Prenatal Testing for Chromosomal Abnormalities and Neural Tube Defects

Prenatal testing is offered to all pregnant women to identify pregnancies with a chromosomal disorder, such as trisomy 21 (Down syndrome), or an open neural tube defect (ONTD).    Most professional guidelines define prenatal genetic testing as encompassing two categories of testing: screening and diagnosis. Maternal serum screening (MSS) and cell-free DNA (cfDNA) screening estimate a patient’s risk of carrying a fetus with a chromosomal disorder.    In addition, MSS assesses risk for ONTDs.  These tests are noninvasive. Of the two tests, cfDNA, also referred to as noninvasive prenatal screening (NIPS) or noninvasive prenatal testing (NIPT), is more sensitive and specific.     Like other screens, NIPT can result in false positives and false negatives, and it is not considered diagnostic. Abnormal results should be followed by confirmatory prenatal genetic diagnostic testing.    This topic focuses on prenatal testing for fetal aneuploidy and neural tube defects; for more information on the use of laboratory tests in carrier screening for genetic disorders, see the ARUP Consult Carrier Screening for Genetic Disorders topic.

Prenatal genetic diagnostic testing is used to confirm whether a fetus has certain genetic disorders before birth. This testing is invasive and carries a small risk of miscarriage. The two most common forms of testing, karyotyping and cytogenomic single nucleotide polymorphism (SNP) microarray, are the most definitive forms of prenatal genetic testing available to assess the risk for chromosome abnormalities and copy number variants in the fetus.     No one prenatal genetic test is superior in all situations, and clinicians and patients should understand the risks, limitations, and benefits of available testing options so that an informed choice about testing can be made.    ,   

Quick Answers for Clinicians

What is the difference between maternal serum screening and noninvasive prenatal testing?

Maternal serum screening (MSS) has long been the standard prenatal testing option for chromosomal abnormalities. MSS measures biochemical markers present in maternal blood serum to assess a pregnant woman’s risk of having a fetus with a fetal aneuploidy or an open neural tube defect (ONTD). Noninvasive prenatal screening (NIPS) or testing (NIPT), also referred to as cell-free DNA (cfDNA) testing, was introduced more recently and is more sensitive and specific than MSS for detecting traditionally screened aneuploidies (trisomies 21, 18, and 13).    ,   NIPT analyzes genomic cfDNA circulating in the maternal bloodstream.

Both tests have advantages and disadvantages to consider. NIPT is more comprehensive and accurate than MSS. NIPT tests for more aneuploidies than MSS and has higher detection rates for trisomies 21 and 18. NIPT reports usually include posttest risks based on positive predictive value (PPV), or the likelihood that a fetus truly has the condition assessed. MSS is less expensive and is less likely than NIPT to produce inconclusive, incidental, or no results.     Reports for MSS include posttest risks for each assessed condition.

Is it necessary to have maternal serum screening or noninvasive prenatal testing before proceeding to prenatal confirmatory testing?

No. Maternal serum screening (MSS) tests and noninvasive prenatal testing (NIPT) are often cheaper, faster, and safer than prenatal genetic diagnostic tests, but they do not evaluate for as many conditions as chromosome or cytogenomic microarray analysis (CMA).     If a pregnancy is believed to be at low risk for fetal abnormalities, MSS or NIPT is an acceptable first step for prenatal testing. However, if a patient would opt for diagnostic testing regardless of the MSS or NIPT result, that patient should only have diagnostic testing performed.    , 

Performing MSS and/or NIPT before prenatal diagnosis may delay obtaining a definitive diagnosis. The limitations of MSS and NIPT are especially relevant for women who have an increased risk of fetal aneuploidy (eg, couples at high risk due to advanced maternal age, carriers of Robertsonian translocations involving chromosomes 21 and 13, and women with fetal ultrasound abnormalities associated with trisomy 21, 18, and 13).    

How is prenatal genetic testing performed?

