The procedures used for prenatal diagnosis may be classified as invasive and noninvasive. The invasive procedures include amniocentesis, CVS, fetoscopy, and fetal biopsy. These procedures are performed to obtain cells or cell-free amniotic fluid. The common noninvasive procedure, ultrasonography, is especially valuable for the intrauterine diagnosis of a variety of congenital anomalies not associated with known chromosomal or metabolic defects.
Amniocentesis is the most common means of obtaining amniotic fluid and fetal cells for analysis of a variety of genetic disorders: chromosomal, metabolic, neural tube defects (NTDs), and those detectable by DNA studies. Transabdominal needle aspiration under ultrasonographic guidance can provide 20 to 40 mL of amniotic fluid for cell culture and cell-free fluid. Traditionally, the procedure is performed at 15 to 20 weeks' gestation. At this stage, sufficient amniotic fluid is present and the ratio of viable to nonviable cells is greatest. Because only a small percentage of amniotic fluid cells are viable, and the different cell types grow at different rates, the viable amniotic fluid cells must be cultured to provide sufficient quantities of actively dividing cells for analysis.
The safety, accuracy, and efficacy of amniocentesis has been studied in the United States and Europe (1,2,3,4,5 and 6). These studies concluded that the procedure is relatively safe and complications such as fetal injury and bacterial infection are extremely infrequent. The U.S. study documented that rates of fetal loss in amniocentesis and nonrandom controls were not significantly different (3.5% versus 3.2%) (3). Amniocentesis at an earlier stage of pregnancy was reported by several groups (7,8 and 9). According to those studies, amniocentesis can be performed at 9 to 14 weeks, when 1 mL of amniotic fluid per gestational week can be withdrawn. Early amniocentesis may provide an alternative for CVS when passage through the cervical canal is not advisable because of cervical-vaginal infections or myomas. It also has an advantage over CVS in that it allows the determination of a-fetoprotein (AFP), which is important for the diagnosis of NTDs, ventral wall defects, and other malformations associated with elevated amniotic fluid a-fetoprotein (AFAFP). However, the safety of early amniocentesis has been debated. Shulman and colleagues (10) and Brumfield and colleagues (11) reported a greater number of complications and pregnancy loss with early amniocentesis when compared with transabdominal CVS and second-trimester amniocentesis. Others also questioned the accuracy of cytogenetic analysis with an increased number of cases of pseudomosaicism (9.9%) and structural anomalies reported (2.8%) (12).
First-trimester genetic diagnosis by CVS has been developed to overcome the need to wait until the 16th week of pregnancy for prenatal testing. The introduction of CVS permitted the diagnosis of numerous genetic disorders by the use of DNA from fetal tissue samples in the first trimester and reduced the maternal risk when pregnancy termination was chosen. It also made prenatal diagnosis feasible for those ethnic groups in which religious and social objections to mid-trimester amniocentesis made prenatal diagnosis unacceptable, even for families at high risk.
Chorionic villus sampling can be performed by a transcervical or transabdominal approach. Both techniques involve aspiration of chorionic villi under ultrasonographic guidance (13,14 and 15). A single good aspiration could yield 10 to 25 mg of wet tissue, which provides abundant material for cytogenetic, enzymatic, and DNA analysis (16,17,18 and 19). Transcervical CVS was first attempted by Hahnemann and Mohr in 1968 (20). Their studies suggested that the 9th to 11th weeks of gestation are the most suitable for obtaining placental biopsy samples.
Chorionic villus sampling is currently performed in an increasing number of specialized centers in the United States and Europe. An international registry, a CVS newsletter, World Health Organization (WHO) sponsorship, and other clinical studies have made it possible to evaluate CVS as a standard fetal-diagnostic approach (21,22,2.3,2.4,25,2.6,27 and 28). As with amniocentesis, the main considerations were whether the procedure is safe for the patient, the fetus, and continuation of the pregnancy, as well as how accurately the chorionic villi represent the fetal condition. Most published reports have demonstrated that first-trimester diagnosis by CVS is safe and reliable enough to be offered in specialized centers. Comparisons of the transcervical and transabdominal approaches showed no difference in rates of pregnancy loss between the two techniques (2.5% versus 2.3%) (29). Furthermore, a prospective randomized study by Smidt-Jensen and colleagues showed no difference in pregnancy loss between transabdominal CVS and second-trimester amniocentesis (30). In 1991, a report by Firth and colleagues (31) identified five cases of severe limb abnormalities in children whose mothers had CVS at 56 to 66 days of gestation. This report was followed by several others, which raised the concern that previous studies might have missed this specific malformation (32). To address this concern, in 1992 the WHO initiated an international registry of post-CVS limb defects. The analysis of data from 138,996 pregnancies has indicated no increased risk of limb defects after CVS compared with the incidence in the general population (33).
Fetal Blood Sampling, Fetoscopy, and Fetal Biopsy
Prior to the availability of direct DNA analysis, fetal blood sampling was the only method by which sickle cell anemia, thalassemia, and hemophilia A could be diagnosed prenatally (34). At present, fetal blood sampling does not require fetoscopy. It is performed by ultrasound-directed percutaneous umbilical cord blood sampling (PUBS) at 18 to 20 weeks. Fetal blood sampling can help distinguish most cases of true mosaicism from pseudomosaicism in amniotic fluid cultures. It also can be used when workup is initiated late in the second trimester and there is a need for rapid karyotyping, or in twin pregnancies (with a single amniotic sac), where it may be especially important when one twin is found to be abnormal by ultrasonographic evaluation (35,36).
