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Noninvasive Prenatal Fetal Screening Tests

By

Jeffrey S. Dungan

, MD, Northwestern University, Feinberg School of Medicine

Reviewed/Revised Jan 2024
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Topic Resources

Noninvasive prenatal screening for genetic disorders, unlike invasive testing, has no risk of test-related complications. Noninvasive maternal screening can help women decide whether to have invasive testing. Noninvasive maternal screening for fetal chromosomal abnormalities should be offered to all pregnant women who have not already decided to have amniocentesis or chorionic villus sampling (CVS). However, even if CVS is to be done, maternal serum screening should still be offered to check for fetal neural tube defects Maternal serum screening for neural tube defects Noninvasive prenatal screening for genetic disorders, unlike invasive testing, has no risk of test-related complications. Noninvasive maternal screening can help women decide whether to have... read more Maternal serum screening for neural tube defects .

Normal values vary with gestational age. Corrections for maternal weight, diabetes mellitus, race, and other factors may be necessary. Screening can be done during the

  • First trimester

  • Second trimester

  • Both trimesters (called sequential or integrated screening)

Any of the three approaches is acceptable. Maternal levels of alpha-fetoprotein should be measured during the second trimester to check for neural tube defects. The American College of Obstetricians and Gynecologists (ACOG) provides recommendations for screening for fetal chromosomal abnormalities and a chart to show the timing of prenatal testing for chromosomal abnormalities (see ACOG: Prenatal Genetic Testing Chart).

Pearls & Pitfalls

  • Measure maternal levels of alpha-fetoprotein during the second trimester to check for neural tube defects regardless of other tests planned and the timing of those tests.

Screening in multiple gestations

All forms of screening in singleton pregnancies are available to patients with a twin pregnancy. For twin pregnancies, screening performance using traditional methods (triple Maternal serum screening for neural tube defects Noninvasive prenatal screening for genetic disorders, unlike invasive testing, has no risk of test-related complications. Noninvasive maternal screening can help women decide whether to have... read more Maternal serum screening for neural tube defects , quad) has lower sensitivity and specificity than in singleton pregnancies. Cell-free DNA First-Trimester Screening Noninvasive prenatal screening for genetic disorders, unlike invasive testing, has no risk of test-related complications. Noninvasive maternal screening can help women decide whether to have... read more First-Trimester Screening (cfDNA) screening performance appears to be comparable for singleton and twin pregnancies. Because most dichorionic twin gestations are discordant for chromosome abnormalities, diagnostic testing is required to distinguish which twin is affected. However, screening for sex chromosome abnormalities in twin pregnancies is usually not available.

No serum screening or cfDNA screening protocols are validated for triplet or higher-order pregnancies.

First-Trimester Screening

First-trimester screening should be offered to all pregnant women. It provides information early so that a definitive diagnosis can be made with CVS. An important advantage of first-trimester screening is that termination of pregnancy is safer during the first rather than the second trimester.

One method of screening for fetal Down syndrome Down Syndrome (Trisomy 21) Down syndrome is an abnormality of chromosome 21 that can cause intellectual disability, microcephaly, short stature, and characteristic facies. Diagnosis is suggested by physical anomalies... read more Down Syndrome (Trisomy 21) , trisomy 18 Trisomy 18 Trisomy 18 is caused by an extra chromosome 18 and is usually associated with intellectual disability, small birth size, and various congenital anomalies, including severe microcephaly, heart... read more Trisomy 18 , and trisomy 13 Trisomy 13 Trisomy 13 is caused by an extra chromosome 13 and causes abnormal forebrain, midface, and eye development; severe intellectual disability; heart defects; and small birth size. Diagnosis is... read more Trisomy 13 is analysis of cell-free DNA First-Trimester Screening Noninvasive prenatal screening for genetic disorders, unlike invasive testing, has no risk of test-related complications. Noninvasive maternal screening can help women decide whether to have... read more First-Trimester Screening (cfDNA) in maternal plasma, which can be done as early as 10 weeks of gestation. Detection rates using this technology are higher than those with older methods. Another method, called analyte screening, uses multiple maternal serum markers Maternal serum screening for neural tube defects Noninvasive prenatal screening for genetic disorders, unlike invasive testing, has no risk of test-related complications. Noninvasive maternal screening can help women decide whether to have... read more Maternal serum screening for neural tube defects (alpha-fetoprotein, beta-human chorionic gonadotropin [beta-hCG], estriol, inhibin A) to detect neural tube defects Maternal serum screening for neural tube defects Noninvasive prenatal screening for genetic disorders, unlike invasive testing, has no risk of test-related complications. Noninvasive maternal screening can help women decide whether to have... read more Maternal serum screening for neural tube defects , Down syndrome Down Syndrome (Trisomy 21) Down syndrome is an abnormality of chromosome 21 that can cause intellectual disability, microcephaly, short stature, and characteristic facies. Diagnosis is suggested by physical anomalies... read more Down Syndrome (Trisomy 21) (and other chromosomal abnormalities), and some other birth defects. Analyte screening is done at 15 to 20 weeks gestation.

