PRENATAL DIAGNOSTIC TESTS	Section 5 of 9
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Diagnosis-directed tests are invasive and pose some risk to the mother and fetus, but they directly analyze the fetal material and confirm the diagnosis. Special circumstances indicate the use of these tests, which are not screening tests.

Indications for Diagnostic Tests

Conditions that increase the risk of chromosomal anomaly include the following:

• Advanced maternal age (>35 y), the most common indication

• Previous offspring with chromosomal anomalies or other birth defects

• Parental balanced translocation, inversion (manifests as recurrent pregnancy loss), or both

• Suggestive fetal ultrasonographic findings

• Positive maternal screening test findings

• Mother having a disease or being exposed to drugs, medications, or infections known to be associated with congenital malformations in the fetus

• Mendelian genetic trait in the parents

• Molecular DNA diagnosis (cystic fibrosis, homozygous hemoglobin sickle disease [HbSS], fragile X)

• Enzymatic activities in villi, amniocytes, or both (Tay-Sachs disease, Refsum disease)

• Precursor levels in cell-free amniotic fluid (17-OH-progesterone in congenital adrenal hyperplasia)

Genetic counseling

As a prerequisite and as follow-up to prenatal diagnosis, families must be informed about the diagnosis, severity, prognosis, and available options for treatment and continuation of pregnancy.

Diagnostic Tests in the First Trimester

Chorionic villous sampling

Chorionic villous sampling (CVS) is the technique of choice for prenatal diagnosis prior to 12 weeks' gestation for detection of a chromosomal anomaly, DNA molecular diagnosis of classic genetic disorders, and the detection of defects in lysosomal enzymes or mucopolysaccharidoses. A diagnosis of enzymatic defects, such as 21-hydroxylase deficiency that causes congenital adrenal hyperplasia, can be made with an allele-specific amplification analysis technique of DNA obtained with CVS.

A preliminary ultrasound is performed to establish fetal viability, gestation, and anatomy and to determine the placental location. A sample of placental tissue is obtained using a 16-gauge polyethylene catheter for analysis under ultrasonographic guidance. The test is usually performed at 8-12 weeks' gestation.

The approach is based on the placental location. A transabdominal approach is preferred for anterior and fundal placentas after 13 weeks' gestation and in active vaginal and cervical infections. The sample is smaller than that obtained with the transcervical method. The transcervical approach is indicated in cases with interposed bowel loops or uterine retroversion and a posterior or low-lying placenta. The transvaginal approach has limited application and is used when the placenta is placed posteriorly, the uterus is retroverted and retroflexed, and the cervical canal points towards the abdomen.

Chromosomal analysis of the sample is performed in 2 ways. The direct method evaluates the metaphysis from the outer layer of cytotrophoblasts in the chorionic villi. This method provides results in 2 days. A long-term culture of the inner mesenchymal layer of trophoblasts provides results in 10-14 days. These results more closely correlate with the true karyotype.

An abnormal direct result has to be confirmed with long-term cultures of trophoblasts, amniotic cells, or fetal lymphocytes. Rarely, a normal direct result is followed by abnormal culture findings, which are confirmed with fetal tissue results. Chromosomal mosaicism occurs in 1.2-2.5% of the samples and may cause diagnostic error. The mosaicism is purely extraembryonic in 70-80% of the cases, and it is more common in direct preparations. If found in both direct preparation and long-term cultures, a follow-up ultrasonographic level 2 screening for anomalies and amniocentesis or cordocentesis are indicated to verify mosaicism in the fetal blood. Maternal cell contamination may also distort the results.

Complications associated with CVS include the following:

• Pregnancy loss is 0.6-0.8% more common than the natural pregnancy loss rate in the first trimester and more common that the rate in midtrimester amniocentesis. Limb reduction defects and oromandibular malformations are more likely in some studies, especially if the procedure is performed prior to 10 weeks' gestation.

