Article

Presence of Fetal DNA in Maternal Plasma and Serum

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Abstract

The potential use of plasma and serum for molecular diagnosis has generated interest. Tumour DNA has been found in 'the plasma and serum of cancer patients, and molecular analysis has been done on this material. We investigated the equivalent condition in pregnancy-that is, whether fetal DNA is present in maternal plasma and serum. We used a rapid-boiling method to extract DNA from plasma and serum. DNA from plasma, serum, and nucleated blood cells from 43 pregnant women underwent a sensitive Y-PCR assay to detect circulating male fetal DNA from women bearing male fetuses. Fetus-derived Y sequences were detected in 24 (80%) of the 30 maternal plasma samples, and in 21 (70%) of the 30 maternal serum samples, from women bearing male fetuses. These results were obtained with only 10 microL of the samples. When DNA from nucleated blood cells extracted from a similar volume of blood was used, only five (17%) of the 30 samples gave a positive Y signal. None of the 13 women bearing female fetuses, and none of the ten non-pregnant control women, had positive results for plasma, serum or nucleated blood cells. Our finding of circulating fetal DNA in maternal plasma may have implications for non-invasive prenatal diagnosis, and for improving our understanding of the fetomaternal relationship.

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... Cell-free DNA (cfDNA) in maternal circulation was first reported by Lo et al. in 1997, and this discovery brought up the development of a noninvasive prenatal approach as a screening test for fetal chromosomal abnormalities (9). Detection rates in a recent meta-analysis evaluating cfDNA screening were higher than 99% for trisomy 21, 98% for trisomy 18, and 99% for trisomy 13, with a combined false-positive rate of 0.13% (10). ...
... Cell-free DNA (cfDNA) in maternal circulation was first reported by Lo et al. in 1997, and this discovery brought up the development of a noninvasive prenatal approach as a screening test for fetal chromosomal abnormalities (9). Detection rates in a recent meta-analysis evaluating cfDNA screening were higher than 99% for trisomy 21, 98% for trisomy 18, and 99% for trisomy 13, with a combined false-positive rate of 0.13% (10). ...
... Cell-free DNA (cfDNA) from the peripheral blood of pregnant women has been widely used to screen for fetal chromosome aneuploidies, including Down syndrome (trisomy 21, T21), Edwards syndrome (trisomy 18, T18), Patau syndrome (trisomy 13, T13), and sex chromosome aneuploidies (SCA). Lo et al., in 1997, was the first to describe fetal cfDNA in the plasma of pregnant women [1], and a prenatal testing method based on cfDNA was introduced in 2008 [2]. Compared with traditional serum screening, cfDNA testing has a higher sensitivity, lower false-positive rate, and higher positive predictive value (PPV) [3,4]. ...
... Cell-free DNA (cfDNA) from the peripheral blood of pregnant women has been widely used to screen for fetal chromosome aneuploidies, including Down syndrome (trisomy 21, T21), Edwards syndrome (trisomy 18, T18), Patau syndrome (trisomy 13, T13), and sex chromosome aneuploidies (SCA). Lo et al., in 1997, was the first to describe fetal cfDNA in the plasma of pregnant women [1], and a prenatal testing method based on cfDNA was introduced in 2008 [2]. Compared with traditional serum screening, cfDNA testing has a higher sensitivity, lower false-positive rate, and higher positive predictive value (PPV) [3,4]. ...
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Background This paper evaluated the clinical utility of massively parallel sequencing-based non-invasive prenatal testing (NIPT) for detecting trisomy 21 (T21), T18, T13, sex chromosome aneuploidies (SCA), and rare chromosome aneuploidies (RCA) among the data collected by a clinical laboratory in southern China. Methods In a 3-year period between January 2017 and December 2019, over 40,000 pregnant women underwent NIPT clinical screening test for fetal T21, T18, T13, SCA, and RCA in our laboratory. NIPT samples were processed using the NextSeq CN500 platform. The positive results were confirmed by karyotyping, and chromosomal microarray analysis (CMA) or copy number variants (CNV) sequencing. Details of the pregnancy outcomes were collected via telephone interview. Results NIPT results were available for 41,819 cases; 691 positive cases were reported. The overall sensitivity for detection of T21, T18, T13, SCA, and RCA was 99.21, 100.00, 100.00, 98.55, and 100.00%, and the specificity was 99.95, 99.94, 99.98, 99.69, and 99.92%, respectively. The positive predictive values (PPVs) for detection of T21, T18, T13, SCA, and RCA were 85.62, 45.24, 40.00, 34.17, and 13.51%, respectively, and those for detection of 45,X, 47,XXY, 47,XXX, 47,XYY, and 46,XY(delX) 20.00, 59.18, 28.95, 61.54, and 25.00%, respectively. Regarding pregnancy outcomes, 92.38% of the pregnancies with confirmed aneuploidies were terminated, and 91.20% of those identified as having a false-positive result were carried to term. Among 252 unconfirmed cases, 24.60% of the pregnancies were terminated and 38.10% carried to term, while 37.30% declined interview. Conclusions NIPT is widely used to screen fetal aneuploidies based on its high sensitivity and specificity. However, in this study, the PPVs of NIPT in terms of detecting T18, T13, XO, XXX and RCA were < 50%. In addition, more than one-third of NIPT-positive women did not accept invasive prenatal diagnosis. Confirmatory diagnosis is strongly recommended for women with positive NIPT outcomes before any further decision is made.
... In 1997, Lo et al., reported the detection of fetal DNA in maternal peripheral blood [5]. This discovery ushered in a new era of NIPT; cffDNA-based NIPT has been in commercially available and widely used since 2011 [5]. ...
... In 1997, Lo et al., reported the detection of fetal DNA in maternal peripheral blood [5]. This discovery ushered in a new era of NIPT; cffDNA-based NIPT has been in commercially available and widely used since 2011 [5]. The cffDNA-based NIPT is a non-invasive procedure with high sensitivity and specificity for the detection of fetal aneuploidies, including trisomy 21, trisomy 18, trisomy 13, and monosomy X [6,7]. ...
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The purpose of the present review is to try to highlight recent advances in the application of microfluidic technology on non-invasive prenatal diagnosis (NIPD). The immunoaffinity based microfluidic technology is the most common approach for NIPD, followed by size-based microfluidic methods. Immunoaffinity microfluidic methods can enrich and isolate circulating fetal extravillous trophoblasts (fEVTs) or fetal nucleated red blood cells (fnRBCs) for NIPD by using specific antibodies, but size-based microfluidic systems are only applied to isolate fEVTs. Most studies based on the immunoaffinity microfluidic system gave good results. Enough fetal cells were obtained for chromosomal and/or genetic analysis in all blood samples. However, the results from studies using size-based microfluidic systems for NIPD are less than ideal. In conclusion, recent advances in microfluidic devices make the immunoaffinity based microfluidic system potentially a powerful tool for cell-based NIPD. However, more clinical validation is needed.
... (11,12). A metaanalysis of the clinical validation and implementation of the non-invasive prenatal testing (NIPT) method revealed a high sensitivity and specificity (92-99%) for trisomy 21, 18, and 13 (4,13,14). NIPT has been recently recommended by several professional societies, such as the International Society for Ultrasound in Obstetrics and Gynecology (ISUOG) and the American College of Obstetricians and Gynecologists (ACOG), International Society of Prenatal Diagnosis (ISPD), and the Royal College of Obstetricians and Gynecologists (RCOG). ...
... Fetal cfDNA in maternal peripheral blood originates from trophoblasts and is mainly composed of placental DNA (11)(12)(13). NIPT is widely used as an alternative to ultrasonography or invasive fetal testing. ...
