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Open Access

Review

Cell-free placental DNA: What do we really know?

Abstract

Cell-free placental DNA (cfpDNA) is present in maternal circulation during gestation. CfpDNA carries great potential as a research and clinical tool as it provides a means to investigate the placental (epi)genome across gestation, which previously required invasive placenta sampling procedures. CfpDNA has been widely implemented in the clinical setting for noninvasive prenatal testing (NIPT). Despite this, the basic biology of cfpDNA remains poorly understood, limiting the research and clinical utility of cfpDNA. This review will examine the current knowledge of cfpDNA, including origins and molecular characteristics, highlight gaps in knowledge, and discuss future research directions.

Author summary

Cell-free DNA are fragments of free-floating DNA released by cells into blood circulation. During pregnancy, the placenta releases cell-free placental DNA (cfpDNA) into maternal blood circulation. CfpDNA can be collected through a routine blood draw and is used for noninvasive prenatal testing (NIPT) to detect risk of the fetus having genetic abnormalities. There remains great untapped potential for the use of cfpDNA to screen not only for a wider range of fetal genetic abnormalities, but also for pregnancy complications affecting the health of both the fetus and pregnant person. This is currently hindered by a poor understanding of the “normal” characteristics of cfpDNA and how exactly it is released from placenta cells. In this review, we will summarize what is currently known about cfpDNA, highlight what remains unclear, and discuss future research directions for advancing the utility of cfpDNA in both research and the clinic.

Citation: Yuen N, Lemaire M, Wilson SL (2024) Cell-free placental DNA: What do we really know? PLoS Genet 20(12): e1011484. https://doi.org/10.1371/journal.pgen.1011484

Editor: Michael Snyder, Stanford University School of Medicine, UNITED STATES OF AMERICA

Published: December 9, 2024

Copyright: © 2024 Yuen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The authors received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Cell-free placental DNA (cfpDNA) is shed into maternal circulation throughout pregnancy [1]. CfpDNA has been detected as early as the fourth week of gestation and has been estimated to comprise up to approximately 40% of total maternal plasma cell-free DNA [2–4]. Since its discovery in 1997, cfpDNA has been used in the clinical setting for noninvasive prenatal testing (NIPT) to screen for fetal genetic aberrations, most notably chromosomal aneuploidies. However, the understanding of the basic biology underlying cfpDNA remains limited, placing constraints on the information that can be obtained from analyzing cfpDNA. Gaps in knowledge include cfpDNA fragmentation processes, release mechanisms, form, and function. CfpDNA carries the potential for real-time study and monitoring of placental health and development. Further characterization of physical, genetic, and epigenetic characteristics of cfpDNA will pave the way for further basic research and clinical applications. In this review, we will examine the current understanding of cfpDNA, highlight gaps in knowledge, and discuss future research directions (Fig 1). While this review focuses on pregnancy, much of what is discussed applies to the cell-free DNA field in general, including other clinical contexts such as cancer.

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Fig 1. The current knowledge surrounding cfpDNA.

https://doi.org/10.1371/journal.pgen.1011484.g001

Placental development, anatomy, and dysfunction

The placenta is a transient organ that forms during gestation. It facilitates the exchange of molecules between the fetal and maternal bloodstreams to support the physiological needs of the developing feto-placental unit. The placenta arises from the trophectoderm layer of the blastocyst, which further differentiates into cytotrophoblasts, syncytiotrophoblasts, and extravillous trophoblasts [5]. Trophoblasts invade and reconstruct the innermost layer of the uterus, the endometrium (Fig 2A). Within the decidua, the differentiated and reconstructed layer of the endometrium, the invading trophoblasts develop into tree-like projections called chorionic villi (Fig 2B). Chorionic villi are composed of an inner stromal core and outer trophoblast layer [6]. Mesenchymal cells in the stromal core form capillaries which circulate fetal blood from the umbilical blood vessels [7]. The spaces between chorionic villi form the intervillous space which is eventually filled with maternal blood. Extravillous trophoblasts invade and remodel uterine spiral arteries, veins, and glands in the endometrium to establish maternal blood circulation and glandular secretion in the intervillous space [8]. Maternal blood circulation to the intervillous space is initially blocked by trophoblast plugs in the uterine vessels. At around 10 to 12 weeks of gestation, trophoblast plugs are displaced as spiral artery remodeling progresses and maternal blood flow to the intervillous space is initiated [9]. The layers of the chorionic villi, namely the trophoblasts and fetal capillary endothelium, form the placental barrier, which maintains separation of the fetal and maternal bloodstreams [6]. At the placental barrier, molecules including nutrients, gases, and hormones are exchanged between fetal and maternal blood. Various mechanisms are used by molecules to cross the placental barrier including passive and facilitated diffusion, active transport, and vesicular transport [10]. Through this exchange, oxygen and nutrients are supplied to the fetus while waste products, such as carbon dioxide, exit the fetal bloodstream to be excreted by the mother. The placenta also carries out an endocrine function by secreting hormones to signal the maternal body to maintain an environment that supports feto-placental growth throughout gestation [11].

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Fig 2. Placental development and anatomy.

(A) Early invasion of the endometrium by trophoblasts during blastocyst implantation. (B) Chorionic villus and surrounding structures at the fetal–maternal interface.

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Abnormal placental morphology and function is observed in various pregnancy complications. Preeclampsia is the new onset of maternal hypertension and proteinuria after 20 weeks of gestation which can lead to multi-organ system failure [12]. The condition is heterogeneous in the range and severity of symptoms and placental lesions. Therefore, there is not one specific cause of preeclampsia, rather multiple disease subtypes with different etiologies that are currently being elucidated [13–16]. Shallow trophoblast invasion, improper spiral artery remodeling, and the resulting poor placental perfusion has been a long-standing model for the etiology of preeclampsia [17]. It is believed that this causes placental hypoxia which leads to a systemic maternal response and endothelial dysfunction [18]. Other factors including maternal cardiovascular health are also believed to contribute to preeclampsia pathogenesis [17,18]. Preeclampsia poses serious short- and long-term risks to both the pregnant person and offspring including fetal growth restriction, cardiovascular disease later in life, and mortality [19]. Fetal growth restriction is characterized by pathologically poor fetal growth [20]. Among other factors, it can be caused by inadequate oxygen and nutrient transport to the fetus due to abnormal placentation and poor placental perfusion [21]. Fetal growth restriction impacts the development of the fetus’ organs and body systems, increasing risks of morbidities across the lifespan [22]. Therefore, proper placental development and function are critical for maternal and offspring health both during and after pregnancy.

