Version July 2025
INTRODUCTION —
Prenatal screening for trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), trisomy 13 (Patau syndrome), and selected sex chromosome aneuploidies can be performed using next-generation sequencing of cell-free DNA (cfDNA) in maternal blood. This is possible because cfDNA from the fetal-placental unit can be detected in maternal blood as early as five weeks of gestation and is completely cleared from maternal blood soon after delivery [1-4].
Tests that use cfDNA to screen for trisomy 21 detect approximately 99 percent of affected pregnancies, at a false-positive rate (FPR) of approximately 1 or 2 per 1000 pregnancies, in patients who do not experience a test failure (ie, no call or no result). Detection rates (DRs) for trisomy 18 and 13 are slightly less, with similar rates of false-positive results. An invasive procedure (eg, amniocentesis or chorionic villus sampling) to obtain a sample for karyotyping or microarray analysis is the gold standard for prenatal diagnosis of fetal aneuploidy and should be offered to patients who screen positive by cfDNA testing: cfDNA test results are not considered diagnostic.
This topic will discuss prenatal screening for the common fetal aneuploidies using cfDNA in maternal plasma. Other methods of screening for these aneuploidies and expanded use of cfDNA for prenatal screening are reviewed separately:
●(See "Down syndrome: Overview of prenatal screening".)
●(See "First-trimester combined test and integrated tests for screening for Down syndrome and trisomy 18".)
●(See "Sonographic findings associated with fetal aneuploidy".)
●(See "Cell-free DNA screening for fetal conditions other than the common aneuploidies".)
CELL-FREE DNA: BASIC PRINCIPLES
Maternal and fetal-placental origins — Both the mother and the fetal-placental unit produce cfDNA. The source of most maternal cfDNA is maternal hematopoietic cells. The primary source of so-called "fetal" cfDNA in the maternal circulation is apoptosis of placental syncytiotrophoblast cells [1-3]. A lesser source is apoptosis of erythroblasts in the fetal circulation, which generates cfDNA that can cross the placenta and enter the maternal circulation [1,5,6]. The fetus and placenta are usually genetically identical, but differences can occur and are important sources of false-positive and false-negative cfDNA test results.
Circulating cfDNA is highly fragmented with variability in size based on tissue of origin or processing and metabolism [7,8]. Importantly, these patterns differ between the maternal and fetal cfDNA. For example, most maternal fragments are slightly longer than most fetal fragments (maternal fragments: predominant size 166 base pairs versus fetal fragments: predominant size 142 base pairs [8]). Size and genomic regions available for analysis can be used both to screen for specific disorders, such as aneuploidy, and to determine the fetal fraction.
Significance of the fetal fraction — The fetal fraction is the percentage of cfDNA in maternal blood that is derived from the fetal-placental unit and generally comprises 10 to 20 percent of the cfDNA in maternal plasma in the first and second trimesters [9]. Fetal-placental cfDNA can be detected in maternal blood as early as five weeks of gestation and almost always by nine weeks [10]. From 10 to approximately 20 weeks of gestation, the relative concentration increases modestly (0.1 percent per week) and then increases rapidly (1 percent per week) until term [11].
An adequate amount of fetal-placental cfDNA must be present to obtain a reliable cfDNA screening result. In general, a minimum of 1 to 3.5 percent of the total circulating cfDNA should be derived from the fetal-placental unit for successful screening of common aneuploidies. Higher minimum fetal fraction levels may be required to test for microdeletions. At least one laboratory in the United States routinely enriches the fetal fraction so that failures due to low fetal fraction are uncommon [12].
Factors that can systematically reduce the fetal fraction, which can lead to an assay failure (a report of "no call" or "no result") or result in a false-negative result, are discussed below.
Causes of a low fetal fraction
●Early gestational age when maternal blood is drawn – The fetal fraction in maternal blood is substantially less before 10 weeks of gestation, so most laboratories require that patients wait until at least 9 to 10 weeks of gestation to ensure an adequate fetal fraction for testing.
●Suboptimal sample collection/handling – Appropriate sample collection and stabilization of fragmented cfDNA are important to preserve the fetal fraction since a small number of degraded maternal white blood cells will greatly reduce the fetal fraction by increasing the maternal fraction. Traditionally, the sample was collected in a purple top (EDTA) tube and centrifuged within six hours; the resulting plasma was stable with -80°C freezer storage. A special cfDNA collection tube (eg, Cell-Free DNA BCT) that stabilizes the sample for up to five days at room temperature is now available for clinical use. These tubes should not be refrigerated or frozen. Other sample stabilizing tubes are being developed.
An incomplete sample draw (eg, half-filled tubes, <10 mL) may be rejected by the laboratory or may increase the likelihood of test failure due to insufficient plasma volume for testing for fetal cfDNA.
●Maternal weight – As maternal weight (and to a lesser extent body mass index [BMI]) increases, the fetal fraction systematically decreases and may be insufficient for prenatal screening in obese patients. The low fetal fraction in these individuals results from dilution of fetal-placental cfDNA in the larger maternal blood volume associated with obesity and an increased contribution of maternal cfDNA from apoptosis of adipose tissue [13] and possibly from cell death related to chronic inflammation [14]. In one study of 1482 patients with euploid pregnancies, a low fetal fraction (<3.5 percent) was noted in 1.1 percent of all samples, 10.5 percent of samples from patients weighing >110 kg (242 pounds), and 0.2 percent of samples from patients weighing <60 kg (132 pounds) [13]. Other studies have reported that the risk for a low fetal fraction increases at maternal weights as low as 81 kg (180 pounds) [13,15]
The laboratory is not able to mathematically adjust the cfDNA result to correct for maternal weight, in contrast to serum biomarker screening. Waiting until the second trimester to perform the test will increase the fetal fraction and reduce the rate of no call results, but not to the level in patients without obesity. In a study of individuals >181 kg (400 pounds), the rate of "no call" was approximately 18 percent in the first trimester and fell to 6.45 percent in the second trimester [16] (by comparison, 1 and 3 percent of the general obstetric population has a no call result). Clinical approaches to address this problem are discussed below. (See 'Maternal obesity' below.)
●Fetal aneuploidy – Some aneuploidies are associated with a low fetal fraction compared with euploid fetuses. For example, trisomy 18 is associated with a lower average fetal fraction at 10 to 20 weeks of gestation compared with euploidy (average fetal fraction: 9 versus 11 to 13 percent) [17]. Fewer data are available for other aneuploidies, but it appears that the fetal fractions in both trisomy 13 and Turner syndrome are also lower than in euploid fetuses [17]. Triploid fetuses have extremely low fetal fractions, usually below 4 percent [18,19].
In contrast, trisomy 21 is associated with a higher fetal fraction compared with euploidy (average fetal fraction: 13 to 15 percent versus 11 to 13 percent) [17]. This may partially explain why detection rates (DRs) for trisomy 21 are higher than for trisomy 18, especially when test failures are considered.
●Less common factors – A low fetal fraction has also been associated with:
•Maternal use of low molecular weight heparin before 20 weeks of gestation [20-22].
•Conception by in vitro fertilization (IVF) [23]. Increasing data show that the fetal fraction is lower, and the test failure rate is approximately two or three times higher for pregnancies conceived with IVF compared with naturally conceived pregnancies [23,24]. One laboratory with a 2 percent failure rate in naturally conceived pregnancies reported a 5 percent failure rate in IVF conceived pregnancies [23].
•Twin gestation, because the per fetus fetal fraction is lower in these gestations [25]. (See 'Multiple gestations' below.)
Clearance of cfDNA after delivery — Maternal clearance of fetal-placental cfDNA occurs rapidly after giving birth. In healthy pregnant people, the half-life is approximately one hour, with essentially all fetal-placental cfDNA eliminated within two days of giving birth [26,27]. Thus, future pregnancies are not affected by cfDNA in the circulation from prior pregnancies.
