Non-invasive Prenatal Diagnosis Of Aneuploidies Research Articles

Establishment of the fetal-maternal interface: developmental events in human implantation and placentation.

Early pregnancy is a complex and well-orchestrated differentiation process that involves all the cellular elements of the fetal-maternal interface. Aberrant trophoblast-decidual interactions can lead to miscarriage and disorders that occur later in pregnancy, including preeclampsia, intrauterine fetal growth restriction, and preterm labor. A great deal of research on the regulation of implantation and placentation has been performed in a wide range of species. However, there is significant species variation regarding trophoblast differentiation as well as decidual-specific gene expression and regulation. Most of the relevant information has been obtained from studies using mouse models. A comprehensive understanding of the physiology and pathology of human implantation and placentation has only recently been obtained because of emerging advanced technologies. With the derivation of human trophoblast stem cells, 3D-organoid cultures, and single-cell analyses of differentiated cells, cell type-specific transcript profiles and functions were generated, and each exhibited a unique signature. Additionally, through integrative transcriptomic information, researchers can uncover the cellular dysfunction of embryonic and placental cells in peri-implantation embryos and the early pathological placenta. In fact, the clinical utility of fetal-maternal cellular trafficking has been applied for the noninvasive prenatal diagnosis of aneuploidies and the prediction of pregnancy complications. Furthermore, recent studies have proposed a viable path toward the development of therapeutic strategies targeting placenta-enriched molecules for placental dysfunction and diseases.

Cell-free nucleic acid based non-invasive prenatal diagnosis of fetal aneuploidies

Prenatal detection of fetal aneuploidies is one of the main goals of the prenatal diagnostic approach. As a benefit of the development of advanced ultrasound equipment and advances in molecular biology in the last decade, there is a significant progress in screening methods for fetal aneuploidies, although invasive methods remain the gold standard for aneuploidy detection. Non-invasive prenatal diagnosis has substantial medical impact as it targets the development of safer and more effective methods to avoid the risk of fetal loss associated with currently used invasive methods. Identification of fetal-specific messenger ribonucleic acids, digital polymerase chain reaction and next-generation sequencing give the real chance for non-invasive prenatal diagnosis of fetal aneuploidies. Although all these methods have both advantages and limitations, some of them are moving closer to clinical implementation. In this review the authors highlight the most recent advances in methods for non-invasive prenatal diagnosis of aneuploidies.

Klinefelter twins presenting with discordant aneuploidies, acardia, forked umbilical cord and with different gonadal sex despite monozygosity

A higher frequency of twin births in sibships of Klinefelter syndrome patients and also monozygotic or dizygotic twins, themselves being affected by Klinefelter syndrome have been noted repeatedly. To address this issue, we evaluated type and frequency of twinning among Klinefelter fetuses that we had received for autopsy within a 'Prenatal Diagnosis' program. We performed fetal autopsies, and genetic analyses on DNA extracted from stained histological slides. Among 41 prenatal diagnoses of a 47, XXY karyotype we observed four twin pairs. One was dizygotic with discordant Klinefelter and Down syndrome. Three twin pairs were monozygotic as concluded from monochorial placentation. In two monozygotic pairs one twin partner was an acardiac monster and in one of these the acardiac twin showed a female gonadal sex and missing Y-chromosomal SRY-sequences as confirmed by polymerase chain reaction. There is a high rate of twinning and twin reversed arterial perfusion sequence among our Klinefelter fetuses. Forked umbilical cords at the placental insertion site in one case allowed classification as conjoined twins in the sense of a 'funiculopagus'. Anaphase lagging or semidizygosity by second polar body twinning are proposed as explanations for the gonadal sex discordance and the excessive developmental disadvantage in the one acardiac. Problems may arise with regard to non-invasive prenatal diagnosis of aneuploidies in twin pregnancies.

Non-invasive prenatal diagnosis of aneuploidies: new technologies and clinical applications

Non-invasive prenatal diagnosis (NIPD) has substantial medical importance as it targets the development of safer and more effective methods to avoid the risk of fetal loss associated with currently used invasive methods. Several approaches have been demonstrated as being proof-of concept for NIPD of chromosomal aneuploidies. These approaches include cell-based and cell-free detection methods, involving the investigation of fetal cells in the maternal circulation, formaldehyde treatment of maternal plasma, DNA methylation studies using sodium bisulfite or restriction enzymes, protein-based studies, identification of fetal-specific mRNAs and digital polymerase chain reaction (PCR) approaches, and recently next-generation sequencing and methylated DNA immunoprecipitation real-time quantitative PCR-based approaches. Although all these NIPD methods have both advantages and limitations, some are moving closer to clinical implementation. Biotechnology companies dedicated to the development of NIPD tests such as the sequencing- or methylation-based approaches are finalizing large clinical trials. It is expected that these new technologies will facilitate safer, more sensitive and accurate prenatal diagnostic tests in the near future. In this review, we highlight the most recent advances in methods for NIPD of aneuploidies, and we discuss their future implications in clinical practice.

