Introduction

The first prenatal screening test was introduced in the 1970s: a single second-trimester serum test for maternal serum alpha-fetoprotein, a marker of neural tube defects. Aneuploidy screening using maternal serum markers was introduced in the 1980s, and the number and complexity of offered screening tests have been on an upward trajectory ever since.[1] Prenatal genetic screening is used to assess whether there is an increased risk of the fetus being affected by a genetic disorder. Originally, prenatal genetic testing primarily focused on trisomy 21 (Down syndrome), but now it can detect a broad range of genetic disorders.[2] Today, prenatal genetic screening falls into 4 categories: ultrasonography, maternal carrier status of specific genetic disorders, maternal serum assays looking for specific biochemical markers indicative of aneuploidy, and most recently, maternal plasma fetal cell-free fetal deoxyribonucleic aid (cffDNA), which has been used for aneuploidy, microdeletion, and copy number variants (CNVs). Maternal serum assays include first-trimester screening, the triple screen, the quadruple screen, and the penta screen. There is also the option of combining first-trimester and second-trimester screening with either integrated, sequential, or contingent screening protocol. This provides a higher detection rate than a one-step screening.[3]

Specimen Collection

Maternal blood is collected through a standard blood draw. Maternal serum levels of maternal serum alpha-fetoprotein (MSAFP), pregnancy-associated plasma protein-A (PAPP-A), free beta-human chorionic gonadotropin (beta-hCG), inhibin A, and unconjugated estriol are measured. Cell-free fetal DNA is isolated and purified from maternal plasma. Maternal carrier screening involves collecting a sample of blood, saliva, or buccal tissue.

Procedures

Regardless of maternal age, all women should be given information regarding an option to pursue aneuploidy screening and follow-up diagnostic testing. Women should discuss these options with their providers to decide the best test for them. Factors affecting the choice of screening include but are not limited to, the mother’s desire for prenatal information, previous pregnancies, family history, gestational age at the first visit, cost, and desire to pursue follow-up pregnancy care or termination in the case of an abnormal diagnostic test.[3] Individual screening procedures are dependent upon the gestational age of the fetus and the type of screening being done. The earliest genetic screen is carrier screening, which ideally would be done preconception, but is also offered prenatally. The cffDNA can be reliably detected after 10 weeks of gestation.

There are several options for first-trimester screening. Maternal serum analytes can be measured alone or in combination with a nuchal translucency measurement. Maternal serum markers measured in the first trimester are PAPP-A and free beta-hCG. Nuchal tissue thickness is measured in a lateral view using the caliper function of the ultrasound (US) machine.[4]  Prenatal diagnostic testing for sickle cell disease is available. DNA-based tests can be performed using chorionic villi obtained by chorionic villus sampling or using cultured amniotic fluid cells obtained by amniocentesis. One option is preimplantation genetic diagnosis in combination with in vitro fertilization may be an alternative to avoid termination of an affected pregnancy. Preimplantation genetic diagnosis has been successfully performed for sickle cell disease.

Second-trimester serum screening options are the triple, quadruple screen, and penta screen. In addition, there are combinations of first and second-trimester screening that include the integrated screen, the stepwise screen, and the contingent screen. The triple screen measures hCG, MSAFP, and unconjugated estriol.[5] The quadruple (quad) screen measures hCG, MSAFP, inhibin A, and unconjugated estriol, and is particularly useful in patients who do not present for their first prenatal visit until the second trimester. The integrated screen is a combination of the results of the first-trimester combined screen and the second-trimester quad-screen into 1 result given after the second-trimester screening. The stepwise screen is like the integrated screen in that it combines first and second-trimester screenings; however, patients are given their first-trimester screen results early. The patient may go straight to diagnostic testing if the first-trimester screen is positive, at which point the stepwise screening would be discontinued. A patient with a negative first-trimester screen is notified of the result and continues to the second-trimester quad screen.[3] The contingent screen gives patients their results after the first-trimester screen and stratifies patients into low, intermediate, and high-risk groups. Low-risk receive no further testing, and high-risk are offered diagnostic testing. Only intermediate-risk groups undergo the second-trimester quad screen.[6] The penta screen measures hCG, MSAFP, inhibin A, unconjugated estriol, and hyperglycosylated hCG.[3]

