Approximately 3% to 5% of pregnancies are affected by birth defects or genetic disorders. Chromosomal abnormalities are found in about 1 in 150 live births, and congenital malformations remain a primary cause of infant and childhood mortality. These abnormalities include aneuploidy (an abnormal number of chromosomes), translocations, duplications, and deletions.
The most prevalent chromosomal disorder is trisomy 21 (Down syndrome), occurring in 1 per 800 live births. Trisomy 18 and 13 can also result in live births, though less frequently. Sex chromosome aneuploidies are less common than autosomal aneuploidies. Monosomy X (Turner syndrome) is the only viable monosomy known. The incidence of common aneuploidies is detailed in Table 1.
Table 1. Incidence of Common Aneuploidies
| Aneuploidy | Incidence |
|---|---|
| Trisomy 21 (Down Syndrome) | 1 in 800 live births |
| Trisomy 18 (Edwards Syndrome) | 1 in 7,500 live births |
| Trisomy 13 (Patau Syndrome) | 1 in 15,000 live births |
| Monosomy X (Turner Syndrome) | 1 in 5,000 girls |
| Trisomy X | 1 in 1,000 girls |
| XXY (Klinefelter Syndrome) | 1 in 1,000 boys |
| XYY (Jacobs Syndrome) | 1 in 1,000 boys |
Source: Adapted from Nussbaum RL, McInnes RR, Willard HF. Thompson & Thompson genetics in medicine. 7th edition. Philadelphia: Saunders/Elsevier; 2007.
The risk of aneuploidy increases with maternal age, as shown in Table 2. Other factors influencing risk include ultrasound findings (birth defects or soft markers) and obstetric history (previous pregnancy with aneuploidy or genetic disorder). Family history of aneuploidy, especially parental balanced Robertsonian translocation, also elevates risk, although most cases are sporadic due to chromosomal nondisjunction.
Table 2. Risk of Aneuploidy by Maternal Age
| Maternal Age at EDD (Years) | Risk of Trisomy 21 | Risk of Other Chromosomal Abnormality |
|---|---|---|
| 20 | 1:1480 | 1:525 |
| 25 | 1:1340 | 1:475 |
| 30 | 1:940 | 1:384 |
| 35 | 1:353 | 1:178 |
| 40 | 1:85 | 1:62 |
| 45 | 1:35 | 1:18 |
Abbreviation: EDD, Estimated Date of Delivery.
Source: Adapted from Practice bulletin no. 163: screening for fetal aneuploidy. Obstet Gynecol 2016; 127(5): e124.
Patients pursue prenatal aneuploidy screening or diagnosis for various reasons. Some may opt for pregnancy termination if a defect is diagnosed early. Others seek screening or testing to prepare for caring for an affected child and to access specialized clinical support. Certain conditions, like neural tube defects, may benefit from prenatal treatment, improving neonatal outcomes. All patients considering screening or testing require counseling on the risks, benefits, and limitations of their chosen plan from healthcare providers or genetic counselors. Aneuploidy screening and testing decisions are deeply personal, driven by individual values. Open discussions about test benefits, risks, and limitations are crucial for patient-centered care.
Historical Overview of Prenatal Screening
Prenatal screening for birth defects began in the 1950s with ultrasound and has become integral to obstetric care. Real-time gray-scale imaging in the 1970s enhanced prenatal diagnosis, enabling earlier pregnancy evaluations. Ultrasonography serves to determine gestational age, fetal number, assess for malformations, evaluate fetal well-being, and guide invasive procedures. Amniocentesis, the first prenatal chromosomal diagnostic test, was introduced in the 1950s. It has become safer and is used for genetic screening and infection assessments. Chorionic villus sampling (CVS), another diagnostic test, can be performed earlier in pregnancy.
Noninvasive screening tests, including serum analyte screening and cell-free DNA screening, emerged later for genetic abnormality screening.
In 2007, the American College of Obstetricians and Gynecologists (ACOG) issued “ACOG Practice Bulletin No. 77,” recommending aneuploidy screening or invasive testing be available to all pregnant women, ideally at the first prenatal visit. This was a significant shift, as previously these tests were mainly offered to high-risk women.