The etiologies of many pregnancy complications remain poorly understood. Work over the past several years has revealed the roles of various molecular networks in placental development and function in both healthy and complicated pregnancies [23–25]. Advancements in understanding the molecular basis of pregnancy complications are invaluable for the creation of treatments and screening and diagnosis tools for earlier and more targeted intervention. Further research utilizing new technologies and models, such as cfpDNA, will help to drive these necessary advancements.

CfpDNA origins

Despite commonly being referred to as “cell-free fetal DNA,” there is a large body of evidence demonstrating that “cell-free fetal DNA” mainly originates from trophoblasts, the major cell type of the placenta. Therefore, it is more accurate to refer to “cell-free fetal DNA (cffDNA)” as “cell-free placental DNA (cfpDNA).” Reports of normal first trimester cfpDNA levels in anembryonic pregnancies [26], the presence of cfpDNA before the establishment of fetal circulation [27], cell-free DNA release in in vitro placental explant studies [28–32], and cases of confined placental mosaicism in which cfpDNA corresponds to trophoblast karyotypes [33–40] provide direct evidence supporting the trophoblastic origin of cfpDNA. Additionally, detection of cell-free DNA carrying placenta-specific DNA methylation patterns in maternal plasma [41–44] and correlation between the placental and cfpDNA methylomes [45, 46] solidify the placenta as the main source of cfpDNA. Reports of increased cfpDNA concentrations in conditions involving placental dysfunction, such as preeclampsia [47–49], invasive placenta (placenta accreta and increta), and placenta previa [50] point towards the placental origin of cfpDNA as these observations are hypothesized to be due to increased trophoblast cell death. In this review, we will use the term “cffDNA” when referring specifically to DNA originating from the fetus and use “cfpDNA” when referring to DNA originating from the placenta. The term “cell-free DNA” will be used to describe cell-free DNA in general, regardless of tissue of origin and both within and beyond the context of pregnancy.

Both fetal- and placenta-derived messenger RNA transcripts have been detected in maternal plasma, suggesting contributions from both the fetus and placenta to cell-free nucleic acids in maternal circulation [51–53]. While it is possible that fetal tissues and cells release cffDNA into maternal circulation, they are likely not major contributors to total maternal plasma cell-free DNA. Fetal cells such as erythroblasts have previously been hypothesized to be a source of cffDNA. However, fetal cells are present in maternal plasma in significantly lower quantities than cfpDNA (1.2 fetal cells/ml of blood versus 25.4 copies of cfpDNA/ml of plasma) [54,55]. Concentrations of fetal erythroblasts and cfpDNA are not correlated and change independently of each other [48,56]. In vitro release of cffDNA from fetal membranes with fragment sizes consistent with plasma cfpDNA has been demonstrated [32,57]. However, fetal membranes were reported to release less cell-free DNA than placental explants (15.5 ng cell-free DNA/mg of fetal membrane versus 73.7 ng cell-free DNA/mg of placenta after 1 h in culture) [32]. These findings demonstrate that fetal cells are not a major source of cell-free DNA in maternal circulation. It is difficult to differentiate fetal and placental DNA since genetic sequences are typically identical between the two. Distinction relies on comparisons of tissue-specific markers, such as gene expression and DNA methylation, or cases where there is confined placental mosaicism. Whether fetal membranes and other fetal tissues contribute a significant amount of cffDNA in vivo can be further explored by comparing fetal tissue, placental tissue, and maternal plasma cell-free DNA methylation profiles. Notably, the mechanism through which cffDNA and fetal mRNA would enter maternal circulation is unclear. It is unknown how fetal cell-free nucleic acids would cross through placental tissue (i.e., chorionic plate, chorionic villi) and into the intervillous space to access maternal circulation. The amniotic membrane is impermeable to large molecules such as cell-free DNA; therefore, cffDNA present in amniotic fluid (“CffDNA and cfpDNA in other body fluids” section) cannot leave the amniotic sac [58].

Most studies are focused on linear nuclear cfpDNA; however, mitochondrial DNA (mtDNA) is also present in maternal circulation during pregnancy. Quantitative aberrations of circulating mtDNA have been observed in pregnancy complications [59]. Using plasma from surrogate pregnancies, Ma and colleagues [60] demonstrated that placental mtDNA is present in maternal circulation. Placental mtDNA was identified by the presence of single-nucleotide variants that are not carried by the surrogate mother and therefore are specific to the oocyte donor. A majority (88%) of circulating placental mtDNA fragments were linear, in contrast to circulating maternal mtDNA, around 50% of which were circular [60]. Placental extrachromosomal circular DNA (eccDNA) is also present in maternal circulation, identified using placenta-specific single-nucleotide polymorphisms (SNPs) [61]. Fragment size distributions and DNA methylation levels of placental cell-free eccDNA have been characterized and are discussed in later sections of this review.

Detecting and quantifying cfpDNA

CfpDNA is present in maternal circulation alongside a major fraction of background maternal cell-free DNA mainly derived from white blood cells [4]. Differentiating maternal and placental cell-free DNA remains a challenge. Various methods have been developed to identify and quantify cfpDNA in maternal circulation. Some approaches directly target placenta-specific genetic markers (i.e., Y chromosome sequences [54] and SNPs [62]), while others utilize physical and epigenetic characteristics that differ between placental- and maternal-derived cell-free DNA (i.e., fragment size [63] and DNA methylation [64]). There are 2 metrics that have generally been used for quantifying cfpDNA in the literature: (1) fetal fraction; and (2) concentration. Fetal fraction is the proportion of total plasma cell-free DNA comprised of cfpDNA (% placental DNA) and is routinely measured as a quality control step for NIPT. CfpDNA concentration (copies or genome equivalents per ml of plasma) can be measured using quantitative polymerase chain reaction (qPCR). Methods used for identifying and quantifying cfpDNA are described below and summarized in Table 1.

