According to the Centers for Disease Control vital statistics, there were about 3.8 million live births in the United States in 2018. Furthermore, in 77.5% of pregnancies prenatal care was initiated in the first trimester. While prenatal care has been around for centuries in one form or another, prenatal screening in comparison, was introduced somewhat recently. Prenatal screening is a method of assessing pregnancies for birth defects, chromosomal conditions, and genetic disorders that could affect the unborn child. Technologies used in prenatal screening have rapidly expanded and increased in accuracy over the last several decades. The aim of this article is to review the history of prenatal screening and current technologies available.

The purpose of prenatal screening is to put the pregnancy into a risk category that a particular condition that could affect the fetus exists, i.e., increased risk or no increased risk. Screening is not diagnostic testing and a screening test cannot provide a definitive diagnosis.  Prenatal screening provides a probability that a particular condition exists. The nature of screening means there will be false positive and false negative risks determined; however, the idea is to keep those false results to an acceptable minimum rate Prenatal screening should not be repeated if there is an informative result. Following a positive screening test, further diagnostic tests may be recommended.  Not all pregnancies that are classified as high risk have a fetus with a birth defect or genetic condition (this would be a false positive screening result) and not all affected pregnancies will be in the high-risk group (false negative). Some women with high-risk pregnancies may decline diagnostic testing. Similarly, women who have average-risk pregnancies may opt in for diagnostic testing.

The chance for a pregnancy to be affected with a chromosomal condition increases with maternal age. These are usually aneuploidies – the addition or subtraction of an entire chromosome. Most aneuploidies are lethal, but some may make it to term. The word “trisomy” refers to an extra chromosome where there are three copies of a chromosome rather than the typical pair of chromosomes, and “monosomy” describes a missing chromosome where there is only one copy. The most common chromosome conditions are Down syndrome (trisomy 21); Edwards syndrome (trisomy 18); Patau syndrome (trisomy 13); Turner syndrome (monosomy X); and Klinefelter syndrome (XXY). Women who are 35 years or older at delivery, are considered “high risk” for aneuploidies.  Prior to prenatal screening, maternal age alone was used to assess for fetal aneuploidy, which had a low sensitivity of only about 30%. There was a need for a non-invasive, inexpensive, reliable pregnancy screening method that could be incorporated into routine prenatal care.

Biomarkers

Several biomarkers have been identified, researched, validated, and commercialized as maternal serum screening tests:

  • Alpha-fetoprotein (AFP) can be measured in the second trimester, typically between 15.0-22.6 weeks of gestation. Interestingly, it was the first maternal serum biomarker that became available in the 1980s. AFP is produced by the fetal liver and yolk sac and can assess for open neural tube defects and ventral wall defects in the heart. It may be measured independently or can be a part of a combined screen with other serum markers.
  • Human chorionic gonadotropin (hCG) is exclusively made by the outer most part of the placenta. It’s concentration typically increases through the first 8 weeks of gestation, then slowly decreases and plateaus in the 20th This biomarker can be used in both first and second trimester maternal serum screening. At first total hCG was used, and then it was determined that free beta-hCG was more sensitive.
  • Unconjugated estriol (uE3) is secreted by the fetal liver and increases throughout pregnancy.
  • Dimeric inhibin A (DIA) is produced by the placental and fetal tissues and increases in concentration until about 10 weeks’ gestation. However, levels are relatively stable between 15-25-week gestation, afterwards it increases until delivery.
  • Pregnancy associated plasma protein A (PAPP-A) is produced by the outer part of the placenta and has a crucial role in fetal and placental the growth. It is informative in the first trimester only.

Typically, a blood draw is performed at a specific time during gestation, and serum is analyzed for these biomarkers, depending on the test ordered. Results are reported out as a multiple of the median (MoM), which is a statistical calculation that considers patient’s ethnicity, gestational age, weight, and maternal age. MoM of 1.0 is considered “average.” Results interpretation depends on the combination of concentration patterns of these markers and are reported as a fraction for risk. Specific risk thresholds must be met to be screened positive (increased risk). First trimester screening may also include a nuchal translucency (NT) ultrasound which measures the fluid-filled space in the back of the fetal neck. Specialized certification is required for ultrasonographers to perform the NT evaluation, which increases detection rates compared to serum screening alone. However, not all facilities are certified for NT ultrasounds, so this may not be available.