TEST METHODOLOGY
Background
●All of the following cfDNA methodologies require obtaining at least one 10 mL maternal blood sample using an appropriate collection tube (purple top [EDTA] tube or special cfDNA collection tube) and appropriate sample handling. (See 'Causes of a low fetal fraction' above.)
●DNA fragments are extracted from maternal plasma and amplified
●Multiple techniques and platforms are utilized for chromosomal screening using fetal cfDNA [28]. While each is unique, they share high sensitivity and specificity for detecting common aneuploidies. Most laboratories can now examine the whole genome but some target the chromosomes of most interest: 21, 18, 13, X, and Y.
●The preliminary assumption is that the mother is euploid. If results are abnormal, this assumption is revisited. (See 'False-positive cfDNA test results' below.)
Whole genome sequencing — The most common method of cfDNA screening sequences cfDNA fragments over the entire genome. In this method, both maternal and fetal cfDNA fragments are sequenced and the chromosomes of origin are identified through alignment with the human genome. This method is sometimes called "massive parallel" or "shotgun" sequencing and may require several million mapped fragments to obtain a reliable test result [4].
The counting method is the most common method laboratories use to analyze fragments of cfDNA in the maternal circulation. In this method, the fragment is evaluated and the chromosome of origin is determined. This is then compared with the expected percent contributed by that chromosome in a euploid pregnant patient carrying a euploid fetus. As an example, in euploid nonpregnant females, approximately 1.3 percent of cfDNA fragments are derived from chromosome 21 (eg, chromosome 21 contains approximately 1.3 percent of the human genome). In pregnancy, the expected percentage of chromosome 21 fragments remains at 1.3 percent when both the fetus and mother are euploid. However, if the fetus has three copies of chromosome 21, the proportion of chromosome 21 fragments in maternal blood will be slightly higher than the expected 1.3 percent; how much higher depends on the proportion of aligned fragments of fetal origin. For example, if the fetus has trisomy 21 and the fetal fraction is 10 percent, the expected proportion of chromosome 21 fragments will be 1.365 percent (1.30 x (1 + [0.10/2]). When the fetal fraction is lower (<10 percent), there is less of an increase in chromosome 21 fragments and it becomes more difficult to detect trisomy 21.
Targeted methodologies — Targeted methodologies focus on the chromosomes (or chromosome regions) that are of most interest (typically 21, 18, and 13). By targeting select chromosomes, fewer sequences need to be aligned and fewer resources are required. Potential targets on other chromosomes are possible but require modifications for each new target.
●Targeted genome sequencing – The massive parallel sequencing method (described above) can be performed for only select chromosomes of interest ("chromosome selective sequencing"), such as chromosome 21.
●Single nucleotide polymorphisms (SNPs) method – A single nucleotide polymorphism represents a common change in a single base. This method evaluates the sequences of the SNPs on cfDNA fragments from selected fetal and maternal chromosomes and compares them with a reference euploid pregnancy. This method relies on reading tens of thousands of highly polymorphic SNPs located on the chromosomes of interest (typically 21, 18 and 13) and produces specific patterns based on the maternal and fetal genotypes. If an additional fetal chromosome is present, as in trisomy 21, the SNP pattern will reflect this. The SNP method can also identify triploidy in singleton pregnancies and aneuploidy in twin pregnancies, twin zygosity, and a vanished dizygotic twin [29].
As with the other methods, only a maternal sample is needed. However, SNP testing cannot be used in the relatively uncommon instances of pregnancy achieved by egg donation, pregnancy in a bone marrow or organ transplant recipient, or a gestational carrier because the maternal plasma contains additional confounding chromosomes. In these instances, the clinician needs to ensure that samples are sent to a laboratory that utilizes another methodology.
●Microarray – This method uses a microarray platform that quantifies several hundred unique loci on each of the targeted chromosomes (typically 21, 18, and 13) [30]. The resulting products are sequenced; sequence counts for each of the targeted chromosomes are adjusted to reduce bias. As with the whole genome methodology, the counts increase when the fetus has a trisomy for one of the targeted chromosomes.
●Rolling circle amplification (RCA) – This method targets selected fragments of cfDNA [28,31]. Specifically designed probes to these fragments bind for each targeted chromosome (typically 21, 18, and 13). Using RCA, these products are amplified into fluorescent products that can be viewed on an automated microscope plate and counted. As with other methods, the counts increase when the fetus has a trisomy for one of the targeted chromosomes.
SCREENING PERFORMANCE IN SINGLETON PREGNANCIES —
The screening performance of tests used for fetal aneuploidy screening is best described by the detection rate (DR; ie, sensitivity) and the false-positive rate (FPR; ie, 1-specificity). It is important to note that a small proportion of cfDNA screening tests fail to provide a useable clinical result (ie, no result, test failure, or no call), which must be considered when interpreting reports of test performance. (See 'Test failures (no result)' below.)
Trisomy 21, 18, and 13 — cfDNA is the most sensitive screening test for these trisomies, which comprise 71 percent of all prenatally detected chromosomal abnormalities [32]. Performance varies by trisomy, but not by methodology, and is similar in both high- and low-risk pregnant patients [33,34].
●DRs and FPRs – DRs and FPRs based on multiple meta-analyses are estimated to be [35-38]:
•Trisomy 21 – DR 99.5 percent, FPR 0.05 percent
•Trisomy 18 – DR 97.7 percent, FPR 0.04 percent
•Trisomy 13 – DR 96.1 percent, FPR 0.06 percent
These DRs are likely an overestimate because the data did not account for test failures in either aneuploid or euploid samples, and many of the studies included in the meta-analyses did not have complete follow-up of all pregnancies. Test failure rates are important to consider when reporting test performance since higher failure rates may decrease actual DRs, increase FPRs, and decrease positive predictive value (PPV) [39]. The direction and magnitude of these changes will depend on the actions taken when the test fails (eg, no further testing, repeat cfDNA testing, ultrasound, diagnostic testing).
●Positive predictive value (PPV) and negative predictive value (NPV) – The PPV and NPV of cfDNA screening for trisomy 21, trisomy 18, and trisomy 13 in a low-/average-risk population (ie, general obstetric population) and a higher risk population (ie, ≥35 years of age) are shown in the table (table 1). The performance of first-trimester biomarker/ultrasound combined screening is also included for comparison. This table was created to provide a reasonable estimate of expected rates, but we acknowledge that some cfDNA tests have slightly higher (or lower) predictive values due to minor differences in estimates of DR and FPR. The first two rows of the table show that cfDNA screening has a higher PPV for trisomy 21 than combined serum biomarker/ultrasound screening in both the general obstetric and high risk population. All NPVs for cfDNA screening are quite high: ≥99.9 percent. This is due to the low a priori prevalence of these three disorders. These very high modeled NPVs for cfDNA screening were confirmed in a large study where only two false-negative results were identified in over 100,000 pregnant patients screened [40]. The figure (figure 1) expands upon this analysis by creating a flowchart of the three common trisomies screened for by first-trimester cfDNA screening. The prevalence and the DR are highest for trisomy 21, and both characteristics are lower for trisomies 18 and 13.
After testing is completed, the laboratory usually provides a patient-specific risk for trisomy 21 (and often for other screening targets) and this is the most appropriate result to be used for patient counseling. It is based on both the patient's a priori risk and cfDNA results. The report should indicate whether this patient-specific risk is for the time of testing (eg, first trimester) or at delivery; the latter will be slightly lower because the prevalence of aneuploidy decreases as the pregnancy advances since all of the trisomies are associated with higher-than-expected fetal loss rates [41]. The laboratory may also provide a PPV and NPV on the report: these are not patient specific (they are population-based), so they should not be used for counseling. Commercial marketing of cfDNA screening for aneuploidy has not emphasized the difference between DR (sensitivity), PPV, and patient-specific risk. Patients who receive a positive result should understand that there is a high likelihood that the fetus has trisomy 21 (PPV ≥85 percent) but it is not certain that the fetus is affected; confirmation with a diagnostic test should be performed. Patients with a negative result should understand that there is a high likelihood that the fetus does not have trisomy 21 (NPV ≥99.9 percent) but that absence of trisomy 21 is not certain. The discrepancy between DR (sensitivity), PPV, and patient-specific risk may be the reason for the initial "surprise" at false-positive results, especially for trisomy 18 [42].