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We aimed to identify the causes of inconsistent results between non-invasive prenatal testing (NIPT) and invasive testing methods for trisomy 21. In the first case, NIPT was performed at 11 weeks of pregnancy, and the result showed a high risk of trisomy 21 [fetal fraction (FF) = 6.98%, 21 chromosome Z-score = 3.6]. The patient underwent quantitative fluorescent (QF)-PCR and karyotyping at 14 + 0 weeks of pregnancy through CVS showing mosaicism of 47, XX, + 21[11] and 46, XX [39] in karyotyping. The patient underwent amniocentesis at 15 + 6 weeks, showing a normal pattern in QF-PCR and 46, XX karyotyping in long term culture. The second case underwent NIPT at 16 + 5 weeks of pregnancy (FF = 7.52%, 21 chromosome Z-score = 2.503). She underwent an invasive test at 19 weeks through amniotic fluid sampling. As a result, trisomy 21 was detected by QF-PCR, and mosaicism of XX, +21[22]/46, XX [4] was identified by karyotyping. Despite significant advances in fetal chromosome analysis using NIPT, invasive testing is still needed as placenta-derived DNA does not reflect 100% fetal genetic information. Placental mosaicism can be detected by NIPT, but more research is needed to increase its sensitivity. Therefore, if the NIPT result is positive, an invasive test can confirm the result, and continuous monitoring is required even if the NIPT result is negative.
... Non-Invasive Prenatal Testing (NIPT) is a challenging technology that analyzes the maternal blood for cell-free fetal DNA (cffDNA) to estimate the possibility of fetal aneuploidy [1]. It was first introduced in Hong Kong in 2011 and became available in more than 60 countries by 2014 [2]. ...
... The NIPT analysis is varied according to the supplier; for instance, it can detect trisomy 13,18,21, and aneuploidies of the sex chromosome [3]. Yet, NIPT is not advised as the first choice for screening; it is used clinically as a second-tier test in high-risk pregnancies [1,[3][4][5]. For instance, the NIPT is performed only after a positive result of enhanced first-trimester screening or maternal serum screening. ...
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Introduction: The Non-Invasive Prenatal Testing (NIPT) guideline was issued and applied in 2013 by the Japanese Medical Association. Since being issued, the NIPT practice in Japan still has some problems related to indication, access, cost coverage and uniformity. Therefore, our study aimed to identify the Japanese challenges of adopting NIPT into prenatal diagnosis by comparing the system and process with other countries. Method: The United Kingdom, Germany, Italy, Sweden, and Taiwan were purposefully selected for comparison. All the countries, including Japan, introduced NIPT. The literature and information searches were conducted using PubMed, SCOPUS, Google Scholar, CiNii and Google searching engine. Results: The process of NIPT in Japan was very different from the other countries. Japan is the only country that indicated NIPT for only pregnant women over 35 years old in certificated facilities and did not have a policy regarding providing information on prenatal screening and NIPT to all women. Japan also did not have a policy regarding abortion due to fetal abnormalities. The practice of NIPT guidelines is different between non-certified and certified facilities. NIPT fee was the highest in Japan and was not covered by insurance. Conclusion: Pregnant women in Japan suffered from disparities in information access, economic burden, geographic location, and practice of NIPT guidelines between the certified and the non-certified facilities. Pregnant women-centered prenatal diagnosis policy, including NIPT, should be established in Japan by learning cases from other countries.
... The discovery of cell-free fetal DNA (cffDNA) in the mother's plasma by Lo et al. (1997) in 1997 has paved the way for developing new applications in clinical practice that rely on analyzing fetal genetic material (Rather et al., 2019). cffDNA comprises approximately 3%-13% of the mother's cellfree DNA (cfDNA) and is released into maternal circulation from placental cells undergoing apoptosis. ...
... Following the discovery of cffDNA by Lo et al. (1997) in pregnant women, NIPT with high-throughput sequencing has been widely adopted for the clinical detection of chromosomal abnormalities. This high-throughput DNA sequencing technique can rapidly and effectively detect large-scale genetic alterations with high accuracy and specificity for trisomies T21, T18, and T13 (Gregg et al., 2016). ...
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Background: In pregnant women at risk of autosomal recessive (AR) disorders, prenatal diagnosis of AR disorders primarily involves invasive procedures, such as chorionic villus sampling and amniocentesis. Methods: We collected blood samples from four pregnant women in their first trimester who presented a risk of having a child with an AR disorder. Cell-free DNA (cfDNA) was extracted, amplified, and double-purified to reduce maternal DNA interference. Additionally, whole-genome amplification was performed for traces of residual purified cfDNA for utilization in subsequent applications. Results: Based on our findings, we detected the fetal status with the family corresponding different genes, i.e., LZTR1, DVL2, HBB, RNASEH2B, and MYO7A, as homozygous affected, wild-type, and heterozygous carriers, respectively. Results were subsequently confirmed by prenatal amniocentesis. The results of AmpFLSTR™ Identifiler™ presented a distinct profile from the corresponding mother profile, thereby corroborating the result reflecting the genetic material of the fetus. Conclusion: Herein, we detected AR disease mutations in the first trimester of pregnancy while surmounting limitations associated with maternal genetic material interference. Importantly, such detection strategies would allow the screening of pregnant women for common AR diseases, especially in highly consanguineous marriage populations. This technique would open avenues for the early detection and prevention of recessive diseases among the population.
... Non-invasive prenatal testing (NIPT) is emerging as a robust technique to screen for trisomies in 13, 18, 21, and sex chromosome (Lo et al., 1997;Lo et al., 1998;Song et al., 2013;Yin et al., 2015;Gross et al., 2016). Furthermore, its ability in screening for subchromosomal abnormalities such as Cri-du-chat deletions, 1p36 deletion syndrome, Wolf-Hirschhorn syndrome, Prader-Willi deletions, or Angelman deletions, has been proven as shown by high predictive positive value (Liu et al., 2016;Liang et al., 2019). ...
... NIPT may detect CNVs with a size greater 10 Mb with high sensitivity and specificity (Lo et al., 1997;Lo et al., 1998;Song et al., 2013;Yin et al., 2015;Gross et al., 2016). Additionally, it has good performance in detecting known microduplication/ microdeletion syndromes (MMS), which are smaller than 10 Mb, such as DiGeorge (93%) and 22q11.22 ...
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Tetrasomy 9p is a rare syndrome characterized by fetal growth restriction, Dandy-Walker malformation, cardiac anomalies, and facial abnormalities and is discovered by ultrasound during the prenatal examination. Herein, we report a fetus of tetrasomy 9p without obvious phenotypic manifestations during the first trimester that was identified by non-invasive prenatal testing (NIPT). NIPT revealed that the gain of 9p24.3–9p11 that was approximately 46.36 Mb in size. Karyotyping of amniocytes indicated an additional marker in all metaphase. Chromosome microarray and fluorescence in situ hybridization on uncultured amniocytes revealed tetrasomic of 9p24.3q13, and that the supernumerary chromosome is a dicentric isochromosome consisted of two copies of the 9p arm. Taken together, it was indicated that the fetal karyotype was 47,XY,+idic (9) (q13), and that multiple techniques are crucial to the prenatal diagnosis.
... Leon and Shapiro, using a radioimmunoassay, showed that the serum of cancer patients contained a significantly higher quantity of cell-free DNA [11]. In 1997, Lo's team demonstrated the presence of circulating fetal DNA in maternal blood during pregnancy [12]; as a result, the first method of diagnosing trisomy 21 was established [13]. ...
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Breast cancer is clinically and biologically heterogeneous and is classified into different subtypes according to the molecular landscape of the tumor. Triple-negative breast cancer is a subtype associated with higher tumor aggressiveness, poor prognosis, and poor response to treatment. In metastatic breast cancer, approximately 6% to 10% of new breast cancer cases are initially staged IV (de novo metastatic disease). The number of metastatic recurrences is estimated to be 20–30% of all existing breast tumor cases, whereby the need to develop specific genetic markers to improve the prognosis of patients suffering from these deadly forms of breast cancer. As an alternative, liquid biopsy methods can minutely identify the molecular architecture of breast cancer, including aggressive forms, which provides new perspectives for more precise diagnosis and more effective therapeutics. This review aimed to summarize the current clinical evidence for the application of circulating tumor DNA in managing breast cancer by detailing the increased usefulness of this biomarker as a diagnostic, prognostic, monitoring, and surveillance marker for breast cancer.
... To date, diagnostic genetic testing of the fetus during early pregnancy requires invasive procedures such as Chorionic Villus Sampling (CVS) and amniocentesis (also called amnio) associated with miscarriage risk. In 1997, it was discovered that maternal plasma contains cell-free fetal DNA (cffDNA) [1]. Most cffDNA comes from villous cells, with its concentration increasing proportionally with gestational age, enabling the chance to obtain fetal genetic information from maternal plasma. ...