Prenatal diagnostic testing (as opposed to prenatal screening testing) requires an invasive procedure—either chorionic villi sampling (CVS) or amniocentesis—to identify chromosomal, molecular, and biochemical abnormalities in the pregnancy and determines whether a pregnancy is affected with the assessed condition(s). CVS, typically performed between 10 and 13 weeks of gestation, is a first-trimester procedure for prenatal diagnosis. CVS is performed by inserting a catheter through the mother’s cervix or abdomen, with ultrasound guidance, to sample fetal villi tissue from the placenta. Amniocentesis refers to the procedure of inserting a thin needle through the mother’s abdomen, with ultrasound guidance, to withdraw amniotic fluid from the amniotic sac. Amniocentesis is performed after 15 weeks of gestation in the second or third trimester.   

If prenatal genetic test results are normal, could a fetus still have a birth defect or genetic syndrome?

Yes. Babies may be born with birth defects and/or genetic syndromes, even when the most extensive testing yields negative results. Karyotyping, microarray, and sequence analysis all target specific types of genetic abnormalities. In a significant number of affected pregnancies, all three tests will be negative.   Medical genetics experts are incorporating new ways to test for genetic abnormalities, such as whole exome sequencing (WES) and whole genome sequencing (WGS). The nuances of new methodologies must be well understood before they are offered to families in a prenatal setting; WES or WGS may be considered when more routine tests fail to yield results.  

How does a twin pregnancy affect the interpretation of maternal serum screening and noninvasive prenatal testing?

Twin pregnancies present unique challenges when it comes to interpreting prenatal testing results. The accuracy of maternal serum screening (MSS) is reduced for twins because the concentrations of measured markers in maternal blood are produced by both fetuses, and this confounds assigning individual risks for each twin.    Noninvasive prenatal testing (NIPT) provides higher positive predictive values among twin pregnancies when compared with MSS, but NIPT is still associated with test failures. Although total fetal fraction (FF) is higher in a twin pregnancy, the FF per twin is lower than in singleton pregnancies. Insufficient FF may affect test results.  NIPT is not recommended when a patient has had a twin demise/vanishing twin. The demised twin’s placenta may still shed aneuploid cell-free DNA (cfDNA), and thus NIPT may lead to false-positive results.    

Indications for Testing

The American College of Obstetricians and Gynecologists (ACOG), Society for Maternal-Fetal Medicine (SMFM), and American College of Medical Genetics and Genomics (ACMG) recommend offering MSS and NIPT to all pregnant women.   ACOG, SMFM, and ACMG also recommend offering both prenatal screening and diagnostic testing to all pregnant patients. Cytogenomic microarray analysis (CMA) is recommended for fetuses with abnormalities detected by ultrasound.    ,  Refer to Follow-Up Strategies for Ultrasound Findings table for further testing suggestions. Patients with structurally normal fetuses who are undergoing invasive prenatal diagnostic testing may have either fetal karyotyping or CMA testing.    

Follow-Up Strategies for Ultrasound Findings Table
Ultrasound Result Follow-Up Tests to Consider

Vanishing twin

cfDNA (or NIPT) screening is contraindicated

Soft marker for aneuploidy detected

Offer screening (MSS or NIPT) and diagnostic testing; if screening was previously performed, discuss risk in context of ultrasound finding and screening result

Multiple soft markers detected

Offer genetic counseling, MFM consultation, and diagnostic testinga

Increased NT detected on first trimester ultrasound

Offer genetic counseling, diagnostic testing,a and detailed anatomy ultrasound at 18-22 weeks GA; refer for cardiac ultrasoundb,c

Anomaly Detected

Offer genetic counseling and diagnostic testinga instead of screening; if patient declines diagnostic testing, offer NIPTc AND MSS AFPd

Ultrasound Result Follow-Up Tests to Consider

Anomaly suggestive of aneuploidy

Offer diagnostic testinga by either chromosome analysis (with or without FISH testing) OR reflex testing that begins with FISH OR chromosome analysis AND AFP with reflex to AChE and fetal hemoglobind,e

Anomaly not suggestive of aneuploidy

Offer diagnostic testinga by fetal genomic microarray OR reflex testing (see “Anomaly suggestive of aneuploidy” for test options) AND AFP with reflex to AChE and fetal hemoglobind,e

High-risk NIPT for aneuploidy with no anomaly detected by ultrasound

Offer diagnostic testinga (see “Anomaly suggestive of aneuploidy” for test options); for mosaic CVS results, patients should be counseled that both results may represent confined placental mosaicism

