Prenatal Testing for Chromosomal Abnormalities and Neural Tube Defects

Medical Experts

Author

Bosworth

Michelle Bosworth, MS, LCGC

Genetic Counselor, ARUP Laboratories

Medical Reviewer

Andersen

Erica F. Andersen, PhD, FACMG

Associate Professor of Pathology (Clinical), University of Utah

Head of Clinical Operations for Cytogenetics and Molecular Genetics, ARUP Laboratories

Medical Reviewer

Genzen

Jonathan R. Genzen, MD, PhD, MBA

Professor of Pathology (Clinical), University of Utah

Chief Medical Officer and Senior Director of Governmental Affairs, ARUP Laboratories

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

LaGrave

Danielle LaGrave, MS, LCGC

Faculty, Genetic Counseling Graduate Program, Human Genetics, University of Utah

Genetic Counselor, ARUP Laboratories

Prenatal testing is offered to all pregnant individuals to identify pregnancies with a chromosomal disorder, such as trisomy 21 (Down syndrome) or an open neural tube defect (ONTD).^,^, Prenatal genetic testing comprises two categories of testing: screening and diagnosis, which are offered in addition to or in conjunction with an ultrasound performed at 11 to 13 weeks.^, Maternal serum screening (MSS) and prenatal 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. Prenatal cfDNA screening, previously referred to as noninvasive prenatal testing (NIPT), is more sensitive and specific than MSS.^,^,^, Abnormal results should be followed by confirmatory 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 conditions, refer to the ARUP Consult Carrier Screening for Genetic Disorders topic.

Prenatal genetic diagnostic testing is used to determine 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 identify chromosome abnormalities and copy number variants in a 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 prenatal cell-free DNA screening?

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

Both tests have advantages and disadvantages to consider. Prenatal cfDNA screening is more comprehensive and accurate than MSS and can detect more aneuploidies than MSS. It also has higher detection rates for trisomies 21 and 18. Reports for prenatal cfDNA screening usually include posttest risks based on positive predictive value (PPV), or the likelihood that a fetus actually has the condition assessed. By contrast, MSS is less expensive and is less likely than prenatal cfDNA screening to produce inconclusive, incidental, or no results.^,^,^, Reports for MSS include posttest probabilities for each assessed condition.

Is it necessary to have maternal serum screening or prenatal cell-free dNA screening before proceeding to prenatal genetic diagnostic testing?

No. Although maternal serum screening (MSS) tests and prenatal cell-free DNA (cfDNA) screening are often cheaper, faster, and safer than prenatal genetic diagnostic tests, they do not evaluate for as many conditions as chromosome or cytogenomic microarray (CMA) analysis.^,^,^, If there are no abnormal findings on an initial ultrasound (performed at 11 to 13 weeks) in a pregnancy believed to be at low risk for fetal abnormalities, MSS or prenatal cfDNA screening is an acceptable first step for prenatal testing.^,

The limitations of MSS and prenatal cfDNA screening are especially relevant for pregnant individuals who have an increased probability of fetal aneuploidy.^,^,^, If a patient would opt for diagnostic testing regardless of the MSS or cfDNA result, or if abnormal findings are detected on ultrasound, then it is reasonable to proceed directly to diagnostic testing.^,^,^,^,^,^, Performing screening before diagnostic testing may delay diagnosis and has the potential to provide false reassurance if results are negative.

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; however, WES or WGS may be considered when more routine tests fail to yield results.^,

Should expanded forms of prenatal cell-free DNA screening be considered for routine screening?

No. Research is ongoing to determine the utility of “expanded” prenatal cell-free DNA (cfDNA) screening in detecting conditions not traditionally screened for, such as rare autosomal trisomies, subchromosomal imbalances, and microdeletion and microduplication syndromes. However, currently, there is insufficient evidence to support these use cases. As such, use of expanded prenatal cfDNA screening in routine prenatal screening is not recommended.^,

How does a twin pregnancy affect the interpretation of maternal serum screening and prenatal cell-free DNA screening?