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Table 1. Methods for detecting and quantifying cfpDNA.

https://doi.org/10.1371/journal.pgen.1011484.t001

Y chromosome sequences

qPCR quantification of Y chromosome genes including SRY and DYS14 were used in the earliest detection and measurement of cfpDNA [54]. The proportion of reads mapped to the Y chromosome by whole genome sequencing is used for fetal fraction calculation. Various approaches and algorithms have been developed based on this principle [3,65,66,68]. The proportion of Y chromosome reads may be measured relative to the total reads from that plasma cell-free DNA sample and/or compared to the percentage of Y chromosome reads in plasma cell-free DNA from adult males or pregnancies carrying XX fetuses. The major drawback of this method is that it is only applicable for pregnancies carrying XY fetuses.

SNPs and genetic variation

Placental SNP genotype can be inferred based on knowledge of parental genotypes, allowing direct identification and interrogation of individual cfpDNA fragments. Placenta- and maternal-specific SNP alleles have been used to identify and study cfpDNA separately from background maternal cell-free DNA [45,90]. Placenta-specific SNP alleles are present at SNP loci that are heterozygous in the placental genome and homozygous in the maternal genome, and vice versa for maternal-specific SNP alleles. Fetal fraction can be estimated based on the frequency of sequencing reads carrying placenta-specific SNP alleles [62,69–72,91]. Fetal fraction can also be estimated based on frequencies of insertion/deletion mutations [73].

DNA methylation

Tissue-specific DNA methylation profiles can be used to classify the tissue of origin of cell-free DNA [92]. Targeting of placenta-specific DNA methylation markers allows for identification of cfpDNA fragments, independent of fetal sex or genotype. Methylation-specific treatment (i.e., methylation-sensitive restriction digestion (MSRD), bisulfite conversion) in combination with DNA quantification methods (i.e., matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) [64], qPCR [41,42,74–76], droplet digital polymerase chain reaction (ddPCR) [75,77,78]) targeting placental differentially methylated regions have been used to quantify cfpDNA. DNA methylation-based tissue of origin deconvolution algorithms have been applied to whole genome bisulfite sequencing data from maternal plasma cell-free DNA samples to estimate cfpDNA proportions [4,93]. Limitations of these methods include sample degradation by bisulfite conversion and dependence on restriction enzyme digestion efficiency. Long-read sequencing platforms (i.e., single-molecule real-time (SMRT) sequencing) allow for bisulfite- and restriction enzyme-free interrogation of DNA methylation status (i.e., the holistic kinetic model) [94]. Yu and colleagues [79] demonstrated the use of long-read sequencing and the holistic kinetic model to identify individual placental and maternal cell-free DNA fragments based on the similarity of their DNA methylation profiles with reference methylomes.

Fragmentomics

The coverage of the genome by cell-free DNA molecules reflects the nucleosome positioning in their cells of origin (“CfpDNA fragmentomics”—“Genome coverage and nucleosome footprinting” section) [95]. Fetal fraction can be measured based on nucleosome positioning inferred from cell-free DNA sequencing data, according to differential nucleosome positioning between maternal and placental DNA [80]. Various algorithms have been developed for fetal fraction measurement that utilize differential sequencing coverage across genomic regions between cfpDNA and background maternal cell-free DNA [81–83]. Genome coordinates that are overrepresented in cell-free DNA fragment start and end coordinates, termed “preferred ends,” also demonstrate tissue specificity (“CfpDNA fragmentomics”—“Preferred ends” section) and can be used to estimate fetal fraction [84–86].

CfpDNA is on average, shorter than maternal cell-free DNA (“CfpDNA fragmentomics”—“Fragment size” section) [96]. CfpDNA size distributions show a major peak at 143 bp while maternal cell-free DNA size distributions show a major peak at 166 bp [62]. Utilizing this physical difference, fetal fraction can be measured as a function of the ratio of short and long cell-free DNA fragments in a sample [63,87]. Fragment size can be measured using gel electrophoresis [97], qPCR [96], paired-end sequencing [63], capillary electrophoresis [63], or semiconductor sequencing [87]. Size selection can also be used to enrich cfpDNA [97].

Combined methods

Studies have also demonstrated the benefit of combining predictors to estimate fetal fraction. Gazdarica and colleagues [89] reported improved accuracy of fetal fraction estimates using a model that combines sequence read counts, fragment length, gestational age, maternal body mass index, and DNA library concentration as predictors.

Absolute cfpDNA quantification

Currently published studies of cfpDNA quantity measure fetal fraction (% placental DNA) or concentration (copies or genome equivalents per ml of plasma). Fetal fraction is affected by both placental and maternal cell-free DNA quantities, and it is difficult to determine which of these factors account for changes in fetal fraction. Therefore, fetal fraction is not a measurement of cfpDNA concentration. Current methods of measuring cfpDNA concentration permit direct investigation of the dynamics of cfpDNA quantity, independent of background maternal cell-free DNA. However, quantities measured using these methods only describe the concentration of the targeted genomic region rather than the total cfpDNA concentration in maternal plasma. The latter would involve inferring total cfpDNA concentration based on the quantity of a targeted region, which works under the assumption that all genomic regions are equally represented in circulating cell-free DNA. Individual fragment-level identification and quantification of cfpDNA beyond placenta-specific genetic and methylated loci requires further characterization of tissue-specific characteristics of cell-free DNA. Absolute quantification of cfpDNA (nanograms or picomoles of DNA) would allow for direct characterization of cfpDNA quantities across gestation and in pregnancy pathologies. Absolute quantification of cell-free DNA can be achieved by using spike-in controls during sequencing protocols [98,99].