Sex chromosome aneuploidies — Sex chromosome aneuploidies are common, affecting up to one in 400 newborns [43]. The cfDNA DRs for these disorders are lower and FPR rates are higher than for the common autosomal trisomies. However, performance is sufficiently accurate to offer this screening at the time of screening for the common autosomal trisomies, with specific pretest counseling and consent [44].
A meta-analysis evaluating cfDNA screening for fetal sex chromosome aneuploidy reported the following findings [45]:
●45,X – DR 98.8 percent (95% CI 94.6-100), specificity 99.4 percent (95% CI 98.7-99.9), PPV 14.5 percent (95% CI 7.0-43.8)
●47,XXX – DR 100 percent (95% CI 96.9-100), specificity 99.9 percent (95% CI 99.7-100), PPV 61.6 percent (95% CI 37.6-95.4)
●47,XYY – DR 100 percent (95% CI 91.3-100), specificity 100 percent (95% CI 100-100), PPV 100 percent (95% CI 76.5-100)
These results should be considered cautiously for two reasons. First, the analyses did not consider cfDNA test failures in the calculated performance estimates even though several of the included studies failed to produce a result in pregnancies with a known sex chromosome aneuploidy. The omission of these cases means that these DRs are overestimated. In one study, the PPV of cfDNA screening for all sex chromosome aneuploidies combined was only 37 percent [46].
Second, the computed PPVs are dependent on the prevalence estimates, which can vary widely based on how the population is ascertained. Compared with the common autosomal trisomies (PPV 73 percent overall for the three common autosomal trisomies in the general obstetric population), the lower PPV for sex chromosome aneuploidies and especially 45 monosomy (Turner syndrome) likely also reflects innate characteristics of the karyotype. For example, 45,X is associated with a higher frequency of fetal, placental, and even maternal mosaicism (45,X/46,XX) compared with autosomal trisomy. It is not associated with maternal age, and, therefore, the PPVs are expected to be the same in both the general pregnancy population and patients ≥35 years of age at delivery [47]. However, specific ultrasound findings, such as an enlarged nuchal translucency (NT) measurement, are associated with a much higher risk of Turner syndrome. A confounding factor is the increasing prevalence of 45,X cells in females as they age. In one study of 187 cfDNA tests that screened positive for fetal sex chromosome aneuploidy, 125 of the patients had loss of chromosome X themselves [48]. In 10 of these patients, it was determined to be the cause of the monosomy X screen-positive result.
False-positive and false-negative results — Although the PPV for cfDNA screening for the common autosomal trisomies in the population is approximately 90 percent in large studies or modeling exercises, this still means that 10 percent of patients with positive cfDNA results will not have an affected pregnancy [36,46]. There are several reasons why the result of a diagnostic test on amniocytes or fetal blood might not agree with the cfDNA test result. The cfDNA test result might be analytically correct (eg, correctly defines the placental genotype) while being clinically incorrect (eg, does not correctly define the fetal genotype). Although analytic test performance is important, the clinical test performance is the key component for patient care.
For the sex chromosomes, the genotype may be accurate but discordant with the phenotype. Genotype-phenotype discordance has been attributed to laboratory error, a vanishing twin, complex disorders of sexual differentiation, sex chromosome aneuploidy with/without mosaicism, and maternal mosaicism [49,50].
False-positive cfDNA test results — Reasons for false-positive cfDNA (defined as the fetus is unaffected, but cfDNA testing indicates aneuploidy) include [51]:
●Confined placental mosaicism – Since the primary source of "fetal" cfDNA in the maternal circulation is placental cells (syncytiotrophoblast), the cfDNA test will provide results relevant to the placenta, which may be discordant with fetal tissue. In these cases, the cfDNA test is analytically correct but clinically incorrect. Experience gained from chorionic villus sampling indicates that this may occur in up to 1 to 2 percent of pregnancies [52-55] and is more likely with monosomy X and trisomy 13 than for trisomy 21 or 18 [56]. (See "Chorionic villus sampling", section on 'Confined placental mosaicism'.)
●Demised twin – A demised twin can cause an FPR if, for example, the demised twin was aneuploid [57,58]. This is because the placenta from the demised twin (which is also more likely to be aneuploid) is still present at the time of testing and continues to shed DNA weeks after the demise. A twin rather than a singleton pregnancy may not have been recognized if the demise occurred very early in gestation, hence the term "vanishing twin." In cases of recognized first-trimester fetal demise, cfDNA from the demised fetus has been detected for 8 to 13 weeks after the demise [57,59], and for up to 16 weeks after a second-trimester fetal demise [60].
●Maternal mosaicism – Most cfDNA testing methods assume that the mother has a normal karyotype, but this is not always true. For example, with advancing age, an increasing proportion of pregnant people have a small percentage of cells that have lost an X chromosome, and these can lead to a false-positive cfDNA result for laboratories reporting sex chromosome aneuploidies [61]. In such phenotypically normal females, the lower X chromosome signal on cfDNA testing would be attributed to the fetus and reported as fetal Turner syndrome. Follow-up fetal diagnostic testing would identify a euploid fetus. Previously unidentified maternal Turner syndrome mosaicism can be diagnosed by karyotyping peripheral blood lymphocytes [48]. The management of such patients incidentally detected because of cfDNA screening in pregnancy has not been standardized but guidelines are available [62]. These patients probably require additional evaluation during and after pregnancy. (See "Management of Turner syndrome in adults".)
Although uncommon, some patients may have a maternal nonmosaic sex chromosomal abnormality (eg, 47,XXX) and appear to have a normal phenotype [63].
●Maternal cancer and other neoplasms (fibroids) – Individuals with a malignancy may shed measurable quantities of cell-free tumor DNA into the circulation [64-66]. In such pregnant individuals, fetal cfDNA, maternal cfDNA, and tumor cfDNA contribute to total cfDNA. The result may be multiple aneuploidies, single autosomal monosomy, or subchromosomal gains and losses on multiple chromosomes [44,64,67-69].
The ability of cfDNA screening to identify maternal cancer was first reported in 2015 [64]. Results from this study and six additional publications were summarized in a 2023 report that included nearly 4.7 million screens [70]. Results from these studies can be stratified by whether they focused on only chromosomes 21, 18, and 13 (targeted screening) or covered all chromosomes (whole genome screening). The weighted overall screen-positive rate was higher for whole genome screening (29 in 100,000, range 12 to 33 in 100,000) than targeted screening (5 in 100,000, range 0.4 to 52 in 100,000). Follow-up identified 119 maternal cancers (true positives) among the 86 percent of screen-positive patients who underwent follow-up. The PPVs were lower for whole genome screening (17 percent, range 8 to 93 percent) than for targeted screening (37 percent, range 18 to 67 percent). The estimated prevalence of cancer among screened pregnancies was higher for whole genome screening (1 in 23,000, range 1 in 8000 to 1 in 40,000) than for targeted screening (1 in 53,000, range 1 in 9000 to 1 in 390,000). However, the 1 in 390,000 upper confidence limit is due to a small study size. The types of cancers identified varied widely and in at least one study [71], noncancerous uterine fibroids were the majority of identified cases. In most studies, a proportion of identified cancers had already been diagnosed.