Current Prenatal Screening Tests
Most prenatal tests are screening tools, including serum screening, carrier screening, and ultrasound. These aim to identify pregnancies at higher risk for chromosomal abnormalities or birth defects. While ultrasound can be diagnostic (e.g., open neural tube defects), serum screening primarily flags pregnancies with increased risk. Various serum screening options exist, differing in criteria and timing (Table 3).
Table 3. Characteristics of Serum Screening Options for Aneuploidy
| Screening Test | Gestational Age (Weeks) | Trisomy 21 Detection Rate (%) | Screen Positive Rate (%) | Analytes/Measurements |
|---|---|---|---|---|
| First-Trimester Screen | 10–13 | 82–87 | 5 | Nuchal translucency, Papp-A, hCG |
| Triple Screen | 15–22 | 69 | 5 | hCG, AFP, uE3 |
| Quad Screen | 15–22 | 81 | 5 | hCG, AFP, uE3, DIA |
| Integrated Screen | 10–13 & 15–22 | 96 | 5 | First-trimester screen analytes + quad screen analytes |
| Sequential Stepwise Screen | 10–13 & 15–22 | 95 | 5 | First-trimester screen, then quad screen |
| Contingent Screen | 10–13 & 15–22 | 88–94 | 5 | First-trimester screen, then quad screen (if needed) |
| Cell-free DNA (cfDNA) | Any age after 9-10 | 99 | 0.5 | Molecular evaluation of cell-free fetal DNA |
Abbreviations: AFP, alpha-fetoprotein; DIA, dimeric inhibin-A; hCG, human chorionic gonadotropin; Papp-A, pregnancy-associated plasma protein A; uE3, unconjugated estriol.
*Detection rate varies with gestational age, improving at earlier ages.
Source: Adapted from Practice bulletin no. 163: screening for fetal aneuploidy. Obstet Gynecol 2016; 127(5): e126.
First-Trimester Screening: Nuchal Translucency and Serum Markers
The first-trimester screen combines serum screening and nuchal translucency ultrasound between 10 and 13 weeks 6 days of gestation. Serum markers, including free beta-hCG and pregnancy-associated plasma protein A (PAPP-A), are collected via blood sample between 9 and 13 weeks 6 days. A risk estimate is calculated using maternal age, pregnancy history, number of fetuses, weight, race, serum markers, and nuchal translucency measurement. Some risk assessments also include nasal bone visualization. The risk is expressed as a ratio, e.g., 1 in 10. A 1 in 300 cutoff is common for high-risk results, but this varies by lab. Trisomy 21 detection rates range from 82% to 87% with a 5% screen positive rate.
Nuchal translucency exceeding 3 mm is linked to both aneuploidy and structural malformations. Initial studies showed 35% of patients with nuchal translucency over 3 mm had confirmed aneuploidy. Later studies confirmed increased cardiac defects in chromosomally normal pregnancies with nuchal translucency over 3.5 mm. Risk of other anomalies, including single gene defects and central nervous system, cardiac, skeletal, and abdominal wall defects, also rises significantly. Targeted ultrasound and fetal echocardiogram are recommended for women with thickened nuchal translucency, regardless of aneuploidy status.
First-trimester screening benefits include early result availability, allowing patients and providers time for decisions on further care: diagnostic testing, genetic counseling, maternal-fetal medicine consultation, or termination. Drawbacks include reliance on certified sonographers for nuchal translucency measurement. A mere 0.5 mm measurement discrepancy can reduce test sensitivity. While more sensitive than the quad screen at 11 weeks, its performance is similar at 13 weeks.
Quadruple Marker Screening: The Quad Screen
The quad screen, the initial serum screening test from the 1990s, remains common, especially for patients starting care after the first trimester (over 25% in public health clinics). Performed between 15 and 22 weeks, it measures serum proteins from the pregnancy: hCG, alpha-fetoprotein (AFP), inhibin A, and unconjugated estriol. These measurements, combined with patient factors (age, race, weight, number of fetuses, diabetes, gestational age), yield a risk estimate. Detection rate is slightly lower than the first-trimester screen, at 81% with a 5% screen positive rate.
