ACOG/SMFM released a new practice bulletin (August 2020) that addresses prenatal screening for fetal chromosomal anomalies. The guidance clearly states that both aneuploidy screening and diagnostic testing “should be discussed and offered to all patients regardless of maternal age or risk for chromosomal abnormality”

• Aneuploidy screening
  • Serum screening with or without NT ultrasound or
  • Cell-free DNA screening
• Diagnostic testing (CVS or amniocentesis)

Notes

• Standard serum screening remains a first line option along with cell-free DNA screening, also known as noninvasive prenatal screening (NIPS), because additional chromosomal and single gene defects may be picked up with NT and performance may be more comparable to NIPS if the higher NIPS test failure rates are considered
• While younger women may have a higher risk for fetal microdeletion syndromes than aneuploidy, ACOG does not recommend NIPS for microdeletions | Women who want information regarding microdeletions should be offered microarray testing using CVS or amniocentesis  

What Is NIPS?

NIPS is a blood test that utilizes cell-free DNA technology (cfDNA) to predict the risk for fetal genetic disorders during pregnancy. In 2011, NIPS was introduced as a screen for T21 (trisomy 21 or Down syndrome). Today, NIPS cover the most common aneuploidies (T21, T13 and T18), as well as sex chromosomes and may also include some microdeletions and single gene genetic disorders

How Does It Work?

• DNA fragments (outside of cells) can be found floating in the blood of all individuals
• In every pregnant woman, there are fragments from
  • Her own DNA
  • Fragments of placental DNA (generally thought to derive from the outer cytotrophoblast rather than the inner mesenchymal layer)
• A maternal blood sample is obtained ≥10 weeks (with some labs offering testing beginning at 9 weeks) of pregnancy
  • Usually performed at 11 to 13 weeks
• Fetal Fraction
  • The percentage of fetal DNA found in maternal blood is known as the fetal fraction | Typical range 3 to 13% of maternal cfDNA
  • The fetal fraction is critical to the success of NIPS | Minimum required approximately 2 to 4%
  • Sensitivity drops with lower fetal fraction and if too low, test failure will result

Technologies

• Generally, NIPS utilizes next generation sequencing and bioinformatics algorithms to interrogate DNA fragments that have been extracted from maternal samples
  • SNP-based: Bioinformatic algorithms combine risk from individual targeted single nucleotide polymorphisms (SNPs) to differentiate maternal from placental DNA fragments
    • SNP is the only analysis that can report on zygosity and individual fetal fractions in the case of twins (dependent on laboratory)
  • Quantification method: Bioinformatic algorithms determine the amount of DNA from chromosomal regions of interest | E.g. if too much chromosome 21 DNA is detected, then a ‘screen positive’ result for T21 will be generated

What Disorders Are Included?

• T13, T18 and T21
  • Most effective at screening for T21
• Chromosomes X and Y (fetal sex usually available)
• Sex chromosome conditions (depending on the lab)
  • Monosomy X (Turner syndrome)
  • 47,XXX (Triple X syndrome)
  • 47,XXY (Klinefelter syndrome)
  • 47,XYY (Jacob’s syndrome)
• Microdeletions (also known as copy number variants or CNVs) are available with more extensive panels | CNVs occur in 0.4% of pregnancies and are not related to maternal age
  • 22q11.2 deletion syndrome (DiGeorge syndrome) | 1,3000 to 1,4000 live births, but may be more prevalent prenatally and cases may be missed at birth (see ‘Related ObG Topics)
  • 1p36 deletion syndrome | 1 in 5,000 to 1 in 10,000 live births
  • 4p16.3 deletion syndrome (Wolf-Hirschhorn syndrome) | 1 in 50,000 live births
  • 5p15.2 deletion syndrome (Cri du Chat syndrome) | 1 in 20,000 to 1 in 50,000 live births
  • 15q11.2 deletion syndrome (Angelman syndrome/Prader-Willi syndrome) |1 in 12,000 to 20,000 (AS) | 1 in 10,000 to 30,000 (PWS)