High-risk NIPT for copy number variant with no anomaly detected by ultrasound

Offer diagnostic testinga by fetal genomic microarray

aBy CVS or amniocentesis.

bConsider if NT is 3.0-3.4 mm; recommended if NT is 3.5 mm or greater or if cystic hygroma is present.

cConsider NIPT only if patient will decline diagnostic testing after low-risk result. Performing screening tests before diagnostic procedures will delay definitive diagnosis. Screening has risk of false-negative results and will also miss fetuses affected with nonscreened conditions.

dFor assessing neural tube defect risk, detailed ultrasound capable of detecting ONTD is an alternative.

eBy amniocentesis.

AChE, acetylcholinesterase; AFP, alpha-fetoprotein; cfDNA, cell-free DNA; CVS, chorionic villi sampling; FISH, fluorescent in situ hybridization (aneuploidy panel); GA, gestational age; MFM, maternal-fetal medicine; MSS, maternal serum screening; NIPT, noninvasive prenatal testing; NT, nuchal translucency; ONTD, open neural tube defect

Sources: ACOG and SMFM, 2020 ; Gregg, 2016 ; Salomon, 2014 

Laboratory Testing

Prenatal genetic tests vary greatly by conditions that are tested for, the benefits and limitations of such testing, and the risks of collecting the specimen. MSS and cfDNA testing are used to assess whether a patient is at increased risk of having a fetus affected by certain chromosomal abnormalities, and, in the case of MSS, ONTDs. Neither MSS nor cfDNA are considered diagnostic because both tests carry the possibility of false-positive and false-negative results.    Karyotyping and CMA determine whether chromosome abnormalities and/or copy number variants are present in the fetus. This testing is performed on samples obtained through CVS or amniocentesis, both of which carry a small risk of miscarriage.

Maternal Serum Screening

MSS is performed during the first trimester, second trimester, or both trimesters by measuring biochemical markers present in maternal blood serum to provide risks for fetal aneuploidy and ONTDs. This testing may be performed in combination with an early ultrasound to assess for nuchal translucency (NT).  

The gestational age windows for measurement of biochemical markers are specific. Results in the first trimester are reported in combination with either a measurement of NT by ultrasound and/or second trimester MSS. MSS tests that include both first- and second-trimester markers, also referred to as integrated or sequential screening, have the highest detection rate. Sequential screening includes an NT measurement, and women at intermediate or low risk after the first draw continue with the second draw and the complete screen. Integrated screening may (or may not) include NT measurement, and interpretation is not provided until after the second draw, which increases the detection rate and lowers the false-positive rate. Refer to the First and Second Trimester Maternal Serum Screening Options for more information about ARUP’s MSS offerings.   If errors in clinical information are discovered (eg, there is a new due date based on ultrasound measurement), reinterpretation of results may be necessary.   

If the NT component of testing is abnormal, biochemical test results are likely to indicate high risk. Pregnant patients with these results should be offered genetic counseling and diagnostic (invasive) testing to further assess risk. A comprehensive anatomy survey ultrasound at 18-22 weeks of gestation is critical and cannot be replaced by prenatal screening for aneuploidies; referral for fetal cardiac ultrasonography may also be beneficial.    Sonographers assessing NT must be certified by the Fetal Medicine Foundation (FMF) or the Nuchal Translucency Quality Review (NTQR) board to ensure the measurement is valid. 

Patients with low-risk test results whose fetus has ultrasound abnormalities should also be offered genetic counseling and confirmatory testing.  Residual risks should be discussed when test results are negative.  

Offer patients with high-risk MSS results genetic counseling, a comprehensive anatomy survey ultrasound, and confirmatory testing. Discuss the natural history and revised risk for the condition.    Performing NIPT before confirmatory testing delays diagnosis and misses chromosome abnormalities not assessed by MSS.     

Noninvasive Prenatal Testing/Cell-Free DNA Testing    

NIPT, also referred to as cfDNA testing, is more sensitive and specific than MSS for traditionally screened aneuploidies (trisomies 21, 18, and 13).    NIPT analyzes genomic fetal cfDNA circulating in the maternal bloodstream, and testing can be performed from as early as 10 weeks of gestation throughout the end of pregnancy. Professional societies endorse this test for women with average or increased risk of having a baby with a chromosome abnormality.   

