Nucleic Acids In Maternal Plasma Research Articles

Circulating Nucleic Acids in Maternal Plasma and Serum in Pregnancy Complications: Are They Really Useful in Clinical Practice? A Systematic Review.

A systematic review was carried out to summarize the available evidence to assess whether circulating nucleic acids in maternal plasma and serum (CNAPS) have the potential to serve as extra and independent markers for the prediction and/or progression monitoring of the most common and severe complications of pregnancy, including preeclampsia, intrauterine growth restriction, preterm delivery, morbidly adherent placenta, gestational diabetes, antiphospholipid syndrome, threatened abortion, intrahepatic cholestasis of pregnancy, and hyperemesis gravidarum. A comprehensive literature search of the MEDLINE (PubMed), EMBASE, and ISI Web of Knowledge databases was conducted to identify relevant studies that included amounts of CNAPS in the abovementioned pregnancy complications. Eighty-three studies met the eligibility criteria. The vast majority of studies were conducted on the quantity of total circulating cell free DNA (cfDNA) and cell free fetal DNA (cffDNA), and some were conducted on messenger RNA (mRNA) species. A few studies have instead evaluated the cell free DNA fetal fraction (cfDNAff), but only in a limited number of pregnancy complications. Despite the growing interest and the abundance of the papers available, little information is available for other new CNAPS, including microRNA (miRNA), long noncoding RNA (lncRNA), mitochondrial DNA (mtDNA), and circular RNA. Due to the heterogeneity of the populations enrolled, the scarcity of the studies that adjusted the CNAPS values for possible confounding factors, and the difficulty in interpreting the published data, no conclusion regarding the statistical robustness and clinical relevance of the data can be made at present. If assayed at the third trimester, the CNAPS have, however, shown better performance, and could be used in populations already at risk of developing complications as suggested by the presence of other clinical features. Other CNAPS, including miRNA, are under investigation, especially for the screening of gestational diabetes mellitus, but no data about their clinical utility are available. Circulating DNA (cfDNA, cffDNA, and cfDNAff) and mRNA have not been properly evaluated yet, especially in patients asymptomatic early in pregnancy but who developed complications later, perhaps because of the high cost of these techniques and the availability of other predictors of pregnancy complications (biochemical, biophysical, and ultrasound markers). Therefore, from the analysis of the data, the positive predictive value is not available. As regards the new CNAPS, including miRNA, there are still no sufficient data to understand if they can be promising markers for pregnancy complications monitoring and screening, since CNAPS are statistically weak and expensive. It is reasonable to currently conclude that the use of the CNAPS in clinical practice is not recommended.

Fetal nucleic acids in maternal plasma: from biology to clinical translation.

Exposure to environmental contaminants during the critical window of pregnancy results in deregulation of highly coordinated genetic and epigenetic mechanisms involved in prenatal growth. Such disturbances significantly alter the fetal programming, and lead to various developmental disorders immediately, over the lifetime, or transgenerationally. During the process of placental development, fetal nucleic acids enter maternal plasma as a result of necrotic, apoptotic, and inflammatory mechanisms. These nucleic acids reflect normal or abnormal ongoing cellular changes during prenatal fetal development. Here, we critically review the utility of maternally circulating cell free fetal nucleic acids towards developing reliable biomarkers for widespread screening of environmentally-associated fetal abnormalities. We further discuss the most recent developments in the fetal nucleic acid analysis, quantification methodologies, challenges involved in their accurate detection and their potential applications in fetomaternal medicine.

Prenatal Diagnosis Innovation: Genome Sequencing of Maternal Plasma.

Noninvasive prenatal testing (NIPT) is accomplished by analysis of circulating cell-free fetal nucleic acids in maternal plasma. The advent of massively parallel sequencing (MPS) has enabled NIPT of chromosomal aneuploidies with unprecedented robustness, and these tests are now widely available for clinical use. Moreover, MPS-based NIPT of subchromosomal deletions/duplications and single-gene disorders has also been achieved, and the number of applications is growing. In addition to specific fetal genetic disorders, the whole fetal genome, transcriptome, and methylome have been revealed by deep sequencing of maternal plasma. The analysis of the fetal transcriptome and methylome may yield valuable information on fetal and maternal health. With continued improvement in sequencing technology and reduction in sequencing costs, the analysis of cell-free nucleic acids would play an increasingly important role in prenatal screening, diagnosis, monitoring, and risk stratification of fetal as well as maternal conditions.

