There are multiple approaches for analyzing cfDNA, including genome-wide massively parallel shotgun sequencing (MPSS), targeted MPSS for particular chromosomal segments, and directed single nucleotide polymorphism (SNP) sequencing. These methods all test DNA fragments from the pregnant patient's blood stream, representing a combination of placental (fetal) and patient's DNA. The methods differ in the selectivity of what is being tested within those fragments and the comparisons used to determine the presence of aneuploidy.

Each method has its strengths and limitations, and reporting may be slightly different. For example, SNP-based testing can provide information about triploidy or a health condition in the patient but is unsuitable for pregnancies created through egg donation. However, all methods provide similar information regarding the chance of a chromosome abnormality. Consult with the lab for specific test parameters.

All forms of prenatal assessments for fetal genetic conditions have benefits and limitations with respect to risk to the pregnancy, what can be detected, and accuracy. (See comparison table resource for more detail.) cfDNA screening has some advantages over other prenatal tests, both screening and diagnostic. However, cfDNA screening is not a comprehensive genomic test, and remains a screening test that does not provide certainty.

Benefits

High predictive value compared to other screening tests for aneuploidy

• High predictive value for Down syndrome, including twin pregnancies
• Better predictive value for common chromosome aneuploidies such as trisomy 13 and 18 than other screening tests, in both low and high-risk pregnancies
• False-negative results are rare

Safety

• Non-invasive
• No risk of miscarriage or other fetal complications

Early availability

• Larger timing window than other prenatal screens; it can be performed at any time after 10 weeks gestation

Increasing scope

• cfDNA screening may provide sex chromosome analysis
• Some cfDNA screening provides analysis for less common microdeletion/duplication syndromes, unlike other prenatal screening options
• cfDNA screening for single-gene disorders is an emerging option available through select labs

Limitations

Limited predictive value compared to diagnostic testing

• Not diagnostic; false-negative results and false-positive results occur
• Lower positive predictive value in patients in low-risk groups compared to high-risk groups
• Lower positive predictive value for less common chromosome disorders (e.g., microdeletion syndromes, sex chromosome abnormalities)

Targeted scope

• Not a comprehensive chromosome or genome-wide test
• Does not evaluate for anatomical abnormalities like open neural tube defects
• Does not assess the cause of trisomy (whole aneuploidy vs. translocation), which is helpful for assessing risk to future pregnancies
• May not detect mosaicism

Secondary findings

• May detect patient health concerns such as chromosome abnormalities or an undiagnosed cancer
• May detect parental consanguinity

Limited application in certain situations

• Only certain labs are validated for twin pregnancies
• Only certain labs are able to analyze pregnancies using donor eggs or surrogates
• Not validated for triplet or higher-order multiple gestation pregnancies
• Higher failure rate in women with high BMI

Results may be uninformative due to insufficientlow fetal fraction or unclear fetal DNA profile.

The specific laboratory offering cfDNA screening may identify additional limitations or considerations for cfDNA screening based on the test technology used. See Screening Methods in Dig Deeper for more information about how specific limitations of different sequencing technologies impact conditions that can be detected.

Indications

• Down syndrome (trisomy 21)
• Edwards syndrome (trisomy 18)
• Patau syndrome (trisomy 13)
• Sex chromosome abnormalities

While screening is possible and included in tests by some labs, ACOG states there is limited evidence for recommending the following:

• Rare trisomies (e.g., T16, T22)
• Microdeletions

ACMG recommends informing patients of the availability of microdeletion screening through cfDNA; if the patient also receives appropriate pre-test counseling about the benefits, limitations and possible outcomes of such screening.

Contraindications

• Higher-order gestations (triplet or greater)
• Vanishing twin syndrome
• Concurrent cfDNA and serum screening

Depending on the cfDNA screening technology used, the lab may identify other contraindications.

Patients making decisions about assessment for common chromosome abnormalities during pregnancy consider many factors in their decision, including (but not limited to):

• Concern about having a child with a chromosomal abnormality
• Concern about putting the pregnancy at risk for miscarriage
• How results will be used
• Family values
• Religious beliefs
• Availability of family/social support
• Insurance coverage
• Timing for reproductive decision-making

Providers counseling patients about screening and testing options should be sensitive to these issues and present accurate and balanced information about the risks and benefits of cfDNA screening. Up-to-date, unbiased information about what it means to have a child with a chromosome abnormality should be available. Genetic counseling to help patients understand the options, including when screening may not be an option (e.g., lack of insurance coverage, gestational age) and the option to decline any or all testing, may be helpful to many women. ACOG/ SMFM, ACMG, ISPD and the NSGC reinforce the importance of genetic counseling either prior to cfDNA screening or for all patients with an abnormal result.

It is critical to counsel patients about the importance of confirmatory testing if cfDNA screening is positive. Patients who choose to terminate a screen-positive pregnancy, believing that the results are diagnostic, may unknowingly terminate an unaffected fetus due to a false-positive cfDNA screen.

Clinicians can prepare patients for what to expect if they receive a positive result, which will include a discussion of diagnostic testing to confirm the cfDNA result, during pregnancy or at birth. Amniocentesis is the preferred confirmatory test since CVS is examining cells from the same material (placenta) contributing "fetal" cfDNA. If placental mosaicism is present, it is likely to be responsible not only for a false-positive cfDNA result but also for a false-positive CVS result. Amniocentesis examines fetal cells directly and is thus more appropriate as a follow-up procedure.

In addition to providing important information for the current pregnancy, diagnostic testing will identify the etiology of the aneuploidy. Most cases of Down syndrome, for example, are due to an extra copy of chromosome 21, which is usually a sporadic occurrence that does not increase risk to future children. Some cases of Down syndrome are the result of a chromosome translocation, which can be inherited and future children may be at risk.

If confirmatory testing is discordant with cfDNA screening results, there may be an underlying fetal, placental or patient condition causing the false-positive result.

Potential reasons for a false-positive result

Fetal and placental conditions

• Confined placental mosaicism. The fetus and placenta may have different genetic compositions. The placenta may have some cells with aneuploidy while the fetus is unaffected
• Twin demise (vanishing twin). If a co-twin dies early in gestation, residual cfDNA from the deceased co-twin may persist in the maternal blood stream. Screening may detect this extra cfDNA, but may not be able to distinguish the source.

Patient conditions

• Previous organ transplantation. If a patient has received an organ transplant from a male donor, the presence of the donor’s Y chromosome may be detected in cfDNA screening, resulting in misattributed fetal sex or a false-positive for a sex chromosome condition.
• Mosaicism. The pregnant patient may have cells with different genetic make-up, such as cells with a normal chromosome composition and cells with aneuploidy.
• Chromosome condition. Previously undetected sex chromosome conditions, microdeletion syndromes, and other chromosome conditions in the pregnant patient may be identified through cfDNA screening as a false-positive result and/or an incidental finding.
• Cancer. Cancer cells often have unusual chromosome arrangements. Rarely, cfDNA screening may identify cancer in the patient as a false-positive and/or a secondary finding