Non-Invasive Prenatal Testing (NIPT): Current guidelines

Aneuploidies, such as translocations, whole or segmental chromosomal duplications, and deletions, are examples of chromosomal abnormalities that are estimated to affect approximately 1 in 150 live births [1]. However, due to their association with early pregnancy loss, the prevalence of aneuploidies in conceptions is generally higher. Prenatal ultrasonographic abnormalities, a history of chromosomal abnormalities in prior pregnancies, and advanced maternal age are some factors that increase the possibility of fetal aneuploidy. However, several aneuploidies, such as microdeletions, are independent of maternal age and have the same risk of occurring in all pregnancies [2].

Prenatal testing

The era of prenatal tests began with ultrasound. Subsequently, amniocentesis was used to confirm ultrasound abnormalities, primarily for high-risk pregnancies. Nowadays, it is recommended that all pregnant women should be offered prenatal tests to make informed decisions throughout their pregnancy. Prenatal tests are categorized into non-invasive screening tests (ultrasounds, biochemical testing, and non-invasive prenatal testing, known as NIPT) and invasive, diagnostic tests (chorionic villus sampling (CVS) or amniocentesis). The primary purpose of a screening test is to identify whether a fetus has an increased risk of having a specific condition. Pregnancies found to be high-risk are referred for confirmatory, invasive diagnostic testing, which has a small risk of miscarriage. Thus, the more accurate a screening test is, the fewer women need to be referred for diagnostic testing.

Non-invasive prenatal testing

NIPT, also known as cell-free fetal DNA (cffDNA) testing or non-invasive prenatal screening (NIPS), was introduced to the market in 2011. It is performed using a blood test from the expectant mother with a subsequent analysis of fetal DNA fragments, which are placental in origin, found in the maternal bloodstream. It is a pregnancy-specific genetic test which can be performed early during pregnancy and is a safe and accurate method of testing a number of fetal aneuploidies. NIPT is available in over 60 countries, and is one of the fastest-growing genetic technologies globally [3].

NIPT screens not only for the common autosomal aneuploidies that can be detected through conventional screening tests, but also for other aneuploidies such as sex chromosome aneuploidies (SCAs), rare autosomal trisomies (RATs), and copy number variations (CNVs) such as subchromosomal imbalances, microdeletions, and microduplications syndromes.

• Autosomal aneuploidies refer to changes in the number of chromosome pairs one through twenty-two, excluding the sex chromosomes. The most common autosomal aneuploidies are trisomy 21, trisomy 18, and trisomy 13.
• SCAs involve changes in the number of sex chromosomes and can affect up to 1 in 400 newborns [4]. Before NIPT, SCAs were not consistently or uniformly screened, so NIPT has opened new doors in prenatal screening with the detection of SCAs. For example, Turner syndrome (45, X) is the only SCA with a consistent prenatal phenotype which can be detected through ultrasound, while the rest of the SCAs are not routinely picked up in screening methods apart from NIPT if there are no incidental findings.
• RATs are autosomal trisomies which occur rarely on chromosome pairs one to twenty-two.
• Subchromosomal imbalances affect a part of the chromosome, not the entire chromosome. They can involve extra copies of a section of chromosome (duplication), a missing or deleted part of the chromosome (deletion), or swapped parts of different chromosomes (translocations).
• Microdeletions and microduplication syndromes also occur from deletions and duplications but the size of the deletion or duplication is smaller.

During the past decade, international societies have focused on the role and clinical utility of NIPT, with guidelines and recommendations evolving almost on an annual basis, as more data became available and the underlying technologies became more efficient, accurate and accessible. Recognizing its superiority and utility, the latest guidelines from the International Society for Prenatal Diagnosis (ISPD), the American College of Medical Genetics and Genomics (ACMG), and the American College of Obstetricians and Gynecologists (ACOG) collectively endorse NIPT as the most accurate screening test for detecting common fetal aneuploidies [2, 5, 6].

While there are some discrepancies in guidelines among different medical organizations, these can be attributed to variations in clinical evidence of their studies, different countries where their studies were conducted, and the different factors they took into consideration.

Current guidelines

Article 1: Position statement from the International Society for Prenatal Diagnosis on the use of non-invasive prenatal testing for the detection of fetal chromosomal conditions in singleton pregnancies. ISPD, 2023

• Autosomal aneuploidies

Through several systematic reviews, it has been demonstrated that NIPT is the most accurate screening test for common autosomal aneuploidies trisomy 21, 18, and 13 in unselected singleton pregnancies, and those with increased probability of having aneuploidy. The ISPD Board states that NIPT’s performance is sufficient to be offered as a primary or secondary screening option. It also notes that there are some biological factors (confined placental mosaicism), technical challenges, and statistical reasons that can contribute to a false positive result in NIPT, therefore genetic counseling and diagnostic testing should follow high-risk NIPT results, particularly in cases where termination of pregnancy is being considered.