Current controversies in prenatal diagnosis 2: prediction and prevention of adverse pregnancy outcomes requires a genomic rather than proteomic solution.

From both presentations, it is clear that understanding the APO associated with placental dysfunction represents one of the greatest challenges in the field of prenatal screening, diagnosis, and therapy. Their clinical impact on the health of the mother and child was well recognized by the debaters, and both have agreed that there is a paucity of knowledge in the etiophysiopathology of placental dysfunction and the associated clinical phenotypes. They also agreed that this marked limitation in our understanding is a significant problem when designing a research protocol, and both stressed the importance of proper study designs. When focusing on the specific topics of the debate, they showed different opinions about the role of genomics in the search for relevant answers. Leslie Myatt pointed out that the genome does not define the cellular phenotype, and although the proteome itself does not define phenotype, it is much closer to it than the genome. Conversely, Rossa Chiu suggested that genomic approaches offer a better chance of achieving the answers than by proteomics alone. Actually, she hypothesized that through genomic approaches, or rather through systems biology, that is, including genomics, epigenomics, transcriptomics, and proteomics, there would be a better chance of obtaining the best answers. She also raised the possibility of the potential use of cell-free fetal nucleic acids in maternal plasma, which in turn are mainly of placental origin. Finally, both debaters and the audience agreed that there was not an exclusive proteomic or genomic 'solution', but that we need a larger spectrum of research strategies to include both proteomics and genomics and other systems biology approaches, combined with detailed and standardized clinical, laboratory, and epidemiological criteria in appropriately designed studies in order to start filling the significant gaps in our knowledge about this highly complex area of placental mediated adverse pregnancy outcomes.

Recent advances in the analysis of fetal nucleic acids in maternal plasma

Noninvasive prenatal diagnosis can be achieved by analyzing cell-free fetal DNA in maternal plasma. The fact that circulating fetal DNA represents only a minor fraction of the DNA that is present in maternal plasma has presented analytical challenges for a number of applications. In this review, we discuss such challenges and how they have been resolved by recent developments in the field. Digital molecular counting methods, such as digital PCR and massively parallel sequencing, have enabled high quantitative precision for maternal plasma DNA analysis. Noninvasive prenatal analysis of monogenic disease mutations has been achieved by identifying small quantitative differences between the mutant and wild-type alleles in maternal plasma. By measuring the small increment in the fractional concentrations of DNA derived from potentially aneuploid chromosomes in maternal plasma, fetal chromosomal aneuploidies have been detected with high diagnostic accuracies. Recently, advances in molecular technologies have enhanced the diagnostic applications of maternal plasma DNA analysis for noninvasive prenatal diagnosis. We foresee that this technology could play an increasingly important role in prenatal investigations.

Detección de ácidos nucleicos fetales en plasma materno: hacia un diagnóstico prenatal no invasivo

The discovery of circulating free fetal nucleic acids in maternal plasma has sparked wide interest in their origin, characteristics and possible medical uses. This review provides a comprehensive and concise summary of the results of studies of free fetal DNA and RNA in maternal plasma and discusses future possibilities for their use, mainly aimed at non-invasive prenatal diagnosis, an area where this discovery is expected to have a major impact in the very near future. Promising results have been reported in the assessment of placental function and in the use of these nucleic acids as predictive markers of the severity of pregnancy complications.

Non-invasive prenatal diagnosis using cell-free fetal nucleic acids in maternal plasma: Progress overview beyond predictive and personalized diagnosis

The discovery of circulating cell-free fetal DNA (cffDNA) in maternal plasma allowed for the development of alternative methodologies that may facilitate safe non-invasive prenatal diagnosis (NIPD). The low concentration of cffDNA in maternal plasma, however, and the coexistence of maternal DNA limit its clinical application to the detection or exclusion of fetal targets that are not present in the mother, such as Y chromosome sequences, the RHD gene in a RhD-negative woman and genetic conditions inherited from the father. Strategies for NIPD of monogenic disorders and fetal chromosomal aneuploidies have also been achieved using next-generation sequencing and could be introduced to the clinics as soon as cost-effective and high throughput protocols are developed.

