Besides giving information on fetal well-being, amniocentesis makes it possible to test for various congenital anomalies via biochemical analysis of amniotic fluid and tissue culture chromosome studies of fetal cells (see Chapter 34). In addition, certain substances of fetal origin may appear in maternal serum. In some cases it is possible to detect certain fetal malformations by screening tests in maternal serum.

Maternal serum alpha-fetoprotein

One of the most widely publicized tests for congenital anomalies is the alpha-fetoprotein (AFP) test in maternal serum for detection of open neural tube defects. Although neural tube defects are much more common in infants born to families in which a previous child had such an abnormality, about 90% occur in families with no previous history of malformation. AFP is an alpha-1 glycoprotein with a molecular weight of about 70,000. It is first produced by the fetal yolk sac and then mostly by the fetal liver. It becomes the predominant fetal serum protein by the 12th or 13th week of gestation but then declines to about 1% of peak levels by delivery. It is excreted via fetal urine into amniotic fluid and from there reaches maternal blood. After the 13th week both fetal serum and amniotic fluid AFP levels decline in parallel, the fetal blood level being about 200 times the amniotic fluid level. In contrast, maternal serum levels become detectable at about the 12th to 14th week and reach a peak between the 26th and 32nd week of gestation. Although maternal serum screening could be done between the 15th and 20th weeks, the majority of investigators have decided that the interval between the 16th and 18th weeks is optimal, since the amniotic fluid AFP level is still relatively high and the fetus is still relatively early in gestation. Maternal AFP normal levels differ for each week of gestation and ideally should be determined for each laboratory. Results are reported as multiples (e.g., 1.5Ч, 2.3Ч) of the normal population mean value for gestational age. In any patients with abnormal AFP values it is essential to confirm fetal gestational age by ultrasound, since 50% of abnormal AFP results are found to be normal due to ultrasound findings that result in a change being made in a previously estimated gestational date. Some reports suggest that maternal weight is also a factor, with heavier women tending to have lower serum AFP values (one group of investigators does not agree that maternal AFP values should be corrected for maternal weight). There are also some reports that AFP values are affected by race, at least when comparing values from Europeans and African Americans.

Maternal AFP levels reportedly detect about 85%-90% (literature range, 67%-97%) of open neural tube defects; about one half are anencephaly and about one half are open or closed spinabifida. There is an incidence of about 1-2 per 1,000 live births. The test also detects a lesser (but currently unknown) percentage of certain other abnormalities, such as fetal ventral wall defects, Turner’s syndrome, pilonidal sinus, hydrocephalus, duodenal atresia, multiple hypospadias, congenital nephrosis, and cystic hygroma. In addition, some cases of recent fetal death, threatened abortion, and Rh erythroblastosis produce elevated maternal AFP levels, as well as some cases of maternal chronic liver disease and some maternal serum specimens obtained soon after amniocentesis. A theoretical but unlikely consideration is AFP-producing tumors such as hepatoma. More important, twin pregnancies cause maternal values that are elevated in terms of the reference range established on single-fetus pregnancies. A large minority of elevated maternal AFP levels represent artifact due to incorrect estimation of fetal gestational age, which, in turn, would result in comparing maternal values to the wrong reference range. There is also the possibility of laboratory error. Most authorities recommend a repeat serum AFP test 1 week later to confirm an abnormal result. If results of the second specimen are abnormal, ultrasound is usually suggested to date the age of gestation more accurately, to examine the fetus for anencephaly, and to exclude twin pregnancy. However, even ultrasonic measurements may vary from true gestational age by as much as 5-7 days. Some perform ultrasonic evaluation if the first AFP test result is abnormal; if ultrasound confirms fetal abnormality, a second AFP specimen would be unnecessary. In some medical centers, about 40%-59% of elevated maternal AFP levels can be explained on the basis of technical error, incorrect fetal gestation date, and multiple pregnancy.

Some conditions produce abnormal decrease in maternal serum AFP values. The most important is Down’s syndrome (discussed later). Other conditions that are associated with decreased maternal serum AFP levels include overestimation of fetal age and absence of pregnancy (including missed abortion).

Amniotic fluid alpha-fetoprotein

Amniocentesis is another technique that can be used to detect open neural tube defects. It is generally considered the next step after elevated maternal AFP levels are detected and confirmed and the age of gestation is accurately determined. As mentioned previously, amniocentesis for this purpose is generally considered to be optimal at 16-18 weeks of gestation. Assay of amniotic fluid AFP is said to be about 95% sensitive for open neural tube defects (literature range, 80%-98%), with a false positive rate in specialized centers less than 1%. Most false positive results are due to contamination by fetal blood, so a test for fetal red blood cells or hemoglobin is recommended when the amniotic fluid AFP level is elevated. Amniotic fluid AFP normal values are age related, similar to maternal serum values.