Article
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In 1997, it was discovered that maternal plasma contains Cell-Free Fetal DNA (cffDNA). cffDNA has been investigated as a source of DNA for non-invasive prenatal testing for fetal pathologies, as well as for non-invasive paternity testing. While the advent of Next Generation Sequencing (NGS) led to the routine use of Non-Invasive Prenatal Screening (NIPT or NIPS), few data are available regarding the reliability and reproducibility of Non-Invasive Prenatal Paternity Testing (NIPPT or NIPAT). Here, we present a non-invasive prenatal paternity test (NIPAT) analyzing 861 Single Nucleotide Variants (SNV) from cffDNA through NGS technology. The test, validated on more than 900 meiosis samples, generated log(CPI)(Combined Paternity Index) values for designated fathers ranging from + 34 to + 85, whereas log(CPI) values calculated for unrelated individuals were below -150. This study suggests that NIPAT can be used with high accuracy in real cases.
... Plasma cell-free DNA (cfDNA) molecules circulating in human peripheral blood are first discovered in 1948 1 . Following this discovery, the presence of tumor-, fetus-, and donor-derived DNA molecules in cancer patients, pregnant women, and organ-transplantation recipients, respectively, has led to a new era of blood-based liquid biopsy that utilizes cfDNA to perform noninvasive cancer diagnosis, prenatal testing, as well as transplantation monitoring [2][3][4][5] . Despite the impressive success in translational medicine 5 , the molecular biology of cfDNA is much less explored. ...
Article
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Plasma cell-free DNA (cfDNA) are small molecules generated through a non-random fragmentation procedure. Despite commendable translational values in cancer liquid biopsy, however, the biology of cfDNA, especially the principles of cfDNA fragmentation, remains largely elusive. Through orientation-aware analyses of cfDNA fragmentation patterns against the nucleosome structure and integration with multidimensional functional genomics data, here we report a DNA methylation – nuclease preference – cutting end – size distribution axis, demonstrating the role of DNA methylation as a functional molecular regulator of cfDNA fragmentation. Hence, low-level DNA methylation could increase nucleosome accessibility and alter the cutting activities of nucleases during DNA fragmentation, which further leads to variation in cutting sites and size distribution of cfDNA. We further develop a cfDNA ending preference-based metric for cancer diagnosis, whose performance has been validated by multiple pan-cancer datasets. Our work sheds light on the molecular basis of cfDNA fragmentation towards broader applications in cancer liquid biopsy.
... Currently, non-invasive prenatal screening is performed in the first trimester as an alternative to chorionic villus sampling for prenatal screening of Down syndrome. This procedure involves sampling and sequencing the cell-free fetal DNA (cffDNA) fraction from blood of pregnant mothers [130,131]. cffDNA originates from the trophoblasts of the placenta, based on genetic evidence from cases of anembryonic pregnancies or confined placental mosaicism [132][133][134]. DNA methylation evidence also has demonstrated that cffDNA contains partially methylated domains [135,136] which are uniquely characteristic of placenta [137,138]. ...
Article
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Autism spectrum disorder (ASD) comprises a heterogeneous group of neurodevelopmental outcomes in children with a commonality in deficits in social communication and language combined with repetitive behaviors and interests. The etiology of ASD is heterogeneous, as several hundred genes have been implicated as well as multiple in utero environmental exposures. Over the past two decades, epigenetic investigations, including DNA methylation, have emerged as a novel way to capture the complex interface of multivariate ASD etiologies. More recently, epigenome-wide association studies using human brain and surrogate accessible tissues have revealed some convergent genes that are epigenetically altered in ASD, many of which overlap with known genetic risk factors. Unlike transcriptomes, epigenomic signatures defined by DNA methylation from surrogate tissues such as placenta and cord blood can reflect past differences in fetal brain gene transcription, transcription factor binding, and chromatin. For example, the discovery of NHIP (neuronal hypoxia inducible, placenta associated) through an epigenome-wide association in placenta, identified a common genetic risk for ASD that was modified by prenatal vitamin use. While epigenomic signatures are distinct between different genetic syndromic causes of ASD, bivalent chromatin and some convergent gene pathways are consistently epigenetically altered in both syndromic and idiopathic ASD, as well as some environmental exposures. Together, these epigenomic signatures hold promising clues towards improved early prediction and prevention of ASD as well genes and gene pathways to target for pharmacological interventions. Future advancements in single cell and multi-omic technologies, machine learning, as well as non-invasive screening of epigenomic signatures during pregnancy or newborn periods are expected to continue to impact the translatability of the recent discoveries in epigenomics to precision public health.
... The discovery of cell-free DNA (cfDNA) of fetal origin in blood plasma of pregnant women paved a new way for non-invasive prenatal screening (NIPS) (Lo et al., 1997). With advances in next-generation sequencing (NGS) technology, tens of millions of short sequence tags can be generated from cfDNA in a single maternal plasma sample. ...
Article
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Non-invasive prenatal screening (NIPS) is a DNA sequencing-based screening test for fetal aneuploidies and possibly other pathogenic genomic abnormalities, such as large deletions and duplications. Validation and quality assurance (QA) of this clinical test using plasmas with and without targeted chromosomal abnormalities from pregnant women as negative and positive controls are required. However, the positive plasma controls may not be available for many laboratories that are planning to establish NIPS. Limited synthetic positive plasmas are commercially available, but the types of abnormalities and the number/quantity of synthetic plasmas for each abnormality are insufficient to meet the minimal requirements for the initial validation. We report here a method of making synthetic positive plasmas by adding cell-free DNA (cfDNA) isolated from culture media of prenatal cells with chromosomal abnormalities to the plasmas from non-pregnant women. Thirty-eight positive plasmas with various chromosomal abnormalities, including autosomal and sex chromosomal aneuploidies, large deletions and duplications, were synthesized. The synthetic plasmas were characterized side-by-side with real positive plasmas from pregnant women and commercially available synthetic positive plasmas using the Illumina VeriSeq NIPT v2 system. All chromosomal abnormalities in the synthetic plasmas were correctly identified with the same testing sensitivity and specificity as in the real and commercial synthetic plasmas. The findings demonstrate that the synthetic positive plasmas are excellent alternatives of real positive plasmas for validation and QA of NIPS. The method described here is simple and straightforward, and can be readily used in clinical genetics laboratories with accessibility to prenatal cultures.
... Later, the presence of circulating free DNA was found successively in cancer patients [2], SLE patients [3], and organ transplant recipients [4]. In 1997, Dennis Lo demonstrated the presence of fetal DNA in the plasma and serum of pregnant women for the first time [5]. Cell-free DNA (cfDNA) is the free fragment of DNA molecules in plasma derived from apoptotic and necrotic cells with different topologies [6], and circulating tumor DNA (ctDNA) is derived from tumor cells [7]. ...
Article
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Cell-free DNA molecules are released into the plasma via apoptotic or necrotic events and active release mechanisms, which carry the genetic and epigenetic information of its origin tissues. However, cfDNA is the mixture of various cell fragments, and the efficient enrichment of cfDNA fragments with diagnostic value remains a great challenge for application in the clinical setting. Evidence from recent years shows that cfDNA fragmentomics’ characteristics differ in normal and diseased individuals without the need to distinguish the source of the cfDNA fragments, which makes it a promising novel biomarker. Moreover, cfDNA fragmentomics can identify tissue origins by inferring epigenetic information. Thus, further insights into the fragmentomics of plasma cfDNA shed light on the origin and fragmentation mechanisms of cfDNA during physiological and pathological processes in diseases and enhance our ability to take the advantage of plasma cfDNA as a molecular diagnostic tool. In this review, we focus on the cfDNA fragment characteristics and its potential application, such as fragment length, end motifs, jagged ends, preferred end coordinates, as well as nucleosome footprints, open chromatin region, and gene expression inferred by the cfDNA fragmentation pattern across the genome. Furthermore, we summarize the methods for deducing the tissue of origin by cfDNA fragmentomics.
... Previous studies that analyzed fetal circulating DNA in maternal plasma were essential for the development of non-invasive prenatal diagnostic tools [21]. These fetal DNA tests are used to analyze the most common trissomies and numerical alterations of the sex chromosomes. ...
