Amniocentesis

Amniocentesis involves the aspiration of 10 to 20 ml of amniotic fluid through the abdominal wall under ultrasonographic guidance (Figure 21.1). This is usually performed around the 16th week of gestation. The sample is spun down to yield a pellet of cells and supernatant fluid. The fluid can be used in the prenatal diagnosis of neural tube defects by assay of α-fetoprotein (p. 328). The cell pellet is resuspended in culture medium with fetal calf serum, which stimulates cell growth. Most of these cells in the amniotic fluid, that have been shed from the amnion, fetal skin, and urinary tract epithelium, are non-viable, but a small proportion will grow. After approximately 14 days, there are usually sufficient cells for chromosome and DNA analysis, although a longer period may be required before enough cells are obtained for biochemical assays. Increasingly, sensitive polymerase chain reaction (PCR) techniques make direct DNA analysis possible without the need for culture.

FIGURE 21.1 Diagram of the technique of amniocentesis.

When a couple is considering amniocentesis, they should be informed of the 0.5% to 1% risk of miscarriage associated with the procedure, and that if the result is abnormal they will be facing the possibility of having to consider a mid-trimester termination of pregnancy that involves an induction of labor.

Trials of amniocentesis earlier in pregnancy, at 12 to 14 weeks’ gestation, yielded comparable rates of success in obtaining results with a similar risk of miscarriage. However, concerns have been expressed regarding the reduction in amniotic fluid at this early stage of pregnancy, and early amniocentesis is not widely practiced. Although it has the advantage of allowing a result to be given earlier in the pregnancy, a mid-trimester termination of pregnancy is still usually required if the fetus is found to be affected.

Chorionic Villus Sampling

In contrast to amniocentesis, chorionic villus sampling (CVS), first developed in China, enables prenatal diagnosis to be undertaken during the first trimester. This procedure is usually carried out at 11 to 12 weeks’ gestation under ultrasonographic guidance by either transcervical or, more usually, transabdominal aspiration of chorionic villus (CV) tissue (Figure 21.2). This tissue is fetal in origin, being derived from the outer cell layer of the blastocyst (i.e., the trophoblast). Maternal decidua, normally present in the biopsy sample, must be removed before the sample is analyzed. Placental biopsy is the term used when the procedure is carried out at later stages of pregnancy.

FIGURE 21.2 Diagram of the technique of transvaginal chorionic villus sampling.

Chromosome analysis can be undertaken on CV tissue either directly, looking at metaphase spreads from actively dividing cells, or after culture. Direct chromosomal analysis of CV tissue usually allows a provisional result to be given within 24 hours. Nowadays rapid, direct fluorescent in-situ hybridization (FISH) probing (p. 34), or DNA analysis by the multiplex ligation-dependent probe amplification technique (p. 66), is used to test for common chromosome aneuploidies prior to a standard karyotype following culture of CV tissue; this may also detect other chromosome abnormalities and balanced rearrangements. For single-gene disorders, sufficient CV tissue is usually obtained to allow prenatal diagnosis by immediate biochemical assay or DNA analysis using uncultured CV tissue.

The major advantage of CV sampling is that it offers first-trimester prenatal diagnosis, although it has the disadvantage that even in experienced hands the procedure conveys a 1% to 2% risk of causing miscarriage. There is also evidence that this technique can cause limb abnormalities in the embryo if carried out before 9 to 10 weeks’ gestation; for this reason is not performed before 11 weeks’ gestation.

Ultrasonography

Ultrasonography offers a valuable means of prenatal diagnosis. It can be used not only for obstetric indications, such as placental localization and the diagnosis of multiple pregnancies, but also for prenatal diagnosis of structural abnormalities not associated with known chromosomal, biochemical or molecular defects. Ultrasonography is particularly valuable because it is non-invasive and conveys no known risk to the fetus or mother. It does, however, require expensive equipment and a skilled, experienced operator. For example, a search can be made for polydactyly as a diagnostic feature of a multiple abnormality syndrome, such as one of the autosomal recessive short-limb polydactyly syndromes that are associated with severe pulmonary hypoplasia—invariably lethal (Figure 21.3). Similarly, a scan can reveal that the fetus has a small jaw, which can be associated with a posterior cleft palate and other more serious abnormalities in several single-gene syndromes (Figure 21.4).