Cell-free fetal nucleic acid testing

Cell-free DNA (cfDNA) testing is a type of noninvasive fetal screening that can identify fetal chromosomal abnormalities in singleton pregnancies by analyzing circulating cell-free fetal nucleic acids in a maternal blood sample. This test can be done as early as 10 gestational weeks and has replaced traditional first- and second-trimester noninvasive screening in many medical centers. Cell-free DNA screening is more accurate than serum marker screening Maternal serum screening for chromosomal abnormalities Noninvasive prenatal screening for genetic disorders, unlike invasive testing, has no risk of test-related complications. Noninvasive maternal screening can help women decide whether to have... read more Maternal serum screening for chromosomal abnormalities and does not depend on gestational age. CfDNA involves collecting fetal cells, but it is considered a screening test, not a definitive fetal diagnostic test.

Cell-free fetal nucleic acids, most commonly DNA fragments, are shed into the maternal circulation during normal breakdown of placental trophoblast cells. Variation in amounts of fragments from particular chromosomes predicts fetal chromosomal abnormalities with higher accuracy than traditional first- and second-trimester combined screening using serum analytes and ultrasonography. Also, sex chromosomal abnormalities (X, XXX, XYY, and XXY) can be identified in singleton pregnancies, although with somewhat lower accuracy. Early validation trials reported > 99% sensitivity and specificity for the identification of Down syndrome (trisomy 21) and trisomy 18 Trisomy 18 Trisomy 18 is caused by an extra chromosome 18 and is usually associated with intellectual disability, small birth size, and various congenital anomalies, including severe microcephaly, heart... read more Trisomy 18 in high-risk pregnancies. Trisomy 13 Trisomy 13 Trisomy 13 is caused by an extra chromosome 13 and causes abnormal forebrain, midface, and eye development; severe intellectual disability; heart defects; and small birth size. Diagnosis is... read more Trisomy 13 can also be detected, although the sensitivity and specificity are somewhat lower (1 First-trimester screening references Noninvasive prenatal screening for genetic disorders, unlike invasive testing, has no risk of test-related complications. Noninvasive maternal screening can help women decide whether to have... read more First-trimester screening references ).

Cell-free DNA (cfDNA) screening was historically recommended only for women with preexisting risk factors for fetal trisomy. However, it is now commonly used in both average-risk and high-risk patients. The American College of Obstetricians and Gynecologists recommends offering cell-free DNA screening to all pregnant women (2 First-trimester screening references Noninvasive prenatal screening for genetic disorders, unlike invasive testing, has no risk of test-related complications. Noninvasive maternal screening can help women decide whether to have... read more First-trimester screening references ). The American College of Medical Genetics and Genomics issued an evidence-based guideline advocating for cell-free DNA screening as the preferred method for all singleton and twin pregnancies (3 First-trimester screening references Noninvasive prenatal screening for genetic disorders, unlike invasive testing, has no risk of test-related complications. Noninvasive maternal screening can help women decide whether to have... read more First-trimester screening references ).

  • Trisomy 21: sensitivity 99%; specificity 100%

  • Trisomy 18: sensitivity 98%; specificity 100%

  • Trisomy 13: sensitivity 91%; specificity 100%

Abnormal results from cfDNA screening should be confirmed with diagnostic karyotyping using fetal specimens obtained through invasive techniques. Negative results from cfDNA screening has reduced the use of routine invasive testing.

Traditionally, first-trimester combined screening includes measurement of

  • Maternal serum beta-hCG (total or free)

  • Pregnancy-associated plasma protein A (PAPP-A)

  • Sometimes, fetal nuchal translucency (by ultrasonography)

Fetal Down syndrome is typically associated with high levels of beta-hCG, low levels of PAPP-A, and enlarged fetal nuchal translucency. Although enlarged nuchal translucency is associated with increased risk of fetal Down syndrome Down Syndrome (Trisomy 21) Down syndrome is an abnormality of chromosome 21 that can cause intellectual disability, microcephaly, short stature, and characteristic facies. Diagnosis is suggested by physical anomalies... read more Down Syndrome (Trisomy 21) , no threshold value for nuchal translucency is considered diagnostic.