SYNOPSIS:

The preferred nomenclature is NIPS (‘S’ for screening) to emphasize that this test is used for screening only and not diagnostic. Cell-free DNA (cfDNA) is also commonly used with an understanding that the DNA is derived from placenta and not the fetus. NIPS utilizes next generation sequencing and bioinformatics algorithms to look at the DNA fragments in the mother and fetus, as a way of determining the likelihood of certain genetic conditions in the fetus.  While there are multiple panels available, there is consensus regarding the clinical utility of NIPS screening for T13, T18 and T21. Patient education, especially around the concept of positive predictive value (PPV) is a priority. Calculator tools are available from professional societies (see ‘Learn More – Primary Sources’ below) or ideally laboratories should be able to provide obstetric professionals with real world test performance results.

NIPS Screening Performance

Detection rates

• The following detection rates are based on recent meta-analysis (see ‘Learn More -Primary Resources’ below)
  • T21: >99% detection rate for T21
  • 98% detection rate for T18
  • 99% detection rate for T13
• Combined false positive rate of 0.13%
  • cfDNA is the most sensitive and specific screen for T21, T18 and T13
• Sensitivity and specificity are superior to standard screening for T21 and other common aneuploidies

Positive Predictive Value (PPV)

Trisomies

• NIPS generally has very high negative predictive values (NPV) | From ages 20 to 45, NPV is >99% (PQF NSGC calculator)
• PPV is also generally high but can vary based on age | Lower background risk will lower PPV | PQF NSGC calculator can be used to determine PPVs
  • PPVs have been reported up to approximately 90% for T21 but tend to be lower for T18, T13 and monosomy X
  • Abnormal ultrasound will increase PPV

Microdeletions

• NIPS for microdeletion syndromes can have high sensitivity and specificity but still have very low PPV because the particular syndrome is a priori so rare | For example
  •  Cri du Chat Syndrome (Using PQF calculator – see ‘Learn More – Primary Sources’ below)
    • Even if sensitivity and specificity are both >99%, the chance of a positive results being a true positive (i.e. PPV) is ≤1%
    • Negative predictive value will generally always be high for a rare disorder
  • 22q11.2 deletion syndrome (22q11.2DS) is the most common microdeletion syndrome, found in 1 in 3000 to 4000 live births | Appears to be more common prenatally with a prevalence of approximately 1 in 1000 of otherwise normal pregnancies (see ‘Learn More – Related Entries)
    • PPV in a low risk population (see ‘Learn More – Primary Sources’) has been reported in a low risk population to be approximately 1/20 and higher in pregnancies at higher risk (e.g. congenital heart disease consistent with 22q11.2DS)

Note: NIPS is a screening test and not diagnostic | Regardless of PPV, screen positive results require patients be offered invasive diagnostic testing to confirm results

• Some women may opt for a cfDNA screen after a positive serum analyte screen instead of an invasive test. This is a valid option for patients who do not want an invasive test
  • However, patients should be counseled that a cfDNA screen will not give a diagnosis and may delay a formal diagnosis
  • cfDNA will not identify all babies with chromosomal abnormalities
  • Residual risk of a chromosomal abnormality after an abnormal traditional screen followed by a normal cfDNA screen is around 2%

Reasons for a False Positive Result

• There is a high risk that a positive T21 NIPS screen is truly positive (approximately 90%)
• However, confirmatory testing is necessary because false positive results are possible
  • Confined placental mosaicism (since NIPS only looks at the placental DNA)
  • Vanishing twin that was aneuploid but surviving twin is normal
  • Maternal condition such as cancer (NIPS results will usually show multiple chromosomal aneuploidies)
  • Unknown