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.^,^, Prenatal cell-free DNA (cfDNA) screening provides higher positive predictive values among twin pregnancies when compared with MSS but is still associated with test failures. Insufficient fetal fraction (FF) may affect test results. Prenatal cfDNA screening is not recommended when a patient has had a twin demise/vanishing twin. The demised twin’s placenta may still shed aneuploid cfDNA and thus cause 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 prenatal cfDNA screening to all pregnant individuals.^, ACOG, SMFM, and ACMG also recommend offering both prenatal screening and diagnostic testing to all pregnant patients. Cytogenomic microarray (CMA) analysis is recommended for fetuses with abnormalities detected by ultrasound.^,^,^,^, Patients with structurally normal fetuses who are undergoing invasive prenatal diagnostic testing may have either fetal karyotyping or CMA testing.^,^,^,

Laboratory Testing

Prenatal genetic tests vary greatly in terms of the conditions included, the performance characteristics and limitations, and the risks of specimen collection. MSS and prenatal cfDNA screening tests are used to assess whether a pregnant individual is at increased risk of having a fetus affected by certain chromosomal abnormalities, and, in the case of MSS, ONTDs. Neither MSS nor prenatal cfDNA screening are considered diagnostic because both tests carry the possibility of false-positive and false-negative results.^,^, Regardless of the results of prenatal screening, diagnostic testing should be offered to all patients with abnormal ultrasound findings (including nuchal translucency [NT] of ≥3.5 mm at 11 to 13 weeks).^, Karyotyping and CMA, both diagnostic methods, determine whether chromosome abnormalities and/or copy number variants are present in a fetus. This testing is performed on samples obtained through chorionic villus sampling (CVS) or amniocentesis, both of which carry a small risk of miscarriage.

Patients with negative test results should be further educated about their risk, which depends on the clinical context and the testing performed.^,^,^,

Maternal Serum Screening

MSS is performed during the first trimester, second trimester, or both by measuring biochemical markers present in maternal blood serum to assess risk for fetal aneuploidy and ONTDs. MSS may be performed in combination with an early ultrasound to assess for NT. MSS tests that include both first- and second-trimester markers (ie, stepwise, contingency, or integrated screening) have the highest detection rate.^,

Stepwise Screening^a	Contingency Screening	Integrated Screening
All patients screened in first trimester^b NT measurement required Pregnancies with low to moderate risk: rescreen in second trimester Combined result reported^e	All patients screened in first trimester^b NT measurement required Pregnancies with intermediate risk: rescreen in second trimester Combined result reported^e	All patients screened in first and second trimesters^c NT measurement not required^d Combined result reported
^aCVS should be available if this option is offered. ^bFirst trimester results are reported immediately. ^cFirst trimester results are withheld for later calculation. This increases the likelihood of detection and reduces the potential for a false-positive result. ^dNT measurement may be included (ie, full integrated testing). ^eFor individuals who receive second trimester screening based on risk. Sources: Driscoll, 2009; Palomaki, 2009

Stepwise Screening^a

Contingency Screening

Integrated Screening

All patients screened in first trimester^b

NT measurement required

Pregnancies with low to moderate risk: rescreen in second trimester

Combined result reported^e

All patients screened in first trimester^b

NT measurement required

Pregnancies with intermediate risk: rescreen in second trimester

Combined result reported^e

All patients screened in first and second trimesters^c

NT measurement not required^d

Combined result reported

^aCVS should be available if this option is offered.

^bFirst trimester results are reported immediately.

^cFirst trimester results are withheld for later calculation. This increases the likelihood of detection and reduces the potential for a false-positive result.

^dNT measurement may be included (ie, full integrated testing).

^eFor individuals who receive second trimester screening based on risk.