CfpDNA release and clearance

Apoptosis

Apoptosis is involved in the normal development and cellular turnover of the placenta [100] and it is hypothesized that this contributes cfpDNA into maternal circulation. Mechanistic links between apoptosis and cfpDNA release have been investigated in vitro using placental explant studies. Parallel increases in apoptosis and cfpDNA release over time in mouse placental explants under physiologic oxygen levels (8% O2) have been reported [29]. Hypoxia-induced oxidative stress (hypoxia (0.5% O2) followed by reoxygenation (10% O2)) was found to significantly increase apoptosis and cfpDNA release, compared to normoxic conditions (10% O2) in human term placental explants [28]. Hydrogen peroxide- and hyperoxia (21% O2)-induced oxidative stress did not increase apoptosis or cfpDNA release in mouse placental explants [29]. One study reported significantly increased apoptosis and cfpDNA release in mouse placental explants following stimulation of inflammation by lipopolysaccharide [29], while other studies found no increase in either apoptosis nor cfpDNA release in human term placental explants following lipopolysaccharide treatment [30,31]. Induction of sterile inflammation in human placental explants significantly increased cfpDNA release without a significant increase in apoptosis, indicating non-apoptotic contributions to cfpDNA release [31]. Apoptosis stimulation (doxorubicin) significantly increased apoptosis and cfpDNA release in human term placental explants [31]. Suppression of apoptosis by caspase inhibition significantly decreased apoptosis and cfpDNA release in mouse placental explants [29]. These studies demonstrate that apoptosis, among other release mechanisms, generates cfpDNA and that factors inducing apoptosis can lead to increased cfpDNA shedding from the placenta. Notably, placental hypoxia and inflammation are characteristics of preeclampsia [101,102]. Increased placental apoptosis in response to these factors may contribute to the increased cfpDNA levels in preeclampsia [47]. The presence of cfpDNA in apoptosis-derived placental extracellular vesicles (syncytiotrophoblast microparticles and apoptotic bodies) that were generated in vitro [103,104] or collected from maternal plasma [103,105] provide further evidence for the apoptotic release of cfpDNA. The fragment size of cfpDNA (∼150 to 200 bp) is consistent with the conserved DNA fragmentation patterns of apoptosis. Specifically, the modal fragment size of cfpDNA (143 to 200 bp) is equivalent to the length of DNA that wraps around a nucleosome, reflecting the internucleosomal cleavage that occurs during apoptosis [96,106].

Necrosis

Necrosis is known to be another release mechanism of cell-free DNA. Placental necrosis is implicated in pregnancy pathologies such as preeclampsia [107]. Whether necrosis contributes to the increased cfpDNA in preeclamptic pregnancies [47] and the extent of this contribution is unknown. In vitro studies investigating cfpDNA release from placental explants have suggested necrosis-mediated cfpDNA generation [28,29,103]. In contrast to apoptosis, necrosis involves random DNA fragmentation and is believed to generate longer cell-free DNA fragments (>10,000 bp) [108]. The relationship between fragment length and cell death mechanism (apoptosis versus necrosis) may not be as clear cut as it is presented in the literature. Long-read sequencing has uncovered a significant quantity of long plasma cell-free DNA (>500 bp) that was previously undetectable using short-read sequencing platforms [79,109]. Notably, long cell-free DNA shows size distribution patterns (200 bp periodicity) reflective of multi-nucleosomal units, therefore consistent with apoptotic internucleosomal DNA fragmentation [79,109]. Therefore, the relationships between cell-free DNA fragmentation patterns and cell death mechanisms need to be further investigated.

Exosomes

Exosomes are actively released by cells to carry out intracellular communication and are another source of cell-free DNA [110]. Placental exosomes can be detected in maternal circulation from the sixth week of gestation and quantities increase with gestational age [111,112]. Placental exosomes are identified by detection of the surface protein, placental alkaline phosphatase (PLAP) [112,113]. Y chromosome sequences can be detected in exosome-bound DNA isolated from maternal plasma, demonstrating the presence of exosome-bound cfpDNA in maternal circulation, although concentrations of exosome-bound cfpDNA were on average, 10 to 11 times lower than free cfpDNA concentrations [114–116]. Exosomes are attractive sources of cfpDNA for NIPT due to the increased stability of extracellular vesicle-bound cell-free nucleic acids [117]. Preliminary studies have demonstrated successful noninvasive fetal sex determination, RHD genotyping, and detection of fetal trisomy and monogenic diseases using exosomal DNA isolated from maternal plasma [114,118]. Placental exosomes have been suggested to be involved in the onset of parturition [119–121]. Whether the cfpDNA encapsulated in placental exosomes is involved in this process is unknown.

CfpDNA clearance

Following delivery, cfpDNA is rapidly cleared from maternal plasma [122,123]. CfpDNA clearance occurs in a biphasic pattern. The first phase takes place during the first 2 h after delivery and involves rapid clearance, with an average cfpDNA half-life of 1 h [123]. The second phase involves slower clearance, with an average cfpDNA half-life of 13 h [123]. This pattern suggests the involvement of different cfpDNA clearance mechanisms across the 2 phases [123]. CfpDNA generally reaches undetectable levels by 2 days postpartum [123]. Cell-free DNA clearance is believed to be mediated by plasma endonucleases and organs such as the liver, kidney, and spleen [124]. Plasma endonucleases and transrenal clearance were found to both be minor contributors to the elimination of cfpDNA [123]. CfpDNA clearance is compromised in preeclampsia, as demonstrated by increased cfpDNA half-lives (median half-life of 114 min in preeclampsia versus 28 min in healthy controls) and persistence of cfpDNA postpartum at time points when normally undetectable in maternal plasma [125]. This clarifies a mechanism for the increased cfpDNA levels observed in preeclampsia [47] and provides support for roles of the kidneys and liver, which are affected by preeclampsia, in normal cfpDNA clearance. Further research on the clearance mechanisms of cfpDNA is required. Placental cell-free eccDNA also demonstrates rapid postpartum clearance from maternal plasma and has comparable half-lives to linear cfpDNA [126].

CfpDNA release and clearance mechanisms: Future research directions

Despite widespread clinical implementation, the exact mechanisms underlying cfpDNA generation and release remain largely unknown. Further in vitro and in vivo studies directly studying cfpDNA release are required to more definitively identify the cell death and release mechanisms involved, the proportions which they contribute, and how these change across different conditions. Other forms of cell death aside from apoptosis and necrosis, such as pyroptosis [127,128], ferroptosis [129], aponecrosis [103], and necroptosis [130] have been implicated in placental dysfunction and should also be investigated as potential sources of cfpDNA. In vivo studies will be beneficial for understanding dynamics of cfpDNA release and clearance and how the balance between these processes affect cfpDNA levels across gestation and in pregnancy pathologies. Knowledge of “normal” cfpDNA characteristics and dynamics in healthy pregnancies is required to properly characterize aberrant patterns in pregnancy complications. Characterization of the specific cfpDNA features generated by different release mechanisms can be leveraged to provide real-time insights into the state of the placenta. This can be a powerful research tool for longitudinal studies of placental development and disease, which are currently hindered by ethical constraints and limited availability of early- and mid-gestation tissue samples. This will open avenues for many more clinical applications of cfpDNA beyond the current NIPT repertoire, which is limited to genetic screening of the fetus. Overall, an improved understanding of the basic biological mechanisms underlying cfpDNA is required to fully leverage it as a tool for assessing placental health and screening for complex pregnancy complications.