• Fetomaternal transfusion can occur regardless of the approach used. Thus, Rh isoimmunization is considered a relative contraindication, and Rh immunoprophylaxis is administered to Rh-negative women after the procedure.

Early amniocentesis

This technique, performed at 12-14 weeks' gestation, assists in the diagnosis of chromosomal anomalies by providing fetal cells for karyotyping. It can also lead to the diagnosis of structural anomalies such as neural tube defects and omphalocele through the measurement of AFP and acetylcholinesterase levels. It is preferred to CVS in situations in which CVS is not reliable, such as twin pregnancy with fused placentae and in certain biochemical disorders.

The procedure consists of the aspiration of amniotic fluid (approximately 1 mL/wk of gestation) from an amniotic fluid pocket with a 22-gauge needle and ultrasonographic guidance.

Complications associated with amniocentesis include uterine bleeding (1.9%), uterine cramping, leakage of amniotic fluid (2.9%), pregnancy loss (1.4-4.2%), increased risk of club foot when performed prior to 12 weeks' gestation, failed procedure due to tenting of the membranes ahead of the needle, and culture failure rates of 1% overall and 5% if the procedure is performed prior to 12 weeks' gestation. Pseudomosaicism and maternal contamination are less likely than with CVS.

Preimplantation biopsy

This controversial procedure is performed for preimplantation diagnosis in a fetus of parents with substantial risk of a known genetic disorder and in women with repeated miscarriages due to chromosomal translocation.

At the 8-cell stage of the embryo, a single cell is removed and analyzed (blastomere biopsy) for X-linked recessive diseases. Only XX embryos are transferred following in vitro fertilization. More trophectodermal cells can be removed from the blastocyst for analysis. Because it has the same genetic constitution as the ovum, the second polar body can also be analyzed for diseases with known gene defects such as cystic fibrosis, hemophilia, and a1-antitrypsin deficiency.

Coelocentesis

Coelocentesis, defined as coelomic fluid aspiration, can be performed as early as 6-10 weeks' gestation but is still considered investigational because of the high rate of pregnancy loss reported.

Fetal cells from maternal blood

The isolation and analysis of nucleated fetal cells from maternal blood is a novel noninvasive technique of prenatal diagnosis of chromosomal aneuploidies. A direct micromanipulator isolation of histochemically identified fetal hemoglobin (HbF)–positive nucleated red cells is followed by fluorescent in situ hybridization (FISH) analysis for chromosomal aneuploidies. The results are obtained in 72 hours and correlate highly with amniocentesis results.

Diagnostic Tests in the Second Trimester

Midtrimester amniocentesis

Amniocentesis performed at 16-18 weeks' gestation is the criterion standard of prenatal diagnostic techniques in both efficacy and safety. It is offered to all women older than 35 years or with an elevated serum AFP level. Genetic counseling to evaluate genetic risks and detailed ultrasonography to estimate gestation, placental location, and amount of amniotic fluid are important prior to the procedure.

The procedure is the same as that of amniocentesis in early gestation, except that 20-30 mL of amniotic fluid is aspirated for analysis.

The amniotic fluid phase can be analyzed for several substances.

• AFP and acetylcholinesterase levels are used to identify a fetus with a neural tube defect, with 98% sensitivity.

• Bilirubin levels in amniotic fluid are elevated in isoimmune hemolysis, and the risk to the fetus can be predicted based on the bilirubin level and gestational age.

• Pulmonary surfactant and surface-active phospholipids are measured in amniotic fluid to evaluate fetal pulmonary maturity. A lecithin-to-sphingomyelin ratio of greater than 2, as measured chromatographically in a noncontaminated sample, suggests lung maturity, except in fetuses of mothers with diabetes. Other fetal lung maturity assays measure the surfactant-to-albumin ratio with fluorescent polarization technology and provide early results. Both of these tests are affected by contamination with blood or meconium, and results are unreliable in fetuses of mothers with diabetes or preeclampsia or in cases of intrauterine asphyxia. The presence of phosphatidyl glycerol in amniotic fluid indicates lung maturity, particularly in fetuses of mothers with diabetes. Saturated phosphatidylcholine is unaffected by contamination with blood. A combination of these tests provides a more accurate indication of lung maturity.

