Noninvasive prenatal screening options have been offered to pregnant individuals for almost 40 years, giving families the opportunity to optimize pregnancy outcomes that align with their values and preferences (American College of Obstetricians and Gynecologists [ACOG], 1996). Noninvasive prenatal screening using cell-free DNA (cfDNA) (also referred to as noninvasive prenatal testing and noninvasive prenatal screening) became available in the United States in 2011 and was developed with the goal to make noninvasive prenatal screening more sensitive, reducing the false-positive rate of test results (Palomaki et al., 2011).

Noninvasive prenatal screening using cell-free DNA: basic facts

Noninvasive prenatal screening using cfDNA is the most sensitive and specific prenatal screening option for common fetal aneuploidies (ACOG, 2020; Gregg et al., 2016). It is less time sensitive than other options and can be offered to all pregnant individuals with a singleton or twin pregnancy as early as 10 weeks' gestation to determine if their fetus has an increased risk for three chromosomal disorders, trisomy 21 (Down syndrome), trisomy 13 (Patau syndrome), and trisomy 18 (Edwards syndrome). Multiple meta-analysis of returned results indicate a 99.5% detection rate for trisomy 21, 96.1% detection rate for trisomy 13, and 97.7% detection rate for trisomy 18, with a combined false-positive rate of 0.15% (Palomaki et al., 2021). The cfDNA test may be used to screen for fetal sex; however, prescreen counseling is recommended, so pregnant individuals understand that the test is intended to screen for fetal sex chromosomal abnormalities and that they will be informed of such findings along with fetal sex (ACOG, 2020; Palomaki et al., 2021). Studies, however, suggest detection and false-positive rates are less robust for these abnormalities. Detection rates range from 90.3% to 93% and false-positive rates range from 0.23% to 0.14% (Mackie et al., 2017). Similar to all noninvasive prenatal screening options, the cfDNA test is only a screening test addressing risk for a medical condition. It is not a diagnostic test.

The cfDNA test involves analysis of a sample of maternal blood for fragments of cfDNA from the fetal-placental unit, which are typically smaller than 150 base pairs and derived primarily from placental trophoblasts released into the maternal blood stream from cells undergoing programmed cell death (ACOG, 2020; Palomaki et al., 2021). Three general approaches using next-generation sequencing technology can be implemented to examine the fetal-placental cfDNA fragments. These laboratory processes incorporate specific computerized algorithms to determine if there are more than the expected number of cfDNA fragments or single-nucleotide polymorphisms associated with chromosomes 21, 18, 13, X, and Y (Allyse & Wick, 2018; Palomaki et al., 2021). Successful testing is dependent on adequate fetal fraction in the maternal blood sample.

Fetal fraction

The fetal-placental component in maternal blood is known as the fetal fraction and comprises approximately 10% of the total cfDNA in maternal blood in the late first trimester and increases throughout gestation (Wang et al, 2013). The quantity of fetal fraction must be at least 4% to avoid test failure (ACOG, 2020; Palomaki et al., 2021). Many factors can contribute to a low fetal fraction, including early gestational age (<10 weeks), high maternal body mass index (maternal weight greater than 180-250 pounds), inadequate blood sample, abnormal fetal chromosome number, such as trisomy 13 and 18 and Turner syndrome, maternal use of heparin before 20 weeks, fetal or maternal mosaicism, and a pregnancy with more than one fetus (ACOG, 2020; Palomaki et al., 2021).