Article
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Cell-free DNA fragments detected in blood and in other biological fluids are released from apoptotic/necrotic cells. In this study, we analyzed cfDNA levels in follicular fluid (FF) samples from patients with infertility. Samples were collected from 178 infertile women and cfDNA was extracted and quantified by qPCR, using ALU115 and ALU247 primers, and statistical correlations were performed. We found that cfDNA concentration was significantly higher in FF pools from women aged 35 and over than in women under 35 years of age (p = 0.017). We also found that q247 cfDNA levels were significantly higher in women with an associated female factor, such as endometriosis, PCOS and POF, compared with women with no specific cause of infertility (p = 0.033). The concentration of cfDNA did not vary significantly in each group of women with an associated female factor. The concentration of cfDNA was significantly higher in the FF of women that obtained embryos with a high fragmentation rate, compared to embryos with a low fragmentation rate (p = 0.007). Finally, we found that women who did not become pregnant during IVF treatments had higher q247 cfDNA levels (p = 0.043). The quantification of cfDNA could be an important biomarker of follicular micro-environment quality to predict embryo quality and the success of IVF, making them more specific and effective.
... The combination of NGS technologies and cell-free fetal DNA detection in maternal blood (Lo et al., 1997) enabled newly designed non-invasive prenatal testing (NIPT) for trisomy and other chromosomal abnormali-ties in fetal tissue. For example, Tatyana Ivashchenko and colleagues (2019) from Ott Research Institute of Obstetrics, Gynecology and Reproductology (St. ...
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The review highlights various methods for deciphering the nucleotide sequence (sequencing) of nucleic acids and their importance for the implementation of the three main principles of personalized medicine: prevention, predictability and personalization. The review, along with its own practical examples, considers three generations of sequencing technologies: 1) sequencing of cloned or amplified DNA fragments according to Sanger and its analogues; 2) massive parallel sequencing of DNA libraries with short reads (NGS); and 3) sequencing of single molecules of DNA and RNA with long reads. The methods of whole ge-nome, whole exome, targeted, RNA sequencing and sequencing based on chro-matin immunoprecipitation are also discussed. The advantages and limitations of the above methods for diagnosing monogenic and oncological diseases, as well as for identifying risk factors and predicting the course of socially significant multifactorial diseases are discussed. Using examples from clinical practice , algorithms for the application and selection of sequencing technologies are demonstrated. As a result of the use of sequencing technologies, it has now become possible to determine the molecular mechanism of the development of monogenic, orphan and multifactorial diseases, the knowledge of which is necessary for personalized patient therapy.
... It was earlier used for pre-implantation genetic analysis to accurately screen and evaluate abnormal embryos, thereby discarding abnormal embryos and selecting normal embryo implantation, which provided a reduction in infant birth defects and improved quality of life [213]. Later researchers used NGS technology to detect trophoblast cells [214], cffDNA [215], and fNRBCs [209] for the analysis of fetal aneuploidy and SNVs. Hua et al. [212] used NGS techniques to detect the aneuploidy of fNRBCs by taking 4 cell samples of known aneuploidy and using Illumina MiSeq for sequencing. ...
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Noninvasive prenatal diagnosis (NIPD) aims to detect fetal-related genetic disorders before birth by detecting markers in the peripheral blood of pregnant women, holding the potential in reducing the risk of fetal birth defects. Fetal-nucleated red blood cells (fNRBCs) can be used as biomarkers for NIPD, given their remarkable nature of carrying the entire genetic information of the fetus. Here, we review recent advances in NIPD technologies based on the isolation and analysis of fNRBCs. Conventional cell separation methods rely primarily on physical properties and surface antigens of fNRBCs, such as density gradient centrifugation, fluorescence-activated cell sorting, and magnetic-activated cell sorting. Due to the limitations of sensitivity and purity in Conventional methods, separation techniques based on micro-/nanomaterials have been developed as novel methods for isolating and enriching fNRBCs. We also discuss emerging methods based on microfluidic chips and nanostructured substrates for static and dynamic isolation of fNRBCs. Additionally, we introduce the identification techniques of fNRBCs and address the potential clinical diagnostic values of fNRBCs. Finally, we highlight the challenges and the future directions of fNRBCs as treatment guidelines in NIPD. Graphical Abstract
... These programs serve as a primary approach to detecting hearing loss in neonates and can thereby facilitate early interventions. In China, two representative examples of large NBHS program have been described by Wang et al. 18 24 and since then, there has been a relatively rapid development and clinical implementation of related tests. In China, noninvasive prenatal testing (NIPT) using cell-free DNA (cfDNA) for aneuploidy screening has been widely applied. ...
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Objectives: Numerous diseases and disorders are associated with mitochondrial DNA (mtDNA) mutations, among which m.1555A > G and m.1494C > T mutations in the 12 S ribosomal RNA gene contribute to aminoglycoside-induced and nonsyndromic hearing loss worldwide. Methods: A total of 76,842 qualified non-invasive prenatal (NIPT) samples were subjected to mtDNA mutation and haplogroup analysis. Results: We detected 181 m.1555A > G and m.1494C > T mutations, 151 of which were subsequently sequenced for full-length mitochondrial genome verification. The positive predictive values for the m.1555A > G and m.1494C > T mutations were 90.78% and 90.00%, respectively, a performance comparable to that attained with newborn hearing screening. Furthermore, mitochondrial haplogroup analysis revealed that the 12 S rRNA 1555A > G mutation was enriched in sub-haplotype D5[p = 0, OR = 4.6706(2.81-7.78)]. Conclusions: Our findings indicate that the non-invasive prenatal testing of cell-free DNA obtained from maternal plasma can successfully detect m.1555A > G and m.1494C > T mutations.
... invasive prenatal testing (NIPT) of circulating cell-free fetal (cff)DNA in the maternal plasma, enabling detection of paternally inherited markers (1). NIPT is based on discoveries led by Professor Lo et al. from late last century, that placental derived cffDNA circulates in the maternal plasma against a background of maternal cell-free deoxyribonucleic acid (DNA), defined as mcfDNA (2,3). The concentration of cffDNA increases with gestation and is cleared rapidly after delivery, thereby representing fetal markers specific for that pregnancy. ...
... For example, the quantification of blood hCG, PP13/galectin-13, and PSG1 has emerged as crucial in the detection of pregnancy or being pregnancy complications, together with preeclampsia, from maternal blood [71,72]. Since the discovery of cell-free fetal DNA (cffDNA) in the maternal circulation by Lo et al. in 1997 [73], the fast-evolving non-invasive prenatal diagnostics (NIPT) technologies have revolutionized prenatal screening of genetic defects primarily based on the detection of cffDNA in a small amount of maternal blood [74]. Shortly after, Lo et al. additionally identified circulating placental/fetal RNA (cpRNA) in the maternal circulation [75] and determined the earliest gestational age (4th week) at which these cpRNAs are present in the maternal circulation. ...
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Preeclampsia is being pregnant-specific, and notably contributes to maternal, and perinatal morbidity and mortality worldwide. An effective predictive test for preeclampsia could facilitate early diagnosis, focused surveillance and well timed delivery; however, restrained alternatives presently exist. A first-trimester screening algorithms has been evolved and demonstrated to expect preterm preeclampsia, with poor utility for term disease, wherein the greatest burden lies. Biomarkers consisting of sFlt-1 and placental growth factor also are now getting used clinically in cases of suspected preterm preeclampsia; their high negative predictive value allows assured exclusion of disease in women with normal results, however sensitivity is modest. There has been a concerted attempt to become aware of ability novel biomarkers that could enhance prediction. These in large part originate from organs concerned in preeclampsia’s pathogenesis, which includes placental, cardiovascular and urinary biomarkers. This review outlines the clinical imperative for an effective test and those already in use and summarises modern-day preeclampsia biomarker studies.
... In 1997, Lo et al. (1) discovered the Y chromosome in the maternal peripheral blood of pregnant male fetuses, which proved the existence of fetal cell-free-DNA (cf-DNA) in the peripheral blood of pregnant women. In recent years, fetal cf-DNA has been widely used in fetal aneuploid screening in the early second-trimester as a biomarker for a non-invasive prenatal test (NIPT). ...
