Prenatal Genetic Screening

Specimen Collection

Prenatal genetic screening typically involves collecting a maternal blood sample via venipuncture to measure serum levels of various biomarkers, including MSAFP, pregnancy-associated plasma protein-A (PAPP-A), free beta-human chorionic gonadotropin (β-hCG), inhibin A, and unconjugated estriol. As part of first-trimester screening, a transabdominal ultrasound is usually performed between 11 and 14 weeks of gestation to assess nuchal translucency, following established measurement criteria.[6] 

CfDNA testing and carrier screening are also performed using maternal blood obtained via venipuncture. CfDNA analysis is most commonly conducted using methods based on single-nucleotide polymorphisms. The fetal fraction of cfDNA originates from placental trophoblasts, which release DNA fragments into the maternal circulation. These fragments are then isolated and purified from maternal plasma for further analysis.[3]

Screening for thalassemias and hemoglobinopathies is performed using a complete blood count (CBC) obtained via maternal venipuncture. As part of this evaluation, red blood cell indices should be assessed, and hemoglobin electrophoresis should be conducted to identify potential hemoglobinopathies.

Procedures

Regardless of maternal age, all patients should be informed of the option to pursue prenatal genetic screening and diagnostic testing. This information should be communicated through an open and objective discussion that considers various factors influencing the choice of screening modality. These factors may include maternal age, the desire for specific prenatal information, prior obstetric and family history, gestational age at the time of screening, cost considerations, and the turnaround time required for results to support pregnancy-related decision-making.[3]

Screening modalities vary in terms of the gestational age at which they can be performed and the type of information they provide. First-trimester screening is limited to the window between 10 and 13 weeks, 6 days of gestation. Second-trimester triple, quadruple, and penta screening are typically conducted between 15 and 22 weeks of gestation. CfDNA testing can be performed at any time from 10 weeks of gestation onward.

First-trimester genetic screening options include the first-trimester screen, nuchal translucency measurement, and cfDNA testing. Maternal serum analytes can be measured alone or in combination with nuchal translucency. The key serum markers assessed in the first trimester are pregnancy-associated PAPP-A and free β-hCG. Nuchal translucency measurement is performed using a transabdominal ultrasound, with the fetus in a sagittal view.[7] To ensure accuracy, the fetal crown-rump length must be between 45 and 84 mm. The Fetal Medicine Foundation outlines specific criteria for obtaining accurate nuchal translucency measurements (Fetal Medicine Foundation-Nuchal Translucency Scan).

Second-trimester serum screening options include the triple, quadruple (quad), and penta screens. First- and second-trimester screenings may also be combined into integrated, sequential-stepwise, or contingent screening protocols to improve detection rates.

The triple screen measures β-hCG, MSAFP, and unconjugated estriol.[8] The quadruple screen adds inhibin A to the triple screen panel and is especially beneficial for patients who do not attend their first prenatal visit until the second trimester. The penta screen further includes hyperglycosylated hCG in addition to β-hCG, MSAFP, inhibin A, and unconjugated estriol.[3]

The integrated screen combines results from the first-trimester screening and the second-trimester quad screen into a single risk assessment, which is reported after the second-trimester screening is completed. The sequential-stepwise screen follows a similar approach but provides patients with their results from the first trimester. If the first-trimester screen is positive, diagnostic testing is offered, and further screening is typically discontinued if the test is pursued. Patients with a negative first-trimester result proceed with the second-trimester quad screen to complete the assessment.[3]

The contingent screen also provides first-trimester results but categorizes patients into low-, intermediate-, and high-risk groups based on their screening outcomes. Low-risk patients require no further testing, while high-risk patients are offered diagnostic testing. Those classified as intermediate-risk are offered additional screening with the second-trimester quad screen.[9] 

Carrier screening is ideally performed during the preconception period to allow for comprehensive counseling on reproductive risks and options for future pregnancies.[10] When performed during pregnancy, screening is typically performed on the pregnant patient first. If the patient is identified as a carrier of a specific condition, the partner should be offered targeted carrier screening for that condition to assess the risk of having an affected child. In cases where time is limited, concurrent screening of both partners may be considered.[5] If both partners are found to be carriers of the same genetic condition, genetic counseling should be offered.

Indications

Prenatal screening guidelines have been issued by several major organizations, including the ACOG, the Society for Maternal-Fetal Medicine, the American College of Medical Genetics and Genomics (ACMG), and the United States Preventive Services Task Force (USPSTF). ACOG recommends that all patients, regardless of maternal age or risk factors, be offered prenatal genetic screening and diagnostic testing options. ACMG advises that patients be informed about the availability of cfDNA screening for trisomies T21, T18, and T13, as well as sex chromosome abnormalities.[11] 

Prenatal genetic screening should be discussed early in pregnancy, with patients being counseled on their risk factors, the conditions being screened for, the accuracy of the screening test, and the importance of confirmatory diagnostic testing to guide pregnancy management.[2][3] Diagnostic testing is universally recommended following a positive screening result, regardless of the screening modality used.[3] If a screening test returns a low-risk or negative result, no further screening should be offered, as this may increase the likelihood of a false-positive result. 