Indications

Prenatal screening guidelines have been issued by several major organizations, including the American College of Obstetricians and Gynecologists (ACOG), the Society for Maternal-Fetal Medicine, the American College of Medical Genetics and Genomics (ACMG), and the United States Preventive Services Task Force. ACOG recommends that prenatal screening should be done for aneuploidy, and follow-up diagnostic testing should be offered to all patients regardless of maternal age or risk factors. Such testing should be discussed early in pregnancy, and patients should be counseled on the individual risk factors, screening sensitivity, and the importance of diagnostic tests.[3][2]

Ultrasound findings are considered soft markers and should not be used exclusively to screen for aneuploidy.[3][7] Carrier screening should ideally be offered preconceptionally. Patients may be offered ethnic-specific, pan-ethnic, or expanded carrier screening according to their provider’s preferences. If a woman is found to be a carrier, her partner should also be offered to screen.[8] The ACMG recommends that patients should be educated on the availability of cffDNA as a screening method for Down syndrome, Edwards syndrome, and Patau syndrome as well as its utility in screening for sex chromosome aneuploidy.[9] Further screening should not be offered to women who have a previous negative screening test, as this increases the likelihood of a false-positive result. Diagnostic testing is universally recommended in the occurrence of a positive screening test regardless of screening modality.[3]

Potential Diagnosis

Carrier screening is commonly offered for spinal muscular atrophy, cystic fibrosis, hemoglobinopathies, fragile X syndrome, and Tay-Sachs disease.[10]  Aneuploidy screening using maternal serum biomarkers is offered for chromosomes 21, 18, and 13 aneuploidy for Down syndrome, Edwards syndrome, and Patau syndrome, respectively. Aneuploidy screening using cffDNA is available for the above aneuploidies as well as Turner syndrome and Klinefelter syndrome. It is technically possible to screen for copy number variants, such as DiGeorge syndrome, using cffDNA.

Normal and Critical Findings

Carrier screens will come back either negative or positive. A positive result indicates carrier status.

Maternal serum biochemical markers include the following:

• Down syndrome is associated with low MSAFP, low estriol, high hCG, high inhibin A, and low PAPP-A
• Edwards syndrome is associated with low MSAFP, low estriol, low hCG, normal inhibin A, and low PAPP-A
• Patau syndrome is associated with normal MSAFP, normal estriol, normal hCG, normal inhibin A, and low PAPP-A

An increased nuchal translucency is a soft marker for aneuploidy but is not diagnostic. Other anatomical anomalies, such as small or absent nasal bone and cardiac defects, are also indicators of possible aneuploidy. The ccfDNA screening results can be reported as either positive or negative, as high risk or low risk. They can also return as no-call. Reports should include the fetal fraction.[11]

Interfering Factors

A ccfDNA screen is less predictive in cases of maternal obesity leading to low fetal fractions, multiple pregnancies, and less than 10 weeks gestational age. It has, in rare cases, given false results due to occult maternal malignancies.[12] In the case of multiple pregnancies, no screening test is as accurate as it is in singleton pregnancies. Screening tests for aneuploidy should not be performed if there is evidence of fetal demise or if one of the fetuses has abnormalities identified on ultrasound.[3] Ultrasound interpretation is dependent upon the skill of the technician.

Complications

Standard phlebotomy to obtain the maternal blood samples has the risk of phlebitis, bruising, bleeding, and localized pain. Ultrasonography has been used for over 20 years and is considered a very low risk to both the mother and fetus. There may be some discomfort due to the pressure of the probe.

Patient Safety and Education

It is important that the patient understands the purpose of screening tests and the difference between screening and diagnostic tests. A positive screen needs to be followed by a diagnostic test before any irreversible decisions are made. Likewise, patients need to understand that a negative screening test is not a guarantee. To achieve this end, patients should be counseled on test sensitivity, specificity, and positive predictive values. Patients with concerns over the implications of genetic testing and privacy of their results should be given information on the Genetic Information Nondiscrimination Act of 2008.[13]

Clinical Significance

Identification of fetal anomalies and genetic conditions during the prenatal period has its advantages. A specific diagnosis allows parents and practitioners to prepare and explore all options. This could mean emotionally preparing for a child with special needs, planning for delivery in a hospital best able to meet the neonate’s needs, or terminating a pregnancy.[14]