TABLE 1. Risk of Having a Live-Born Child with Chromosomal Abnormalities

TABLE 2. Indications for Prenatal Diagnosis

In 1952, metabolic disorders were first demonstrated to be the result of an absence of a normally functioning enzymatic or structural protein.⁵ Most of these metabolic disorders are inherited in an autosomal-recessive manner; approximately 0.8% of newborns have such a disorder.⁶ Couples who are both heterozygous carriers of an autosomal-recessive genetic trait have a 25% risk of a homozygous affected fetus in each pregnancy. The metabolic conditions amenable to prenatal diagnosis include mucopolysaccharidoses, mucolipidoses, sphyngolipidoses, lysosomal-storage forms of carbohydrate metabolic disorders, aminoacidopathies with cultured cell expression of a known enzymatic defect, and a growing list of heterogeneously classified disorders.^1,7

Women who are either known or suspected carriers of an X-linked recessive disorder may wish to use prenatal diagnosis because of the potential risk of 50% of male offspring having the disorder in question. Carrier tests for X-linked conditions are difficult and in many cases are not completely reliable. Therefore, many women may choose to consider themselves carriers when test results are in doubt. Presently, only a few X-linked conditions (e.g., Fabry's syndrome, Hunter's syndrome, Menkes ' kinky hair syndrome, and Lesch-Nyhan syndrome) can be diagnosed by enzymatic analysis.^1,5 Duchenne-type muscular dystrophy and classic hemophilia can be diagnosed by fetal blood aspiration and serum creatine phosphokinase determination.⁸ Other mothers may choose to undergo amniocentesis in order to identify the fetal gender by chromosomal study or to determine testosterone levels, electing to bear only girls, who will be unaffected.⁹

METHODS OF SCREENING AND DIAGNOSIS

Maternal Screening

Maternal serum screening can identify pregnant women who are at an increased risk for having a baby with certain birth defects. Patient-specific risks for open spina bifida, Down syndrome and trisomy 18 (Edwards syndrome) (Fig. 1) can be determined by measuring the levels of certain proteins in maternal serum and combining those data with the patient's maternal age and clinical information.¹⁰ Women with a positive screen should be offered a definitive diagnostic test.

Serum Alpha-Fetoprotein

Until the mid-1980s, there was no way to identify younger women at risk of having children with Down syndrome. Down syndrome screening for younger women was initiated when researchers discovered that the mean level of maternal serum alpha-fetoprotein (AFP) in pregnancies complicated by Down syndrome is 0.7 multiples of the normal medium (MOM).² AFP can also be used to detect at least 80% of open neural tube defects, such as spina bifida.

Alpha-Fetoprotein P X-tra/Triple Screen Test

Shortly after the associated between AFP and Down syndrome was established, it was found that higher levels of human chorionic gonadotropins (hCG) and lower levels of unconjugated estriol levels (uE3) were also associated with Down syndrome.² These three markers were combined to make the Triple Screen Test. Together, these markers are combined with gestational age, maternal age, weight, race, number of fetuses (up to twins), and presence of maternal diabetes to provide patient-specific risks for open spina bifida, Down syndrome, and trisomy 18. The Triple Screen Test detects at least 80% of open neural tube defects and at least 60% of Down syndrome and trisomy 18.¹⁰ Although serum screening does not detect other aneuploidies with great frequency, the aneuploidies likely to be missed by serum screening usually are ultimately lethal (e.g., trisomy 13) or are gender-chromosome abnormalities not associated with profound mental retardation or other severe physical or developmental limitations.

Maternal blood sampling can be performed between 15 and 20 weeks of gestation but is most accurate when performed between 16 and 18 weeks of gestation. Accurate pregnancy dating is essential.

Alpha-Fetoprotein Tetra/Quad Screen

AFP tetra adds a fourth marker, dimeric inhibin A (DIA), to the AFP X-tra. AFP Tetra increases the detection efficiency of Down syndrome by approximately 15%, while slightly lowering the false-positive rate. DIA is a glycol protein hormone made by the ovary and placenta. DIA levels are twice as high in Down syndrome pregnancies.¹¹ Unlike AFP, hCG, and uE3, DIA does not vary with gestational age, resulting in greater screening accuracy.