• Enzyme analysis and measurement of metabolite levels, used for the diagnosis of urea cycle disorders and of other inborn errors of metabolism, may also be performed on the amniotic fluid at this stage.

Fetal cells can be extracted from amniotic fluid samples and analyzed for the following chromosomal and genetic disorders:

• Chromosome analysis through direct metaphase visualization is the traditional method; results are obtained in 1-2 weeks. FISH, when used in addition to standard cytogenetics, can analyze fetal cells for abnormalities in chromosomes 21, 18, 13, X, and Y and provide results in 48-72 hours. FISH can also detect microdeletions found in Prader-Willi, DiGeorge, Williams, and Angelman syndromes.

• Direct DNA analysis is done with polymerase chain reaction (PCR) gene amplification, followed by Southern blot analysis to detect gene deletions. Allele-specific oligonucleotide (ASO) analysis measures the specific binding of labeled probes to normal DNA or mutant sequences to detect gene mutations. This technique is important in identifying disorders in which multiple mutations have to be screened for, such as cystic fibrosis and thalassemia, or in which a restriction site is not created, such as Duchenne muscular dystrophy, Tay-Sachs disease, and phenylketonuria.

• Indirect DNA methods, such as linkage analysis with restriction fragment length polymorphisms (RFLPs), are performed in affected individuals and multiple other family members. This can help in making the diagnosis of diseases in which the exact gene defect and location are not known. Cross-over changes between the gene and the RFLP probe can distort the results. Survival motor neuron (SMN) analysis for gene deletion in the family of an affected patient is useful in the prenatal diagnosis of spinal muscular atrophy. Molecular analysis of the fibroblast growth factor receptor 3 gene with direct and restriction enzyme analysis can help to diagnose thanatophoric dysplasia. Fetal DNA obtained by amniocentesis can be analyzed for the same deletion as that of the index case.

Complications of second trimester amniocentesis include the following:

• The risk of pregnancy loss is 0.3-1%.

• The risk of amniotic fluid leakage is 1-2%. In rare cases, this may lead to oligohydramnios, arthrogryposis, and pulmonary hypoplasia.

• Amnionitis occurs in 0.1%.

• As the risk of Rh isoimmunization is increased 1% above the baseline risk, Rh immunoprophylaxis is recommended for Rh-negative nonsensitized women.

• Mosaicism on cytogenetic analysis is seen in 1% of samples. True fetal mosaicism is relatively rare, and fetal blood sampling is required for confirmation.

Percutaneous umbilical blood sampling or cordocentesis

The greatest advantage of this technique is that it provides a direct fetal sample and access to the fetus for in utero treatment.

With ultrasound guidance, a sample of fetal blood is obtained from the umbilical vessel close to the cord insertion near the placenta. A 20- to 27-gauge needle is used, and the approach can be transplacental in an anterior placenta or transamniotic in a posterior placenta.

Diagnostic studies that can be performed include the following:

• Direct karyotyping of fetal lymphocytes can provide results within 24-72 hours.

• DNA studies can be used to diagnose metabolic diseases.

• Hematologic problems are especially amenable to this technique. The diagnoses of thalassemias, sickle cell disease, hemophilia, von Willebrand disease, and alloimmune thrombocytopenia can be made. In Rh-isoimmunized fetuses, diagnosis and treatment of fetal anemia and thrombocytopenia is possible, as is intrauterine transfusion.

• Immunologic tests can be performed on blood samples for diagnosis of fetal infections (eg, specific immunoglobulin M [IgM] for toxoplasmosis, rubella, cytomegalovirus [CMV], varicella zoster, HIV). Viral DNA can be detected using PCR for certain infections, such as parvovirus B19.

• Determine fetal PaO₂, PCO₂, and pH levels, as these can provide critical indicators of fetal well-being in a small-for-date or compromised fetus. These data can guide management decisions.