NIPT has high detection rates and low false-positive rates, but results are not equivalent to a diagnosis. The accuracy, or positive predictive value (PPV), of NIPT varies depending on the pretest risk for the assessed condition. For example, a high-risk result for trisomy 21 in a mother who is 40 years of age is more likely to be accurate than the same result in a mother of 22 years, due to the increasing risk for trisomy as maternal age increases. Similarly, a high-risk NIPT result for trisomy 21 is more likely to be an accurate result than a high-risk result for trisomy 13 because NIPT is more accurate for trisomy 21.   

The most commonly used platforms for NIPT are either massively parallel shotgun sequencing or chromosome-selective sequencing to detect overrepresentation of targeted chromosomes, or SNP-based analysis to detect nondisjunction of targeted chromosomes. The NIPT platform used for testing may inform test-specific limitations and follow-up approaches in the case of test failure.

Several factors may contribute to test failure or false-positive test results. NIPT requires an adequate amount of fetal fraction (FF), the proportion of fetal cfDNA, to obtain a reliable test result. When FF is too low, a NIPT result cannot be accurately determined and repeat NIPT is not recommended. Factors like maternal weight and chromosome abnormalities (trisomies 18 and 13 and triploidy) may cause low FF, thus impacting the interpretation of the test failure.    NIPT may also yield uninterpretable test results when the fetal cfDNA analysis yields a result that is consistent with neither a normal fetus nor trisomy. Individuals who have no-call and uninterpretable test results are at increased risk for chromosomal abnormalities.    These and other limitations are particularly important to consider in pregnancies with abnormal ultrasounds or very high-risk results by MSS. In these situations, a comprehensive anatomy survey ultrasound, genetic counseling, and diagnostic testing are more appropriate than NIPT.    False-positive NIPT results may be caused by confined placental mosaicism (CPM), twin demise, maternal mosaicism, and maternal medical conditions (eg, cancer).   

Patients with a high-risk NIPT result or no result should be offered genetic counseling, a comprehensive anatomy survey ultrasound, and confirmatory testing.    Conversely, patients with low-risk test results whose fetus has ultrasound abnormalities should also be offered these options.  Residual risks should also be discussed.   Irreversible clinical decisions should never be based solely on NIPT results.

Cytogenomic Single Nucleotide Polymorphism Microarray

Cytogenomic SNP microarray (CMA) detects aneuploidy and can also detect copy number variants across the genome (deletions and duplications) that are below the resolution of karyotype analysis. CMA is recommended for pregnancies with a fetal structural abnormality detected by ultrasound, unless the observed anomaly is strongly associated with an aneuploidy syndrome.    ,  However, CMA may be performed as a first-line test in pregnancies with an absence of structural abnormalities or other risk factors (eg, advanced maternal age).   Patients at high risk for microdeletion syndromes due to family history or NIPT screening should have CMA.    In most cases, CMA replaces the need to perform fetal karyotyping.   CMA of prenatal samples usually has a faster turnaround time compared to karyotyping.

CMA testing does have limitations. CMA does not identify balanced chromosome rearrangements, nor does it characterize abnormalities. CMA also carries a higher chance for incidental and uncertain findings than karyotype analysis. Incidental findings are defined as clinically significant results unrelated to the indication for testing, and uncertain findings are variants (DNA changes) of uncertain clinical significance for which insufficient data are available to categorize the finding as benign or pathogenic. Refer to the ARUP Constitutional Copy Number Variant Assertion Criteria for more information.

Patients with negative test results should be further educated about their risk, which depends on the clinical context and the test performed.     For example, a normal CMA result eliminates the risk of a microdeletion in the fetus of a carrier parent but does not eliminate the risk of a genetic syndrome in a fetus.

If there are concerning ultrasound findings and CMA results are normal, further testing may be considered. Most often, additional testing would consist of a panel of specific genes to target differential diagnoses based on clinical findings. However, some patients may opt for whole exome sequencing (WES) or whole genome sequencing (WGS) that target DNA alterations in all genes, or all nuclear DNA, not assessed by CMA. This WES/WGS analysis is performed at specialty centers with informed consent, including detailed discussions of the limitations and significant chance for incidental and uncertain findings.   Conversely, if a fetus is at high risk for a specific condition due to family history, targeted testing of the familial gene variants may be performed. 

