After the first detection of cell-free DNA (cfDNA) in the maternal plasma in the 70s, no one could imagine the tremendous impact of this discovery on the clinical practice. The present article aims at analyzing the actual knowledge about cfDNA from the clinician point of view. In the first part, the origin, kinetics and metabolization of cfDNA are explained. Afterwards, the major applications and limitations of cfDNA are presented. A vast part of the review is dedicated to the different techniques used for trisomy and sex chromosome detection and to their performance. The current international and local guidelines are presented at the end of the article.
După prima detectare a ADN-ului fetal liber (cfDNA) în plasma maternă, în anii ’70, nimeni nu şi-ar fi putut imagina impactul acestei descoperiri asupra practicii clinice. Acest articol îşi propune să analizeze cunoştinţele actuale despre cfADN din punctul de vedere al clinicianului. În prima parte sunt explicate originea, kinetica şi mobilizarea cfADN-ului. Ulterior sunt prezentate aplicaţiile şi limitele utilizării cfADN-ului. O mare parte a review-ului este dedicată diferitelor tehnici utilizate pentru diagnosticarea trisomiilor şi gonozomilor şi analizării performanţei acestora. Ghidurile internaţionale şi locale sunt descrise la sfârşitul articolului.
The screening for Down syndrome has entered a new era since the discovery of cell-free fetal DNA (cfDNA). The detection of cfDNA in the clinical practice is also known as “noninvasive prenatal screening” (NIPS) or “noninvasive prenatal testing” (NIPT). The latter test has the advantage of being noninvasive and highly sensitive, but expensive for the routine screening(1).
Classically, the screening for Down syndrome was made by detailed first-trimester ultrasound scan, including nuchal translucency measurement, crown-rump length, nasal bone, ductus venosus flow pattern, maternal age and serum markers (b-human chorionic gonadotrophin hormone [b-HCG], and pregnancy-associated plasma protein A [PAPP-A]). In case of a positive result, fetal karyotype was obtained using invasive procedures such as first-trimester chorionic villous sampling or second-trimester amniocentesis. The latter procedure has a low but confirmed risk of miscarriage varying between 0.1% and 1.3%(2). This explains the high interest in validating a reliable, noninvasive, but sensitive technique for Down syndrome screening.
Despite the belief that the placenta forms an impervious barrier between the mother and her fetus, nowadays it is clear that fetal cells are present and flow freely in the maternal blood(3). The discovery of cell-free fetal DNA and its widespread utility led prenatal medicine to a completely new era. Given the enormous progress in the area of molecular biology and gene sequencing, it is now possible to detect with an accuracy of 99% the presence of most common trisomies (21, 18, 13, 22, 9 and 6), deletions and duplication syndromes, and sex chromosome aneuploidies(4). Today, all of the aforementioned anomalies can be determined easily from maternal serum starting from the 10th week of gestation(5,6).
In the following sections, we aim at reviewing the current knowledge about cfDNA and its current implications for the clinical practice.
The origin and kinetics of cell-free fetal DNA
Although more than 20 years have passed since Lo et al. proved for the first time the existence of fetal DNA in the serum of pregnant women, there is an ongoing debate regarding its exact source(7). In their pioneering experiment, Lo et al. proved that it is possible to detect the presence of Y chromosome from the maternal serum(7). The authors chose the amplification of Y-specific fetal sequence because Y chromosome was virtually absent in pregnant women karyotype, therefore any detection of a Y chromosome DNA should have a fetal origin. Moreover, they compared their serum-detected fragments to the fetal karyotype obtained by amniocentesis. The conclusion firmly demonstrated that fetal DNA is circulating in the pregnant women blood, but its origin was speculative. In addition, the Y chromosome was absent in plasma of all pregnant women with female fetuses.