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IntroductionThis study aimed to determine the correlation between fetal fraction (FF) of cell-free DNA (cf-DNA) and pregnancy complications related to placental dysfunction in Twin Pregnancy.Methods This retrospective cohort study analyzed twin pregnant women who underwent non-invasive prenatal testing (NIPT) at 12+0–26+6 weeks of gestation from April 2017 to April 2021. Low fetal fraction (LFF) was defined individually as less than the 25th, 10th, 5th, and 2.5th percentile among all fetal fractions in the cohort. Primary outcomes included gestational hypertension (GH), preeclampsia (PE), gestational diabetes mellitus (GDM), and small for gestational age (SGA). Logistic regression analysis was used to assess the relationship between LFF and pregnancy complications.ResultsA total of 500 twin pregnancies (male-male twins, 245; female-female twins, 255) were included in this study. In LFF group (FF < 25th percentiles), maternal BMI was significantly higher than FF > 75th percentiles (23.6 kg/m2 vs. 21.3 kg/m2; P < 0.001). The risk of SGA increased gradually from FF < 25th percentiles [adjusted odds ratio (OR), 1.71; 95% confidence interval (CI), 1.07–2.99; P = 0.016] to FF < 2.5th percentiles (adjusted OR, 4.44; 95% CI,1.33–14.82; P < 0.015). In addition, the risks of SGA in both fetuses were higher than the risks of at least one fetus SGA in LFF group. LFF had no correlation with GH, PE, and GDM in twin pregnancy.ConclusionLFF has a strong association with increased risk of SGA in twin pregnancy. Moreover, FF of cf-DNA may provide a new idea for the early screening of diseases related to placental dysfunction in twin pregnancy.
... The addition of extra ultrasound markers at the time of a nuchal translucency scan increases the detection rate to 96% and decreases the false-positive rate to 2.5% [4,5]. Cell-free fetal DNA (cffDNA) found circulating in maternal plasma [6] represents the entire fetal genome [7] and can be used to screen for fetal aneuploidy [8,9]. Non-invasive prenatal screening (NIPS) for the three common trisomies is becoming increasingly routine as a screening test in many countries [10] and is extremely accurate [11,12]; there has been an increasing uptake of NIPS for additional chromosomal abnormalities, although with much lower positive predictive value [13,14]. ...
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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.
... Since the discovery of cell-free fetal nucleic acids in maternal blood (Lo et al., 1997), non-invasive prenatal testing (NIPT) has been widely used to detect fetal chromosomal aneuploidy (Chiu et al., 2008;Chiu et al., 2010). Furthermore, with the advancement of next-generation sequencing (NGS) technology, new methods for the large-scale analysis of sequencing data have been developed. ...
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With advances in next-generation sequencing technology, non-invasive prenatal testing (NIPT) has been widely implemented to detect fetal aneuploidies, including trisomy 21, 18, and 13 (T21, T18, and T13). Most NIPT methods use cell-free DNA (cfDNA) fragment count (FC) in maternal blood. In this study, we developed a novel NIPT method using cfDNA fragment distance (FD) and convolutional neural network-based artificial intelligence algorithm (aiD-NIPT). Four types of aiD-NIPT algorithm (mean, median, interquartile range, and its ensemble) were developed using 2,215 samples. In an analysis of 17,678 clinical samples, all algorithms showed >99.40% accuracy for T21/T18/T13, and the ensemble algorithm showed the best performance (sensitivity: 99.07%, positive predictive value (PPV): 88.43%); the FC-based conventional Z-score and normalized chromosomal value showed 98.15% sensitivity, with 40.77% and 36.81% PPV, respectively. In conclusion, FD-based aiD-NIPT was successfully developed, and it showed better performance than FC-based NIPT methods.
... Constituting about 10% of cell free (cf) DNA, cffDNA can be detected as early as 5 weeks of gestation and cleared rapidly from maternal circulation within 2 hours after delivery of the baby 39 . Hence, results are not affected by previous pregnancy complications 40 . Both in house and kit based methods receiver operating characteristic (ROC) curve analysis of cffDNA content was done to identify cutoff for preeclampsia. ...
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This gender-independent detection of cell-free fetal DNA in maternal plasma using RASSF1A/beta-actin has curtained off a new dimension regarding its utility to predict the adverse pregnancy outcomes. Recent efforts have been directed at developing sequences from cell-free fetal DNA (cffDNA) as markers for pregnancy outcomes. The utility of cffDNA using the methylation-dependent DSCR3 and RASSF1A markers along with total cell-free DNA (cf-DNA) in maternal serum by HYP2 marker are useful in predicting adverse pregnancy outcomes. Indigenously developed low-cost method of the gender-independent sequence markers from cffDNA was investigated and evaluated with the standardized commercial kits as predictive markers for adverse pregnancy outcomes. In the present study, we have tested whether the elevated amount of cffDNA in maternal plasma is associated with adverse pregnancy outcomes and development of new marker by the low-cost method to predict adverse pregnancy outcomes. 210 pregnant women within the age group of 20-30 years attending for routine antenatal checkups after 20 weeks with fulfilling the diagnostic criteria of adverse pregnancy outcomes were included in our study. Age-matched pregnant women without adverse pregnancy outcomes were included as controls (n=210). Identification of cell-free fetal DNA (cffDNA) in maternal plasma by using in-house methods (Guanidium isothiocyanate) was found comparable with commercial kit and its content (GE/ µl) in adverse pregnancy outcomes subjects were significantly higher than the normotensive subjects. Our results indicated that indigenously developed method for detection of gender-independent cffDNA can be applicable for screening test of adverse pregnancy outcome.
... The field of non-invasive prenatal testing was enabled by the discovery that circulating cell free (ccf) fetal DNA is present pregnant female plasma [1]. It does, however, only present the minority species of DNA in total ccf DNA obtained from pregnant women. ...
Preprint
Background Circulating cell free fetal DNA has enabled non-invasive prenatal fetal aneuploidy testing without direct discrimination of the genetically distinct maternal and fetal DNA. Current testing may be improved by specifically enriching the sample material for fetal DNA. DNA methylation may allow for such a separation of DNA and thus support additional clinical opportunities; however, this depends on knowledge of the methylomes of ccf DNA and its cellular contributors. Results Whole genome bisulfite sequencing was performed on a set of unmatched samples including ccf DNA from 8 non-pregnant (NP) and 7 pregnant female donors and genomic DNA from 7 maternal buffy coat and 5 placenta samples. We found CpG cytosines within longer fragments were more likely to be methylated, linking DNA methylation and fragment size in ccf DNA. Comparison of the methylomes of placenta and NP ccf DNA revealed many of the 51,259 identified differentially methylated regions (DMRs) were located in domains exhibiting consistent placenta hypomethylation across millions of consecutive bases, regions we termed placenta hypomethylated domains (PHDs). We found PHDs were consistently located within regions exhibiting low CpG and gene density. DMRs identified when comparing placenta to NP ccf DNA were recapitulated in pregnant ccf DNA, confirming the ability to detect differential methylation in ccf DNA mixtures. Conclusions We generated methylome maps for four sample types at single base resolution, identified a link between DNA methylation and fragment length in ccf DNA, identified DMRs between sample groups, and uncovered the presence of megabase-size placenta hypomethylated domains. Furthermore, we anticipate these results to provide a foundation to which future studies using discriminatory DNA methylation may be compared.
... In addition to the primary drawback of amniocentesis and CVS being invasive techniques, they can only be completed later in the gestational period, after genital virilization has already begun. In 1997, Lo et al. discovered circulating cell-free fetal DNA (cffDNA) in maternal plasma which shifted the direction of prenatal testing (19). Obtaining cffDNA from maternal plasma is non-invasive and is done via a blood draw. ...
... The use of cfDNA in the diagnosis of tumors began in 1977, but was not very effective, due to the limitations of the existing technology [3]. The real-time polymerase chain reaction allowed the detection of RhD and the fetal sex in maternal plasma in 1997 [4]. The real expansion of non-invasive fetal genetic disease detection began in 2011, with the introduction of massive parallel sequencing [5]. ...