Chromosome Analysis (Giemsa Band Karyotype)

Karyotyping is performed to detect changes in chromosome number associated with aneuploidy (eg, trisomy 21) and to characterize the arrangement of structural changes (eg, translocations, inversions, and large deletions and duplications). Chromosome analysis also identifies balanced rearrangements, which cannot be detected by CMA.

Karyotyping has limited resolution compared to CMA but is the preferred testing methodology in two situations: first, when a patient would like definitive testing but does not want CMA due to the small chance of incidental findings, variants of uncertain significance, and/or for financial reasons, and second, when a fetus may be at risk for a specific aneuploidy syndrome (eg, trisomy 21 or trisomy 13) due to family history or ultrasound findings.    

Prenatal FISH Analysis

Prenatal genetic testing by fluorescence in situ hybridization (FISH) assesses small regions within five chromosomes: 21, 18, 13, X, and Y. Performing FISH without karyotype or cytogenomic SNP microarray analysis is not recommended. This testing provides limited information and may rarely return false-positive or false-negative results. Clinical decision-making should not be based on FISH results alone, except when there is corroborating evidence of the diagnosis, such as ultrasound anomalies. 

Amniotic Fluid Alpha-Fetoprotein

When a fetus has an ONTD, such as meningomyelocele or anencephaly, the amniotic fluid has an elevated alpha-fetoprotein (AFP) concentration and acetylcholinesterase (AChE) activity. The best option for testing is reflexive and involves measuring AFP from amniotic fluid first. If the amniotic fluid AFP is elevated, then reflexive testing is performed to determine if AChE activity is present. Reflexive testing increases the specificity for ONTD detection because elevated AFP without the presence of AChE activity can be caused by other conditions and pregnancy complications, such as fetal renal disease and intrauterine bleeding. This reflex pattern offers a sensitivity and specificity for anencephaly of 98% and <1%, respectively, and for open spina bifida, 96% and <1%.  Alternatively, spina bifida and anencephaly in a fetus may be confirmed by targeted ultrasound exam. 

Comparison of Prenatal Genetic Tests

Conditions Detected by Different Prenatal Genetic Testing Methods
Condition(s) Method
MSS cfDNA Screening CMA Karyotype Analysis

Neural tube defects

Detected

Not detected

Not detected

Not detected

Aneuploidy

Detecteda

Detectedb

Detected

Detected

Chromosomal copy number variants (deletions/duplications)

Not detected

Detected (in some cases)

Detected

Not detected

Absence of heterozygosityc

Not detected

Not detected

Detected

Not detected

Uniparental disomy

Not detected

Not detected

Indirectly detectedd

Not detected

Single-gene disorders

Not detected

Not detected

Not detected

Not detected

Mosaicism

Not detected

Not detected

Detected (in some cases)

Detectede

aTrisomy 21 and 18.

bTrisomy 21, 18, 13 and sometimes additional trisomies, depending on version.

cDue to common ancestry or uniparental disomy.

dMay be suspected due to absence of heterozygosity.

eThreshold determined by colony count.

Patient Resources

Resources for Positive Prenatal Test Results and Patient Education
Resource Type Link Source

Patient support and education

Lettercase

National Center for Prenatal and Postnatal Resources

Patient education videos

Prenatal genetic testing videos

Genetic Support Foundation, Patient Library

Patient education

What is noninvasive prenatal testing (NIPT) and what disorders can it screen for?