FIGURE 21.3 Ultrasonographic image of a transverse section of the hand of a fetus showing polydactyly.

FIGURE 21.4 Longitudinal sagittal ultrasonographic image of the head and upper chest of a fetus showing micrognathia (small jaw) (arrow).

Until a few years ago, detailed ultrasonography for structural abnormalities was offered only to couples who had a child with a genetic disorder or syndrome for which there was no chromosomal, biochemical, or molecular marker. Increasingly, however, detailed ‘fetal anomaly’ scanning is being offered routinely to all pregnant women at around 18 weeks’ gestation as screening for structural abnormalities such as neural tube defects or cardiac anomalies. This technique can also identify features that suggest the presence of an underlying chromosomal abnormality. Such a finding would lead to an offer of amniocentesis or placental biopsy for definitive chromosome analysis.

The future of fetal scanning holds the prospect of three-dimensional imaging and magnetic resonance imaging being used more widely and routinely. Although this will clearly enable the unborn baby to be visualized in far greater detail, it will also generate bigger challenges for the dysmorphologist, who might be expected to diagnose serious disorders on the basis of very subtle features.

Nuchal Translucency

In addition, the observation that increased nuchal translucency (NT) is seen in fetuses who are subsequently born with Down syndrome has resulted in the introduction of measurements of nuchal pad thickness (Figure 21.5) in the first and second trimesters as part of screening for Down syndrome (p. 273). In fact, the finding is not specific and may be seen in various chromosomal anomalies as well as isolated congenital heart disease.

FIGURE 21.5 Nuchal thickening—an accumulation of fluid at the back of the neck. The greater the thickness, the more likely there will be a chromosomal abnormality (e.g., Down syndrome) and/or cardiac anomaly. This finding leads to detailed fetal heart scanning and, usually, fetal karyotyping.

(Courtesy Dr. Helen Liversedge, Exeter.)

Fetoscopy

Fetoscopy involves visualization of the fetus by means of an endoscope. Increasingly, this technique is being superseded by detailed ultrasonography, although occasionally fetoscopy is still undertaken during the second trimester to try to detect the presence of subtle structural abnormalities that would point to a serious underlying diagnosis.

Fetoscopy has also been used to obtain samples of tissue from the fetus that can be analyzed as a means of achieving the prenatal diagnosis of several rare disorders. These have included inherited skin disorders such as epidermolysis bullosa and, before DNA testing became available, metabolic disorders in which the enzyme is expressed only in certain tissues or organs, such as the liver—e.g., ornithine transcarbamylase deficiency (p. 172).

Unfortunately, fetoscopy is associated with a 3% to 5% risk of miscarriage. This relatively high risk, coupled with the increasing sensitivity of ultrasonography and the availability of either linked DNA markers or specific mutation analysis, means that fetoscopy is used only infrequently and in highly specialized prenatal diagnostic centers.

Cordocentesis

Although fetoscopy can also be used to obtain a small sample of fetal blood from one of the umbilical cord vessels in the procedure known as cordocentesis, improvements in ultrasonography have enabled visualization of the vessels in the umbilical cord, allowing transabdominal percutaneous fetal blood sampling. Fetal blood sampling is used routinely in the management of rhesus iso-immunization (p. 205) and can be used to obtain samples for chromosome analysis to resolve problems associated with possible chromosomal mosaicism in CV or amniocentesis samples.

Radiography

The fetal skeleton can be visualized by radiography from 10 weeks onwards, and this technique has been used in the past to diagnose inherited skeletal dysplasias. It is now employed only occasionally because of the dangers of radiography to the fetus (p. 26) and the widespread availability of detailed ultrasonography.