Complications associated with cordocentesis are more common in posterior placentae and when the procedure is performed prior to 19 weeks' gestation. These include fetal loss (1-2.3%), preterm labor (5-9%), hematoma of the umbilical cord and placental abruption, chorioamnionitis (0.6%), fetal exsanguination from the procedure site, and Rh isoimmunization. Rh immunoprophylaxis is mandatory in all Rh-negative nonsensitized women after the procedure.

Late chorionic villus sampling

The technique of placental biopsy is equally effective in the second and third trimesters, and karyotyping is possible with small amounts of placental tissue. It has the advantage of being as accurate as amniocentesis, and it provides rapid results.

Fetal muscle and liver biopsy

Muscle biopsy is used in rare cases of Duchenne muscular dystrophy in which findings from all previous investigations are nondiagnostic. Dystrophin levels are measured in myoblasts by in situ hybridization. Fetal liver biopsies have also been performed to measure enzyme levels of glucose-6-phosphatase and ornithine transcarbamylase in patients with suspected glycogenesis and urea cycle disorders when direct DNA techniques are not sensitive.

Diagnostic Tests in the Third Trimester and During Labor

The purpose of prenatal diagnosis in the third trimester is to confirm fetal growth, well-being, and lung maturity. For timely and appropriate intervention, fetal well-being needs to be assessed in the third trimester and particularly in labor. Fetal movement is monitored based on maternal perception. Lack of fetal movement for longer than 30 minutes suggests possible fetal compromise. Fetal lung maturity is determined as discussed previously in case of an impending preterm delivery and in making the decision to induce labor for any indication.

Nonstress test

Nonstress testing is a simple low-risk procedure in which the fetal heart rate is monitored with Doppler ultrasonography or electrodes on the maternal abdomen or a fetal scalp electrode placed after rupture of membranes, along with the simultaneous recording of uterine activity with a tocodynamometer. After 32 weeks' gestation, the fetus responds to uterine contractions with tachycardia. The criteria for reactive test results are the following:

• Heart rate of 120-160 beats per minute (bpm): Fetal tachycardia may be due to fever, drugs, or fetal arrhythmias or hypoxemia.

• Normal beat-to-beat variability of more than 5 bpm: Decreased beat-to-beat variability suggests fetal hypoxia, sleep, prematurity, maternal sedation, or narcotic use.

• Two accelerations of more than 15 bpm lasting more than 20 seconds each within a 15-minute test period: A reactive test is reassuring, with a high chance of intrauterine survival over the next 7 days. A nonreactive test, which does not meet these criteria, necessitates further testing for confirmation. The disadvantage of the test is variable reproducibility; nonreactivity may be a late sign of fetal hypoxia, a benign pattern, or the result of a prior asphyxial event.

Contraction stress test

The contraction stress test (CST) is used to monitor fetal heart rate in response to uterine contractions that are spontaneous or induced with oxytocin. The contraction should occur within 30 minutes and last 40-60 seconds with a frequency of 3 in 10 minutes. In a healthy fetus, uterine contractions cause transient hypoxia and hypoperfusion of the intervillous space, which is relatively well tolerated. Early decelerations start with the onset of uterine contractions, reach the nadir at the time of peak of the contraction, and end simultaneously. These are benign and are seen in late labor from fetal head compression.

Variable decelerations vary in their timing and relation with uterine contractions and occur in response to cord compression. They are benign unless they are associated with severe or prolonged bradycardia, are less than 60 bpm, last more than 60 seconds, are associated with an overshoot acceleration lasting more than 1 minute after a variable deceleration, or have poor beat-to-beat variability. Under conditions of uteroplacental insufficiency, a late deceleration is induced. A late deceleration begins 10-30 seconds after the onset of uterine contraction, the nadir is later than the peak of the contraction, and it returns to baseline after the contraction ceases.