Screening for Down’s syndrome

While maternal serum AFP screening was being done to detect neural tube defects, it was noticed that decreased AFP levels appeared to be associated with Down’s syndrome (trisomy 21, the most common multiple malformation congenital syndrome). Previously, it had been established that women over age 35 had a higher incidence of Down’s syndrome pregnancies. In fact, although these women represent only 5%-8% of pregnancies, they account for 20%-25% (range, 14%-30%) of congenital Down’s syndrome. Since it was discovered that mothers carrying a Down’s syndrome fetus had AFP values averaging 25% below average values in normal pregnancy, it became possible to detect about 20% of all Down’s syndrome fetuses in pregnant women less than age 35 years in the second trimester. Combined with approximately 20% of all Down’s syndrome fetuses detected by amniocentesis on all possible women over age 35, the addition of AFP screening to maternal age criteria potentially detected about 40% of all Down’s syndrome pregnancies. Later, it was found that serum unconjugated estriol (uE3) was decreased about 25% below average values seen in normal pregnancies, and hCG values were increased at least 200% above average normal levels; both were independent of maternal age. Addition of hCG and uE3 to AFP screening raised the total detection rate of all Down’s syndrome patients to about 60%. Later, there was controversy whether including uE3 was cost effective. Even more recently it was found that substituting beta-hCG for total hCG increased the total Down’s syndrome detection rate to 80%-86%. Also, it was found that screening could be done in the first trimester as well as the second trimester (although AFP was less often abnormal). Finally, it was found that if AFP, uE3, and beta-hCG were all three decreased (beta-hCG decreased rather than elevated), about 60% of fetal trisomy 18 could be detected. Trisomy 18 (Edward’s syndrome) is the second most common congenital trisomy. Decreased AFP can also be caused by hydatidiform mole, insulin-dependent diabetes, and incorrect gestational age estimation.

Amniotic fluid acetylcholinesterase

Acetylcholinesterase (ACE) assay in amniotic fluid has been advocated as another way to detect open neural tube defects and to help eliminate diagnostic errors caused by false positive AFP results. Acetylcholinesterase is a major enzyme in spinal fluid. Results from a limited number of studies in the late 1970s and early 1980s suggest that the test has 98%-99% sensitivity for open neural tube defects. Acetylcholinesterase assay has the further advantage that it is not as dependent as AFP on gestational age. It is not specific for open neural tube defects; amniotic fluid elevations have been reported in some patients with exomphalos (protrusion of viscera outside the body due to a ventral wall defect) and certain other serious congenital anomalies and in some patients who eventually miscarry. Not enough data are available to properly evaluate risk of abnormal ACE results in normal pregnancies, with reports in the literature ranging from 0%-6%. There is also disagreement as to how much fetal blood contamination affects ACE assay. The test is less affected than AFP assay, but substantial contamination seems capable of producing falsely elevated results.

Chromosome analysis (cytogenetic karyotyping) on fetal amniotic cells obtained by amniocentesis is the standard way for early prenatal diagnosis of fetal trisomies and other congenital abnormalities. However, standard karyotyping is very time-consuming, requires a certain minimum number of fetal cells that need culturing, and usually takes several days to complete. A new method called fluorescent in situ hybridization (FISH) uses nucleic acid (deoxyribonucleic acid, DNA) probes to detect certain fetal cell chromosomes such as 13, 18, 21, X, and Y, with identification accomplished by a fluorescent dye coupled to the probe molecules. Correlation with traditional cytogenetics has generally been over 95%, with results in 24 hours or less. FISH modifications have made it possible to detect fetal cells in maternal blood and subject them to the same chromosome analysis. One company has a combined-reagent procedure that can be completed in 1 hour. Disadvantages of FISH include inability to detect abnormalities in chromosomes other than the ones specifically targeted by the probes and inability to detect mosaic abnormalities or translocations, thereby missing an estimated 35% of chromosome defects that would have been identified by standard karyotyping methods.

Preterm labor and placental infection

It has been estimated that about 7% of deliveries involve mothers who develop preterm labor. It has also been reported that chorioamnionitis is frequently associated with this problem (about 30%; range, 16%-82%). Less than 20% of infected patients are symptomatic. Diagnosis of infection has been attempted by amniotic fluid analysis. Amniotic fluid culture is reported to be positive in about 20% of cases (range, 4%-38%). Mycoplasmas are the most frequent organisms cultured. Amniotic fluid Gram stain is positive in about 20% of patients (range, 12%-64%). Amniotic fluid white blood cell count was reported to be elevated in 57%-64% of cases. However, there was great overlap between patients with or without infection and also between those with proven infection. In three reports, the most sensitive amniotic fluid test for infection was amniotic fluid interleukin-6 (IL-6) assay (81%-100%). However, at present most hospitals would have to obtain IL-6 assay from large reference laboratories.