Overwhelming evidence suggests that the primary source is the placenta(3). In the first week of embryonic development, flat squamous epithelial cells form the outer layer of the blastocyst. This layer of cells, called the trophoblast layer, are the first to differentiate in the human embryo. Upon the contact with the uterine wall, they differentiate into the cytotrophoblastic cells. The mononuclear cells fuse together to form the syncytiotrophoblast, representing the main site of specific hormonal secretion during pregnancy. Cellular parts shed from this unit into the maternal circulation. As postulated by Bischoff et al., the majority of cfDNA originates from the placenta and fetal cells apoptosis(8) since fetal cells are quite scarce in the maternal plasma (about one per milliliter)(3). This apoptotic process in the placenta seems to be a part of the fusion process described before(9). The increasing levels of cfDNA as the pregnancy progresses are also supporting this theory(10). Among the fetal sources of cfDNA, fetal erythroblasts apoptosis generates DNA particles that cross the placenta and can be detected in the maternal plasma(11). Hristoskova et al. demonstrated by TUNEL assay that the apoptotic process is initiated in most of these erythroblasts while being still in the fetal bloodstream(12).
In the early stages of implantation, the syncytiotrophoblast begins the invasion and dissolution of the endometrial vessels. The erosion of the spiraled arteries marks the beginning of the feto-maternal circulation(13). At 28-30 days of gestation (9 GWs+0 days – 9 GWs+2 days), a complete circulation is established. At this point, 80% of patients have detectable levels of fetal DNA(10). Using a quantitative real-time PCR assay, Guibert et al. demonstrated the presence of cfDNA at 18 days post-conception (6 GWs + 2 days) in a twin pregnancy and on day 22 (7 GWs + 2 days) in a singleton pregnancy(10).
The available clinical tests used for trisomy diagnosis are indicated after the 10th week of gestation(14). This gestational age is chosen because in the majority of cases the cfDNA fraction is above 4%, the minimal value required for sample validation; however, cfDNA can be detected earlier, but the levels are lower(14).
From 10 weeks of gestation (WG), when the median percent of cfDNA is 10.2%, the levels of cfDNA increase by 0.1% every week until the 20th week(15). After 21 WG, the amount raises with 1% until term when the fetal fraction represents 10-25% of all plasma DNA(16). Analyzing 22,384 pregnant women’s serum samples, the largest cohort study so far, Wang et al. showed that the medium amount of cfDNA in the maternal serum varies between 11% and 13.4% regardless of the type of assay used(15). Another study performed by Dar et al. revealed a large variability of cfDNA ranging from 0.6% to 50%, with an average of 10.2%(17). In the same study, insufficient amounts of fetal DNA were correlated with higher maternal Body Mass Index and lower gestational age(15).
Typically, most laboratories perform the analysis earliest after 10 weeks of gestation, when the fetal fraction reaches the minimum amount required (4%). When examining the amount of circulating fetal fraction, variations were observed in certain disorders. In Down syndrome, the amount of fetal fraction is higher compared to euploid pregnancies screened at the same gestational age. As proven by Palomaki et al., in trisomy 18 pregnancies, or in the case of a triploid fetus, the amount of cfDNA is lower(18). Lower cfDNA levels were also reported in pregnancies with trisomy 13 and monosomy X(18).
Also, as mentioned before, maternal weight has a tremendous impact upon cfDNA levels. In obese patients, cfDNA levels are lower due to the higher circulating volume of blood in these patients. In fact, with every 4.5-5% increase in the maternal weight, the cfDNA fraction decreases with 0.5%(19).
Ashoor et al. found that cfDNA fraction correlates directly with fetal crown-rump length, serum pregnancy-associated plasma protein-A, serum free b-human chorionic gonadotropin, smoking and trisomy 21 karyotype(19). An alternative for Down syndrome detection in obese pregnancy is represented by the amplification of specific single-nucleotide polymorphism (SNP) from chromosome 21 using polymerase chain reaction (PCR), which requires lower amount of cfDNA while maintaining a sensitivity of 100%(20).