Professional clinical recommendations

Professional recommendations have been published by ACOG and the American College of Medical Genetics and Genomics (ACMG). The ACOG (2020) states the cfDNA test is an optional test that should be routinely offered to pregnant individuals. ACMG states that individuals' preferences and values should play an important role in guiding the use of the cfDNA test (Gregg et al., 2016). The importance of prescreen and postscreen counseling for individuals who are considering the cfDNA test is emphasized by both professional organizations. Before testing, counseling should include the purpose of the screening, its limitations, the screening process, possible results of the screening (positive, negative, and no call) and their implications, residual risk (risk of having an inherited condition that was not screened), and possibility of incidental findings that affect the mother, such as medical conditions reflecting mosaicism, malignancy, or an abnormality in her chromosome(s). In postscreen counseling, the test result and its clinical implications should be explained. The ACOG (2020) recommends a baseline ultrasound before the cfDNA test to detect findings that may affect accuracy of results, such as an earlier-than-expected gestational age, fetal viability, number of fetuses, presence of a nonviable twin, and fetal anomalies. Moreover, use of the cfDNA test to assess for single-gene disorders or other autosomal chromosomal abnormalities, such as microdeletions, large deletions, and duplications, is available through some laboratories; however, this screening lacks clinical validation and is not currently recommended (ACOG, 2020; Gregg et al., 2016).

Screen results

Results from the cfDNA test are commonly reported as screen positive, screen negative, or no call or no result, which means the interpretation of the test could not be done. Because this is a screening test, false-positive and false-negative results can occur; however, they are less common than with other prenatal screening options (Palomaki et al., 2021). Potential causes of false-positive and false-negative results include mosaicism, in which there are cells in the fetus, placenta, and/or pregnant individual that have abnormal chromosomes and other cells have normal chromosomes, maternal chromosome duplication or deletion, technical issues or labeling errors, and chance (ACOG, 2020; Palomaki et al., 2021). Additional potential causes of false-positive results include a deceased twin, maternal cancer, and the mother is a transplant recipient or recently received donor blood. Low fetal fraction can also cause a false-negative result (ACOG, 2020; Palomaki et al., 2021); consequently, all laboratories should report the fetal fraction on result report (Gregg et al., 2016).

Screen positive result indicates the fetus has an increased risk for the chromosomal abnormality presented in the report. Patients should be counseled regarding their revised risk, given information about the condition, offered genetic counseling services if available, reminded of the possibility of false-positive result, and offered a comprehensive ultrasound evaluation with an opportunity for diagnostic testing, such as chorionic villi sampling or amniocentesis to collect fetal cells, to confirm results with karyotype analysis (ACOG, 2020; Gregg et al., 2016; Palomaki et al., 2021). Critical decisions, such as termination of the pregnancy, should never be based on results of the cfDNA test (ACOG, 2020). If the patient declines diagnostic testing, pregnancy management should be based on serial ultrasounds and patient preferences, as well as appropriate intrapartum preparation to care for a potentially affected newborn (ACOG, 2020; Palomaki et al., 2021).

Screen negative results indicate that a fetus is at reduced risk for abnormalities of chromosomes tested and further evaluation is not usually necessary. The negative result, however, does not guarantee the fetus is free from the conditions screened due to a false-negative result, or from other chromosome and genetic abnormalities and noninherited conditions not tested. This is known as residual risk (Gregg et al., 2016; Palomaki et al., 2021). It is extremely important that the provider fully explains the limitations of the screening test.

Approximately 1-5% of cfDNA tests do not produce a result and are reported as no call or no result (Palomaki et al., 2021). Possible reasons include low fetal fraction and its potential causes, as well as in vitro fertilization (Palomaki et al., 2021). With no call results, ACOG (2020) recommends the patient be informed such results can be associated with increased risk for chromosomal abnormality, be referred to genetic counseling, and be offered comprehensive ultrasound evaluation and diagnostic testing. ACMG recommends all laboratories include the reason for the no call on the report, diagnostic testing be offered if result is due to low fetal fraction, and/or maternal blood be drawn at an appropriate gestational age. In cases of high maternal obesity, consider other screening options, such as first and/or second trimester screening, for abnormal fetal chromosomes (Gregg et al., 2016).

Conclusions

Noninvasive prenatal screening using cfDNA is the most sensitive and specific screening for trisomy 21, 13, and 18 and sex chromosome aneuploidies in pregnancy after 10 weeks' gestation. Although laboratories offer cfDNA screening for single-gene disorders and other chromosomal abnormalities, it lacks clinical validation and is not currently recommended. Positive screen results need further evaluation for diagnosis to confirm a medical condition, certainly before critical decisions regarding pregnancy are made. Professional organizations provide clinical recommendations to providers regarding prescreen and postscreen counseling and management of results.

References