CfpDNA genomics

CfpDNA is representative of the full placental genome [62], providing a means for noninvasive genome-wide characterization of the placenta. NIPT has been widely implemented to screen for common fetal aneuploidies including Trisomies 13, 18, and 21 [131] by assessing for overrepresentation of the affected chromosomes in maternal plasma cell-free DNA [3,132]. The effectiveness of NIPT for aneuploidy screening has recently been reviewed elsewhere [133–135].

Efforts are being made to expand NIPT to include the detection of sub-chromosomal aberrations such as copy number variations and microdeletions [87,135–137]. Methods for detecting fetal de novo mutations, paternal inheritance, and maternal inheritance for the diagnosis of monogenic disorders have been summarized elsewhere [138,139], and these methods have been used in efforts to reconstruct full placental genomes [62,84,140,141]. The ongoing development of long-read sequencing platforms will open opportunities for detecting larger-scale genomic aberrations such as recombination events, structural variants, and repeat expansions [79,109,142].

Most studies using cfpDNA to noninvasively investigate the placental genome are focused on NIPT applications. Notably, they are geared towards using cfpDNA to determine fetal genotypes. While fetal and placental genome sequences are generally identical, confined placental mosaicism can occur [33]. In such cases, the fetal and placental genomes do not match, highlighting the importance of making the distinction between cell-free “fetal” DNA and cell-free “placental” DNA, especially when discussing clinical applications of cfpDNA. A future research direction is to explore the use of cfpDNA for noninvasive investigation of placental genomic aberrations in the context of placental dysfunction and pregnancy complications [143].

CfpDNA methylation

DNA methylation is an epigenetic modification involved in the modulation of gene expression. It demonstrates tissue specificity [144] and plays an important role in development, health, and disease [145,146].

The placenta methylome is characterized by global hypomethylation, tissue-specific methylated CpG islands, large spans of intermediate methylation called partially methylated domains, and placenta-specific imprinted regions [147]. Studies have demonstrated concordance between cfpDNA and placental methylomes [45,46,148]. Lun and colleagues [45] used whole genome bisulfite sequencing to construct a cfpDNA methylome from a maternal plasma cell-free DNA sample by sub-setting cell-free DNA reads carrying placenta-specific alleles at informative SNP loci. Similarly, they constructed the “shared” methylome by identifying cell-free DNA reads containing alleles carried by both the fetus and mother at informative SNP loci [45]. Consistent with known patterns of placental DNA methylation, overall DNA methylation levels were lower in cfpDNA relative to shared cell-free DNA, and increased with gestational age [45]. This pattern was observed genome-wide, across repeat and non-repeat regions, autosomes, and the X chromosome [45]. CpG methylation densities were correlated between cfpDNA and placenta, and the shared methylome and maternal blood cells [45]. This is consistent with the placental origin of cfpDNA and the hematopoietic origin of background maternal cell-free DNA [26,46]. At the time of writing this review, this is the only publication that has separated cfpDNA from background maternal cell-free DNA to study cfpDNA methylation. Consistent with these findings, Jensen and colleagues [46] reported reduced genome-wide CpG methylation in total maternal cell-free DNA compared to non-pregnant cell-free DNA using whole genome bisulfite sequencing. Pregnant and non-pregnant cell-free DNA were differentially methylated in regions that were also differentially methylated between placenta and non-pregnant cell-free DNA [46]. Chu and colleagues [148] reported similar findings using targeted bisulfite sequencing in first trimester pregnant and non-pregnant cell-free DNA samples. Their study revealed a reduction in overall cell-free DNA CpG methylation in pregnant plasma compared to non-pregnant plasma. The identity, magnitude, and directionality of differentially methylated CpGs identified between pregnant cell-free DNA and non-pregnant cell-free DNA were consistent with those identified when comparing chorionic villus and maternal leukocyte DNA [148]. More dramatic differences were observed in intron and exon regions compared to regulatory elements, consistent with previous reports of increased DNA methylation in regulatory elements in placental DNA, despite global hypomethylation compared to other tissues [148]. Placenta-derived cell-free eccDNA displays lower methylation density compared to maternal-derived cell-free eccDNA, consistent with the patterns observed in linear cfpDNA [126].

DNA methylation-based tissue of origin deconvolution algorithms have been developed using reference methylomes for different tissues to estimate proportions of tissue contributions to circulating cell-free DNA. Inclusion of placenta reference methylomes allows for estimation of the proportion of cfpDNA in maternal plasma and investigation of how relative tissue contributions to cell-free DNA change across gestation and in pregnancy pathologies [4,93,149]. DNA methylation patterns can also be used to identify the placental or maternal origin of individual cell-free DNA molecules sequenced using long-read sequencing platforms [79].

Identification of DNA methylation markers for the detection of cfpDNA has been an active area of research ever since its discovery. Such markers are applicable for identifying cfpDNA in all pregnancies regardless of fetal sex and genotype. Evidence points towards the hematopoietic origin of background maternal cell-free DNA [4,150], therefore, genomic regions that are differentially methylated between placenta and white blood cells are investigated as candidate markers. Table 2 lists cfpDNA methylation markers that have been validated in maternal plasma. CfpDNA carries placental genome imprinting status, and this has also been proposed as a method for differentiating cfpDNA from background maternal cell-free DNA [45,151].

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Table 2. CfpDNA methylation markers validated in maternal plasma.

https://doi.org/10.1371/journal.pgen.1011484.t002

CfpDNA methylation carries great potential as a tool for research and noninvasive screening. Utilizing cfpDNA to characterize and detect disease-specific placental DNA methylation changes across gestation is a future research direction that would advance the understanding of placental pathologies and greatly broaden the potential applications of NIPT [161–163]. This is significant since it is difficult to obtain placental tissue samples during pregnancy for research and screening purposes as this requires invasive procedures such as chorionic villus sampling. Investigation of other epigenetic modifications, such as hydroxymethylation, in cfpDNA is another research direction to pursue [164].