Using real-time PCR techniques, Lo et al. showed that fetal fraction clearance after labor or caesarean section always occurs in the 48 hours after delivery(4,21).
Massively parallel genomic sequencing – also called next-generation sequencing or second-generation sequencing – screens thousands of loci from a chromosome in order to find a pathogenic mutation. DNA sequencing was first described in 1977 by Maxam, Gilbert and Sanger et al.(22,23) Since then, important improvements have been made in the field of sequencing instruments, programs and bioinformatics, allowing larger-scale production of genomic sequences.
For clinical purposes, three main approaches are used: massively parallel shotgun sequencing (MPSS), targeted massively parallel sequencing (t-MPS), and the single nucleotide polymorphism (SNP)-based approach(24).
MPSS is based on the amplification and sequencing of both fetal and maternal DNA extracted from maternal plasma. The number of copies of the chromosome of interest is later compared to the reference. The test concludes if a certain chromosome is nullisomic (2n-2), monosomic (2n-1), euploid (2n) or trisomic (2n+1). The method has the advantage that it does not require the separation of the fetal fraction from the whole DNA extracted from the maternal plasma. The method has three major limitations: the minimum amount of 4% of the fetal fraction necessary for test validation, the need of a comparison with a reference chromosome, and the high price(24).
t-MPS was developed in order to simplify MPSS and to reduce the costs. It is based on the amplification of selected regions of the chromosome of interest(24).
SNP amplification followed by sequencing of the chromosome of interest extracted from the father’s plasma are compared against maternal and fetal fraction, allowing the estimation of fetal chromosome amount(24). SNP cannot be used in pregnancies from egg donation or for pregnancies in patients with organ transplantation.
Performance of cfDNA in aneuploidy detection
Various studies have analyzed the performance of cfDNA in the clinical practice. The calculated sensibility and specificity of the most relevant meta-analyses for singleton pregnancies are presented in Table 1. Most of the authors reported difficulties in including the studies due to different methods of performing cfDNA and on selected (high-risk) or unselected population. Most of the studies focused on trisomies detection. The detection of trisomy 21 was reported by all studies and methods above 99% in terms of sensibility and specificity regardless the detection method. Good sensitivity has been reported also for trisomy 18 – the lowest value of sensitivity was 90.9 by TMPS in unselected pregnant patients, and up to 100% by MPSS in the same category(25). The sensitivity was even lower for trisomy 13 – 65.1% in unselected populations by TMPS, but 100% in high-risk populations using the same method(25).
Few studies have included sex determination; in France, cfDNA sex determination is not available due to ethical reasons(26). The meta-analysis of Mackie et al. found a very good sensibility (98.9%), with a 99.6% specificity for fetal sex determination(27). Also, the performance of fetal Rhesus D determination had a good sensibility (99.3%), with a 98.4% specificity. The monosomy X detection rates were 92.9 in Mackie’s analysis, or 91.7% in high-risk population, but with very good specificity (99.8-100%).
Limitations of cfDNA
In about 2% of cases, the test failed to furnish a result. Three approaches are possible: to perform a new cfDNA test at a more advanced gestational age, to accomplish a standard serum screening test (especially in obese women), and extensive ultrasound or invasive diagnostic test (chorionic villus sampling [CVS] or amniocentesis)(28). The most frequent reason for an inconclusive result is a low fetal fraction, below 4%. The fetal fraction seems to be lower in IVF pregnancies(29). Also, uniparental disomy or pregnancies from consanguine couples can have a borderline result(30).