Quad screen advantages include screening for open neural tube defects and aneuploidy. Serum AFP, produced by the fetus and present in amniotic fluid and maternal serum, aids neural tube defect detection. It also does not require specialized sonographers, increasing accessibility.
Variations like the triple screen (without inhibin) or penta screen (with hyperglycosylated hCG) exist but do not demonstrably improve test characteristics.
Integrated, Stepwise Sequential, and Contingent Screening Strategies
Several screening approaches combine first-trimester and quad screens: integrated, stepwise sequential, and contingent screening. Integrated screening performs both screens, but results are only released after the quad screen, providing a comprehensive second-trimester aneuploidy risk estimate. It has the highest serum screen detection rate (96%) with a 5% positive rate. However, delayed results limit decision-making time.
Stepwise sequential and contingent screening provide first-trimester results. Stepwise sequential screening releases results after the first-trimester screen and again after the quad screen, enabling earlier counseling and diagnosis for high-risk patients. Contingent screening stratifies women after the first-trimester screen into high-, medium-, and low-risk groups. High-risk women are offered diagnostic testing; low-risk, no further testing; intermediate-risk, quad screening. Contingent screening detection rates range from 80% to 94% with a 5% positive rate.
Cell-Free Fetal DNA (cfDNA) Screening: Noninvasive Prenatal Testing (NIPT)
Cell-free DNA (cfDNA) screening, or noninvasive prenatal screening (NIPS), became commercially available in 2011. This technology analyzes cell-free DNA fragments from the pregnancy isolated from a maternal serum sample. Primarily placental in origin, this DNA is released from apoptotic trophoblasts. Fetal fraction increases with gestational age, reliably exceeding 10% by 10 weeks. A fetal fraction over 4% is needed for reliable analysis. cfDNA is analyzed via massive parallel sequencing or single nucleotide polymorphism interrogation, depending on the lab. Results are reported as aneuploidy detected/not detected or high/low risk, with optional sex chromosome information.
cfDNA screening offers the highest trisomy 21 detection rate (99%) among screening tests, according to a recent meta-analysis. Detection rates for trisomy 18, 13, and sex chromosome abnormalities are lower (Table 4). ACOG currently recommends cfDNA screening only for women with high pretest aneuploidy risk (Box 1). Studies defining test characteristics excluded patients with insufficient fetal fraction. Inconclusive results due to low fetal fraction significantly increase aneuploidy risk, particularly trisomy 13 and 18. Factors like obesity and lower gestational age can reduce fetal fraction.
Table 4. Estimated Detection Rate and Positive Predictive Value (PPV) of cfDNA for Aneuploidy by Maternal Age
| Aneuploidy | Pooled Detection Rate (%) | PPV at 25 Years (%) | PPV at 35 Years (%) | PPV at 45 Years (%) |
|---|---|---|---|---|
| Trisomy 21 | 99.2 | 51 | 79 | 98 |
| Trisomy 18 | 96.3 | 15 | 39 | 90 |
| Trisomy 13 | 91.7 | 7 | 21 | Data insufficient |
| Monosomy X | 90.3 | 41 | 41 | 41 |
Abbreviation: PPV, positive predictive value.
Source: Predictive values calculated via the Perinatal Quality Foundation calculator. Available at perinatalquality.org; retrieved July 22, 2016.
Box 1. Indications for cfDNA Screening
- Maternal age ≥ 35 years at delivery
- Ultrasound findings indicating increased aneuploidy risk
- History of prior trisomy-affected pregnancy
- Parental balanced Robertsonian translocation increasing trisomy 13 or 21 risk
- High-risk first- or second-trimester aneuploidy screening results
Source: Cell-free DNA screening for fetal aneuploidy. Committee Opinion No. 640. American College of Obstetricians and Gynecologists. Obstet Gynecol 2015; 126(3): e31–7.