Circulating placental nucleosomes and histone modifications

Cell-free DNA can circulate in multiple forms: naked DNA, circulating nucleosomes, or bound to extracellular vesicles, proteins, and histones [114,165]. Circulating nucleosomes and associated histone modifications have been studied as noninvasive biomarkers for cancer, liver transplant rejection, and idiopathic pulmonary fibrosis [166–168]. Histone modifications play roles in placental development and pregnancy complications [169–173]. Investigation of circulating placental nucleosomes and associated histone modifications can provide tools for studying placental health and development noninvasively throughout gestation. Bouvier and colleagues [174] demonstrated that circulating nucleosome levels in maternal plasma increases with gestational age and in preeclampsia, although proportions of maternal- and placenta-derived nucleosomes were unknown. While knowledge of circulating placental nucleosomes in maternal plasma is limited, information on placental nucleosome positioning is carried by cfpDNA (“CfpDNA fragmentomics”—“Genome coverage and nucleosome footprinting” section). Patterns of cell-free DNA fragmentation are reminiscent of nucleosome structure [62,79,85], and genome coverage by cell-free DNA is informative of nucleosome positioning in the tissue of origin [95].

CfpDNA fragmentomics

Fragmentomics is a new area of research that studies the mechanisms underlying cell-free DNA fragmentation and the specific characteristics that are generated. As demonstrated by tissue- and disease-specific fragmentomic patterns, the process of cell-free DNA fragmentation is non-random. Fragmentomic characteristics that have been identified and studied include fragment size, end motifs, genome coverage, preferred ends, and jagged ends.

Fragment size

The fragment size distribution of cfpDNA is distinct from that of background maternal cell-free DNA. CfpDNA is generally shorter than background maternal cell-free DNA, made evident by a shift towards a shorter size distribution and depletion of longer fragments [3,62,63,96,97,175]. This trend is observed not only in linear nuclear cfpDNA, but also circulating eccDNA [61] and mtDNA [60]. The size distribution of background maternal cell-free DNA shows a major peak at 166 bp, corresponding to the length of DNA wrapped around a nucleosome core (∼147 bp) plus adjacent linker regions (modal length of ∼20 bp) [62,176]. The cfpDNA size distribution shows a reduced 166 bp peak and a major peak is observed at 143 bp instead [62]. This corresponds to the length of DNA wrapping around a nucleosome core only [62,176]. The 10 bp periodicity observed below 150 bp in cfpDNA is hypothesized to be due to trimming of nucleosome-bound DNA by nucleases [62,176]. A minor peak at approximately 320 bp has also been observed in cfpDNA size distributions, likely representing dinucleosomal units [79,175].

Using long-read sequencing, Yu and colleagues [79] discovered a population of long cell-free DNA (>500 bp) in maternal plasma. Detection of these fragments is typically beyond the technical limits of short-read sequencing platforms. Long cell-free DNA fragments were present in both maternal and placental cell-free DNA pools, which were distinguished using SNP genotyping [79]. Consistent with previous observations of cfpDNA being shorter than background maternal cell-free DNA, the proportion of long cell-free DNA was reduced in cfpDNA compared to background maternal cell-free DNA [79]. Long fragments comprised greater than 20% of cfpDNA and this proportion increased with gestational age [79]. The longest cfpDNA molecule was 23,635 bp [79]. Long cell-free DNA (>250 bp) can be recovered using DNA repair prior to sequencing [142]. Notably, this process enriches cfpDNA but not background maternal cell-free DNA, pointing towards differences in the types of DNA damage between placental and maternal cell-free DNA, and differences in the mechanisms underlying their fragmentation [142]. While often stated in the literature that apoptosis generates short cell-free DNA while necrosis generates long cell-free DNA [108], findings from the recent long-read sequencing studies may indicate otherwise. Size distribution peaks corresponding to multi-nucleosomal units suggest apoptotic origins of long cell-free DNA [109]. Therefore, the relationships between cell-free DNA fragment size and cell death mechanisms remain poorly understood.

Cell-free DNA fragment size and DNA methylation levels are positively correlated [45,46,126,177,178]. Total maternal plasma cell-free DNA methylation and fragment size both increase with gestational age [177], consistent with previous observations of placental DNA methylation increasing with advancing gestational age [179]. These findings also point towards the role of DNA methylation in regulating cell-free DNA fragmentation and generating the distinct size distribution of cfpDNA. The role of DNA methylation in cell-free DNA fragmentation may be related to nucleosome packaging and nuclease activity (i.e., DNASE1L3) [178].

Clinical applications of cfpDNA fragment size are being investigated. The size distribution of cell-free DNA is altered in preeclampsia, with a decrease in the proportion of long fragments [79,180,181]. Whether this decrease is in background maternal cell-free DNA, cfpDNA, or both has not been determined. Researchers were able to identify pregnancies with preeclampsia based on the proportion of long cell-free DNA (>170 bp) in maternal plasma using long-read sequencing [79] and ddPCR [180]. The distinct size distributions of background maternal cell-free DNA and cfpDNA have been investigated for NIPT applications such as fetal fraction measurement [63] and cfpDNA enrichment [97,182].

Fragment end motifs

Cell-free DNA fragment end motifs are informative of the nucleases and fragmentation processes by which they were generated. Associations between specific fragment end motifs and nucleases have been identified. DNASE1L3, DNASE1, and DFFB have been found to have preferences to cleave 5’ of C, T, and A nucleotides, respectively [183–185]. End motifs are also associated with cell-free DNA fragment size, likely due to the stepwise activity of endonucleases [79,184]. Shorter cell-free DNA fragments (<1,000 bp) predominantly carry C- and G- ends while longer fragments (>1,000 bp) predominantly carry A- and G-ends [79]. The frequency of T- and C-ends decrease as fragment size increases beyond 1,000 bp [79].