Regardless of whether screening or diagnostic testing is performed, all patients should be offered a second-trimester ultrasound to evaluate for potential fetal structural abnormalities.[3] This ultrasound is ideally conducted between 18 and 22 weeks of gestation. Some ultrasound findings are considered soft markers and should not be used solely to screen for aneuploidy, as these markers may also appear as normal variants in euploid fetuses (see Image. Soft Marker Findings on Fetal Ultrasound).[3][12] 

Each soft marker should be evaluated in the clinical context of the patient, considering her baseline risk factors and prior screening results for aneuploidy. Depending on the soft marker identified, a detailed Level II anatomic survey should be performed to assess for any coexisting structural anomalies. Genetic screening and/or diagnostic options should be reviewed if already completed or offered if not yet conducted.[3]

Normal and Critical Findings

First-trimester maternal serum analyte screening, with or without nuchal translucency screening, provides results categorized as high-risk or low-risk for trisomies T21, T18, and T13. In general, PAPP-A levels are decreased in the presence of trisomies T21, T18, and T13. β-hCG is typically decreased in T18 and T13 but increased in T21. A nuchal translucency measurement greater than 3 mm is considered abnormal and, when used in combination with serum analyte screening, can help increase the risk assessment for aneuploidy. 

Second-trimester maternal serum analyte screening results are categorized as low-risk or high-risk based on characteristic analyte patterns associated with specific trisomies:

• T21 is associated with low MSAFP, low estriol, high β-hCG, high inhibin A, and low PAPP-A.
• T18 is associated with low MSAFP, low estriol, low β-hCG, normal inhibin A, and low PAPP-A.
• T13 is associated with normal MSAFP, normal estriol, normal β-hCG, normal inhibin A, and low PAPP-A.

cfDNA screening provides individual results reported as either high-risk or low-risk for each specific genetic condition tested, including trisomies T21, T18, and T13, as well as sex chromosome abnormalities and particular microdeletions, including 22q11.2. In some cases, cfDNA results may return as a "no call," indicating that a determination of risk could not be made for any of the conditions tested. The ACMG recommends that all "no call" cfDNA reports include the fetal fraction.[3][13]

Interfering Factors

CfDNA screening is dependent on a sufficient fetal fraction—the proportion of fetal DNA in the maternal blood sample—for accurate analysis. The minimum fetal fraction required for reliable cfDNA screening is generally considered to be between 2% and 4%, depending on the specific screening test and laboratory used. Maternal weight can affect the fetal fraction.

Approximately 10% of women with a body weight of over 250 pounds may have a fetal fraction of less than 4%, which can lead to test failure. In addition to maternal body mass index (BMI), test failure can also occur due to factors such as the type of laboratory analysis used by a specific genetic screening company, early gestational age at the time of collection, advanced maternal age, pregnancies conceived via in vitro fertilization, maternal use of low molecular weight heparin, and certain racial backgrounds, including Black and South Asian women.[3]

Test results that cannot be calculated (no call results) due to these interfering factors should not be considered equivalent to low-risk results.[14] Patients should be counseled about the ongoing possibility of a chromosomal abnormality and offered genetic diagnostic testing as a means to confirm or rule out this possibility. Inaccurate test results may also occur in cases of multiple gestations, particularly when one twin has been lost early (vanishing twin) or when one twin has a genetic abnormality while the co-twin is genetically normal. Specific cfDNA tests are available for use in the context of a vanishing twin.[15]

Complications

Standard phlebotomy for obtaining maternal blood samples carries risks such as bruising, bleeding, phlebitis, and localized pain. Ultrasonography, in use for over 30 years, is considered a very low-risk procedure for both the mother and fetus. The "as low as reasonably achievable" (ALARA) principle has been established to ensure the safety of radiological imaging modalities.[16] 

CfDNA is the most sensitive and specific of the genetic screening modalities, but still carries a small risk of false-negative or false-positive results. Serum analyte screening modalities are generally considered to carry a 5% risk of false-positive results.[3] The potential for patient anxiety and distress resulting from a false-positive result should be thoughtfully considered and addressed during pretest counseling. In cfDNA screening specifically, incidental findings may be reported and can be associated with maternal aneuploidy, mosaicism, or malignancy.[3] This information should also be included in the pretest counseling as a possible complication of prenatal genetic screening.

The information presented so far applies to singleton gestations. Serum aneuploidy screening, regardless of the method used, is less accurate in twin pregnancies compared to singleton gestations. Reliable data regarding genetic screening in higher-order multiples, such as triplets and quadruplets, is lacking. First-trimester screening, including serum analytes and nuchal translucency, as well as second-trimester quad screening and sequential-stepwise or integrated screening, are available options for twin pregnancies.[3] CfDNA can also be performed in twin gestations. However, interpreting the results in twin pregnancies requires careful consideration, as it is not possible to determine which twin may be affected based on blood work alone.[17]

Counseling patients about carrier screening is essential, as it cannot identify all individuals who may be at risk for the condition being screened for. Many gene variations associated with a given condition may not be included in the screening. Patients should be informed that a residual risk remains with any genetic testing result.[4] 

Patient Safety and Education

Patient safety and education play a crucial role in prenatal screening. Pretest and posttest counseling are essential to ensure that patients fully understand the purpose of screening tests and the distinction between screening and diagnostic testing. A positive screening result should always be followed by diagnostic testing before any irreversible decisions are made regarding the pregnancy.

Additionally, patients must be informed that a negative or low-risk result from a screening test does not entirely rule out the possibility of an abnormality, as there may still be residual risk. Counseling should also include a discussion of test sensitivity, specificity, and positive predictive values to help patients interpret the results accurately. For patients with concerns about the implications of genetic testing and the privacy of their results, it is important to provide information about the Genetic Information Nondiscrimination Act of 2008.[18]

Clinical Significance

Identifying fetal anomalies and genetic conditions during the prenatal period offers several significant advantages. Early detection allows parents and healthcare providers to make informed decisions and prepare for potential outcomes. This may include transferring care and planning delivery at a facility equipped with a neonatal intensive care unit and pediatric subspecialists to support a newborn with genetic or structural abnormalities. In addition, early detection also provides families with time for emotional preparation and planning for the potential long-term needs of the child. Additionally, early diagnosis may offer the option of pregnancy termination, if desired.[19]