Programs that offer cfDNA testing that do identify patterns suspicious of maternal cancer should have referral plans in place to deal with these rare abnormal reports. The appropriate clinical evaluation of such patients is currently unclear, in part because there is no proven correlation between any abnormal pattern and the tissue of origin of the malignancy and no professional guidelines address the clinical management of cfDNA results suggestive of maternal malignancy. Various approaches have been suggested [72] but remain unvalidated.
An ongoing clinical trial through the National Institutes of Health (the Incidental Detection of Maternal Neoplasia Through Non-invasive Cell-free DNA Analysis [IDENTIFY] trial) may help develop recommendations for cancer investigations and the best approach for clinical diagnostic work-up [73]. Preliminary results have been published using a uniform cancer-screening protocol that included rapid whole-body magnetic resonance imaging (MRI), laboratory tests, and standardized cfDNA sequencing using a genome-wide platform for research purposes. Cancer screening was performed in pregnant or postpartum persons who received unusual clinical cfDNA-sequencing results or results that were nonreportable (excluding cases with low fetal fraction or other technical/sample issues). Cancer was found in 52 of the 107 participants, most of whom were asymptomatic. Whole-body MRI had sensitivity and specificity of 98.0 percent and 88.5 percent, respectively, in detecting occult cancer, whereas physical examination and laboratory tests were of limited value. Sequencing showed that 49 participants had a combination of copy-number gains and losses across multiple (≥3) chromosomes and 47 of these participants had cancer. Although these data are informative, multiple concerns remain, such as provider and patient education about the possibility of identifying maternal cancer through cfDNA screening, laboratory standardization, reporting suspicious findings, and access to imaging methods such as whole-body MRI [74].
Educational materials and counseling of patients considering cfDNA for fetal aneuploidy screening should include the possibility that maternal cancer may be identified. Patients want to know if maternal cancer is suspected based on cfDNA test results and consider this an added benefit of screening [75]. However, cfDNA testing should not be considered a screening test for maternal malignancy, given the paucity of data on this association, the potential for FPR, and the emotional and medical impact of such results on the patient's well-being.
●Maternal copy number variants – The methodology for cfDNA analysis assumes that every person carries the same proportion of genetic material on a given chromosome, but chromosomes vary slightly among individuals due to inherited or de novo copy number variants (ie, deletion or duplication of a genomic region[s]). In these individuals, cfDNA sequencing might yield a positive result when the size of the maternal duplication was relatively large and it occurred on a chromosome of interest (eg, chromosome 21) [76,77]. In two studies, maternal duplications on chromosome 18 were the likely cause of trisomy 18 FPR in six of seven cases examined [76,77]. Shallow sequencing (eg, a low number of fragments sequenced and limiting the number of referent chromosomes) makes this form of FPR more likely. Shotgun methods are less likely to be influenced by copy number variants on one chromosome if all autosomes are used to normalize counts from the chromosome of interest.
●Transplant recipient – If transplanted tissue (bone marrow or organ) was obtained from a male donor, cfDNA testing may incorrectly identify a female fetus as male due to the release of male cfDNA from the donor tissue into the maternal circulation [78].
●Recent blood transfusion – Maternal blood transfusion from a male donor performed <4 weeks prior to the blood draw for cfDNA may incorrectly identify a female fetus as being male.
●Chance – FPRs can also be the result of statistical chance, as the cutoff for a positive test is often set at +3 standard deviations. Therefore, 1 or 2 per 1000 euploid fetuses might have an FPR by chance alone, and if 100,000 tests were performed, an estimated 100 FPRs would be expected.
●Technical issues – As with all laboratory testing, rare sample mix-ups or other technical errors could lead to false-positive (or false-negative) test results. However, these would likely be identified as part of subsequent follow-up testing.
False-negative cfDNA test results — Reasons for false-negative cfDNA (fetus is affected, but cfDNA testing indicates no chromosomal abnormality) include:
●Confined placental mosaicism – As discussed above, the primary source of "fetal" cfDNA in the maternal circulation is placental cells (syncytiotrophoblast), which may be discordant with fetal tissue. It is possible that a fetus could be aneuploid even though the karyotype of the placenta does not reflect that finding. In these cases, the cfDNA test is analytically correct (ie, detecting those placental cells of the mosaicism that are euploid) but clinically incorrect (ie, the fetus itself is aneuploid). This is recognized to occur for trisomy 13 and 18 and rarely for trisomy 21 [79]. Isochromosome 21q rearrangements are overrepresented among false-negative cfDNA screening results involving trisomy 21 [80]. Postzygotic isochromosome formation leading to placental mosaicism provides a biological cause for the increased prevalence of these rearrangements among false-negative cases. (See "Chorionic villus sampling", section on 'Confined placental mosaicism'.)
●Borderline low fetal fraction – A low but adequate fetal fraction (eg, between 1 and 3.5 percent) results in a very small difference in the expected (normal reference) versus observed percentage of chromosome fragments (eg, chromosome 21 fragments). If a sufficient number of fragments are not sequenced, this difference will not be identified, and the results will be incorrectly reported as screen negative. (See 'Test methodology' above and 'Significance of the fetal fraction' above.)
●Maternal copy number variants – As described above, maternal duplications can cause an FPR. It is also theoretically possible for a maternal deletion to cause a false-negative result. However, this would be a much rarer event, as the fetus must be aneuploid and the maternal deletion would need to be on the same chromosome.
●Technical issues – Technical assay issues can make the identification of some aneuploidies more difficult. For example, the low guanine-cytosine content of chromosome 13 renders the polymerase chain reaction steps and subsequent sequencing counts less reliable. This results in lower DRs than for other aneuploidies. Laboratories attempt to correct for this in the bioinformatics analysis, but this is not always successful. There are also rare sample mix-ups or other laboratory-related issues that could cause a false-negative test result.
CLINICAL USE —
Patients who choose to be screened for trisomy 21 by cfDNA will almost always also receive screening for trisomy 18 and trisomy 13, which are less common (figure 2). They may also choose to be screened for sex chromosome aneuploidies, or these may be included in the baseline set of conditions included in the screen. As discussed above, detection rates (DRs) for these aneuploidies are lower than for trisomy 21. (See 'Trisomy 21, 18, and 13' above and 'Sex chromosome aneuploidies' above.)
Primary cfDNA screening — Primary screening, by definition, is the first screening test for a given disorder or set of disorders. The use of cfDNA as a primary screening test in the United States is increasing but is limited by some practical concerns [81]. A 2022 study identified 20 laboratories in the US that reported cfDNA testing of 2.18 million pregnancies; four of the laboratories contributed nearly 80 percent of all test results [28]. The study estimated that serum biomarker screening was performed in approximately 510,000 pregnancies during the same interval. In the US, most laboratories appeared to be using cfDNA to screen a general pregnancy population. In contrast, at least five laboratories outside of the US reported that 99 percent of cfDNA screening was performed only for high-risk pregnancies. In some countries, the test is available to all pregnant people at a low cost [67]. In Belgium, for example, the cost for members of the Belgian service for public health insurance is 9 euros (approximately USD $9) [82].
Important considerations of cfDNA screening include insurance coverage, concerns about the availability of appropriate pretest counseling, and concerns that some patients will terminate pregnancy after a positive screen without diagnostic testing [83]. In addition, there is a lack of consensus regarding the appropriate follow-up procedures for patients in the general population who have a cfDNA test failure (usually between 1 and 3 percent). Importantly, sufficient expertise and resources are not available in the United States to provide formal genetic counseling for any prenatal screening test in all low-risk patients. This is a concern for all screening tests for aneuploidy, including serum biochemical marker analysis where a discussion of the screen-positive rate (higher than cfDNA) and positive predictive value (PPV; lower than cfDNA) should be provided. (See 'Implementation issues' below.)