Studies have demonstrated that cell-free DNA end motif profiles are associated with tissue of origin and are altered in pathological states such as cancer, likely attributable to changes in nuclease activities and DNA fragmentation processes [90,183]. CfpDNA end motif profiles are distinct from those of background maternal cell-free DNA [90]. The relative contributions of an end motif profile that is hypothesized to be generated by non-enzymatic DNA fragmentation was elevated in cfpDNA in the first trimester compared to the second and third trimesters [183]. This is consistent with the state of increased oxidative stress in the early placenta [183]. Yu and colleagues [79] demonstrated that the cell-free DNA end motif profile of patients with preeclampsia is distinct from control subjects. A reduction in G- and A-ends and increase in T- and C-ends was reported [79]. These observations are consistent with what would be expected given the relationship between cell-free DNA fragment size and end motifs, and the observation of a decreased proportion of long cell-free DNA in preeclampsia [79]. Upon investigating expression levels of endonucleases, DNASE2 was found to be up-regulated in preeclamptic placentas, suggesting a potential role in aberrant cell-free DNA fragmentation in preeclampsia [79]. The relationship between DNASE2 activity and cell-free DNA fragment end motifs has not yet been investigated due to the lethality of Dnase2a deletion in mouse models [79]. The authors developed a classifier to identify preeclamptic pregnancies based on cell-free DNA 4-mer end motifs [79]. This demonstrates the potential application of fragment end motifs as an indicator of placental health and a noninvasive biomarker for pregnancy complications.

Genome coverage and nucleosome footprinting

Cell-free DNA fragmentation is influenced by nucleosome structure and positioning. This is made evident through cell-free DNA size distribution patterns (“CfpDNA fragmentomics”—“Fragment size” section) and the observed relationships between cell-free DNA genome coverage, nucleosome positioning, and gene expression.

Cell-free DNA genome coverage reflects the nucleosome structure and positioning in tissues of origin. This is based on the principle that nucleosome-bound DNA is protected from nuclease activity and is expected to be more abundantly represented in cell-free DNA. On the other hand, naked DNA is more susceptible to nuclease digestion and is expected to have lower representation in cell-free DNA [95]. Accordingly, genome mapping of cell-free DNA sequencing reads shows peaks of increased coverage that demonstrate a ∼190 bp periodicity, consistent with the length of DNA wrapping around a nucleosome plus the linker regions [85,95,176,186]. Based on this principle, patterns of cell-free DNA coverage are consistent with the expected nucleosome positioning in specific genomic regions (i.e., transcriptional start sites [95,187], nucleosome depleted regions [176], open and closed chromatin regions [85,95], and transcription factor binding sites [95]). Cell-free DNA coverage therefore reflects tissue of origin [85,95] and gene expression activity [95,176]. Cell-free DNA coverage also footprints transcription factor binding [95].

Tissue-specific nucleosome positioning inferred using cell-free DNA coverage can measure the fetal fraction in maternal plasma cell-free DNA samples. Straver and colleagues [80] developed a method for estimating fetal fraction based on the proportions of cell-free DNA fragments starting at different positions in the nucleosome structure, according to cfpDNA-specific patterns. Sun and colleagues [85] developed a metric called the “orientation-aware cell-free DNA fragmentation (OCF)” value, which measures tissue contributions to total plasma cell-free DNA based on representation levels of tissue-specific open chromatin region nucleosome positioning. In maternal plasma cell-free DNA, the T cell OCF value was decreased while the placenta OCF value was increased compared to non-pregnant plasma cell-free DNA samples [85].

Gene expression is influenced by nucleosome positioning. Therefore, cell-free DNA coverage can be used to deduce nucleosome positioning at promoter regions, which can in turn infer gene expression patterns in the tissue of origin. Accordingly, cell-free DNA coverage at promoter regions has been found to be inversely associated with gene expression levels [188]. Han and colleagues [187] demonstrated the feasibility and utility of noninvasively inferring placental gene expression using maternal plasma cell-free DNA. The authors reported reduced coverage of placenta-specific genes in pregnant plasma cell-free DNA compared to non-pregnant plasma cell-free DNA [187]. Gestational age-dependent changes in transcription start site coverage of placenta-specific genes (corticotropin releasing hormone (CRH) and chorionic gonadotropin beta polypeptide 5 (CGB5)) in maternal plasma cell-free DNA consistent with their respective gene expression dynamics across gestation were observed [187]. Dynamics of gene isoform expression across gestation could be characterized [187]. Classifiers were built for determining gestational trimester, fetal sex, and trisomy 21 status based on inferred gene expression from maternal plasma cell-free DNA coverage of transcription start sites and transcription factor binding sites [187]. Other researchers have developed classifiers for identifying pregnancies with preeclampsia, gestational diabetes mellitus, fetal growth restriction, and macrosomia based on nucleosome positioning and gene expression deduced using maternal plasma cell-free DNA genome coverage [189–191]. Overall, mapping of nucleosome positioning using cell-free DNA provides a noninvasive method for studying maternal and placental gene expression profiles across gestation [187,192].

Preferred ends

The genomic coordinates at which cell-free DNA fragments start and end are non-random. Fragment end coordinates that are overrepresented in cell-free DNA have been termed “preferred ends.” Preferred ends are associated with tissue of origin [84] and fragment size [86].

Chan and colleagues [84] identified distinct preferred ends for maternal and placental cell-free DNA. They demonstrated that the ratio of fragments carrying placental- and maternal-specific preferred ends was positively correlated with fetal fraction measured using the proportion of Y chromosome reads [84]. The size distribution of fragments carrying the placenta-specific preferred ends was shorter than that of fragments carrying the maternal-specific preferred ends, reflecting the different size distributions of cfpDNA and background maternal cell-free DNA [84]. Notably, tissue-specific preferred ends identified in one pregnancy were robust across samples from other pregnancies [84].

Sun and colleagues [86] identified size-specific preferred ends after separating sequence reads into short (60 to 155 bp) and long (170 to 250 bp) cell-free DNA fragment categories. The ratio of fragments carrying short and long fragment-specific preferred ends was positively correlated with fetal fraction measured using the proportion of Y chromosome reads [86]. Enriching cfpDNA using short fragment-specific preferred ends improved the performance of trisomy 21 detection [86]. Positioning of short and long fragment-specific preferred ends were associated with nucleosome structure, with short fragment-specific preferred ends often being located within the nucleosome core and long fragment-specific preferred ends often being located in the linker regions [86]. This provides further evidence for the role of nucleosome positioning in mediating cell-free DNA fragmentation patterns.