Pregnancy-Associated Plasma Protein-A and β-Human Chorionic Gonadotropin

Many maternal serum analytes have been evaluated for possible use for first-trimester Down syndrome screening.¹² Pregnancy-associated plasma protein-A (PAPP-A) is a highly glycosylated, high molecular weight protein and is produced by the human placenta and released into maternal circulation during pregnancy.¹³ Maternal serum concentrations of PAPP-A have been found to be reduced in pregnancies affected by Down syndrome in the first trimester when compared to normal, unaffected pregnancy at the same gestational period. This reduction is most pronounced before the twelfth week of gestation. Later, serum concentrations in Down syndrome gradually reach the control range and, in the second trimester, PAPP-A does not distinguish between normal and Down syndrome pregnancy. Therefore, accurate gestational dating is needed.

In the less frequent cases of fetal trisomy 18 and 13, first trimester maternal serum PAPP-A is even more strongly reduced than in Down syndrome, and preliminary results indicate that in fetal trisomy 18 PAPP-A continues to be below the normal range, including in the early second trimester (weeks 15 to 20).

Early pregnancy maternal serum PAPP-A is also reduced in nonviable pregnancies and threatened abortion and is absent in the very rare Cornelia de Lange syndrome. In ectopic pregnancies, PAPP-A has also been found to be reduced.

β Subunit of hCG is also produced by the placenta. When combined with PAPP-A, a 65% detection rate for Down syndrome can be achieved, with a 5% false-positive rate. When PAPP-A and β-hCG are combined with an ultrasound obtained nuchal translucency (to be discussed later in the text), an 86% detection rate for Down syndrome with a 5% false-positive rate can be achieved, while providing results earlier in the pregnancy so that a woman may have the choice of chorionic villus sampling (CVS) or amniocentesis.^14–18 Both of these procedures will be discussed in depth later in the chapter. The first trimester only screen also affords the chance for women to make personal decisions regarding prenatal diagnosis and termination earlier in the pregnancy.

In 1956, Fuchs and Riis²³ demonstrated the feasibility of fetal sex determination by examining X-chromatin bodies in amniotic fluid cells. The ability to culture amniotic fluid cells in tissue culture and to acquire sufficient viable cells for karyotype analysis and biochemical studies was demonstrated in 1966.²⁴ A partial list of conditions that lend themselves to prenatal diagnosis by amniocentesis is found in Table 3; all of these disorders have ocular manifestations.

TABLE 3. Disorders Diagnosed by Amniocentesis with Ocular Manifestations

Traditional genetic amniocentesis is usually offered between 15 and 20 weeks' gestation. Amniocentesis performed earlier has a higher complication rate, as well as more amniotic culture failures. It may be offered when prenatal maternal screening results are high risk for a genetic abnormality or as an elective diagnostic test such as in advanced maternal age or prior history of an aneuploidy (see Fig. 2).

Many large, multicenter studies have confirmed the safety of genetic amniocentesis, as well as its cytogenetic diagnostic accuracy (greater than 99%).² The fetal loss rate is approximately 0.5%, and minor complications occur infrequently. Table 4 lists known complications for amniocentesis.

TABLE 4. Complications and Their Incidence in Amniocentesis

The procedure is performed under ultrasound guidance. After obtaining informed consent, an ultrasound examinations is performed to establish fetal viability, placental and fetal location, and depth to the largest pocket of amniotic fluid (Fig. 3). The maternal abdomen is prepped aseptically and a local anesthetic may be administered. A small gauge needle is then used to aspirate approximately 10 to 20 mL of amniotic fluid. The availability of the results is dependent on the amount of time needed for cell culture growth but usually is available within 7 to 10 days. The results received are a full cytogenic karyotype.

Fluorescent in situ hybridization (FISH) is a new technology utilizing fluorescently labeled DNA probes to detect or confirm gene or chromosome abnormalities.^25,26 The sample DNA is first denatured, a process that separates the complementary strands within the DNA double helix structure. The fluorescently labeled probe of interest is then added to the denatured sample mixture and hybridizes with the sample DNA at the target site as it reforms itself back into a double helix. The probe signal can then be seen through a fluorescent microscope and the sample DNA scored for the presence or absence of the signal.