Sources: Driscoll, 2009; Palomaki, 2009

After screening is complete, if errors in clinical information are discovered (eg, there is a new due date based on ultrasound measurement), reinterpretation of results may be necessary.^,^, Residual risks should be discussed when test results are negative.^, Diagnostic (invasive) testing and genetic counseling should be offered to patients with high-risk MSS results or abnormal NT results. A comprehensive anatomy survey ultrasound is indicated in these scenarios and cannot be replaced by prenatal screening for aneuploidies.^,^, Performing prenatal cfDNA screening before confirmatory testing delays diagnosis and misses certain chromosome abnormalities (eg, those detectable by MSS).^,^,^,

Prenatal Cell-Free DNA Screening

Prenatal cfDNA screening is more sensitive and specific than MSS for traditionally screened aneuploidies (trisomies 21, 18, and 13) and has been validated in both high-prevalence and general populations. This form of screening analyzes genomic fetal cfDNA circulating in the maternal bloodstream and can be performed from as early as 10 weeks of gestation until the end of pregnancy. Professional societies endorse this test for women with average or increased risk of having a baby with a chromosome abnormality.^,^,

Although prenatal cfDNA screening has high detection rates and low false-positive rates, its positive predictive value varies depending on prevalence and pretest probability of an assessed condition, and results are nondiagnostic.^,^,^, For more information, refer to Positive Predictive Value of a High-Risk Noninvasive Prenatal Screening Result for Various Increased Pretest Risk Levels and for Various Gestational Ages and Maternal Ages.

Several factors may contribute to test failure or false-positive test results, such as very low fetal fraction (FF, or the proportion of fetal cfDNA detected in a specimen), confined placental mosaicism, twin demise, discordant aneuploidy among twins, maternal mosaicism, or maternal medical conditions (eg, malignancy). Individuals who have no-call results (ie, the test did not yield a result or the lab could not run the test) and uninterpretable test results are at increased risk for chromosomal abnormalities.^,^,^,

Diagnostic testing and genetic counseling should be offered to patients with a high-risk cfDNA result, no result, or a low-risk result in the presence of an abnormal ultrasound.^,^, Residual risks should also be discussed.^, Irreversible clinical decisions should never be based solely on cfDNA results.

Cytogenomic Single Nucleotide Polymorphism 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 no risk factors.^, Pregnant individuals with a high probability of microdeletion syndromes due to family history or prenatal cfDNA 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 with karyotyping.

Notably, CMA cannot be used to identify balanced chromosome rearrangements or characterize abnormalities. CMA also carries a greater chance of incidental findings and variants of uncertain clinical significance compared with karyotype analysis.

If there are concerning ultrasound findings and CMA results are normal, further testing, such as whole exome sequencing (WES), whole genome sequencing (WGS), or a genetic panel that corresponds with clinical findings, may be considered.^, Targeted sequencing may be performed if there is a known familial variant.

Chromosome Analysis (Giemsa Band Karyotyping)

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 with CMA but is the preferred testing methodology in two situations: first, when an individual would like definitive testing but does not want to have CMA due to the small chance of incidental findings, variants of uncertain significance, and/or for financial reasons, and second, when a fetus has an increased probability of 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 CMA 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 >99%, respectively, and for open spina bifida, 96% and >99%. Alternatively, spina bifida and anencephaly in a fetus may be confirmed by targeted ultrasound exam.

Comparison of Prenatal Genetic Tests

Conditions Detected by Various Prenatal Genetic Testing Methods
Condition(s)	Method
Condition(s)	MSS	cfDNA Screening	CMA	Karyotype Analysis
Neural tube defects	Detected	Not detected	Not detected	Not detected
Aneuploidy	Detected^a	Detected^b	Detected	Detected
Chromosomal copy number variants (deletions/duplications)	Not detected	Detected (in some cases)	Detected	Not detected
Absence of heterozygosity^c	Not detected	Not detected	Detected	Not detected
Uniparental disomy	Not detected	Not detected	Indirectly detected^d	Not detected
Single-gene disorders	Not detected	Not detected	Not detected	Not detected
Mosaicism	Not detected	Not detected	Detected (in some cases)	Detected^e
^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.