Jagged ends

The term “jagged ends” describes single-stranded overhangs that are present on most cell-free DNA fragments (87.7% of plasma cell-free DNA fragments from a cohort of 15 pregnant women) [193]. Patterns of jagged end lengths are reminiscent of nucleosome positioning [193]. The jagged end length of cell-free DNA has been hypothesized to be associated with tissue of origin. Accordingly, cfpDNA bears longer jagged ends than background maternal cell-free DNA and jagged end length was positively correlated with fetal fraction [193]. DNASE1, an endonuclease involved in cell-free DNA fragmentation, is implicated in the generation of jagged ends [193]. Consistent with the increased jaggedness of cfpDNA, DNASE1 is more highly expressed in the placenta than in white blood cells [193].

CffDNA and cfpDNA in other body fluids

CffDNA and cfpDNA have been detected in maternal body fluids other than blood such as cerebrospinal fluid [194] and peritoneal fluid [58,195].

Several studies have reported the presence of cell-free DNA carrying Y chromosome sequences or placenta-specific SNP alleles in maternal urine [196–199]. It can be assumed that this is cfpDNA filtered from maternal blood. Fragment size analysis has revealed that urinary cfpDNA is highly fragmented, with size distribution peaks between 29 to 45 bp [199]. CfpDNA is present in lower amounts in maternal urine compared to maternal plasma. The fetal fraction in third trimester maternal urine samples was 1.92% to 4.73%, while the fetal fraction in maternal plasma at the same gestational age was around 20.4% [199].

There is a large fraction of cffDNA present in amniotic fluid. The concentration of cffDNA is 100- to 200-fold higher in amniotic fluid compared to the concentration of cfpDNA in maternal plasma [200] and is correlated with gestational age [201]. Burnham and colleagues [202] found that amniotic fluid cffDNA is highly fragmented and shorter than plasma cfpDNA, with a median fragment size of 108 bp. Analysis of fragmentation patterns in euploid and aneuploid pregnancies revealed fragmentation profiles unique to different karyotypes [201,203]. Amniotic fluid cffDNA is mainly derived from the fetus rather than the placenta, indicated by the lack of correlation with plasma cfpDNA levels [200], absence of placenta-specific mRNA transcripts [204], and low detection of hypermethylated RASSF1A, a placenta-specific DNA methylation pattern [205], in amniotic fluid.

CfpDNA function

Whether cfpDNA plays a functional role during pregnancy is unknown. Sterile inflammation is implicated in parturition and pregnancy complications such as preeclampsia [206,207]. Given that cfpDNA is hypomethylated like microbial DNA, it is hypothesized that cfpDNA can act as a proinflammatory danger-associated molecular pattern and activate Toll-like Receptor 9 (TLR9) [30,208]. Increases in cfpDNA levels near delivery [55], in preterm birth [209], and in inflammation-associated placental pathologies (i.e., preeclampsia) [47] provide further rationale for exploring this hypothesis. In vitro studies have investigated the proinflammatory potential of cell-free DNA released from mouse and human placental explants. Cell-free DNA isolated from explant culture media has been found to induce cytokine secretion from mouse macrophages [29] and human maternal peripheral blood mononuclear cells (PBMCs) [208]. Kazemi and colleagues [208] found that cell-free DNA from human maternal plasma collected at 36 to 38 weeks of gestation induced cytokine secretion from maternal PBMCs, while cell-free DNA from maternal plasma collected postpartum did not. This suggests a proinflammatory role specific to cfpDNA. Cell-free DNA specifically triggered monocytes, which then amplified the immune response by activating neighboring T-cells [208]. They also reported that cell-free DNA only triggered immune cells when transfected in lipid-based transfection reagents, suggesting that extracellular vesicle-bound cfpDNA may specifically be responsible for cfpDNA-mediated inflammation. This is likely related to increased efficiency of cellular uptake and signaling [208]. Active release, protection, and transfection of cfpDNA through extracellular vesicles (i.e., exosomes), rather than passive release through cell death, would be more consistent with a functional role of cfpDNA. The culture media of PBMCs treated with cell-free DNA promoted endothelial cell activation and myometrial contraction, which are both processes involved in parturition [208]. In contrast with the above reports, one study found that cell-free DNA collected from human term placenta culture media did not induce cytokine release from maternal PBMCs [30]. Further investigation of DNA methylation patterns revealed that the collected cell-free DNA was not hypomethylated, making it an unsuitable TLR9 agonist [30]. Further in vitro and in vivo studies will provide more insights into any potential biological roles of cfpDNA.

Future directions

Great progress has been made in developing the clinical utility of cell-free DNA, from NIPT to cancer monitoring. However, many gaps remain in the understanding of the release mechanisms, form, and functions of cell-free DNA. This hinders the potential of cell-free DNA not only as a clinical tool, but also as a research tool for better understanding tissue health, development, and disease. Further investigation of the basic biology of cell-free DNA and characterization of its molecular characteristics will allow researchers to collect more information about the states of their tissues of origin. The ongoing development of in vitro models, sequencing technologies, and bioinformatic tools will help to drive these advancements and open doors for future research directions and subsequent development of clinical applications. Insights into the form and physical properties of cell-free DNA can guide improvements in isolation and analysis methods. General characterization of the effects of physiological and lifestyle factors [210] on cell-free DNA will help deconvolute cell-free DNA signals and increase the power of pathology-specific analyses. Reproductive and women’s health research is underfunded [211,212] and studies investigating cell-free DNA in pregnancy are limited compared to other fields such as cancer. Directing more attention to studying cell-free DNA in pregnancy will improve our understanding of the placental physiological processes that generate cfpDNA, the functional role of cfpDNA in pregnancy, and how cfpDNA can be leveraged to study and monitor placental health and disease. It is also of value to study background maternal cell-free DNA to gain insights into maternal adaptations to pregnancy and any maternal origins of pregnancy complications and the impacts of these conditions on maternal health. Ultimately, this will lead to improvements in perinatal care and reproductive and women’s health.

Acknowledgments

The authors recognize and acknowledge that this work was completed on the traditional territories of the Mississauga and Haudenosaunee nations and within the lands protected by the “Dish with One Spoon” wampum agreement.

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