Screening for the most common sex chromosome aneuploidies is controversial because these individuals have fewer serious physical abnormalities than those with trisomy 21, 18, or 13 and the phenotypic features are much more variable. One rationale for screening is that, in the absence of prenatal or early childhood screening, these disorders are often diagnosed later in life after some options for treatment have passed, such as beginning low-dose testosterone therapy early in life for males with 47,XXY. (See "Down syndrome: Clinical features and diagnosis" and "Congenital cytogenetic abnormalities" and "Sex chromosome abnormalities".)
Secondary cfDNA screening — cfDNA was initially a secondary trisomy 21 screening test, and was also used as a secondary screening test for trisomy 18 and trisomy 13. This is no longer a common practice in the United States. However, it has been discussed in the literature [84-86] and may be used in populations where there is limited access to cfDNA testing.
By definition, secondary screening is a follow-up, nondiagnostic test offered to a population that has already been found to be screen positive (high risk) as a result of a previous screening test. A benefit of such secondary screening is that it will reduce the number of pregnancies for which a diagnostic test, with an associated risk of pregnancy loss, is offered. The downside to a secondary screening approach is that it relies on the sensitivity and specificity of the initial screening test. For example, if the initial screening test has a DR of 85 percent (such as with serum biochemical marker screening), this will mean that 15 percent of the cohort of pregnancies with trisomy 21 will be initial screen negative and secondary screening will not be offered.
For trisomy 21, preliminary screening tests can include maternal age ≥35 years at delivery, abnormal ultrasound findings indicating increased risk (eg, enlarged nuchal translucency [NT]), an abnormal serum screening test (eg, first-trimester combined testing), a positive family history of aneuploidy (eg, previous aneuploid pregnancy), or a parent who carries a relevant Robertsonian translocation (eg, balanced translocation with risk for trisomy 13 or 21) [87].
The purpose of secondary screening in this setting is to take advantage of the high DR and low false-positive rate (FPR) of cfDNA screening. High specificity (low FPR) of cfDNA testing allows for a large reduction in the number of unnecessary invasive diagnostic procedures in initially screen-positive patients (high risk) (figure 2). The high sensitivity (DR) of cfDNA testing helps ensure that the few patients with an affected pregnancy who were initially screen positive will remain correctly classified as being screen positive. Since the majority of patients who undergo cfDNA screening prior to amniocentesis will receive a low-risk result and thus might want to avoid an invasive procedure, the cost of the cfDNA screening test may be justified by savings from averted diagnostic testing (amniocentesis and karyotype). Since 2012, cfDNA screening has resulted in a 40 to 76 percent reduction in the number of invasive procedures for prenatal genetic diagnosis [88,89]. Although cfDNA screening fails to give usable results for some patients (1 to 5 percent), those with test failures who are already classified as being at high risk can still be offered diagnostic testing.
●Contingent and reflexive models – There are two models (contingent and reflexive) that utilize serum and cfDNA screening in combination to screen the general pregnancy population. All patients begin the process with serum screening, but the risk cutoff and follow-up testing can differ. The aims of both models are to increase the DR above that usually found in serum screening and reduce the FPRs usually found with serum screening. These tests approach the screening performance of cfDNA testing in all patients while, at the same time, reducing the costs below that of offering cfDNA testing to all patients [90,91].
•Contingent model – In this model [84], all patients from a general pregnancy population are offered first-trimester combined screening with two risk cutoffs. The "high risk" (eg, >1:150) identifies a group that could choose between going directly to invasive testing or to secondary cfDNA screening (or no further testing). Approximately 3 to 5 percent of patients would have a "high-risk" result. The low-risk group (eg, <1:1000) would receive routine prenatal care with no options for further testing. Approximately 80 to 85 percent of patients having the combined test would be low risk. The newly defined intermediate-risk group (eg, 1:151 to 1:1000) represents approximately 10 to 15 percent of the screened population. These patients are informed of their intermediate risk and offered cfDNA screening after counseling. If the cfDNA test is positive for patients in the high- or intermediate-risk group, those patients would also be offered invasive testing.
An important feature of contingent screening is that the patients receiving an intermediate-risk report must return for counseling and the offer of further testing. This contingent screening has the potential to increase detection because 10 to 15 percent of the population is offered secondary screening. However, the use of cfDNA as the secondary test results in very few "false-positive" findings (0.1 to 0.2 percent) that result in an unnecessary invasive test. The actual DRs and FPRs for a contingent model are dependent on the proportion of patients (and their risks) in the intermediate group that proceeds with further testing. It also requires additional resources and costs to meet with and counsel these patients while collecting the second sample.
•Reflexive model – This model [85] utilizes the same risk cutoff levels described above for contingent screening but collects a plasma sample suitable for cfDNA testing at the time the serum is collected. The "high-risk" patients are offered cfDNA or invasive testing, and the low-risk patients receive routine care. However, the intermediate-risk patients automatically (reflexively) have the plasma sample tested and the result returned, negating the need for a call-back or counseling session. This saves time and resources and provides a predictable increase in detection. However, there is an additional expense added by the upfront collection of plasma samples in 100 percent of patients when only 10 to 12 percent will have that sample reflexively tested.
Overall, the DRs and FPRs are more predictable for the reflexive model than the contingent model. In the contingent model, patients with an intermediate-risk result will need to return for a sample draw and cfDNA testing, and not all will choose to do so. In the reflexive model, a sample is already available for cfDNA testing for all patients in the intermediate-risk group. In the reflexive model, it is important that patients understand and consent to the possibility of the two tests and that the resulting individual risk may be quite high (similar to having cfDNA as a primary screen in the high-risk population).
Combined screening alone results in a DR and FPR of approximately 85 and 5 percent, respectively. Both the contingent and reflexive models will increase detection (by defining a new intermediate-risk group) and reduce the FPR as the highly specific cfDNA is the secondary screen. In a report summarizing the results of a reflexive model implementation in over 22,000 patients, the trisomy 21 DR was 95 percent (69 of 73) at an FPR of 0.02 percent (4 of 22,706) [86]. The DR for the contingent model will be slightly lower as not all patients with intermediate risks will chose secondary screening via cfDNA.
Multiple gestations
●Use in twin gestations – The American College of Obstetricians and Gynecologists (ACOG) [81] and the International Society for Prenatal Diagnosis (ISPD) [25] allow for or recommend use of cfDNA screening for common trisomies in twin pregnancies. The amount of cfDNA for the pregnancy overall is approximately 35 percent higher in twin pregnancies than singleton pregnancies [92]. In turn, the amount of cfDNA contributed by each twin is lower than in a singleton pregnancy and may be quite different for the two fetuses in dizygotic twins [93]. For example, if the total fetal fraction for the twin pregnancy is 8 percent, one fetus may provide 6 percent and the other 2 percent.
One approach, therefore, is to modify the algorithm used for singleton pregnancies to estimate the smallest fetal fraction contribution of the two fetuses, which involves comparing polymorphic loci that will differ in dizygotic twins and maternal loci [94]. A sample from a twin pregnancy with one low fetal fraction would be reported as a test failure (no call result), so the laboratory would not miss a trisomy in the fetus with only 2 percent fetal fraction. However, this type of analysis is not performed by the majority of laboratories providing cfDNA testing. Because it is impossible to determine which twin is abnormal based on cfDNA analysis alone, results are reported for the entire pregnancy, and an invasive procedure is required to obtain chorionic villi or amniocytes for genetic testing and determine which twin, if either one, is affected. Some laboratories that offer cfDNA screening in twin pregnancies use methods that are "blind" to the number of fetuses (ie, the laboratory interpretation for a singleton and known or unknown twins are the same).