NIPS Test Failure

• Low fetal fraction
  • BMI: 10% of patients >250 lbs will have a fetal fraction <4% | May be caused by (1) dilutional effect or (2) elevation in maternal DNA due to inflammatory processes
  • Early Gestational age <9 weeks
• Other reasons for no result include
  • Laboratory failure | IVF pregnancy | Maternal drug exposure to LMWH | Racial background (e.g. Black and South Asian vs white) | Fetal aneuploidy (e.g. T13 or T18 and sex chromosome aneuploidies)

Follow-Up for ‘No Call Result’

• Inform patients that there is an increased risk of aneuploidy | Effect may be due to smaller placentas  
• Offer genetic counseling, detailed ultrasound evaluation and diagnostic testing
• Repeat screening “may be considered’
  • Success rate 75 to 80% (less with high BMI)
• Repeat screening is not advised for the following
  • Ultrasound anomalies present
  • Later gestational age where further delay may complicate access to reproductive options

Note: It is preferred that the laboratory report the fetal fraction

Other Practice Guidelines: ACMG

• All patients should be offered the option of fetal aneuploidy screening or diagnostic testing
  • NIPS is an appropriate option vs standard ultrasound/serum marker screening
• The choice to accept or decline remains that of the patient
• Patients should be provided with adequate information to make an informed decision including
  • Risks
  • Benefits
  • Alternatives
  • An understanding that (1) a positive screening result does not necessarily mean the pregnancy is affected and (2) false negatives are possible
  • There is a baseline risk for birth defects despite testing (approximately 3 to 4%)
• Post-test genetic counseling should be available to patients especially if there is a ‘screen positive’ result
  • Confirmation with CVS or amnio should be offered whether the patient has a screen positive result with NIPS or standard screening
  • Because CVS assesses placental tissue, there is a risk for confined placental mosaicism and follow-up with amniocentesis may be required (especially for T13 or monosomy X)

In addition, the ACMG

• Recommends against testing for other aneuploidies (all other non-sex chromosomes aside from 21, 18 and 13)
• Recommends patients be informed that NIPS can screen for sex aneuploidies
• Recommends informing patients that microdeletion syndrome screening is available with NIPS if the following are discussed with the patient
  • Benefit and risks for use of screening vs diagnostic testing, especially if a patient wants the most fetal genetic information possible
  • Higher chance for false-positive and false-negative results compared to NIPS for common aneuploidies  
  • Prognosis for some of the resultant findings may not be clear
• Recommends the following regarding reports

Laboratory requisitions and pretest counseling information should specify the DR, SPEC, PPV, and NPV of each CNV screened. This material should state whether PPV and NPV are modeled or derived from clinical utility studies (natural population or sample with known prevalence).

Single Gene and NIPS

• NIPS also has the capability to identify single gene disorders
  • Ability to identify single gene variants that would likely not be detected with carrier testing
  • Generally serious autosomal dominant disorders that may be absent in the parents, but pathogenic variants may spontaneously occur in the fetus (de novo) such as Cornelia de Lange syndrome or Achondroplasia
  • Some of these conditions are associated with advanced paternal age (≥40 at time of conception)
• Single gene screening is clinically available, although currently not recommended by ACOG  

Learn More – Primary Sources: 

ACOG Practice Bulletin 226: Screening for Fetal Chromosomal Abnormalities

ACOG Practice Advisory: Cell-free DNA to Screen for Single-Gene Disorders

Analysis of cell‐free DNA in maternal blood in screening for aneuploidies: updated meta‐analysis

Screening for trisomies by cfDNA testing of maternal blood in twin pregnancy: update of The Fetal Medicine Foundation results and meta‐analysis

Prevalence of recurrent pathogenic microdeletions and microduplications in over 9500 pregnancies

Clinical experience with a single-nucleotide polymorphism-based noninvasive prenatal test for five clinically significant microdeletions

NIPT/Cell Free DNA Screening Predictive Value Calculator

NSGC: Abnormal Prenatal Cell-free DNA Screening Results

Noninvasive prenatal screening for fetal aneuploidy, 2016 update:  position statement of the American College of Medical Genetics and Genomics

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

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