FISH can be used in interphase cells to determine the chromosome number or more chromosomes, as well as detect some specific chromosome rearrangements that are characteristic for certain cancers. The primary advantage of interphase FISH is that it can be performed rapidly if necessary, usually within 24 hours, because cell growth is not required.

A good example is the Aneuploid Screen Test that is performed on amniotic fluid cells when there is a strong clinical indication for one of the common trisomies. The sample nuclei are denatured and hybridized with DNA probes for chromosomes 13, 18, 21, X, and Y.

CHORIONIC VILLUS SAMPLING

The indications for CVS are similar for amniocentesis, except for a few rare genetic conditions that require chorionic villi for diagnosis.² CVS is generally performed at 10 to 12 weeks' gestation. Similar to other first trimester methods, CVS allows for results earlier that can provide reassurance or allow for earlier and safer methods of pregnancy termination. Similar to amniocentesis, CVS is performed under ultrasound guidance. It can be performed either transabdominally or transcervically (Fig 4). Table 5 lists the contraindications and relative contraindications for CVS.

TABLE 5. Contraindications and Relative Contraindications for Chorionic Villus Sampling

Patients considering CVS should be counseled that there may be a slightly higher risk of pregnancy loss associated with CVS than with traditional amniocentesis. Pregnancy loss rates are reported to be 0.6% to 0.8% for CVS in excess of traditional amniocentesis. Loss may result from the procedure itself, but may incorporate the expected spontaneous loss rate between 9 and 16 weeks of gestation. According to the World Health Organization, the incidence of limb reduction defects are approximately 6 per 10,000, which is not significantly different from the incidence in the general population.² Oromandibular-limb hypogenesis appeared to be more common with CVS, although highest when CVS is performed before 9 weeks' gestation.^27–29 Similar to amniocentesis, cytogenetics can be available in 7 to 10 days (Fig. 5). FISH can also be used to provide a limited aneuploid screen in 24 hours.

CORDOCENTESIS

Cordocentesis is also known as percutaneous umbilical blood sampling (PUBS). Under direct ultrasound guidance, the umbilical vein is punctured. This procedure cannot be performed before 18 weeks' gestation. A karyotype of fetal blood can be available within 24 to 48 hours. Procedure-related pregnancy loss is less than 2%. Cordocentesis is rarely used for cytogenetics. This procedure is utilized more to evaluate fetal platelets, Rh sensitivity, and to administer fetal medications.²

ULTRASOUND

Diagnostic ultrasound is widely used in the assessment of pregnancy and the fetus. Although clinical benefits of routine ultrasonography during pregnancy have not been established, approximately 70% of pregnancies in the United States undergo ultrasound evaluation.³⁰ Because most instruments used in diagnostic ultrasonography produce energies no greater than 10 to 20 mW cm² (safety defined as less than 100 mW cm²), ultrasound is considered generally safe. No harmful biologic effects on instrument operators, pregnant women, fetuses, or other patients have been found. Infants exposed in utero have shown no significant differences in birth weight or length, childhood growth, cognitive function, acoustic or visual ability, or rates of neurologic deficits (see Fig. 6).

Indications for ultrasound are listed in Table 6. There are several levels of ultrasound. A basic ultrasound suffices for most obstetric patients. Table 7 lists the components of a basic ultrasound. A comprehensive ultrasound may be indicated for a patient who is suspected of carrying a physiologically or anatomically defective fetus by history, clinical evaluation, or prior ultrasound examination. Because of the level of expertise needed, comprehensive examinations are usually performed at a tertiary center.

TABLE 6. Indications for Ultrasonography During Pregnancy

TABLE 7. Components of Basic Ultrasound Examination

FIRST TRIMESTER ULTRASOUND

First trimester ultrasound may be performed transabdominally or transvaginally. Table 7 lists the components of a first trimester ultrasound. A crown–rump length, done between 7 and 13 weeks, can define a gestational age to within 5 days (Fig. 7).