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The basic function of the immune system is the protection of the host against infections, along with the preservation of the individual antigenic identity. The process of self-tolerance covers the discrimination between self and foreign antigens, including proteins, nucleic acids, and larger molecules. Consequently, a broken immunological self-tolerance results in the development of autoimmune or autoinflammatory disorders. Immunocompetent cells express pattern-recognition receptors on their cell membrane and cytoplasm. The majority of endogenous DNA is located intracellularly within nuclei and mitochondria. However, extracellular, cell-free DNA (cfDNA) can also be detected in a variety of diseases, such as autoimmune disorders and malignancies, which has sparked interest in using cfDNA as a possible biomarker. In recent years, the widespread use of liquid biopsies and the increasing demand for screening, as well as monitoring disease activity and therapy response, have enabled the revival of cfDNA research. The majority of studies have mainly focused on the function of cfDNA as a biomarker. However, research regarding the immunological consequences of cfDNA, such as its potential immunomodulatory or therapeutic benefits, is still in its infancy. This article discusses the involvement of various DNA-sensing receptors (e.g., absent in melanoma-2; Toll-like receptor 9; cyclic GMP–AMP synthase/activator of interferon genes) in identifying host cfDNA as a potent danger-associated molecular pattern. Furthermore, we aim to summarize the results of the experimental studies that we recently performed and highlight the immunomodulatory capacity of cfDNA, and thus, the potential for possible therapeutic consideration.
... For example, the quantification of blood hCG, PP13/galectin-13, and PSG1 has emerged as crucial in the detection of pregnancy or being pregnancy complications, together with preeclampsia, from maternal blood [71,72]. Since the discovery of cell-free fetal DNA (cffDNA) in the maternal circulation by Lo et al. in 1997 [73], the fast-evolving non-invasive prenatal diagnostics (NIPT) technologies have revolutionized prenatal screening of genetic defects primarily based on the detection of cffDNA in a small amount of maternal blood [74]. Shortly after, Lo et al. additionally identified circulating placental/fetal RNA (cpRNA) in the maternal circulation [75] and determined the earliest gestational age (4th week) at which these cpRNAs are present in the maternal circulation. ...
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Preeclampsia is being pregnant-specific, and notably contributes to maternal, and perinatal morbidity and mortality worldwide. An effective predictive test for preeclampsia could facilitate early diagnosis, focused surveillance and well timed delivery; however, restrained alternatives presently exist. A first-trimester screening algorithms has been evolved and demonstrated to expect preterm preeclampsia, with poor utility for term disease, wherein the greatest burden lies. Biomarkers consisting of sFlt-1 and placental growth factor also are now getting used clinically in cases of suspected preterm preeclampsia; their high negative predictive value allows assured exclusion of disease in women with normal results, however sensitivity is modest. There has been a concerted attempt to become aware of ability novel biomarkers that could enhance prediction. These in large part originate from organs concerned in preeclampsia's pathogenesis, which includes placental, cardiovascular and urinary biomarkers. This review outlines the clinical imperative for an effective test and those already in use and summarises modern-day preeclampsia biomarker studies.
Article
Objectives This study aims to develop a novel library preparation method, plasma to library express technology (PLET), to construct next-generation sequencing (NGS) libraries directly from plasma without cell-free DNA (cfDNA) isolation. Methods Peripheral blood samples (600) were obtained from a retrospective cohort of 300 pregnant women prior to invasive diagnostic testing. The samples were subsequently distributed between library preparation methodologies, with 300 samples prepared by PLET and 300 by conventional methods for non-invasive prenatal testing (NIPT) to screen for common trisomies using low-pass whole genome next generation sequencing. Results NIPT conducted on PLET libraries demonstrated comparable metrics to libraries prepared using conventional methods, including 100% sensitivity and specificity. Conclusions Our study demonstrates the potential utility of PLET in the clinical setting and highlights its significant advantages, including dramatically reduced process complexity and markedly decreased turnaround time.
Article
The past several decades have witnessed unprecedented progress in basic and clinical cancer research, and our understanding of the molecular mechanisms and pathogenesis of cancers have been greatly improved. More recently, with the availability of high-throughput sequencing and profiling platforms as well as sophisticated analytical tools and high-performance computing capacity, there have been tremendous advances in the development of diagnostic approaches in clinical oncology, especially the discovery of novel biomarkers for cancer early detection. Although tissue biopsy-based pathology has been the "gold standard" for cancer diagnosis, notable limitations such as the risk due to invasiveness and the bias due to intra-tumoral heterogeneity have limited its broader applications in oncology (e.g., screening, regular disease monitoring). Liquid biopsy analysis that exploits the genetic and epigenetic information contained in DNA/RNA materials from body fluids, particularly circulating cell-free DNA (cfDNA) in the blood, has been an intriguing alternative approach because of advantageous features such as sampling convenience and minimal invasiveness. Taking advantage of innovative enabling technologies, cfDNA has been demonstrated for its clinical potential in cancer early detection, including hepatocellular carcinoma (HCC), the most common liver cancer that causes serious healthcare burden globally. Hereby, we reviewed the current advances in cfDNA-based approaches for cancer biomarker discovery, with a focus on recent findings of cfDNA-based early detection of HCC. Future clinical investigations and trials are warranted to further validate these approaches for early detection of HCC, which will contribute to more effective prevention, control, and intervention strategies with the ultimate goal of reducing HCC-associated mortality. This article is categorized under: Cancer > Genetics/Genomics/Epigenetics.
Article
The discovery of cell-free fetal DNA (cffDNA) in maternal blood and the rapid development of massively parallel sequencing have revolutionized prenatal testing from invasive to noninvasive. Noninvasive prenatal screening (NIPS) based on cffDNA enables the detection of fetal trisomy through sequencing, comparison, and bioassays. Its accuracy is better than that of traditional screening methods, and it is the most advanced clinical application of high-throughput sequencing technologies. However, the existing sequencing methods are limited by high costs and complex sequencing procedures. These limitations restrict the availability of NIPS for pregnant women. Many amplification methods have been developed to overcome the limitations of sequencing methods. The rapid development of non-sequencing methods has not been accompanied by reviews to summarize them. In this review, we initially describe the detection principles for sequencing-based NIPS. We summarize the rapidly evolving amplification technologies, focusing on the need to reduce costs and simplify the procedures. To ensure that the testing systems are feasible and that the testing processes are reliable, we expand our vision to the clinic. We evaluate the clinical validity of NIPS in terms of sensitivity, specificity, and positive predictive value. Finally, we summarize the application guidelines and discuss the corresponding quality control methods for NIPS. In addition to cffDNA, extracellular vesicle DNA, RNA, protein/peptide, and fetal cells can also be detected as biomarkers of NIPS. With the development of prenatal testing, NIPS has become increasingly important. Notably, NIPS is a screening test instead of a diagnostic test. The testing methods and procedures used in the NIPS process require standardization.
Preprint
Blood-based, or 'liquid,' biopsies enable minimally invasive diagnostics but have limits on sensitivity due to scarce cell-free DNA (cfDNA). Improvements to sensitivity have primarily relied on enhancing sequencing technology ex vivo. Here, we sought to augment the level of circulating tumor DNA (ctDNA) detected in a blood draw by attenuating the clearance of cfDNA in vivo. We report a first-in-class intravenous DNA-binding priming agent given 2 hours prior to a blood draw to recover more cfDNA. The DNA-binding antibody minimizes nuclease digestion and organ uptake of cfDNA, decreasing its clearance at 1 hour by over 150-fold. To improve plasma persistence and limit potential immune interactions, we abrogated its Fc-effector function. We found that it protects GC-rich sequences and DNase-hypersensitive sites, which are ordinarily underrepresented in cfDNA. In tumor-bearing mice, priming improved tumor DNA recovery by 19-fold and sensitivity for detecting cancer from 6% to 84%. These results suggest a novel method to enhance the sensitivity of existing DNA-based cancer testing using blood biopsies.
Chapter
Retinoblastoma is the prototypic genetic cancer, revealing much about the fundamentals of cancer genomics over the past 50 years. The majority of retinoblastoma tumors are initiated by biallelic loss (M1 and M2) of the retinoblastoma tumor suppressor gene, with progression driven by subsequent recurrent genomic changes (M3 to Mn). Knowledge of retinoblastoma genetics has transformed all aspects of clinical care. High-sensitivity molecular RB1 testing and counseling stratify both ocular and second cancer risks for the proband and family members. Prenatal diagnosis of familial retinoblastoma enables early detection of tumors and improves outcomes. Aqueous humor is a promising “liquid biopsy” with potential diagnostic and prognostic applications. Current advances in retinoblastoma genomics will pave the way toward individualized, precision medicine in which early intervention follows subclinical detection. Outcomes for children and families will be optimized through collaborative, multicenter, prospective clinical trials, targeted molecular therapies, and genotypic prognostication.
