I. GESTATIONAL AGE ASSESSMENT is important to both the obstetrician and pediatrician and must be made with a reasonable degree of precision. Elective obstetric interventions such as chorionic villus sampling (CVS) and amniocentesis must be timed appropriately. When premature delivery is inevitable, gestational age is important with regard to prognosis, the management of labor and delivery, and the initial neonatal treatment plan.

A. The clinical estimate of gestational age is usually made on the basis of the first day of the last menstrual period (LMP). Accompanied by physical examination, auscultation of fetal heart sounds and maternal perception of fetal movement can also be helpful.

B. Ultrasound is the most accurate method for estimating gestational age. During the first trimester, fetal crown-rump length (CRL) can be an accurate predictor of gestational age. At <8 weeks and 6 days if the CRL and the LMP are >5 days different, the ultrasound is the best estimate for gestational age. From 9 0/7 to 15 6/7 weeks, CRL estimation of gestational age is expected to be within 7 days of the true gestational age. After 14 weeks, measurements of the biparietal diameter (BPD), the head circumference (HC), abdominal circumference (AC), and the fetal femur length best estimate gestational age. Strict criteria for measuring the crosssectional images through the fetal head ensure accuracy. Nonetheless, owing to normal biologic variability, the accuracy of gestational age estimated by biometry decreases with increasing gestational age. For measurements made at 16 to 21 6/7 weeks of gestation, the variation is up to 10 days; at 22 to 27 6/7 weeks, the variation is up to 14 days; and at 28 weeks and beyond, the variation can be up to 21 days.

II. PRENATAL DIAGNOSIS OF FETAL DISEASE continues to improve. The genetic or developmental basis for many disorders is emerging, along with increased test accuracy. Two types of tests are available: screening tests and diagnostic procedures. Screening tests, such as a sample of the mother’s blood or an ultrasound, are noninvasive but relatively nonspecific. A positive screening test, concerning family history, or an ultrasonic examination that suggests anomalies or aneuploidy may lead patient and physician to consider a diagnostic procedure. Diagnostic procedures, which necessitate obtaining a sample of fetal material, pose a small risk to both mother and fetus but can confirm or rule out the disorder in question.

A. Screening by maternal serum analysis during pregnancy individualizes a woman’s risk of carrying a fetus with a neural tube defect (NTD) or an aneuploidy such as trisomy 21 (Down syndrome) or trisomy 18 (Edward syndrome).

1. Maternal serum α-fetoprotein (MSAFP) measurement between 15 and 22 weeks’ gestation screens for NTDs. MSAFP elevated above 2.5 multiples of the median for gestation age occurs in 70% to 85% of fetuses with open spina bifida and 95% of fetuses with anencephaly. In half of the women with elevated levels, ultrasonic examination reveals another cause, most commonly an error in gestational age estimate. Ultrasonography that incorporates cranial or intracranial signs such as changes in head shape (lemon sign) or deformation of the cerebellum (banana sign) that are secondary to the NTD increase the sensitivity of ultrasound for the visual detection of open spinal defects.

2. Second-trimester aneuploidy screening: MSAFP/quad panel. Low levels of MSAFP are associated with chromosomal abnormalities. Altered levels of human chorionic gonadotropin (hCG), unconjugated estriol (uE3), and inhibin are also associated with fetal chromosomal abnormalities. On average, in a pregnancy with a fetus with trisomy 21, hCG and inhibin levels are higher than expected and uE3 levels are decreased. A serum panel in combination with maternal age can estimate the risk of trisomy 21 for an individual woman. For women <35 years, 5% will have a positive serum screen, but the majority (98%) will not have a fetus with aneuploidy. Only 80% of fetuses with trisomy 21 will have a “positive” quad screen (MSAFP, hCG, uE3, inhibin). Trisomy 18 is typically signaled by low levels of all markers.

3. First-trimester serum screening. Maternal levels of two analytes, pregnancy-associated plasma protein-A (PAPP-A) and hCG (either free or total), are altered in pregnancies with an aneuploid conception, especially trisomy 21. Similar to second-trimester serum screening, these values can individualize a woman’s risk of pregnancy complicated by aneuploidy. However, these tests need to be drawn early in pregnancy (optimally at 9 to 10 weeks) and, even if abnormal, detect less than half of the fetuses with trisomy 21.