●Screening performance – The DRs and FPRs are similar in twin versus singleton pregnancies. A 2025 meta-analysis evaluating the diagnostic accuracy of cell-free DNA screening for trisomies 21, 18 and 13, monosomy X and/or other sex-chromosome aneuploidies (SCAs) included over 35,000 twin pregnancies [95]. Pooled sensitivity and specificity for trisomy 21 (253 cases) were 98.8 percent (95% CI 96.5-100) and 100 percent (95% CI 99.9-100), respectively; for trisomy 18 (63 cases), 94.9 percent (95% CI 75.9-99.1) and 100 percent (95% CI 99.9-100), respectively; for trisomy 13 (12 cases), 84.6 percent (95% CI 54.6-98.1) and 100 percent (95% CI 99.9-100), respectively. There were no cases of monosomy X. For other SCAs (11 cases), sensitivity was 100 percent (95% CI 71.5-100) and specificity 99.8 percent (95% CI 99.7-99.9). The accuracy of cfDNA in detecting the common trisomies was similar for dichorionic and monochorionic twin pregnancies.
The pooled failure rate was 1.28 percent (95% CI 1.1-1.5). In a previous analysis, the FPR was 0.29 percent and the initial test failure rate ranged from 1.6 to 13.2 percent with a median of 3.6 percent, with insufficient fetal fraction accounting for most of the failures [25]. Five studies provided revised failure rates from a combined population of 2938 twin pregnancies with 179 repeat tests. Between 83 and 100 percent of those offered repeat testing did so, and success rates ranged from 50 to 83 percent (overall 58 percent, 103 of 179). This reduced the median failure rate in these five studies from 5.6 to 3.1 percent, a 45 percent reduction.
●Use in triplet gestations – Given the rarity of triplet pregnancies, it is unlikely that the type of dataset now available for twins will ever be available for triplet pregnancies. For this reason and the lack of other screening options, an ISPD position statement suggests that cfDNA may be a potential option in triplet pregnancies, but diagnostic testing should always be offered and limitations of screening tests stressed [25]. At least one major laboratory in the US offers cfDNA screening for triplet gestations.
•Screening performance – One laboratory that used three times the needed fetal fraction as a minimum to interpret results in triplet gestations reported test failures in 151 of 709 pregnancies (22 percent) [96]. In a series with affected pregnancies, three tests were positive for trisomy 21 of which one was confirmed by an antenatal karyotype, one was confirmed at birth, and one was not confirmed at birth (false positive) [97].
Implementation issues — Several factors need to be considered when offering noninvasive prenatal aneuploidy screening using cfDNA.
Patient pretest education and counseling
●Personnel – Pretest counseling for cfDNA aneuploidy screening is typically offered and performed by the obstetrical provider as part of routine prenatal care. Although further study is needed to determine whether primary care practices can provide adequate counseling to allow for informed choice, at least one study in the United States reported that patient education about cfDNA screening can be conducted successfully through general obstetric providers [98].
Genetic counseling by a genetic counselor should be strongly considered for special circumstances such as a vanishing twin, triplet pregnancy, and/or parental translocation carriers and for all patients who receive a high-risk result.
●Key discussion points
•Screening is optional. Prenatal screening and diagnostic testing for genetic disorders are offered to all pregnant patients. The decision to undergo screening depends on how each patient balances the benefits of obtaining information about aneuploidy with the potential emotional and physical risks of screening and diagnostic testing.
•Clinical features and variability of trisomy 21, 18, and 13 and sex chromosome aneuploidies. (See "Down syndrome: Clinical features and diagnosis" and "Congenital cytogenetic abnormalities", section on 'Trisomy 18 syndrome' and "Congenital cytogenetic abnormalities", section on 'Trisomy 13 syndrome'.)
•Test limitations and differences between screening tests and diagnostic tests. Patients need to understand the difference between a screening test, which classifies patients as at higher or lower risk of specific fetal aneuploidies, versus a diagnostic test, which diagnoses or excludes fetal chromosomal abnormalities. They also need to understand the limits of the test chosen.
The cfDNA screening test for trisomy 21, 18, and 13 and sex chromosome aneuploidy does not screen for other aneuploidies or genetic conditions and has false-positive and false-negative results. Diagnostic procedures, such as amniocentesis, obtain fetal cells, and subsequent testing by karyotyping or microarray can diagnose all aneuploidies; distinguish between full trisomy and trisomy caused by an unbalanced chromosomal rearrangement; detect mosaicism and microdeletions/microduplications (only microarray); and, via amniotic fluid alpha-fetoprotein (AFP) and acetylcholinesterase, detect open neural tube defects. Patients who want to maximize the amount of genetic information they can obtain about their fetus should consider an invasive procedure (amniocentesis, chorionic villus sampling) for diagnostic genetic testing with chromosomal microarray analysis. If there are one or more structural anomalies on ultrasound examination, a microarray on amniocytes is the preferred test [99]. (See "Prenatal diagnosis of chromosomal imbalance: Chromosomal microarray".)
•Basic principles of cfDNA technology, including the gestational age when screening is performed. (See 'Test methodology' above.)
•Potential for incidental findings. Patients should understand that prenatal screening may raise suspicion for conditions other than the fetal aneuploidies for which the test is being performed. These include maternal sex chromosome aneuploidy, mosaicism, and malignancy. (See 'False-positive cfDNA test results' above.)
•Test performance (eg, DR, FPR, test failure rate, patient-specific risk, and/or PPV) and need to confirm abnormal screening results before considering pregnancy termination. (See 'Screening performance in singleton pregnancies' above.)
The terms PPV and patient-specific risk are often used when discussing cfDNA screening and are not interchangeable. Prior to having the test performed, patients can be informed of the test's PPV in a group of individuals (ie, population-based statistic). The PPV is usually presented as a percentage and is impacted by the DR, the FPR, the prevalence, and the screening cut-off level. It shows the patient that the test is not diagnostic (a diagnostic test has a PPV approaching 100 percent) and further evaluation should be performed if a positive screening result occurs.
After the test is performed, the patient-specific risk is available and used for counseling, the "group" PPV is no longer relevant. The patient-specific risk is based on that individual's test results and represents the odds that their pregnancy is affected. A reasonable laboratory practice is to provide individual risks as odds (eg, 3:1 or 3 to 1) rather than a percentage to distinguish them from the PPV. For example, the risk might be 1:2, indicating that for every three pregnancies with this result, only one would be affected. The patient-specific risk can be lower or higher than the PPV, as the latter value can be thought of as the average of all patient-specific risks.
•cfDNA screening versus serum marker screening – Whether prenatal screening based on a combination of serum biomarkers and ultrasound measurements will identify more chromosomal abnormalities than next-generation sequencing of circulating cfDNA is no longer controversial [100]. Although standard serum biomarker screening for aneuploidy can lead to the serendipitous identification of chromosomal abnormalities not targeted by the screening test, it does so only by randomly identifying patients as being false positives. Prior to the widespread use of cfDNA as a primary or secondary screening test, serum screen-positive patients generally chose an invasive procedure for karyotype or microarray. However, many of the additional abnormalities identified by karyotype or microarray after a positive serum screen are rare, some have a very low chance of survival (eg, triploidy), and others are not phenotypically significant.
First-trimester combined testing requires a fetal ultrasound examination, which may detect a fetal anomaly. Since ultrasound is not a component of cfDNA screening, a first-trimester ultrasound examination may not be performed, thus losing the opportunity for early detection of some anomalies [101].
•Cost – Patients need to know whether their insurance covers the cfDNA screening costs completely and, if not, which charges will be their responsibility (eg, copayment, deductible). More insurance policies now cover the cost of cfDNA testing for all pregnant people.
•Turnaround time – The time it takes to perform laboratory testing for a cfDNA result is slightly longer than for serum biochemical markers, but typically not a deciding factor in choosing the screening test. Nearly 80 percent of all cfDNA testing in the United States is performed by four major laboratories, so time in transit adds to the turnaround time [28]. Time in transit is not an issue where local serum biochemical marker screening programs still exist; in this setting, the time from sampling to reporting a result is often only one or two days.