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Zusammenfassung Eine Alloimmunisierung aufgrund einer Blutgruppeninkompatibilität, insbesondere des Rhesusgens, ist eine seltene, meist schwerwiegende Komplikation in der Schwangerschaft, die unbehandelt zum intrauterinen Fruchttod führen kann. Bereits im ersten Trimenon sollten daher eine Blutgruppenbestimmung und ein Antikörpersuchtest durchgeführt werden. Ziel dieser Screeninguntersuchung ist es, Risikoschwangerschaften aufgrund mütterlicher irregulärer Antikörper frühzeitig zu erkennen. Durch die Einführung der standardisierten prä- und postpartalen Anti-D-Immunprophylaxe bei Rh(Rhesus)D-negativen Schwangeren konnte die RhD-Alloimmunisierung deutlich gesenkt werden. Inzwischen bietet zudem die nichtinvasive Testung an „cell free fetal DNA“ im mütterlichen Blut die Möglichkeit, fetale Rh-Antigene bereits präpartal zu bestimmen. Dadurch eröffnet sich die Möglichkeit, das Risikokollektiv weiter einzugrenzen und selektiv eine gezielte Anti-D-Immunprophylaxe anzubieten. Bei positivem fetalem Rh-D-Nachweis wird bei RhD-negativen Schwangeren in der Regel bei 28–30 Schwangerschaftswochen eine Anti-D-Immunprophylaxe verabreicht. Hat trotzdem eine Alloimmunisierung mit erhöhtem Risiko für eine fetale Anämie stattgefunden, ist eine Zuweisung in ein Perinatalzentrum oder zu einen fetomaternalen Spezialisten indiziert. Bei Verdacht auf eine fetale Anämie kann eine fetale Bluttransfusion per Nabelschnurpunktion lebensrettend sein.
Article
Objectives: The aim was to determine the accuracy of cell free DNA testing (cfDNA) for detecting sex chromosome aneuploidies (SCA) in singleton pregnancies. Methods: A systematic review and meta-analysis was performed to assess cfDNA accuracy for prenatal detection of 45,X, 47,XXY, 47,XXX and 47,XYY. Inclusion was restricted to studies published between January 2010 and December 2021 reporting both cfDNA and confirmatory diagnostic test results. Results: For 45,X the sensitivity was 98.8% (95%CI 94.6%-100%), specificity 99.4% (95%CI 98.7%-99.9%), and positive predictive value (PPV) 14.5% (95%CI 7.0%-43.8%). For 47,XXY the sensitivity was 100% (95%CI 99.6%-100%), specificity 100% (95%CI 99.9%-100%), and PPV 97.7% (95%CI 78.6%-100%) . For 47,XXX the sensitivity was 100% (95%CI 96.9%-100%), specificity 99.9% (95%CI 99.7%-100%), and PPV 61.6% (95%CI 37.6%-95.4%). For 47,XYY the sensitivity was 100% (95%CI 91.3%-100%), specificity 100% (95% CI 100%-100%), and PPV 100% (95%CI 76.5%-100%) . All four SCAs had estimated negative predictive values (NPV) exceeding 99.99%, though false negatives were reported. Conclusions: This analysis suggests cfDNA is a reliable screening test for SCA, though both false negatives and false positives were reported. These estimates of test performance are derived from pregnancies at high pretest risk for aneuploidy, limiting the generalizability to average risk pregnancies. This article is protected by copyright. All rights reserved.
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Background: Poor knowledge and the lack of deliberation have been cited as reasons for women making uninformed choices about aneuploidy screening. Adequate pre-test counselling is of particular importance where non-invasive prenatal screening (NIPS) is being increasingly offered as a primary screening test. Design: Women attending the antenatal clinic with a singleton pregnancy below 14 weeks were randomised to receive routine counselling or the intervention-a 16-min educational video on aneuploidy screening before their consult. The primary outcome, rate of informed choice, was assessed using an adapted multidimensional measure of informed choice questionnaire, where informed choice was defined as good knowledge and value-consistent behaviour. Secondary outcomes included informed choice with deliberation, decisional conflict and anxiety. Results: Two hundred and eighty-six women were recruited. 69.8% of women in the intervention group made an informed choice compared with 53.6% in the control group (Risk Ratio [RR] 1.30, p = 0.014). A significantly higher number of women in the intervention group had good knowledge compared to controls (81% vs. 60.9%; RR 1.33, p = 0.001). Decisional conflict did not differ between groups, but women in the intervention group had higher anxiety scores (p < 0.001). Conclusion: The study intervention was effective in helping women make informed choice. Qualitative studies to determine the reason for increased anxiety are needed. Trial registration: Trial registry: ClinicalTrials.gov; Identifier: NCT05492981.
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Pancreatic ductal adenocarcinoma (PDAC) is one of the most cancers. Its 5-year survival rate is very low. The recent induction of neoadjuvant chemotherapy and improvements in chemotherapy for patients with pancreatic cancer have resulted in improved survival outcomes. However, the prognosis of pancreatic cancer is still poor. To dramatically improve the prognosis, we need to develop more tools for early diagnosis, treatment selection, disease monitoring, and response rate evaluation. Recently, liquid biopsy (circulating free DNA, circulating tumor DNA, circulating tumor cells, exosomes, and microRNAs) has caught the attention of many researchers as a new biomarker that is minimally invasive, confers low-risk, and displays an overall state of the tumor. Thus, liquid biopsy does not employ the traditional difficulties of obtaining tumor samples from patients with advanced PDAC to investigate their molecular biological status. In addition, it allows for long-term monitoring of the molecular profile of tumor progression. These could help in identifying tumor-specific alterations that use the target structure for tailor-made therapy. Through this review, we highlighted the latest discoveries and advances in liquid biopsy technology in pancreatic cancer research and showed how it can be applied in clinical practice.
Chapter
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Chapter
With significant improvements in prenatal testing and screening options, structural birth defects and genetic conditions are increasingly identified in utero. This creates an opportunity for expectant parents to make informed decisions about the pregnancy and to optimize obstetrical and neonatal management. Options available to expectant parents can include termination of the pregnancy, alterations in perinatal care and delivery venue to allow for complex neonatal care in the proper setting, and fetal intervention in appropriately selected cases. Pediatric subspecialists, commonly surgical subspecialists, are often involved in prenatal counseling for families whose unborn child has been diagnosed with a structural or genetic abnormality. This chapter should serve as a practical guide to prenatal diagnosis and genetic counseling.
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Cell-free fetal DNA (cffDNA) is released into the maternal circulation from trophoblastic cells during pregnancy, is detectable from 4 weeks and is representative of the entire fetal genome. The presence of this cffDNA in the maternal bloodstream has enabled clinical implementation of non-invasive prenatal diagnosis (NIPD) for monogenic disorders. Detection of paternally inherited and de novo mutations is relatively straightforward, and several methods have been developed for clinical use, including quantitative polymerase chain reaction (qPCR), and PCR followed by restriction enzyme digest (PCR-RED) or next-generation sequencing (NGS). A greater challenge has been in the detection of maternally inherited variants owing to the high background of maternal cell-free DNA (cfDNA). Molecular counting techniques have been developed to measure subtle changes in allele frequency. For instance, relative haplotype dosage analysis (RHDO), which uses single nucleotide polymorphisms (SNPs) for phasing of high- and low-risk alleles, is clinically available for several monogenic disorders. A major drawback is that RHDO requires samples from both parents and an affected or unaffected proband, therefore alternative methods, such as proband-free RHDO and relative mutation dosage (RMD), are being investigated. cffDNA was thought to exist only as short fragments (<500 bp); however, long-read sequencing technologies have recently revealed a range of sizes up to ∼23 kb. cffDNA also carries a specific placental epigenetic mark, and so fragmentomics and epigenetics are of interest for targeted enrichment of cffDNA. Cell-based NIPD approaches are also currently under investigation as a means to obtain a pure source of intact fetal genomic DNA.