Genetics Home Reference, National Institutes of Health

Cell-free DNA prenatal screening test

The American College of Obstetricians and Gynecologists

NIPT positive predictive value online calculator

NIPT/cell free DNA screening predictive value calculator

The National Society of Genetic Counselors and Perinatal Quality Foundation

Down syndrome/trisomy 21

Children with Down syndrome: health care information for families

American Academy of Pediatrics, Healthychildren.org

Health supervision for children with Down syndrome

Pediatrics. 2011;128(2):393-406. PMID: 21788214

Turner syndrome/monosomy X

Clinical practice guidelines for the care of girls and women with Turner syndrome: proceedings from the 2016 Cincinnati International Turner Syndrome Meeting

Eur J Endocrinol. 2017;177(3):G1-G70. PMID: 28705803

Neural tube defects

Facts about neural tube defects

U.S. Department of Health and Human Services, Centers for Disease Control and Prevention

22q11.2 Deletion syndrome (DiGeorge/velocardiofacial syndrome)

Practical guidelines for managing patients with 22q11.2 deletion syndrome

J Pediatr. 2011;159(2):332-339.e1. PMID: 21570089

Towards a safety net for management of 22q11.2 deletion syndrome: guidelines for our times

Eur J Pediatr. 2014;173(6):757-765. PMID: 24384789

ARUP Laboratory Tests

MSS Tests

Conditions screened: T21, T18

Conditions screened: T21, T18, ONTDs

Conditions screened: T21, T18, ONTDs

Conditions screened: T21, T18, ONTDs

Conditions screened: T21, T18

Conditions screened: T21, T18, ONTDs

Conditions screened: ONTDs

NIPT/cfDNA

Conditions screened: T21, T18, T13, Turner syndrome, and sex chromosome trisomies [XXX, XXY, XYY]

Conditions screened: T21, T18, T13, Turner syndrome, and sex chromosome trisomies [XXX, XXY, XYY], microdeletions causing 22q11.2 deletion (DiGeorge or velocardiofacial [VCFS] syndrome), 1p36 deletion, Angelman, Prader-Willi, and cri-du-chat (5p-) syndromes

Conditions screened: T21, T18, T13, Turner syndrome, and sex chromosome trisomies [XXX, XXY, XYY], and microdeletions causing 22q11.2 deletion (DiGeorge or velocardiofacial [VCFS] syndrome)

Prenatal Confirmatory Tests

Conditions assessed: aneuploidy and other chromosomal abnormalities (eg, large deletions/duplications, translocations, inversions, marker chromosomes)

Conditions assessed: T21, T18, T13, and X and Y sex chromosomes

Performing FISH without karyotype or cytogenomic SNP microarray analysis is not recommended

Conditions assessed: T21, T18, T13, and X and Y sex chromosomes, then either karyotype or genomic copy number variants (eg, aneuploidy, microdeletions, and microduplications)

Reflex pattern: If results of aneuploidy FISH panel are normal, genomic microarray is performed; if results are abnormal, chromosome analysis is performed

Conditions assessed: aneuploidy and other chromosomal abnormalities (eg, large deletions/duplications, translocations, inversions, marker chromosomes)

Conditions assessed: aneuploidy and other chromosomal abnormalities (eg, large deletions/duplications, translocations, inversions, marker chromosomes) with reflex to genomic abnormalities (eg, chromosomal abnormalities and copy number variants [microdeletions and microduplications]) if normal

Reflex pattern: If results of chromosome analysis are normal, genomic microarray is performed; in cases with a normal karyotype, microarray studies reveal clinically relevant copy number variations (CNV) in ~6% of fetuses with an US anomaly and ~2% whose indication is advanced maternal age or positive aneuploidy screen

Conditions assessed: T21, T18, T13, and X and Y sex chromosomes

Performing FISH without karyotype or cytogenomic SNP microarray analysis is not recommended

Conditions assessed: T21, T18, T13, and X and Y sex chromosomes, then either karyotype or genomic copy number variants (eg, aneuploidy, microdeletions, and microduplications)

Reflex pattern: If results of aneuploidy FISH panel are normal, genomic microarray is performed; if results are abnormal, chromosome analysis is performed

Conditions screened: copy number variants (eg, aneuploidy, microdeletions, and microduplications)

Related Tests

Condition screened: ONTD

Condition screened: ONTD

Medical Experts

Author
Medical Reviewer
Medical Reviewer
Medical Reviewer

Ji

Yuan Ji, PhD, DABCP, FACMG
Associate Professor of Pathology (Clinical), University of Utah
Section Chief, Molecular Genetics and Genomics, ARUP Laboratories
Medical Reviewer
Medical Reviewer

References

Additional Resources