•Desire for fetal sex result – In the general obstetric population, data suggest that one strong driver of interest in cfDNA screening is earlier identification of fetal sex (at 10 to 13 weeks via cfDNA rather than 17 to 20 weeks via ultrasound) [98]. Parents should understand that the test is intended to evaluate for major fetal chromosomal anomalies and that they will receive this information along with the fetal sex. It is also possible that no result can be obtained for their test or that the fetal sex assignment will be incorrect.
Maternal obesity — As discussed above, as maternal weight increases, the fetal fraction systematically decreases and may be insufficient for prenatal screening in patients with obesity. (See 'Significance of the fetal fraction' above.)
For example, patients over 81 kg (180 pounds) can be informed that their chance of having a test failure or an inaccurate result is at least three or four times higher than in patients of lower weight (3.3 versus 0.5 percent in one study [13]). In a subsequent larger study that considered only test failures due to a low fetal fraction, the rate in patients ≥79 kg (174 pounds) was over six times higher than in patients <79 kg (1.11 versus 0.18 percent), but remained less than 5 percent even in patients ≥136 kgs (300 pounds) [16]. The chance of a false negative is also slightly higher in patients with obesity [13,102].
In patients with a body mass index (BMI) ≥35 kg/m2, postponing the test to a later gestational age does not eliminate the risk of test failure because the fetal fraction rises more slowly in patients with higher weights [96,103]. As described above, in a study of individuals >181 kgs (400 pounds), the rate of "no call" was approximately 18 percent in the first trimester and fell to 6.45 percent in the second trimester [16] but did not achieve the 1 and 3 percent rate in the general obstetric population. Serum/ultrasound screening may be offered to patients with severe obesity as an alternative to primary or repeat cfDNA screening. If a cfDNA is performed and a no-call result is obtained late in the midtrimester (after 16 to 18 weeks) and the patient is high risk, offering diagnostic testing rather than any screening test is an option.
Laboratory issues — In the US, nearly all testing is being performed by commercial companies (private or public) that have active sales and marketing forces. It is not possible to confidently conclude that any one laboratory has a superior combination of DR, FPR, and failure rates than another. It is most likely that decisions regarding choice of laboratory will be based on other factors, such as reimbursement, ease of ordering and receiving results, charges, turnaround time, special circumstances (eg, known twins), method of reporting results, identification of particular microdeletions, and customer service. This last item is crucial, as some report results are cryptic [104], and the availability of knowledgeable support personnel is invaluable in providing easy-to-understand results to patients.
Individual clinicians, practices, and patients are targets for aggressive tactics to obtain market share. A systematic review concluded these companies' internet sites often do not provide supporting evidence for the information cited and may not provide adequate information about the need for an invasive test to definitively diagnose aneuploidy [105].
POST-TEST FOLLOW-UP
Screen positive (high risk) — Even with the high performance of cfDNA screening, an invasive procedure for diagnostic genetic testing must be offered to patients to confirm the fetal karyotype. There is some controversy about whether an early gestation cfDNA positive screen should be confirmed by chorionic villus sampling or postponed until ≥15 weeks when amniocentesis can be performed, as analysis of amniocytes is more accurate since the result is representative of the fetal genotype rather than placental cells and sometimes the two tissues are discordant [56]. Some patients may choose to delay diagnostic testing until after delivery, or even later, if the result would not affect pregnancy management.
A standard G-banded karyotype is optimal to obtain in cfDNA screen-positive cases. Fluorescence in situ hybridization (FISH) and microarray can identify additional copies of aneuploid segments but not their orientation to the other chromosomes. In 5 to 10 percent of pregnancies with a common aneuploidy, one of the parents will be identified with a balanced translocation, and this information will alter reproductive counseling. Thus, if FISH or microarray is the initial diagnostic test and is positive, then a standard karyotype should also be performed.
Screen negative (low risk) — A screen-negative result means the fetus is at a reduced risk of having one of the aneuploidies in the test panel, but it does not eliminate the possibility of an affected fetus or the possibility of a fetus with a chromosomal abnormality not targeted by the screening test but detectable with diagnostic testing. Screen-negative patients are usually not offered an invasive procedure for diagnostic genetic testing because of the high (≥99.9 percent) NPV if cfDNA testing and risk, albeit small, of invasive procedures.
However, screen-negative patients who go on to develop an indication for an invasive procedure for diagnostic genetic testing, such as a fetal anatomic anomaly on ultrasound examination, should be offered this testing. This recommendation does not apply to the fetus found to have an isolated soft marker. (See "Prenatal genetic evaluation of the fetus with anomalies or soft markers".)
Test failures (no result)
●Rate – No result rates generally fall between 1 and 3.5 percent [106,107]. However, a wide range of cfDNA failure rates (ie, no result) has been reported, likely because the rate depends on population characteristics (eg, proportion of patients with obesity), test method, and whether a second sample is routinely tested after an initial failure.
It is important to consider whether the laboratory's reported failure rate includes only total assay failures (eg, the interpretation for trisomy 21 is present, but those for trisomy 18 and trisomy 13 are not); includes only failures related to chromosomes 21, 18, and 13; or includes failures to provide sex chromosome or other interpretations, such as microdeletion syndromes.
●Reasons for test failure
•Less than a specified absolute amount of total and/or fetal-placental DNA, or fetal fraction below an acceptable level (eg, <3.5 percent). Low fetal fraction may be responsible for up to 50 percent of all failures, depending on methodology. Causes of a low fetal fraction are described above. (See 'Significance of the fetal fraction' above.)
•Long stretches of homozygosity (fragments in which identical gene sequences are discovered originating from the maternally and paternally derived chromosomes). When using a single nucleotide polymorphism s(SNP) analysis for testing, the laboratory cannot distinguish between the two similar DNA sources. Examples include uniparental disomy (inheritance of both chromosomes from one parent) or parental consanguinity.
•The laboratory's requirements for cfDNA test performance. Laboratories that prioritize minimizing false-positive and false-negative results may accept a higher rate of failures. This might be most appropriate if most of the samples are already considered to be at high risk. Other laboratories may consider false negatives and false positives to be an inevitable consequence of any screening test in a general pregnancy population and may place more emphasis on a low failure rate. Reporting a test failure may not result in appropriate follow-up.
•Some laboratories will identify results as "borderline" and will not make a screen-positive or screen-negative call even when all quality control parameters are met. In such instances, a "borderline" call should be considered screen positive and not a test failure or screen negative since follow-up action is needed.
●Patient management – There is no standard approach. The patient has three options in this setting:
•Repeat the cfDNA test after seven days (or more), which is around the time that test failure is reported. Some failures (eg, those due to large regions of homozygosity) will always cause the test to fail, so repeat testing is not an option. Repeat testing, when allowed, is successful in approximately 60 to 80 percent of cases [34,107,108]. Laboratory reports should indicate whether or not a repeat sample is recommended for each patient with a failed test.
•Offer an invasive procedure (amniocentesis, chorionic villus sampling) for diagnostic testing (karyotyping/microarray). This is definitive and probably the best option if the pregnancy was already at high risk for aneuploidy prior to cfDNA testing (eg, advanced maternal age, abnormal ultrasound).
A few studies suggest that there may be a higher than expected rate of aneuploidy in patients with cfDNA test failures, and these cases often have very low fetal fractions (eg, <2 percent) [19,34,109-113]. When reviewing this literature, it is important to ensure that the testing was equivalent to that found in clinical practice. For example, the proportion of aneuploid pregnancies among initial test failures may not be representative of that found after the laboratory-specific repeat cfDNA testing protocol is used, as is often the practice.
The American College of Obstetricians and Gynecologists (ACOG) recommends informing patients that test failure is associated with an increased risk of certain aneuploidies, providing additional genetic counseling, and offering comprehensive ultrasound evaluation and diagnostic testing [81].