In a large prospective study that included women of various ages, overall sensitivity for detection of Down syndrome was approximately 85%, with a false-positive rate of 5% (5 First-trimester screening references Noninvasive prenatal screening for genetic disorders, unlike invasive testing, has no risk of test-related complications. Noninvasive maternal screening can help women decide whether to have... read more First-trimester screening references ). Specialized ultrasound training and adherence to rigorous quality-assurance monitoring of nuchal translucency measurements are necessary to achieve this level of screening accuracy.

First-trimester screening references

  • 1. Badeau M, Lindsay C, Blais J, Nshimyumukiza L, et al. Genomics-based non-invasive prenatal testing for detection of fetal chromosomal aneuploidy in pregnant women. Cochrane Database Syst Rev 11:CD011767, 2017. doi: 10.1002/14651858.CD011767.pub2

  • 2. Practice Bulletin No. 162: Prenatal Diagnostic Testing for Genetic Disorders. Obstet Gynecol. 2016;127(5):e108-e122. doi:10.1097/AOG.0000000000001405

  • 3. Dungan JS, Klugman S, Darilek S, et al: Noninvasive prenatal screening (NIPS) for fetal chromosome abnormalities in a general-risk population: An evidence-based clinical guideline of the American College of Medical Genetics and Genomics (ACMG) [published correction appears in Genet Med 2023 Aug;25(8):100874]. Genet Med 25(2):100336, 2023. doi:10.1016/j.gim.2022.11.004

  • 4. Mackie FL, Hemming K, Allen S, et al: The accuracy of cell-free fetal DNA-based non-invasive prenatal testing in singleton pregnancies: a systematic review and bivariate meta-analysis. BJOG 124(1):32-46, 2017. doi:10.1111/1471-0528.14050

  • 5. Malone FD, Canick JA, Ball RH, et al: First-trimester or second-trimester screening, or both, for Down's syndrome. N Engl J Med 353(19):2001-2011, 2005. doi:10.1056/NEJMoa043693

Second-Trimester Screening

Serum marker screening includes

Second-trimester multiple marker screening is used to help assess the risk of Down syndrome Down Syndrome (Trisomy 21) Down syndrome is an abnormality of chromosome 21 that can cause intellectual disability, microcephaly, short stature, and characteristic facies. Diagnosis is suggested by physical anomalies... read more Down Syndrome (Trisomy 21) , trisomy 18 Trisomy 18 Trisomy 18 is caused by an extra chromosome 18 and is usually associated with intellectual disability, small birth size, and various congenital anomalies, including severe microcephaly, heart... read more Trisomy 18 , and a few rarer single-gene syndromes (eg, Smith-Lemli-Opitz syndrome). Maternal serum tests are widely available, but detection rates for Down syndrome are not as high as those obtained with first-trimester serum marker screening or with cfDNA. Also, termination of pregnancy is riskier in the second trimester than in the first trimester.

Maternal serum screening for chromosomal abnormalities

If maternal serum screening suggests Down syndrome, ultrasonography is done to confirm gestational age, and risk is recalculated if the gestational age is corrected. If the original sample was drawn too early based on the previously presumed gestational age, another one must be drawn at the appropriate time. Analysis of cfDNA does not depend on gestational age and thus is not prone to dating errors. In addition, amniocentesis is offered if serum screening indicates that risk of trisomy 21 exceeds a specific prespecified threshold (usually 1 in 270, which is about the same as risk when maternal age is > 35).

Maternal serum screening for neural tube defects

An elevated level of MSAFP may indicate a fetal malformation such as open spina bifida Spina Bifida Spina bifida is defective closure of the vertebral column. Although the cause is often unknown, low folate levels during pregnancy increase risk. Some children are asymptomatic, and others have... read more . Results are most accurate when the initial sample is obtained between 16 and 18 weeks gestation, although screening can be done from about 15 to 20 weeks.