• Sex chromosome aneuploidies (SCAs)

According to the ISPD Broad, NIPT for SCAs is accurate enough to be offered alongside autosomal aneuploidy screening, with appropriate counseling. ISPD also notes the importance of looking into other societal, economic, cultural, and ethical factors when it comes to evaluating the downstream impacts of offering NIPT for SCAs.

• Rare autosomal trisomies (RATs): trisomies other than chromosomes 21, 18, and 13

NIPT for RATs is not currently recommended as a routine screening test for unselected populations. Amniocentesis combined with karyotype analysis has been considered the gold standard for diagnosing fetal karyotype, given the rarity of RATs and the frequent occurrence of CPM [7]. As of today, the effectiveness and clinical utility of NIPT for RATs have not been confirmed, and more studies need to be implemented. ISPD recommends post-test counseling and professional management following a high-risk result for RATs.

• Subchromosomal imbalances: chromosomal imbalances such as duplications, deletions, and unbalanced translocations

NIPT is not recommended as a routine screening test for subchromosomal imbalances in unselected populations . The guidelines specifically focus on how genome-wide NIPT should not be considered a comprehensive screen for all pathogenic CNVs, as many CNVs have a size of less than 7Mb which is below the limits of genome-wide NIPT resolution.

• Microdeletions and microduplication syndromes (MMS): small subchromosomal copy number variants (CNVs), typically <5Mb

Currently, the ISPD board does not endorse NIPT for MMS as a routine screening test for unselected populations, considering the insufficient information on the performance, clinical utility, and cost-effectiveness. They advise conducting more research to evaluate the usage of NIPT screening for MMS.

The ISPD guidelines additionally focus on another important factor influencing the accuracy of NIPT – fetal fraction, which is the proportion of fetal cfDNA in the maternal bloodstream. According to the statement, laboratories should have an internal validation of their detection limit and threshold for “no call” or ‘‘failed’’ results. Fetal fraction is considered an essential metric for quality control to avoid false negative and false positive results. Lastly, the guidelines discuss ethical issues, genetic counseling, and diagnostic testing for high-risk NIPT results. These guidelines replace the 2015 ISPD guidelines, which formed the first position statement of ISPD on NIPT.

Read the position statement here.

Article 2: Non-invasive 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). ACMG, 2022

ACMG evidence-based recommendations, which address the use of NIPS for chromosomal abnormalities in all pregnant patients, are based on the findings of a recent comprehensive evidence review performed by a working group of experts.

The ACMG board strongly recommends:

• NIPS for fetal trisomies 21, 18, and 13 over traditional screening methods for all singleton and twin pregnancies due to its superior performance in the detection of these common trisomies.
• NIPS for SCAs for all singleton pregnancies. The guidelines add “the option of screening for fetal SCAs is unique to NIPS and has not been available through traditional screening”. Screening for SCAs in twin pregnancies is either limited or unavailable based on technology and whether the twins are monochorionic or dichorionic.

The ACMG Board also recommends:

• NIPS for 22q11.2 deletion syndrome (DiGeorge syndrome) for all pregnancies, as it is the most common pathogenic CNV that can be identified prenatally. This is a conditional recommendation, based on moderate certainty of evidence.

Currently, due to the lack of clinically relevant evidence and validation, the ACMG board does not recommend screening for CNVs other than 22q11.2 deletions and (RATs). The statement adds that advancements in technology will likely improve the screening performance for these rarer conditions. Additional points of emphasis in the statement include the dramatic reduction of invasive testing with the introduction of NIPS into clinical practice, the importance of careful and thorough counseling and informative education on the benefits and limitations of screening, concluding with how NIPS has been a “practice-changing advance in prenatal care”.

Read the ACMG practice guidelines here.

Article 3: Screening for Fetal Chromosomal Abnormalities, ACOG Practice Bulletin Number 226. ACOG and SMFM, 2020

In October 2020, the American College of Obstetricians and Gynaecologists (ACOG) in collaboration with the Society for Maternal Fetal Medicine (SMFM), developed a new practice bulletin on screening tests, which replaced the 2018 recommendations. The statement includes a comprehensive and robust set of data, detailing the risk of different aneuploidies per maternal age (age 20, 25, 30, 35, and 40), and comparing different screening methods, NIPT included.