4. First-trimester nuchal lucency screening. Ultrasonographic assessment of the fluid collected at the nape of the fetal neck is a sensitive marker for aneuploidy. With attention to optimization of image and quality control, studies indicate a 70% to 80% detection of aneuploidy
in pregnancies with an enlarged nuchal lucency on ultrasonography. In addition, some fetuses with structural abnormalities such as cardiac defects will also have an enlarged nuchal lucency.

5. Combined first-trimester screening. Combining the two first-trimester maternal serum markers (PAPP-A and β-hCG) and the nuchal lucency measurements in addition to the maternal age detects 80% of trisomy 21 fetuses with a low screen positive rate (5% in women <35 years). This combined first-trimester screening provides women with a highly sensitive risk assessment in the first trimester.

6. Combined first- and second-trimester screening for trisomy 21. Various approaches have been developed to further increase the sensitivity of screening for trisomy 21 while retaining a low screen positive rate. These approaches differ primarily by whether they disclose the results of their first trimester results.

a. Integrated screening. This is a nondisclosure approach that achieves the highest detection of trisomy 21 (97%) at a low screen positive rate (2%). It involves a first-trimester ultrasound and maternal serum screening in both the first and second trimester before the results are released.

b. Sequential screening. Two types of sequential screening tools exist. Both are disclosure tests, which means that they release those results indicating a high risk of trisomy 21 in the first trimester but then go on to either further screen the entire remaining population in the second trimester (stepwise sequential) or only a subgroup of women felt to be in a medium-risk zone (contingent sequential). With contingent sequential screening, patients can be classified as high risk, medium risk, or low risk for Down syndrome in the first trimester. Low-risk patients do not return for further screening as their risk of a fetus with Down syndrome is low. When the two types of sequential tests are compared, they have similar overall screen positive rates of 2% to 3%, and both have sensitivities of >90% for trisomy 21 (stepwise, 95%; contingent, 93%)

7. Cell-free fetal DNA screening for aneuploidy. Newer technology has allowed analysis of cell-free fetal DNA from maternal serum in order to detect trisomies 13, 18, and 21 and sex chromosomal aneuploidies. The fetal DNA detected in maternal serum is placental in origin, can be detected as early as 9 weeks, and can be tested throughout the entire pregnancy. A number of laboratories have commercially available tests; all of which report a high sensitivity and specificity for trisomies 21 and 18. Sensitivity for trisomy 21 is reported at 99.3% and specificity at 99.8%. For trisomy 18, sensitivity is 97.4% and specificity is 99.8%. Sensitivity is lower for trisomy 13 (91%) with a specificity of 99.6%. Importantly, the positive predictive value (PPV) is lower for younger women secondary to the lower prevalence of aneuploidy in this population. For example, for trisomy 21, the PPV is 33% for women <25 years, in comparison to 87% for women >40 years. It is also important to note that cell-free fetal DNA targets specific aneuploidies and will ultimately miss abnormalities in other chromosomes and those with a mosaic karyotype. These abnormalities may have been detected by traditional screening methods. One study estimates up to 17% of significant chromosome abnormalities may go undetected with the use
of cell-free fetal DNA screening alone. For these reasons, cell-free fetal DNA screening for aneuploidy is not recommended for the general obstetric population and currently is recommended for women considered high risk for aneuploidy, including women who are >35 years old, have a history of a fetus or newborn with aneuploidy, carriers of a balanced translocation, or have a positive traditional screening test. Cell-free fetal DNA is considered a screening test, and any positive cell-free fetal DNA result should be followed up with a diagnostic test (CVS or amniocentesis) for confirmation of the diagnosis. Cell-free fetal DNA is also known as noninvasive prenatal testing (NIPT) despite that, as mentioned earlier, this test is considered a screening test and is not diagnostic.

a. Second-trimester ultrasound targeted for the detection of aneuploidy has also been successful as a screening tool. Application of secondtrimester ultrasound that is targeted to screen for aneuploidy can decrease the a priori maternal age risk of Down syndrome by 50% to 60% as well as the risk conveyed by serum screening. Second-trimester ultrasound following first-trimester screening for aneuploidy has likewise been shown to have value in decreasing the risk assessment for trisomy 21.