cfDNA screening should be presented as a screening, not diagnostic, test. Positive predictive value for trisomy 21 is high in recommended populations but lower in average-risk populations due to prevalence. A study at two academic centers found aneuploidy confirmed by karyotype in only 82% of 105 patients with cfDNA results suggestive of autosomal trisomies. Patient misunderstanding of cfDNA screening is common despite counseling. In the same study, 9 patients terminated pregnancies based on cfDNA results without diagnostic confirmation. Genetic counseling is crucial for interpreting cfDNA results and individual risk assessment. Risk calculators are available via UNC’s MomBaby website and the Perinatal Quality Foundation. Ultrasound before cfDNA testing is recommended to confirm gestational age, fetal number, and assess for major anomalies, which can alter aneuploidy risk. Ultrasound findings changed counseling in 16% of patients in a study, revealing incorrect dating, demise, twins, or anomalies. In cases of cystic hygroma or anomalies, diagnostic testing may be preferred over screening for earlier diagnosis.
Additional cfDNA benefits include accurate fetal sex identification and fetal Rh status determination in Rh isoimmunization risk pregnancies. FDA-approved cfDNA tests for anti-Kell and other isoimmunization sources are not yet available in the US. Some labs offer cfDNA screening for other autosomal aneuploidies or microdeletions, but these are not validated and not currently recommended due to low positive predictive values. cfDNA use in multiple gestations is not well-studied and currently not recommended.
Occasionally, cfDNA screening reveals maternal chromosomal abnormalities or concerns, like mosaicism or, rarely, malignancies. Patients should be informed of this possibility. Maternal conditions can cause nonreportable or false-positive results. Other false-positive sources include vanishing twins or confined placental mosaicism. While cfDNA has the best trisomy 21 detection rate, sequential screening has a better overall detection rate for all chromosomal abnormalities, meaning some abnormalities detectable by traditional serum screening may be missed by cfDNA.
Ultrasound Alone for Prenatal Screening
Ultrasound is a routine part of pregnancy care. Most women undergo at least one ultrasound for dating and birth defect surveillance, many have more. Second-trimester transabdominal ultrasound (18-23 weeks) for anatomic anomaly assessment is standard. First-trimester ultrasound (transvaginal or transabdominal) assesses viability, pregnancy number, and major anomalies like anencephaly or cystic hygromas. Some anomalies are associated with specific aneuploidies or chromosomal defects, increasing suspicion when identified.
Diagnostic Prenatal Testing: Confirming Screening Results
Diagnostic testing provides definitive information about whether a pregnancy is affected by a genetic condition. The most common indication in the US is advanced maternal age (≥35 years at delivery). Other indications include positive aneuploidy screening, family history of genetic disorders, or ultrasound anomalies. ACOG recommends diagnostic testing be available to all women, regardless of age, after counseling on pregnancy loss risks.
Chorionic Villus Sampling (CVS): First-Trimester Diagnosis
CVS frequency has decreased with cfDNA screening. It remains the only first-trimester diagnostic test, enabling FISH, karyotype, microarray, molecular testing, and gene sequencing. CVS is performed between 10 and 14 weeks. Earlier CVS (<9 weeks) is linked to limb deformities and is not recommended.
CVS can be transcervical or transabdominal. Chorionic villi are collected under ultrasound guidance, avoiding the amniotic sac. CVS allows earlier diagnosis, reducing uncertainty and enabling earlier (safer) termination if desired. A CVS disadvantage is confined placental mosaicism in 1%-2% of results, which may not reflect fetal chromosomes but can increase small-for-gestational-age infant risk. CVS-related pregnancy loss risk is about 1 in 455 in recent estimates.
Amniocentesis: Second and Third-Trimester Diagnosis
Amniocentesis, like CVS, is less frequent due to cfDNA screening. It’s the only diagnostic test for the second and third trimesters, performed after 15 weeks. A needle is inserted into the amniotic sac under ultrasound guidance to collect amniotic fluid for testing. Besides genetic disorders, amniocentesis can detect intra-amniotic or fetal infection (culture, PCR) and neural tube defects (amniotic fluid AFP and acetylcholinesterase). Complications are more common at earlier gestational ages. Amniocentesis-related pregnancy loss risk is about 1 in 900 in recent estimates.