Sites of Differential DNA Methylation between Placenta and Peripheral Blood: Molecular Markers for Noninvasive Prenatal Diagnosis of Aneuploidies

The use of epigenetic differences between maternal whole blood and fetal (placental) DNA is one of the main areas of interest for the development of noninvasive prenatal diagnosis of aneuploidies. However, the lack of detailed chromosome-wide identification of differentially methylated sites has limited the application of this approach. In this study, we describe an analysis of chromosome-wide methylation status using methylation DNA immunoprecipitation coupled with high-resolution tiling oligonucleotide array analysis specific for chromosomes 21, 18, 13, X, and Y using female whole blood and placental DNA. We identified more than 2000 regions of differential methylation between female whole blood and placental DNA on each of the chromosomes tested. A subset of the differentially methylated regions identified was validated by real-time quantitative polymerase chain reaction. Additionally, correlation of these regions with CpG islands, genes, and promoter regions was investigated. Between 56 to 83% of the regions were located within nongenic regions whereas only 1 to 11% of the regions overlapped with CpG islands; of these, up to 65% were found in promoter regions. In summary, we identified a large number of previously unreported fetal epigenetic molecular markers that have the potential to be developed into targets for noninvasive prenatal diagnosis of trisomy 21 and other common aneuploidies. In addition, we demonstrated the effectiveness of the methylation DNA immunoprecipitation approach in the enrichment of hypermethylated fetal DNA.

Use of the Kleihauer test to detect fetal erythroblasts in the maternal circulation

Non-invasive prenatal diagnosis of aneuploidies on fetal nucleated erythrocytes present in the maternal circulation is hampered by the extremely small cell number of uncertain origin (70% of erythroblasts circulating during pregnancy have a maternal origin). Therefore, a method allowing selection of the fetal cells among the maternal cells is indispensable after the erythroblast enrichment step. In the present study, after an erythroblast enrichment step on a ficoll gradient followed by a positive immuno-magnetic selection with anti-CD71 or anti-GPA antibodies, a rapid, simple and direct chemical staining method adapted from the classical Kleihauer test was developed to select fetal cells. Precise differentiation between fetal and maternal erythroblasts is based on the constitutional difference between fetal and adult haemoglobin (Hb). The fetal cells appear with an intense pink cytoplasmic staining while maternal cells with adult haemoglobin are colourless. Preservation of the cytoplasmic integrity allows one to distinguish morphological characteristics and to visualize simultaneously nuclear hybridization signal by FISH (fluorescent in situ hybridization). This approach was tested by FISH analysis using dual-colour X- and Y-specific DNA probes on blood samples from 15 pregnant women, with the results being compared to cytogenetic or sonographic sex determination. For 12 pregnancies fetal sex was determined successfully (5 XY/7 XX), in two cases in situ hybridization failed, and in one case no fetal erythroblast was observed after the Kleihauer test. The selection method was applied to a pregnancy at risk for cystic fibrosis (CF). After a Kleihauer test, fetal erythroblasts were collected by microdissection, whole genomic DNA was amplified by primer extension pre-amplification (PEP) followed by a nested CF PCR. The fetal genotype was successfully characterized and confirmed by conventional prenatal diagnosis.

Use of the Kleihauer test to detect fetal erythroblasts in the maternal circulation

Non-invasive prenatal diagnosis of aneuploidies on fetal nucleated erythrocytes present in the maternal circulation is hampered by the extremely small cell number of uncertain origin (70% of erythroblasts circulating during pregnancy have a maternal origin). Therefore, a method allowing selection of the fetal cells among the maternal cells is indispensable after the erythroblast enrichment step. In the present study, after an erythroblast enrichment step on a ficoll gradient followed by a positive immuno-magnetic selection with anti-CD71 or anti-GPA antibodies, a rapid, simple and direct chemical staining method adapted from the classical Kleihauer test was developed to select fetal cells. Precise differentiation between fetal and maternal erythroblasts is based on the constitutional difference between fetal and adult haemoglobin (Hb). The fetal cells appear with an intense pink cytoplasmic staining while maternal cells with adult haemoglobin are colourless. Preservation of the cytoplasmic integrity allows one to distinguish morphological characteristics and to visualize simultaneously nuclear hybridization signal by FISH (fluorescent in situ hybridization). This approach was tested by FISH analysis using dual-colour X- and Y-specific DNA probes on blood samples from 15 pregnant women, with the results being compared to cytogenetic or sonographic sex determination. For 12 pregnancies fetal sex was determined successfully (5 XY/7 XX), in two cases in situ hybridization failed, and in one case no fetal erythroblast was observed after the Kleihauer test. The selection method was applied to a pregnancy at risk for cystic fibrosis (CF). After a Kleihauer test, fetal erythroblasts were collected by microdissection, whole genomic DNA was amplified by primer extension pre-amplification (PEP) followed by a nested CF PCR. The fetal genotype was successfully characterized and confirmed by conventional prenatal diagnosis.