Prediction, prevention and personalisation of medication for the prenatal period: genetic prenatal tests for both rare and common diseases

Genetic testing usually helps physicians to determine possible genetic diseases in unborn babies, genetic disorders of patients and the carriers who might pass the mutant gene on to their children. They are performed on blood, tissues or other body fluids. In recent years, the screening tests and diagnostic tests have improved quickly and, as a result, the risks of pregnancy can be determined more commonly and physicians can diagnose several genetic disorders in the prenatal period. Detecting the abnormalities in utero enables correct management of the pregnancy, prenatal and postnatal medical care, and it is also important for making well informed decisions about continuing or terminating a pregnancy. Besides the improvements of conventional invasive diagnostic tests, the discovery of circulating cell-free foetal nucleic acids in maternal plasma has developed a new point of view for non-invasive prenatal diagnosis recently.

Cell-free fetal nucleic acids as prenatal biomarkers

Cell-free fetal nucleic acids in maternal plasma or serum have become important tools in the pursuance of new methods for non-invasive prenatal diagnosis, such as the determination of fetal blood groups and fetal gender. During these pioneering explorations, elevations in the concentration of these new-found biological analytes were noted in several pregnancy-related disorders, including preterm labor, pre-eclampsia and malimplantation. As these elevations appeared to occur before onset of clinical symptoms, it was proposed that such analyses might assist in screening for at-risk pregnancies. A major problem with these early studies is that they relied on the quantitation of Y-chromosome-specific gene sequences, and as such could be applied only in those cases where the fetus was male. Recent developments that might permit gender-independent analysis include epigenetic markers, as well as the analysis of cell-free placentally derived mRNA species. This article focuses specifically on prognostic markers, which enable at-risk pregnancies to be identified, allowing the modification of pregnancy management and in turn improvement of pregnancy outcome. The authors also provide their opinion on the progress and future challenges that lie ahead. Accurate quantification of fetal nucleic acids and the specificity of these elevations for particular disorders remain controversial issues. Regarding the multifactorial etiology of some pregnancy disorders, the use of fetal nucleic acids as prenatal markers is restricted to well-defined high-risk groups.

Quantification of Fetal DNA by Use of Methylation-Based DNA Discrimination

Detection of circulating cell-free fetal nucleic acids in maternal plasma has been used in noninvasive prenatal diagnostics. Most applications rely on the qualitative detection of fetal nucleic acids to determine the genetic makeup of the fetus. This method leads to an analytic dilemma, because test results from samples that do not contain fetal DNA or are contaminated with maternal cellular DNA can be misleading. We developed a multiplex approach to analyze regions that are hypermethylated in placenta relative to maternal blood to evaluate the fetal portion of circulating cell-free DNA isolated from maternal plasma. The assay used methylation-sensitive restriction enzymes to eliminate the maternal (unmethylated) fraction of the DNA sample. The undigested fetal DNA fraction was then coamplified in the presence of a synthetic oligonucleotide to permit competitive PCR. The amplification products were quantified by single-base extension and MALDI-TOF MS analysis. Using 2 independent markers, (sex determining region Y)-box 14 (SOX14) and T-box 3 (TBX3), we measured a mean of 151 copies of fetal DNA/mL plasma and a mean fetal fraction of 0.13 in samples obtained from pregnant women. We investigated 242 DNA samples isolated from plasma from pregnant and nonpregnant women and observed an analytical sensitivity and specificity for the assay of 99% and 100%, respectively. By investigating several regions in parallel, we reduced the measurement variance and enabled quantification of circulating cell-free DNA. Our results indicate that this multiplex methylation-based reaction detects and quantifies the amount of fetal DNA in a sample isolated from maternal plasma.