Thesis
La surveillance de la grossesse, aujourd'hui en France, allie trois dimensions clinique, biologique et échographique concernant la mère et le fœtus. La médicalisation de la société, illustrée par la médicalisation de la grossesse et de la naissance, questionne les choix en matière de politique de santé. Nous pensons que les pratiques médicales et les décisions sociétales, évoluent vers une normalisation. Le suivi de grossesse peut en être une illustration. Cette évolution suscite des questionnements éthiques quant aux objectifs et aux enjeux des politiques périnatales. Quelle place laisserons-nous à la singularité ? Nous souhaitons montrer, par ce travail, les différents facteurs qui transforment les pratiques médicales et les politiques de santé périnatale et comment cette évolution suscite une réflexion d'ordre éthique. Dans chaque partie, la dimension historique nous aidera, à comprendre comment s'est mis en place le suivi prénatal actuel, à la faveur des avancées scientifiques et techniques, mais aussi des transformations sociales, puis, nous évoquerons les questionnements éthiques suscités par cette évolution. Nous nous positionnons, en tant que sage-femme, notre réflexion sur le suivi prénatal des femmes présentant une grossesse de déroulement normal. Pour cela, la distinction est nécessaire entre "dépistage prénatal" et "diagnostic prénatal". Ce dernier concerne les grossesses pathologiques et fait l'objet de nombreux travaux. Dans les deux premières parties, nous évoquons les images du fœtus avec l'essor de l'échographie et les examens biologiques. Les évolutions techniques sont considérables. Les nouvelles pratiques apportent leur lot de nouveaux questionnements : l'échographie créé de nouvelles attentes, lieux de tension et d'incompréhension entre les attentes médicales et parentales ; les marqueurs sériques et tests génétiques favorisent une démarche prédictive, qui suscitent des craintes de dérives eugéniques. Nous finissons en approfondissant les notions de normalité et de pathologie avec Georges Canguilhem et en questionnant l'évolution de la médecine occidentale devenue prédictive au risque d'une normalisation silencieuse
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Cancer treatment is improving, yet patient recovery remains a challenge. Cancer induces the disruption of the negative feedback mechanism of the proliferating signal, excessive proliferative signalling that triggers cell senescence, induction of angiogenesis, and hypoxia. Nanomedicine displays many advantages over conventional cancer therapies and provides new opportunities for early detection, improved treatment, and diagnosis of cancer. Cancer nanomedicines allow to deliver drugs at the target site with lower systemic toxicity. The tumor microenvironment and biological processes influence nanoparticle-protein interactions, blood circulation, and tumor penetration.
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Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a common cause of severe thrombocytopenia in newborns. Intracranial bleeding may lead to severe neurological sequelae and mortality. Current management of pregnancies at risk is suboptimal. Prenatal FNAIT diagnosis commonly requires invasive procedures and therapy is associated with a high treatment burden. The present review explores advances in the field and their potential contribution to modification of the diagnostic and therapeutic landscape. Topics addressed include the role of noninvasive prenatal testing using fetal cell free DNA, insights into novel and prospective therapeutic options achieved through the development of murine models of FNAIT as well as the forecast for the progress in pregnancy risk stratification through advancement in the investigation of biological characteristics of alloantibodies and their association with the risk of fetal bleeding.
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Chapter
Obstetric hematology is a fast-growing area of medicine covering the diagnosis and management of hematological problems of pregnancy. Comprehensive in approach, The Obstetric Hematology Manual addresses the many hematological conditions that can cause serious problems in pregnancy, delivery and the post-partum period for both mother and baby. Written by a team of international authorities, this text provides up-to-date, evidence-based guidelines on best care, as well as sound advice based on the experience and opinion of experts. Where appropriate, basic principles are discussed to clarify the rationale for management, and systems and procedures for disease prevention are highlighted. Many conditions and cases are discussed, including venous thromboembolism, pre-eclampsia, anemia, thrombocytopenia and inherited disorders. This book will appeal to both trainees and practitioners in obstetrics, obstetric medicine, obstetric anesthesia and hematology. It is also an accessible text for midwives, nurses, and laboratory staff.
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Molecular diagnosis of hemoglobin (Hb) Lepore-Boston in the fetus was successfully accomplished using maternal blood as a source for fetal cells in three pregnancies at risk for beta-thalassemia/Hb Lepore disease. Taking advantage of the possibility of amplifying Lepore-specific DNA fragments by polymerase chain reaction and of families in which Hb Lepore was inherited by the paternal side, we demonstrated in two cases and excluded in one case the presence of this hemoglobinopathy in the fetus directly on maternal DNA. The diagnosis was concordant with that obtained by traditional approaches in all three cases. Our results unequivocally show that nucleated fetal cells are present in maternal blood during pregnancy, and demonstrate for the first time that prenatal diagnosis of a genetic disease may be feasible without invasive procedures.
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Molecular diagnosis of hemoglobin (Hb) Lepore-Boston in the fetus was successfully accomplished using maternal blood as a source for fetal cells in three pregnancies at risk for beta-thalassemia/Hb Lepore disease. Taking advantage of the possibility of amplifying Lepore- specific DNA fragments by polymerase chain reaction and of families in which Hb Lepore was inherited by the paternal side, we demonstrated in two cases and excluded in one case the presence of this hemoglobinopathy in the fetus directly on maternal DNA. The diagnosis was concordant with that obtained by traditional approaches in all three cases. Our results unequivocally show that nucleated fetal cells are present in maternal blood during pregnancy, and demonstrate for the first time that prenatal diagnosis of a genetic disease may be feasible without invasive procedures.
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To review the rationale for and progress toward the goal of isolating and analyzing fetal cells circulating in maternal blood, and to explore the feasibility of this method in providing noninvasive prenatal cytogenetic diagnosis. Critical review of data published since the first report (1969) of fetal metaphases in maternal blood. Emphasis is placed on data since the demonstration by polymerase chain reaction (PCR) in 1989 and 1990 that fetal cells indeed exist in maternal blood. Clinical evaluations have not yet been conducted, but it is already clear that molecular technologies have allowed the unequivocal demonstration of fetal cells in maternal blood. Using PCR, our own group and others have demonstrated Y sequences and single gene sequences (eg, hemoglobin LeporeBoston) in maternal blood. Thus, fetal DNA sequences indeed exist in maternal blood. Among the various candidate cells, the most promising appear to be fetal nucleated red blood cells. We isolated nucleated red blood cells on the basis of flow-sorting for the transferrin receptor and glycophorin-A. Enriched samples were then subjected to fluorescence in situ hybridization with chromosome-specific probes. This approach allowed us to detect trisomy 21 and trisomy 18, work later confirmed by others. Isolating and analyzing fetal cells from maternal blood is clearly possible. Several key biologic questions remain--the optimal cells for isolation, frequency of cells in maternal blood, timing during gestation for maternal blood sampling, and the likelihood of persistence of fetal cells after delivery. Clinical evaluations planned by the National Institute of Child Health and Human Development will determine the sensitivity and specificity of this method and its precise role in prenatal cytogenetic diagnosis.
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Microsatellite instability is an important characteristic of many tumor types especially those associated with hereditary non-polyposis colorectal carcinoma (HNPCC) syndrome. Microsatellite alterations in 50% of primary small cell lung carcinoma (SCLC) have been found. These alterations were also found in the sputum. Because neoplastic characteristics such as decreased strand stability9 and ras mutations have been found in the plasma DNA of cancer patients, we looked for microsatellite alterations in the plasma of SCLC patients. A microsatellite alteration was present in 16 out of 21 (76%) SCLC tumors and in 15 out of 21 (71%) plasma samples. In one case, the alteration was present only in the plasma DNA. If confirmed in larger studies, microsatellite analysis of plasma DNA might constitute a new tool for tumor staging, management and, possibly, detection.
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Currently, amniocentesis, chorionic villus sampling (CVS) and fetal blood sampling are used to obtain fetal cells for genetic diagnosis. These invasive procedures pose a small but not negligible risk for the fetus. Efforts have been directed towards the enrichment of fetal cells, such as erythroblasts, from maternal blood and progress has been made in the diagnosis of some chromosomal disorders and in sex determinations. We now report the detection of point mutations in single gene disorders using this method of prenatal diagnosis by enriching fetal cells from maternal blood by magnetic cell sorting followed by isolation of pure fetal cells by microdissection. In two pregnancies at risk for sickle cell anaemia and beta-thalassaemia, we successfully identified the fetal genotypes. Thus, prenatal diagnosis of single gene disorders by recovering fetal cells from maternal circulation appears to be a feasible approach.
Prenatal sex determination by DNA amplification from maternal peripheral blood
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Two-way cell traffic between mother and fetus biologic and clinical implications
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