Another issue is represented by particular situations when a false positive result can be diagnosed: confined placental mosaicism, demised twin, maternal mosaicism, maternal cancer, pregnant women after organ transplant, or even recent maternal transfusion(28,30).
cfDNA in twin pregnancies
Aneuploidy screening using cell-free DNA testing has been successfully applied in singleton pregnancies for more than a decade now. When considering twin pregnancies, the problem of cfDNA testing is more complicated. Ultrasound examination allows the correct identification of the number of amniotic sacs and future placentas in the first trimester, but the discrimination between monozygotic and dizygotic twins is impossible in case of diamniotic dichorionic pregnancies. Monozygotic twins share the same genetic material, making aneuploidy risk assessment very similar to singleton pregnancies. Difficulties arise when testing dichorionic twins. Most of them are dizygotic and, as proven by Leung et al. using ultradeep sequencing, the amount of cfDNA released in the mother’s circulation can differ from one twin to another by approximately 2-fold(31). When the amount of the cfDNA of the affected fetus is below the detection threshold, the result could be false negative. Using chromosome-selective sequencing, Gil et al. analyzed the risk for trisomy 21, 13 and 18 retrospectively in 207 singleton pregnancies and prospectively in 68 twin gestations at 11-13 weeks. Three pregnancies with trisomy were accurately detected in the twin group, deeming the method feasible in case of twin pregnancies(32). Another solution for dizygotic twin testing, proposed by Struble et al., is the use of the smaller fetal fraction for analysis(33). A recent meta-analysis that included 997 pregnancies concluded that the detection rates for trisomies are comparable with those obtained in singletons and superior to first-trimester combined test or second-trimester biochemical testing(34). The detection rates were 98.2%, 88.9% and 66.7%, and the false positive rates were 0.05%, 0.03% and 0.19%, for trisomy 21, trisomy 18 and trisomy 13, respectively(34).
Clinical implications of cfDNA results – current guidelines
Nowadays, noninvasive prenatal test (NIPT) is not considered a diagnostic tests. Therefore, all obstetrical societies emphasize the importance of the ultrasound examination at 11-13 gestational weeks. Beside the detailed fetal examination, this ultrasound examination provides an excellent opportunity for the obstetrician to discuss with the pregnant women the aneuploidy available screening tests, depicting in details the risks and benefits of every method. In most countries, the preferred approach in low-risk patient is to offer the combined first-trimester test. In case of a high-risk result (>1/50), the preferred approach is to perform an invasive procedure (CVS or amniocentesis)(6,35,36). If the calculated risk is intermediate, between 1/50 and 1/250, the patient can choose between an invasive procedure or NIPT. For calculated intermediate test between 1/250 and 1/1000, NIPT should be offered. For patients with low risk (>1/1000), no further test for aneuploidies should be provided. In case of a high-risk patient – maternal age>38 years old or previous pregnancy with aneuploidy (trisomy 21, 13, 18 or X monosomy) – NIPT can be offered at the end of the first trimester, but NIPT result, if positive, should be confirmed by an invasive procedure(6,35,36). There is an important debate upon the use of NIPT as a screening method in low-risk pregnancies. The current international guidelines do not support this approach. However, Gil et al. demonstrated that cfDNA can be reliable for the detection of trisomy 21 (99%), trisomy 13 (99%) and trisomy 18 (98%) when used as a screening test in singletons. Moreover, cfDNA can be used as a screening test for Down syndrome in twins(37,38).
The introduction of cfDNA has revolutionized the prenatal care. The constant evolution of the genetic techniques and their availability have permitted their introduction in the clinical practice. cfDNA have several advantages: noninvasiveness, high sensitivity and the fact that they can be applied early in pregnancy starting from 12 gestational weeks.
Conflict of interests: The authors declare no conflict of interests.
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37. Gil MM, Accurti V, Santacruz B, et al. Analysis of cell-free DNA in maternal blood in screening for aneuploidies: updated meta-analysis. Ultrasound Obstet Gynecol. 2017; 50(3):302-314.
38. Gil MM, Galeva S, Jani J, et al. Screening for trisomies by cfDNA testing of maternal blood in twin pregnancy: update of The Fetal Medicine Foundation results and meta-analysis. Ultrasound Obstet Gynecol. 2019; 53(6):734-742.