Detection of fetal RhD gene from maternal blood

Hemolytic disease of the newborn (HDN) is a clinic phenomenon which occurs during pregnancy due to the Rhesus (Rh) D alloimmunization between a Rh (-) pregnant woman, who has become sensitive to RhD antigens, and her Rh (+) fetus. As a result of the attack of maternal RhD antibodies on fetal RhD antigens, fetal anemia, HDN and fetal death may occur. % 40 of Rh (-) pregnant women carry Rh (-) fetus. However, all Rh (-) pregnant women are offered anti-D Immunoglobulin (Anti-D Ig) at 28 weeks' gestation in case of fetomaternal haemorrhage, so the pregnant women carrying Rh (-) fetus are exposed to blood products unnecessarily. Although the RhD of fetus can be detected, methods used for prenatal diagnosis recently are invasive tests and they can result in abortion in a certain percentage. The discovery of circulating cell-free fetal nucleic acids in maternal plasma has opened up new possibilities for non invasive prenatal diagnosis. The aim of this study was to detect prenatal RhD by analysing the presence of the RhD gene of fetal DNA in maternal blood. Total free DNA was isolated from the blood of 19 Rh (-) pregnant women, who had RhD alloimmunization with their husbands, in the 11-14 th week of their pregnancy. The existence of a gene in isolated DNA was investigated with TaqMan prob and "Real-time PCR" method by using primers belonging to exon 7 of RhD gene. Using a quantitative real-time PCR assay, the presence of RhD gene sequences was evaluated in the serum of patients at the onset of pregnancy. We have analyzed 19 Rh (-) pregnant women. Twelve of them were Rh (-) and the rest of them were 7 Rh (+). After birth the baby's blood groups were concordant with our results. The results obtained by RhD primer were analysed. The possibility of detection of fetal RhD gene in maternal blood contributed to noninvasive prenatal diagnosis.

Detection of fetal chromosomal aneuploidies by ms analysis of cell-free fetal nucleic acids in maternal plasma

Detection of fetal chromosomal aneuploidies by ms analysis of cell-free fetal nucleic acids in maternal plasma

Circulating free fetal nucleic acids in maternal plasma and preeclampsia

Although preeclampsia represents a major threat for many pregnant women, the pathogenesis of this complication is far from being clear. Recent studies suggest that preeclampsia is an autoimmune disorder. Auto-antibodies against angiotensin receptor might explain some of the pathologic findings associated with preeclampsia. However, the origin of the autoimmune reaction is unknown. Here we hypothesize that circulating fetal RNA in maternal plasma might transfect maternal cells. Expression of fetal specific sequences could lead to an immune reaction breaking the immune tolerance against some antigens. Male fetus bearing pregnancies could be at higher risk of preeclampsia due to expression of Y-specific transcripts. This hypothesis is testable by analyzing antibodies and T-lymphocytes of pregnant women with male and female fetuses.

727: Markers for trisomy 21: elucidating sequence variants of the PLAC4 gene

The utility of single nucleotide polymorphisms (SNPs) in the analysis of circulating cell-free fetal (ccff) nucleic acids in maternal plasma is well recognized. The objective of this study was to utilize second generation sequencing of samples pooled from an ethnically diverse population to identify sequence variations within the PLAC4 gene that ultimately will be useful in improving our approach to noninvasive prenatal testing.

Non‐invasive detection of foetal RhD status and other genetic characteristics by circulating nucleic acids in maternal plasma

Since the discovery of cell‐free foetal DNA in maternal plasma in 1997, many circulating foetal DNA markers have been developed for the non‐invasive prenatal assessment of foetal genetic status. Some of the diagnostic applications, such as foetal sex determination and foetal rhesus D blood group genotyping, have already been introduced as routine services in some clinical centres. To extend the spectrum of foetal traits in which circulating foetal DNA can be worked on, advanced molecular technologies, such as mass spectrometry, microfluidic system and digital polymerase chain reaction analysis, have been employed. These newly developed tools have superior detection sensitivities and specificities over conventional methods. They have allowed researchers to overcome many technical challenges regarding the non‐invasive investigations of the foetal genetic characteristics, especially those involving a single nucleotide change. We envision that after validations by large‐scale studies, these new tools would be useful in a clinical setting in the near future.

Detection of circulating fetal nucleic acids: a review of methods and applications

The discovery of cell-free circulating fetal nucleic acids in maternal plasma has opened up new possibilities in non-invasive prenatal diagnosis. The rapid advancement of this field in the past decade...

