gene expression suggest that the cells do not possess stemness properties, despite being primitive in nature. They have limited proliferative activity and highly deacetylated chromatin, but a microdroplet culture system can prolong their viability under normoxic conditions. No apoptosis was seen by 11 weeks' gestation, and there was no enucleation in vitro. CONCLUSION: These properties confirm that viable cells with intact nuclei can be obtained at very early gestation for genetic analysis.

Detection of aneuploidy from single fetal nucleated red blood cells using whole genome sequencing.

OBJECTIVE: The objective of the study was to detect aneuploidy in single fetal nucleated red blood cells (FNRBCs) from placental villi using whole genome amplification (WGA) and next generation sequencing. METHODS: Three single FNRBCs per sample were manually picked from villi collected from ten women undergoing elective first-trimester termination of pregnancy, and one or two cells were picked from each of four aneuploid chorionic villus samples. Following WGA and addition of adaptor and index sequences, samples were sequenced on the Illumina MiSeq. Leading and trailing 15 bases were trimmed, and reads were aligned to the human reference genome. Z-scores were calculated to determine deviation of the mean of the test from reference samples, with a score of 3 used as the threshold for classification of a particular chromosome as trisomic. RESULTS: We successfully made correct diagnoses from ten single cells isolated from villi from two cases of trisomy 21 (one case from a single cell and one from two cells), two cases of trisomy 18 (two cells each), and a case of trisomy 15 (three cells). CONCLUSION: With their faithful representation of fetal genome, diagnosis using single FNRBCs provides a definitive result compared with non-invasive prenatal testing using cell-free fetal DNA, and is a safer alternative to invasive amniocentesis.

Novel approaches to manipulating foetal cells in the maternal circulation for non-invasive prenatal diagnosis of the unborn child.

Due to the risks to the foetus with invasive prenatal diagnosis, non-invasive prenatal diagnosis (NIPD) is gaining tremendous interest but no reliable method that can be widely used has been developed to date. Manipulation of foetal cells and foetal cell-free genetic material in the maternal blood are two promising approaches being researched. The manipulation of foetal cells in the maternal circulation is more popular as it can provide complete genetic information of the foetus particularly the diagnosis of aneuploidies. However, the foetal cell numbers in the maternal circulation are small and their enrichment and ex vivo culture remain two major challenges for NIPD. Primitive foetal erythroblasts (pFEs) have been considered as a good potential candidate for early first trimester NIPD but their nature, properties and manipulation to provide adequate cell numbers remain a challenging task and several approaches need to be meticulously evaluated. In this review we describe the current status of NIPD and suggest some novel approaches in manipulating pFEs for future clinical application of NIPD. These novel approaches include (1) understanding the pFE enucleation process, (2) enriching pFE numbers by individual pick-up of pFEs from maternal blood using micromanipulation and microdroplet culture, (3) expansion of pFEs using mitogens and (4) decondensation of the pFE nucleus with histone deacetylase (HDAC) inhibitors followed by reprogramming using gene delivery protocols with/without small reprogramming molecules to improve reprogrammed pFE proliferation rates for successful NIPD.