Designating a cutoff value to determine whether further testing is warranted involves weighing the risk of a missed abnormality versus the risk of complications from unnecessary testing. Usually, a cutoff value in the 95th to 98th percentile, or 2.0 to 2.5 times the normal pregnancy median (multiples of the median, or MOM), is used. This value is about 80% sensitive for open spina bifida and 95% sensitive for anencephaly Anencephaly Anencephaly is absence of the cerebral hemispheres. It is usually accompanied by a defect in the formation of the skull posteriorly, leaving the back of the head without skeletal protection... read more . False positive rates are between 2 to 5% (4 Second-trimester screening references Noninvasive prenatal screening for genetic disorders, unlike invasive testing, has no risk of test-related complications. Noninvasive maternal screening can help women decide whether to have... read more Second-trimester screening references ). Closed spina bifida is usually not detected.

Amniocentesis Amniocentesis Prenatal procedures that provide a definitive diagnosis of genetic disorders are invasive and involve some fetal risk. Women may choose to have prenatal procedures to know of fetal abnormalities... read more is eventually required in 1 to 2% of women originally screened. Lower cutoff values of MSAFP increase sensitivity but decrease specificity, resulting in more amniocenteses. Women who have been screened for fetal chromosome disorders by cell-free DNA screening should have serum screening with MSAFP alone, not with multiple marker screening.

Ultrasonography is the next step if further testing is warranted. Targeted ultrasonography with or without amniocentesis is done if no explanation can be determined with basic ultrasonography. Ultrasonography can

  • Confirm gestational age (which may be underestimated)

  • Detect multifetal pregnancy, fetal death, or congenital malformations

In some women, ultrasonography cannot identify a cause for elevated alpha-fetoprotein levels. Some experts believe that if high-resolution ultrasonography done by an experienced operator is normal, further testing is unnecessary. However, because this test occasionally misses neural tube defects, many experts recommend further testing by amniocentesis regardless of ultrasonography results.

Amniocentesis with measurement of alpha-fetoprotein and acetylcholinesterase levels in amniotic fluid is done if further testing is needed. Elevated alpha-fetoprotein in amniotic fluid suggests

  • A neural tube defect

  • Another malformation (eg, omphalocele, congenital nephrosis, cystic hygroma, gastroschisis, upper gastrointestinal atresia)

  • Contamination of the sample with fetal blood

Presence of acetylcholinesterase in amniotic fluid suggests

  • A neural tube defect

  • Another malformation

Second-trimester screening references

  • 1. Conde-Agudelo A, Kafury-Goeta AC: Triple-marker test as screening for Down syndrome: a meta-analysis. Obstet Gynecol Surv 53(6):369-376, 1998. doi:10.1097/00006254-199806000-00022

  • 2. Wald NJ, Huttly WJ, Hackshaw AK: Antenatal screening for Down's syndrome with the quadruple test. Lancet 361(9360):835-836, 2003. doi:10.1016/S0140-6736(03)12680-3

  • 3. Breathnach FM, Malone FD, Lambert-Messerlian G, et al: First- and second-trimester screening: detection of aneuploidies other than Down syndrome. Obstet Gynecol 110(3):651-657, 2007. doi:10.1097/01.AOG.0000278570.76392.a6

  • 4. Palomaki GE, Bupp C, Gregg AR, et al: Laboratory screening and diagnosis of open neural tube defects, 2019 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG). Genet Med 22(3):462-474, 2020. doi:10.1038/s41436-019-0681-0

Sequential First- and Second-Trimester Screening

Noninvasive first-trimester and second-trimester quad screening can be combined sequentially, with invasive fetal genetic testing withheld until results of second-trimester screening are available—whether first-trimester test results are abnormal or not. Sequential screening followed by amniocentesis for high-risk patterns increases sensitivity for Down syndrome to 95%, with a false-positive rate of only 5%.

A variation of sequential screening, called contingent sequential screening, is based on the level of risk indicated by first-trimester screening:

  • High risk: Invasive testing is offered without doing second-trimester screening.

  • Intermediate risk: Second-trimester screening is offered.

  • Low risk (eg, < 1 in 1500): Second-trimester screening for Down syndrome is not offered because the first-trimester risk is so low.

Patients with abnormal first-trimester, second-trimester, or sequential screening should be offered diagnostic testing (eg, amniocentesis). However, some patients may choose to pursue further testing for fetal trisomy with cell-free DNA (cfDNA) analysis (1 Sequential first- and second trimester screening references Noninvasive prenatal screening for genetic disorders, unlike invasive testing, has no risk of test-related complications. Noninvasive maternal screening can help women decide whether to have... read more Sequential first- and second trimester screening references ). Results of cfDNA testing may indicate low risk and be reassuring but are not definitive. Also, cfDNA testing may be inordinately expensive, and awaiting results of cfDNA testing delays definitive testing such as chorionic villus sampling or amniocentesis (2 Sequential first- and second trimester screening references Noninvasive prenatal screening for genetic disorders, unlike invasive testing, has no risk of test-related complications. Noninvasive maternal screening can help women decide whether to have... read more Sequential first- and second trimester screening references ).