There are numerous screening tests that use different combinations of maternal serum analytes and the NT ultrasound. Table 1 summarizes different screening protocols, but is not comprehensive:

Test Analyte Conditions Screened Gestation Advantages Disadvantages
First Trimester Screen PAPP-A
hCG
Down syndrome
Trisomy 18
First Trimester Early results
Single blood draw
NT measurement
No ONTD risk
Quad Screen hCG
AFP
uE3
DIA
Down syndrome
Trisomy 18
ONTD
Second Trimester Single blood draw No first trimester
results
MSAFP AFP ONTD Second Trimester Single blood draw ONTD alone
Integrated
Screen*
PAPP-A
hCG
AFP
uE3
DIA
Down syndrome
Trisomy 18
ONTD
Frist Trimester
Second Trimester
High detection rate NT measurement
2 blood samplings
No first trimester results
Serum
Integrated Screen*
PAPP-A
hCG
AFP
uE3
DIA
Down syndrome
Trisomy 18
ONTD
First Trimester
Second Trimester
No NT required 2 blood samplings
No first trimester results

*there are multiple integrated, sequential, and contingent screening protocols, some may not need the second blood draw, depending on results.

Cell-free DNA

The number and complexity of screening protocols increased during the 90s and early 2000s, which could make pre-test counseling cumbersome and possibly confusing to the patient. Thankfully, a newer screening technology was introduced that mitigated the need for multiple blood draws and had a higher detection rate for the most common chromosome conditions: cell-free DNA screening.

Cell-free DNA screening (cfDNA) has rapidly become the standard of care in prenatal screening for fetal aneuploidies. It can be performed as early as the 9th week of gestation, requires a single blood draw, has a very high sensitivity and specificity, and the results are typically available within a week or two. The technology analyzes cell-free DNA, which is shed by the placenta and is present in the maternal blood stream. It increases in concentration throughout the pregnancy and is usually completely absent soon after delivery. cfDNA are short DNA sequences and are unique to chromosomes 1-22, X, and Y. There is an expected amount of DNA that should match to each chromosome. If there is too much or too little DNA from a specific chromosome, then it is interpreted as an increased risk for fetal aneuploidy for that chromosome. Some companies use a single nucleotide polymorphism (SNP) based technology, comparing placental and parental SNPs to assess risk for fetal aneuploidy. Although this is a DNA-based test, it is still screening; therefore, there are false positives and false negatives. Likewise, positive results should be followed up with diagnostic testing or further evaluation. cfDNA screening does not assess for open neural tube defects (ONTDs) risk. Some clinicians may still choose to run maternal serum AFP to obtain this information. Moreover, sometimes serum markers may indicate other fetal health issues besides aneuploidy or ONTD and this information is not captured with cfDNA screening. Given a mixture of placental (fetal) and maternal DNA is analyzed, there may be maternal confounders that can impact results, such as a maternal chromosome condition, blood transfusion or bone marrow transplant, or maternal malignancy.

Prenatal screening technology has improved over the last 50 years. Multiple tests became available in the 90s and early 2000s. However, with these new choices came different testing algorithms, which could be confusing to patients, and even clinicians. The advent of cfDNA screening within the last decade has simplified the prenatal screening process and has increased accuracy. Professional medical society guidelines state that cfDNA screening should be offered to all pregnant women, just as invasive prenatal diagnosis should be offered. The uptake of cfDNA screening has been tremendous in the last decade. It will be interesting to see what new screening technologies or new applications of cfDNA testing become clinically available in the future.

References

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Practice Guidelines:

Palomaki, G.E., Bupp, C., Gregg, A.R. 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, 462–474 (2020). https://doi.org/10.1038/s41436-019-0681-0

American College of Obstetricians and Gynecologists’ Committee on Practice Bulletins—Obstetrics; Committee on Genetics; Society for Maternal-Fetal Medicine. Screening for Fetal Chromosomal Abnormalities: ACOG Practice Bulletin, Number 226. Obstet Gynecol. 2020 Oct;136(4):e48-e69. doi: 10.1097/AOG.0000000000004084. PMID: 32804883.