B. In women with a positive family history of genetic disease, a positive screening test, or at-risk ultrasonographic features, diagnostic tests are considered. When an invasive diagnostic test is performed for a structural abnormality detected on ultrasound, a chromosomal microarray is indicated, which will detect aneuploidy as well as smaller chromosomal deletions and duplications. If an invasive test is performed secondary to a positive screening test, either a chromosomal microarray or a karyotype can be offered. When a significant malformation or a genetic disease is diagnosed prenatally, the information gives the obstetrician and pediatrician time to educate parents, discuss options, and establish an initial neonatal treatment plan before the infant is delivered. In some cases, treatment may be initiated in utero.

1. CVS. Under ultrasonic guidance, a sample of placental tissue is obtained through a catheter placed either transcervically or transabdominally. Performed at or after 10 weeks’ gestation, CVS provides the earliest possible detection of a genetically abnormal fetus through analysis of trophoblast cells. Transabdominal CVS can also be used as late as the third trimester when amniotic fluid is not available or fetal blood sampling cannot be performed. Technical improvements in ultrasonographic imaging and in the CVS procedure have brought the pregnancy loss rate very close to the loss rate after second-trimester amniocentesis, 0.5% to 1.0%. The possible complications of amniocentesis and CVS are similar. CVS, if performed before 10 weeks of gestation, can be associated with an increased risk of fetal limb-reduction defects and oromandibular malformations.

a. Direct preparations of rapidly dividing cytotrophoblasts can be prepared, making a full karyotype analysis available in 2 days. Although direct preparations minimize maternal cell contamination, most centers also analyze cultured trophoblast cells, which are embryologically closer to the fetus. This procedure takes an additional 8 to 12 days.

b. In approximately 2% of CVS samples, a mosaic diagnosis is made, which indicates that both karyotypically normal and abnormal cells are identified in the same sample. Because CVS-acquired cells reflect placental constitution, in these cases, amniocentesis is typically performed as a follow-up study to analyze fetal cells. Approximately one-third of CVS mosaicisms are confirmed in the fetus through amniocentesis.

2. Amniocentesis. Amniotic fluid is removed from around the fetus through a needle guided by ultrasonic images. The removed amniotic fluid (˜20 mL) is replaced by the fetus within 24 hours. Amniocentesis can technically be performed as early as 10 to 14 weeks’ gestation, although early amniocentesis (<13 weeks) is associated with a pregnancy loss rate of 1% to 2% and an increased incidence of clubfoot. Loss of the pregnancy following an ultrasonography-guided second-trimester amniocentesis (16 to 20 weeks) occurs in 0.5% to 1.0% cases in most centers, so they are usually performed in the second trimester.

a. Amniotic fluid can be analyzed for a number of compounds, including alpha-fetoprotein (AFP), acetylcholinesterase (AChE), bilirubin, and pulmonary surfactant. Increased levels of AFP along with the presence of AChE identify NTDs with >98% sensitivity when the fluid sample is not contaminated by fetal blood. AFP levels are also elevated when the fetus has abdominal wall defects, congenital nephrosis, or intestinal atresias. Several biochemical tests of the amniotic fluid are available to assess fetal lung maturity.

b. Fetal cells can be extracted from the fluid sample and analyzed for chromosomal and genetic makeup.

i. Among second-trimester amniocenteses, 73% of clinically significant karyotype abnormalities relate to one of five chromosomes: 13, 18, 21, X, or Y. These can be rapidly detected using fluorescent in situ hybridization (FISH), with sensitivities in the 90% range.

ii. DNA analysis is diagnostic for an increasing number of diseases.

a) Increasingly, direct DNA methodologies can be used when the gene sequence producing the disease in question is known. Disorders secondary to deletion of DNA (e.g., α-thalassemia, Duchenne and Becker muscular dystrophy, cystic fibrosis, and growth hormone deficiency) can be detected by the altered size of DNA fragments produced following a polymerase chain reaction (PCR). Direct detection of a DNA mutation can also be accomplished by allele-specific oligonucleotide (ASO) analysis. If the PCR-amplified DNA is not altered in size by a deletion or insertion, recognition of a mutated DNA sequence can occur by hybridization with the known mutant allele. Rapid advances in molecular technologies have provided many new opportunities for mutation identification which are now applicable to fetal DNA.