Cytogenetic Evaluations: Karyotyping, FISH, and Microarray
Chromosome analysis from CVS and amniocentesis is the most reliable method for identifying chromosomal disorders. However, cytogenetic testing has limitations.
Mosaicism, the presence of multiple cell lines in tissue, is considered true mosaicism when consistent across cultures. Pseudomosaicism, a single cell with a different genetic makeup, is usually not clinically significant. Mosaicism can also arise in cell culture (pseudomosaicism). Confined placental mosaicism in CVS occurs in 1%-2% of pregnancies and, while true mosaicism, has different fetal implications. Confined placental mosaicism can cause false-positive cfDNA results, making amniocentesis preferred over CVS for diagnostic confirmation after positive cfDNA screening. Trisomy rescue, especially in trisomy 15, can lead to a diploid fetus but increases uniparental disomy risk and Prader-Willi/Angelman syndrome risk. Cell culture failure is rare, more common with CVS than amniocentesis.
Various testing methods detect different genetic abnormalities. Karyotyping identifies large deletions/duplications (>5 Mb), while microarray detects smaller ones (≥50 kb). FISH identifies major autosomal aneuploidies or specific deletions/duplications like DiGeorge syndrome. Single-gene disorders require targeted molecular approaches. FISH is often the first test for aneuploidy detection due to rapid results (48 hours) and no cell culture need. Despite being diagnostic, FISH should be confirmed by karyotype due to rare false results. Microarray can also be performed on uncultured cells for rapid turnaround and can analyze nonviable cells, useful in stillbirth cases. Microarray can detect aneuploidy and smaller abnormalities and is now recommended with FISH, instead of karyotype alone, for structural abnormality evaluations.
Increased microarray use has identified more chromosomal abnormalities of uncertain clinical significance (variants of uncertain significance, VUS), potentially causing parental anxiety. Parental studies can help determine if a VUS is inherited and likely benign. About 1.7% of structurally normal pregnancies without aneuploidy have a VUS detected. Patients undergoing amniocentesis with normal anatomy scans should be counseled about potential VUS findings with microarray testing.
Preimplantation Genetic Diagnosis (PGD): Genetic Testing Before Implantation
Preimplantation genetic diagnosis (PGD), now widely available, allows even earlier chromosomal abnormality detection. Performed after in vitro fertilization (IVF), PGD involves embryo manipulation to remove a polar body or blastocyst cell for testing. Only unaffected embryos are transferred. Confirmatory CVS or amniocentesis is recommended for IVF/PGD pregnancies due to possible false-negative PGD results (reported negative predictive value of normal FISH is 81%). PGD has minimal risks beyond cost.
Summary: Informed Choices in Prenatal Diagnosis
All pregnant women should be offered aneuploidy screening or diagnostic testing. Comprehensive explanations of options, risks, benefits, and result interpretation are crucial. Patients choosing cfDNA screening should understand it is a screening test for aneuploidy, and microdeletion testing has low positive predictive value. Multiple screening modalities are not recommended. Women with positive screens needing further testing should be counseled on diagnostic CVS and amniocentesis to avoid diagnosis delays. Amniocentesis and CVS are safe with low pregnancy loss rates and should be available to all women desiring diagnostic testing, regardless of risk factors or anomalies.
Key Points in Prenatal Diagnosis
- Aneuploidy screening should be offered to all women at their first prenatal visit.
- Cell-free fetal DNA (cfDNA) screening is recommended for high-risk populations and is a screening, not diagnostic, test.
- Chorionic villus sampling (CVS) and amniocentesis carry a small pregnancy loss risk but are the only current diagnostic methods.
- Thorough pre- and post-test counseling is essential, covering risks, benefits, options, and individualized result interpretation.
Image of a pregnant woman having an ultrasound
Alt Text: Expectant mother undergoing routine prenatal ultrasound for fetal health assessment.
Image of amniocentesis procedure
Alt Text: Diagram illustrating the amniocentesis procedure for prenatal genetic diagnosis.
Footnotes
Disclosure: The authors have no conflicts of interest to report.