•Although uncommon in the United States, some programs offer standard serum biomarker or combined serum biomarker/ultrasound screening using a lower risk threshold. For example, the screen-positive rate may be increased to 10 or 15 percent of the population. Screen-positive patients are then reflexed or offered cfDNA testing. However, patients should understand that standard serum biomarker/ultrasound screening is a less sensitive test than cfDNA screening and typically screens for trisomies 21 and 18, although some will detection trisomy 13 and sex chromosome aneuploidy (eg, first-trimester screening will identify trisomy 13 as part of the trisomy 18 screen). Although serum biomarker screening may miss some trisomy 18 and most trisomy 13, these diagnoses are associated with multiple structural anomalies that are likely to be detected when a second-trimester ultrasound examination is performed. (See "Sonographic findings associated with fetal aneuploidy", section on 'Trisomy 18 (Edward syndrome)' and "Sonographic findings associated with fetal aneuploidy", section on 'Trisomy 13 (Patau syndrome)'.)
Although a prospective cohort study reported an increased risk for developing preeclampsia and preterm birth in pregnancies with a nonreportable cfDNA and euploid fetus, this association may have been related to the higher prevalence of obesity and chronic hypertension in the nonreportable group [114]. These characteristics are risk factors for both of these adverse outcomes.
Second-trimester screening for fetal structural anomalies — Patients who undergo cfDNA screening should be offered additional second-trimester ultrasound screening and/or maternal serum alpha-fetoprotein (AFP) screening for fetal structural anomalies, including open neural tube defects [81].
SOCIETY GUIDELINE LINKS —
Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Prenatal genetic screening and diagnosis" and "Society guideline links: Down syndrome".)
SUMMARY AND RECOMMENDATIONS
●Biology – Prenatal screening for fetal aneuploidy can be performed by analyzing cell-free DNA (cfDNA) in maternal blood, which derives from both maternal and placental sources during pregnancy. Placental cfDNA is rapidly cleared from the maternal circulation after delivery, thus cfDNA test results in future pregnancies are not affected by prior pregnancies. (See 'Maternal and fetal-placental origins' above and 'Clearance of cfDNA after delivery' above.)
●Advantages compared with other screening approaches – Compared with serum biomarker/ultrasound screening for the common aneuploidies, the main advantages of cfDNA screening include higher detection rates (DRs) and fewer false-positive results (thus fewer unnecessary invasive procedures for diagnostic genetic testing) (table 1). In addition, results are often available at an earlier gestational age. (See 'Primary cfDNA screening' above and 'Secondary cfDNA screening' above.)
●Pretest counseling – Issues that should be discussed with patients when offering prenatal aneuploidy screening using cfDNA include the following:
•The difference between screening and diagnostic testing, and that choosing to undergo screening or diagnostic testing is optional. (See 'Patient pretest education and counseling' above.)
•A discussion of the specific aneuploidies being screened for (trisomy 21 [Down syndrome], trisomy 18, trisomy 13, and common sex chromosome aneuploidies). Patients also should understand the limited scope of the test (ie, it does not screen for other genetic conditions or congenital anomalies). (See 'Trisomy 21, 18, and 13' above and 'Sex chromosome aneuploidies' above.)
•The possibility of identifying maternal conditions of medical significance (eg, malignancy, mosaicism). (See 'False-positive cfDNA test results' above.)
•Test performance (eg, DR, false-positive rate [FPR], test failure rate, patient-specific risk, positive and negative predictive value [PPV, NPV]), and need to confirm abnormal screening results before considering pregnancy termination. (See 'Screening performance in singleton pregnancies' above.)
Patients with obesity should be informed that their chance of having an initial cfDNA test failure is higher than patients without obesity. (See 'Maternal obesity' above and 'Causes of a low fetal fraction' above.)
●Screening performance
•Trisomy 21, 18, and 13 – cfDNA is the most sensitive screening option (highest DR) for trisomy 21, 18, and 13 and the most specific. Performance varies by trisomy. Based on multiple meta-analyses, the consensus DRs and FPRs in successful tests are as follows (see 'Trisomy 21, 18, and 13' above):
-Trisomy 21 – DR 99.5 percent, FPR 0.05 percent
-Trisomy 18 – DR 97.7 percent, FPR 0.04 percent
-Trisomy 13 – DR 96.1 percent, FPR 0.06 percent
The total FPR is the sum of the three rates, or 0.15 percent. However, these data do not account for test failures.
•Sex chromosome aneuploidies – In the largest meta-analysis that evaluated cfDNA test performance for sex chromosome aneuploidies, the DR and FPR for monosomy X (177 cases and 9079 controls) were 90.3 and 0.23 percent, respectively. For the sex chromosome trisomies, 47,XXX; 47,XXY; and 47,XYY (56 cases and 6699 controls), the DR and FPR were 93 and 0.14 percent, respectively. (See 'Sex chromosome aneuploidies' above.)
For the sex chromosomes, the genotype may be accurate but discordant with the phenotype. Genotype-phenotype discordance has been attributed to laboratory error, a vanishing twin, complex disorders of sexual differentiation, sex chromosome aneuploidy with/without mosaicism, and maternal mosaicism. (See 'False-positive and false-negative results' above.)
•Twin gestations – DRs and FPRs are similar for twin versus singleton gestations. Because it is impossible to determine which twin is abnormal based on cfDNA analysis alone, results are reported for the entire pregnancy. Some commercial laboratories can provide zygosity information, which may be helpful in interpretation. Alternative options, such as ultrasound measurement of nuchal translucency (NT) or serum screening, have far lower DRs and higher FPRs. (See 'Multiple gestations' above.)
●Post-test follow-up
•Screen-positive test results
-An invasive procedure (chorionic villus sampling [CVS] or amniocentesis) to obtain samples for diagnostic genetic testing (eg, karyotype or microarray) should be offered to confirm all screen-positive test results. Results will be available sooner if CVS is performed, but amniocytes are more accurate when fetal-placental discordancy is present. Diagnostic testing is particularly important if pregnancy termination is being considered based on these results. For disorders in which definitive diagnosis will not affect the decision to continue the pregnancy or pregnancy management, the parents' choice to delay diagnostic testing until after delivery, or even later, is also reasonable. (See 'Screen positive (high risk)' above.)
-False-positive tests may be due to confined placental mosaicism, demised twin, maternal mosaicism, maternal cancer, maternal copy number variants, technical issues, or chance. (See 'False-positive cfDNA test results' above.)
•Screen-negative test results
-Screen-negative patients are not routinely offered invasive diagnostic testing. The high sensitivity and specificity of cfDNA screening means these patients are at a very low risk of having a fetus affected by one of the aneuploidies in the test panel, although the possibility of an affected fetus cannot be eliminated. If fetal structural abnormalities are found on ultrasound, diagnostic testing with a chromosome microarray should be offered. (See 'Screen negative (low risk)' above.)
-False-negative tests may be due to confined placental mosaicism, borderline low fetal fraction, maternal copy number variants, and technical issues. (See 'False-negative cfDNA test results' above.)
•Test failure (no call result)
-No result rates generally fall between 1 and 3 percent. Maternal plasma must contain an adequate amount of fetal cfDNA (fetal fraction) to obtain a reliable test result. Several factors can reduce the fetal fraction, which can lead to an assay failure (a report of "no result"): screening before 9 to 10 weeks of gestation, suboptimal sample collection or processing, obesity, some types of fetal aneuploidy (eg, trisomy 18, triploidy), maternal use of low molecular weight heparin, conception by in vitro fertilization (IVF), or multiple gestation. (See 'Significance of the fetal fraction' above.)
-Patients whose test does not yield a result can choose to undergo repeat cfDNA testing, an invasive diagnostic procedure, or standard serum marker/ultrasound screening. Repeat testing, when allowed, is successful in approximately 60 to 80 percent of cases. (See 'Test failures (no result)' above.)