While it was known that autosomal aneuploidies increase with maternal age, important points of evidence emerging from the practice bulletin are how:

• The risk of microarray or rare chromosomal abnormality is not associated with maternal age, as there is a 1 in 270 risk in all age groups tested.
• The risk of sex chromosome aneuploidies increases slightly with age, with a risk of 1 in 294 at age 20, 25, and 30; a risk of 1 in 285 at age 35, and a risk of 1 in 196 at age 40.
• Collectively, the risk for all chromosomal aneuploidies is:
  • 1 in 122 for age 20
  • 1 in 119 for age 25
  • 1 in 110 for age 30
  • 1 in 84 for age 35
  • 1 in 40 for age 40

The above data and overall systematic review of ACOG demonstrate how all pregnancies have a risk for certain aneuploidies, and in patients under 36 years of age specifically, the risk of having a microarray (CNV/deletion) aneuploidy, which is not routinely tested prenatally, is higher than the risk of having trisomy 21.

Summary of recommendations and conclusions based on good and consistent scientific evidence, classified by ACMG as ‘Level A’:

• Screening (serum screening with or without nuchal translucency (NT) ultrasound or cell-free DNA screening) and diagnostic testing for chromosomal abnormalities should be offered to all pregnant women of any age or baseline risk as early as possible.
• Cell-free DNA is the most sensitive and specific screening test for common aneuploidies. However, cell-free DNA is not a diagnostic test, and if patients receive a positive screening test result or a “no-call” result because of a test failure, they should confirm their results with a comprehensive ultrasound evaluation, a diagnostic test, and undergo genetic counseling.

Some additional recommendations based on limited or inconsistent scientific evidence, classified as ‘Level B’, are:

• Patients who choose cell-free DNA screening over a diagnostic test as a follow-up test, should be informed about the probability of the test failing to identify some fetuses with chromosomal abnormalities and the chance to delay a definitive diagnosis.
• Although cell-free DNA can be done in twin pregnancies, there is not a lot of evidence available to determine the detection rate of the method in these cases.

The practice bulletin also focused on the importance of fetal fraction, noting how a minimum of approximately 2-4% of fetal fraction is required for accurate NIPT results, and with the recommendation that all laboratories should include a clearly visible fetal fraction result on their reports.

Read the Practice bulletin No.226 here.

Conclusion

The latest guidelines from professional genetic societies endorse NIPT as the most accurate screening test for detecting common fetal aneuploidies. With the rapid evolution of both NGS-based NIPT technologies and recommendations, it’s certain that the introduction of NIPT marked a shift in the era of prenatal screening and care.

References

[1] Nussbaum, Robert L, et al. Principles of clinical cytogenetics and genome analysis. Thompson & Thompson Genetics in Medicine 8^th ed. Philadelphia, Elsevier, 2016. p. 57-74 (Level III). ACOG. “Screening for Fetal Chromosomal Abnormalities.” Www.acog.org, 2020, www.acog.org/clinical/clinical-guidance/practice-bulletin/articles/2020/10/screening-for-fetal-chromosomal-abnormalities

[2] Ravitsky, Vardit, et al. “The Emergence and Global Spread of Noninvasive Prenatal Testing.” Annual Review of Genomics and Human Genetics, vol. 22, no. 1, 31 Aug. 2021, pp. 309–338, https://doi.org/10.1146/annurev-genom-083118-015053

[3] Berglund, Agnethe, et al. “The Epidemiology of Sex Chromosome Abnormalities.” American Journal of Medical Genetics Part C: Seminars in Medical Genetics, vol. 184, no. 2, June 2020, pp. 202–215, https://doi.org/10.1002/ajmg.c.31805

[4] Hui, Lisa, et al. “Position Statement from the International Society for Prenatal Diagnosis on the Use of Non‐Invasive Prenatal Testing for the Detection of Fetal Chromosomal Conditions in Singleton Pregnancies.” Prenatal Diagnosis, vol. 43, no. 7, 16 May 2023, pp. 814–828, https://doi.org/10.1002/pd.6357

[5] Dungan, Jeffrey 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).” Genetics in Medicine, vol. 25, no. 2, 1 Feb. 2023, p. 100336, www.sciencedirect.com/science/article/pii/S1098360022010048, https://doi.org/10.1016/j.gim.2022.11.004

[6] Liu, Yi, et al. Amniotic Fluid Karyotype Analysis and Prenatal Diagnosis Strategy of 3117 Pregnant Women with Amniocentesis Indication. Vol. 12, no. 6, 1 June 2023, https://doi.org/10.57264/cer-2022-0168