Noninvasive prenatal detection of fetal chromosomal aneuploidies by maternal plasma nucleic acid analysis: a review of the current state of the art

Fetal nucleic acids in maternal plasma have opened up new possibilities for noninvasive prenatal diagnosis of chromosomal aneuploidies. One approach is based on the measurement of the allelic ratio of single nucleotide polymorphisms in the coding region of placental mRNA. Another approach is through the analysis of DNA fragments with different patterns of DNA methylation between fetal and maternal DNA. One other alternative is to enrich the fractional concentration of fetal DNA in maternal plasma using physical or chemical methods. Finally, the development of more precise digital polymerase chain reaction-based methods for fetal nucleic acid analysis might further catalyse the developments in this area. It is hoped that plasma-based molecular prenatal diagnosis might ultimately make prenatal testing safer for pregnant women and their fetuses.

Fetal Nucleic Acids in Maternal Plasma

The discovery of cell-free fetal nucleic acids in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. Over the last few years, a number of approaches have been demonstrated to allow such circulating fetal nucleic acids to be used for the prenatal detection of chromosomal aneuploidies. One such approach involves the enrichment of fetal DNA, such as by size fractionation or by the controversial formaldehyde treatment technique. A second approach involves the targeting of fetal-specific nucleic acid molecules, including fetal-specific epigenetic markers and placenta-specific mRNA markers. A third approach involves the development of highly discriminatory quantitative methods for chromosome dosage analysis using digital polymerase chain reaction technology. It is likely that these and other methods yet to be developed would allow noninvasive prenatal diagnosis of chromosomal aneuploidies by maternal plasma nucleic acids to be realized in the near future.

Noninvasive Prenatal Diagnosis of Fetal Chromosomal Aneuploidies by Maternal Plasma Nucleic Acid Analysis

The discovery of circulating cell-free fetal nucleic acids in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. The potential application of this technology for the noninvasive prenatal detection of fetal chromosomal aneuploidies is an aspect of this field that is being actively investigated. The main challenge of work in this area is the fact that cell-free fetal nucleic acids represent only a minor fraction of the total nucleic acids in maternal plasma. We performed a review of the literature, which revealed that investigators have applied methods based on the physical and molecular enrichment of fetal nucleic acid targets from maternal plasma. The former includes the use of size fractionation of plasma DNA and the use of the controversial formaldehyde treatment method. The latter has been achieved through the development of fetal epigenetic and fetal RNA markers. The aneuploidy status of the fetus has been explored through the use of allelic ratio analysis of plasma fetal epigenetic and RNA markers. Digital PCR has been shown to offer high precision for allelic ratio and relative chromosome dosage analyses. After a decade of work, the theoretical and practical feasibility of prenatal fetal chromosomal aneuploidy detection by plasma nucleic acid analysis has been demonstrated in studies using small sample sets. Larger scale independent studies will be needed to validate these initial observations. If these larger scale studies prove successful, it is expected that with further development of new fetal DNA/RNA markers and new analytical methods, molecular noninvasive prenatal diagnosis of the major chromosomal aneuploidies could become a routine practice in the near future.

Detection and Characterization of Placental MicroRNAs in Maternal Plasma

The discovery of circulating fetal nucleic acids in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. MicroRNAs (miRNAs), a class of small RNAs, have been intensely investigated recently because of their important regulatory role in gene expression. Because nucleic acids of placental origin are released into maternal plasma, we hypothesized that miRNAs produced by the placenta would also be released into maternal plasma. We systematically searched for placental miRNAs in maternal plasma to identify miRNAs that were at high concentrations in placentas compared with maternal blood cells and then investigated the stability and filterability of this novel class of pregnancy-associated markers in maternal plasma. In a panel of TaqMan MicroRNA Assays available for 157 well-established miRNAs, 17 occurred at concentrations >10-fold higher in the placentas than in maternal blood cells and were undetectable in postdelivery maternal plasma. The 4 most abundant of these placental miRNAs (miR-141, miR-149, miR-299-5p, and miR-135b) were detectable in maternal plasma during pregnancy and showed reduced detection rates in postdelivery plasma. The plasma concentration of miR-141 increased as pregnancy progressed into the third trimester. Compared with mRNA encoded by CSH1 [chorionic somatomammotropin hormone 1 (placental lactogen)], miR-141 was even more stable in maternal plasma, and its concentration did not decrease after filtration. We have demonstrated the existence of placental miRNAs in maternal plasma and provide some information on their stability and physical nature. These findings open up a new class of molecular markers for pregnancy monitoring.