Maternal Serum Screening

It has been government policy in the UK since 2001 that antenatal Down syndrome screening be available to all women, though it was introduced in the late 1980s. Where it is standard practice, maternal serum screening is offered for NTDs and Down syndrome using a blood sample obtained from the mother at 16 weeks’ gestation. In this way up to 75% of all cases of open NTDs and 60% to 70% of all cases of Down syndrome can be detected.

Neural Tube Defects

In 1972 it was recognized that many pregnancies in which the baby had an open NTD (p. 258) could be detected at 16 weeks’ gestation by assay of AFP in maternal serum. AFP is the fetal equivalent of albumin and is the major protein in fetal blood. If the fetus has an open NTD, the level of AFP is raised in both the amniotic fluid and maternal serum as a result of leakage from the open defect. Open NTDs fulfil the criterion of being serious disorders, as anencephaly is invariably fatal, and between 80% and 90% of the small proportion of babies who survive with an open lumbosacral lesion are severely handicapped.

Unfortunately maternal serum AFP screening for NTDs is neither 100% sensitive nor 100% specific (p. 319). The curves for the levels of maternal serum AFP in normal and affected pregnancies overlap (Figure 21.6), so that in practice an arbitrary cut-off level has to be introduced below which no further action is taken. This is usually either the 95th centile, or 2.5 multiples of the median (MoM); as a result around 75% of screened open spina bifida cases are detected. Those pregnant women with results that lie above this arbitrary cut-off level are offered detailed ultrasonography; which is usually sufficient to diagnose NTD. In fact, ultrasonography has more or less superceded maternal serum screening as a means of diagnosing NTD. Anencephaly shows a dramatic deficiency in the cranium (Figure 21.7) and an open myelomeningocele is almost invariably associated with herniation of the cerebellar tonsils through the foramen magnum. This deforms the cerebellar hemispheres, which then have a curved appearance known as the ‘banana sign’; the forehead is also distorted, giving rise to a shape referred to as the ‘lemon sign’ (Figure 21.8). A posterior encephalocele is readily visualized as a sac in the occipital region (Figure 21.9) and always prompts a search for additional anomalies that might help diagnose a recognizable condition such as Meckel-Gruber syndrome.

FIGURE 21.6 Maternal serum α-fetoprotein (AFP) levels at 16 weeks’ gestation plotted on a logarithmic scale as multiples of the median (MoMs). Women with a value off or above 2.5 multiples of the median are offered further investigations.

(Adapted from Brock DJH, Rodeck CH, Ferguson-Smith MA [eds] 1992 Prenatal diagnosis and screening. Edinburgh, UK: Churchill Livingstone.)

FIGURE 21.7 Anencephaly (arrow). There is no cranium and this form of neural tube defect is incompatible with life.

(Courtesy Dr. Helen Liversedge, Exeter, UK.)

FIGURE 21.8 The so-called banana sign showing the distortion of the cerebellar hemispheres into a curved structure (solid arrow). The forehead is also distorted into a shape referred to as the ‘lemon sign’ (broken arrow).

(Courtesy Dr. Helen Liversedge, Exeter, UK.)

FIGURE 21.9 Posterior encephalocele (arrow), a more rare form of neural tube defect. This may be an isolated finding or associated with polydactyly and cystic renal changes in Meckel-Gruber syndrome.

(Courtesy Dr. Helen Liversedge, Exeter, UK.)

A raised maternal serum AFP concentration is not specific for open NTDs (Box 21.1). Other causes include threatened miscarriage, twin pregnancy and a fetal abnormality such as exomphalos, in which there is a protrusion of abdominal contents through the umbilicus.

As a result of these screening modalities there has been a striking decline in the incidence of open NTDs in liveborn and stillborn babies. Other contributory factors are a general improvement in diet and the introduction of periconceptional folic acid supplementation (p. 258). In England and Wales the combined incidence of anencephaly and spina bifida in liveborn and stillborn babies fell from 1 in 250 in 1973 to 1 in 6250 in 1993.

Down Syndrome and Other Chromosome Abnormalities