Fetoscopic visualization has facilitated sampling of fetal skin and liver for diagnoses that could not be made by cytogenetic or biochemical analysis of cultured amniotic cells or amniotic fluid (37,38 and 39). It is known to cause complications to both fetus and mother and carries a high rate of sampling failure. Even experienced fetoscopists have reported a 5% risk of spontaneous abortion within 48 hours of the procedure and a 2% to 4% risk for prematurity. Amniotic fluid leakage occurs in about 4% of cases.
The introduction of DNA technology and direct gene analysis for prenatal diagnosis has significantly decreased the need for fetoscopic sampling. Fetal liver biopsies were used in the past for the diagnosis of disorders resulting from the deficiency of liver-specific enzymes such as ornithine transcarbamylase deficiency (OTC), phenylketonuria (PKU), and glycogen storage disease type I (Von Gierke's disease) (40,41). Most of these disorders can now be diagnosed by DNA techniques if the specific mutation is known. Approximately 90 different mutations associated with OTC deficiency have been identified (42,43), and an even larger number of mutations are known for PKU (44,45 and 46). Some of the mutations seem to be recurrent, but the majority are private mutations; therefore, family studies of the affected and carrier status of the parents are essential before prenatal diagnosis is attempted. Once a mutation is identified, prenatal diagnosis can be offered.
At present, fetoscopy is limited to prenatal diagnosis of severe congenital autosomal-recessive forms of epidermolysis bullosa and ichthyosis, which require morphologic examination of fetal skin (4.7,4.8,4.9 and 50). Bakharev and colleagues (51) have reported that fetal skin biopsies can be successfully obtained by an endoscopic needle introduced transabdominally under ultrasonographic guidance. This procedure might reduce the unfavorable outcome in pregnancies at risk for the various genodermatoses as well.
State-of-the-art real-time ultrasound scanners provide detailed dynamic images of the fetus. In a routine level I examination, when no fetal abnormality is suspected, gestational age is assessed by measurement of the biparietal diameter and femur length. Confirmation of multiple gestations, assessment of the quantity of amniotic fluid, and localization of the placenta are also performed. A discrepancy in any one of these parameters or the detection of a gross fetal abnormality are indications for additional diagnostic evaluation.
Level II scans are performed to evaluate pregnancies in which an abnormality is suspected or prenatal diagnosis is indicated. High-resolution ultrasonography has led to the rapid expansion of the number of diagnoses that can be made by indirect visual evaluation of the fetal anatomy. Abnormalities such as small facial clefts, polydactyly, and defects in the fetal spine can be visualized (52). Disproportionate growth can be readily assessed for the prenatal diagnosis of skeletal dysplasias (53). Filling and emptying of the fetal bladder in response to maternal diuretic ingestion can be used to assess the functional integrity of the fetal urinary tract. The need for amniocentesis has been the subject of ongoing debate when choroid plexus cysts are seen on ultrasonographic examination because of the association with trisomy (54). Generally, the finding of more than one anomaly increases the risk of a chromosome abnormality, in which case amniocentesis should be offered for cytogenetic studies.
Fetal gender can be determined by about 20 menstrual weeks. In cases in which sex determination is performed for diagnostic reasons (e.g., prenatal diagnosis of X-linked disorders), ultrasonographic determination of sex is suggestive but not conclusive. Karyotype confirmation of fetal gender must be performed in these cases.
The simultaneous use of ultrasonography with other prenatal diagnostic techniques has reduced the associated risk and facilitated the performance of these procedures. Ultrasonographic localization of the placenta, fetus, and amniotic fluid pool prior to amniocentesis has reduced the number of bloody taps. It has also made the assessment of twin pregnancies by amniocentesis feasible. When fetoscopy is performed, ultrasonographic localization of the placenta, cord, and head significantly shortens the time of the procedure and improves fetal tissue sampling. Ultrasonography is also used for visual guidance during chorionic villus biopsy.
Fetal echocardiography has been successful in diagnosing congenital heart disease and dysrhythmias (55). Both M-mode and real-time echocardiograms may be necessary for adequate interpretation of cardiac anatomy (56). Serial echocardiography is performed at mid-gestation on fetuses with a family history of congenital heart disease as well as for the evaluation of fetal ascites and dysrhythmias appreciated by auscultation of fetal heart tones. The association of maternal nongestational diabetes and congenital heart disease makes this group of high-risk patients candidates for fetal echocardiography. Echocardiography also should be performed as part of a complete diagnostic evaluation of fetuses found to have a major structural anomaly, such as an omphalocele. The finding of additional malformations may alert physicians to the possibility of a chromosomal anomaly and optimize intrapartum decision making. The rapid development of high-resolution ultrasonography has made other forms of radiologic imaging virtually obsolete. Amniography, using water-soluble dye to outline the fetus, and fetography, which uses a fat-soluble dye that is miscible with the vernix caseosa, have been replaced by safer, noninvasive ultrasonography.
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