Sequential first- and second trimester screening references

Prenatal Ultrasonography

Most experts recommend ultrasonography Ultrasonography In ultrasonography, a signal generator is combined with a transducer. Piezoelectric crystals in the signal generator convert electricity into high-frequency sound waves, which are sent into... read more Ultrasonography routinely for all pregnant women. Others use ultrasonography only for specific indications, such as checking for suspected genetic or obstetric abnormalities or helping interpret abnormal maternal serum marker levels. If ultrasonography is done by skilled operators, sensitivity for major congenital malformations is high. However, some conditions (eg, oligohydramnios, maternal obesity, fetal position) interfere with obtaining optimal images. Ultrasonography is noninvasive and has no known risks to the woman or fetus.

Basic ultrasonography is done to

  • Confirm gestational age

  • Determine fetal viability

  • Detect a multifetal pregnancy

  • During the second or third trimester, possibly identify major malformations in the fetal intracranial structures, spine, heart, bladder, kidneys, stomach, thorax, abdominal wall, long bones, and umbilical cord

Although ultrasonography provides only structural information, some structural abnormalities strongly suggest genetic abnormalities. Multiple malformations may suggest a chromosomal disorder.

Targeted ultrasonography, with high-resolution ultrasonography equipment, is available at certain referral centers and provides more detailed images than basic ultrasonography. This test may be indicated for patients who have or whose partner has a family history of a congenital malformation (eg, congenital heart defects Overview of Congenital Cardiovascular Anomalies Congenital heart disease is the most common congenital anomaly, occurring in almost 1% of live births ( 1). Among birth defects, congenital heart disease is the leading cause of infant mortality... read more Overview of Congenital Cardiovascular Anomalies , cleft lip and palate Cleft Lip and Cleft Palate An oral-facial cleft is a birth defect in which the lip, the roof of the mouth, or both do not close in the midline and remain open, creating a cleft lip and/or cleft palate. These defects are... read more Cleft Lip and Cleft Palate , pyloric stenosis Hypertrophic Pyloric Stenosis Hypertrophic pyloric stenosis is obstruction of the pyloric lumen due to pyloric muscular hypertrophy. Diagnosis is by abdominal ultrasonography. Treatment is surgical. Hypertrophic pyloric... read more ), particularly one that may be treated effectively before birth (eg, posterior urethral valves with megacystis) or at delivery (eg, diaphragmatic hernia Diaphragmatic Hernia Diaphragmatic hernia is protrusion of abdominal contents into the thorax through a defect in the diaphragm. Lung compression may cause persistent pulmonary hypertension. Diagnosis is by chest... read more Diaphragmatic Hernia ). Targeted ultrasonography may also be used if maternal serum marker levels are abnormal and may also allow detection of the following:

Targeted ultrasonography is used to assess risk of chromosomal abnormalities by searching for structural features associated with fetal aneuploidy (so-called soft markers, such as renal pelvis dilation or hyperechoic bowel). However, no structural finding is diagnostic for a given chromosomal abnormality, and all soft markers may also be seen in fetuses that are chromosomally normal. If results from prior trisomy screening were negative, many of these soft markers have no clinical relevance and may be ignored (1 Second-trimester screening references Noninvasive prenatal screening for genetic disorders, unlike invasive testing, has no risk of test-related complications. Noninvasive maternal screening can help women decide whether to have... read more Second-trimester screening references ). Nonetheless, the discovery of such a marker may lead to offering the woman amniocentesis Amniocentesis Prenatal procedures that provide a definitive diagnosis of genetic disorders are invasive and involve some fetal risk. Women may choose to have prenatal procedures to know of fetal abnormalities... read more to confirm or exclude a chromosomal abnormality. If a major structural malformation is present, a fetal chromosomal abnormality is more likely. Disadvantages include unnecessary anxiety if a soft marker is detected and unnecessary amniocentesis. Several experienced centers report high sensitivity, but whether a normal ultrasound indicates a substantially reduced risk of fetal chromosomal abnormalities is unclear.

Drugs Mentioned In This Article

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Novarel, Ovidrel, Pregnyl
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