Nuchal edema is an echo-free space between the skin line and the soft tissue overlying the cervical spine. Nuchal edema is caused by subcutaneous accumulation of fluid and has diverse etiology, including aneuploidies, cardiovascular and pulmonary defects, skeletal dysplasias, congenital infections, and hematologic and metabolic disorders. A nuchal translucency (NT) is obtained between 10 and 13 weeks' 6 days' gestational age (Fig. 8).³¹ A study at King's College Hospital in London found an NT of 3 mm was associated with a 4-times increase in the maternal age related risk for aneuploidy. An NT greater than 4 mm resulted in a 29 times increased risk for trisomies 21, 18, and 13. Additionally, with a 4 mm or more NT, there was a high incidence of other anomalies and poor prognosis, whereas with just 3 mm and a normal karyotype, the outcome was usually normal. Table 8 lists the disorders associated with an increased nuchal translucency thickness.

TABLE 8. Disorders Associated with Increased Nuchal Translucency

The First Trimester Maternal Serum Biochemistry and Fetal Nuchal Translucency Screening Study is looking at combining NT, maternal age, gestational age, PAPP-A, and β-hCG to calculate a Down syndrome and trisomy 18 risk by using computer software. Currently still under investigation, this method is widely used in Europe. Women who screen positive are then offered CVS or amniocentesis.

SECOND TRIMESTER ULTRASOUND

A second trimester ultrasound is usually done at 20 to 22 weeks' gestational age. The most commonly used fetal measurements are biparietal diameter, length of the femur or other long bones, and abdominal and head circumference. In addition to measurements, an anatomic survey is also done to evaluate the fetal brain (Fig. 9), spine, stomach, heart, kidneys, placental location and assessment of amniotic fluid (Fig. 10). If maternal risk factors are present, tetra screening results are abnormal, or there are abnormal findings on the anatomic survey, the patient is sent for a comprehensive ultrasound. The components of a comprehensive ultrasound are shown in Table 9. The ultrasound findings associated with Down syndrome include cardiac defects or enlargement, cystic hygroma (Fig. 11), duodenal atresia (Fig. 12), omphalocele, polyhydramnios, choroids plexus cyst, and renal calyceal dilation.

TABLE 9. Components of a Comprehensive (Level II) Ultrasound

THREE-DIMENSIONAL ULTRASOUND

Three-dimensional ultrasound is currently investigational. It is most commonly used at tertiary care centers and is commercially available for patients to obtain a keepsake image of their unborn child. Potential advantages include the ability to visualize fetal anatomy better and possibly change a patient's diagnosis through improved visibility (Figs. 13 and 14). No confirmed adverse biologic effects on patients or instrument operators caused by exposure have been demonstrated.³²

MAGNETIC RESONANCE IMAGING

Magnetic resonance imaging (MRI) is currently under investigation for use in prenatal diagnosis. Advantages of MRI over ultrasound include excellent tissue contrast, a large field of view, and relative operator independence.³³ Table 10 lists the indications of fetal MRI. One of the most successful areas has been in the evaluation on the brain and central nervous system.

TABLE 10. Indications of Fetal Magnetic Resonance Imaging

GENETIC COUNSELING/PRECONCEPTION COUNSELING

Genetic counselors serve as the link between the medical communities' increasing knowledge of genetics and a patient's understanding of genetic risk. A genetic counselor helps a health care provider and a patient understand the risks associated with birth defects and hereditary disorders through interpreting family history, laboratory results, and other medical information. Often times, a couple proceeds no further with diagnostic testing after receiving a risk assessment. Ideally, the couple planning pregnancy meets with a health care provider prior to pregnancy in order to assess risk.

Decisions at this time are as difficult as those after diagnosis of a live-born child with a genetic handicap, and similar psychologic reactions can occur.³⁴ After abortion of an affected fetus, both parents—especially the mother—may also require supportive psychological counseling.³⁵

Properly applied, prenatal diagnosis with attendant genetic counseling can be a powerful preventive medical tool. In the more personal sense, it frequently allows at-risk couples to have healthy children when they might otherwise forfeit the opportunity.

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