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.

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.

These properties confirm that viable cells with intact nuclei can be obtained at very early gestation for genetic analysis.

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.