A CST result is positive if late decelerations are present with 50% or more of contractions. A CST finding is equivocal if decelerations are inconsistent. A negative CST result, defined as the absence of late decelerations, is associated with a risk of fetal demise of 0.4 cases per 1000 within the week. Drawbacks of the test are its duration, which is approximately 90 minutes, and the need for oxytocin.

Biophysical profile test

The biophysical profile (BPP) combines the nonstress test with an assessment of amniotic fluid volume, fetal breathing movements, fetal activity, and fetal muscle tone.

A score of 0-2 is given for each parameter. In a reactive nonstress test, each of the following criteria earns 2 points:

• At least one pocket of amniotic fluid greater than or equal to 1 cm depth

• At least one episode of fetal breathing of 60 seconds duration within 30 minutes

• Three or more discrete episodes of fetal movement

• At least one episode of extension and flexion of extremities or spine

Scores greater than 8 indicate a low risk and the need for weekly retesting. A score of 2 is strongly suggestive of hypoxia and indicates the need for immediate delivery (if persistent for 120 min). Intermediate scores need further evaluation. Maternal depressant medication and cerebral or neuromuscular anomalies may result in a low score.

Doppler study

A Doppler study of fetal umbilical arterial blood flow velocity or resistance to flow is another modality used to assess placental function, particularly to monitor high-risk fetuses. Decreased flow velocity during diastole indicates placental insufficiency, and, in severe cases, diastolic flow may stop completely or even reverse. Therefore, a systolic-to-diastolic umbilical blood flow ratio of greater than 3 after 30 weeks' gestation is associated with fetal compromise.

Researchers are still investigating the utility of measuring fetal arterial velocity in assessing redistribution in the hypoxic fetus and as indicators of placental circulation in pathologic placental processes, such as pregnancy-induced hypertension.

Ultrasonography is the single most valuable modality in the identification of fetal structural anomalies. It is also useful in the detection of abnormal growth patterns in the fetus, in estimating gestation, and in assessing fetal well-being in the third trimester and during labor and delivery. It is important in guiding the operator during procedures such as amniocentesis and cordocentesis.

Ultrasonography is widely available and has no known adverse effects. Newer techniques, including high-resolution multiplanar imaging and Doppler imaging, have improved its yield. Disadvantages are beam attenuation with maternal adipose tissue and poor images with an engaged fetal head or oligohydramnios.

Fetal gestation

• Gestation is best estimated in the first trimester, with an error range of 3-5 days. The range increases to 1 week at 12 weeks' gestation and 3 weeks at 36 weeks' gestation.

• In the first trimester, the crown-rump length is the most accurate measure of gestation. This is measured from the top of the head to the bottom of the torso or the longest dimension of the fetus excluding the yolk sac and extremities.

• In the second and third trimesters, parameters used to estimate gestation are the biparietal diameter (BPD), head circumference, abdominal circumference, and femur lengths.
  • BPD is measured on the transaxial view of the head from the outer edge of the cranium nearest the transducer to the inner edge of the cranium farthest from the transducer. BPD, which is measured at the level of the thalami, including the cavum septum pellucidum, should not be used in cases of hydrocephalus, abnormal head shape, or late in the third trimester when the head may be engaged. Measures such as a corrected BPD have been devised to take into account differences in head shape.

  • Abdominal circumference is the length of the outer perimeter of the fetal abdomen measured at the level of the stomach and intrahepatic umbilical vein on a transverse scan. This measure should not be used in cases of fetal growth abnormality in which the head size may be relatively preserved or in a fetus with diaphragmatic hernia.

  • In comparison, femoral length is more affected by caliper placement and technically more difficult. Femoral length may be affected by skeletal dysplasias, Down syndrome, and fetal growth abnormalities. Only the length of the diaphysis is measured for femur length.

  • A combination of these measurements yields the most accurate results.

Abnormal fetal growth patterns

• Intrauterine growth retardation (IUGR): Serial ultrasonography can be used to monitor the rate of increase in fetal biparietal diameter, abdominal circumference, and femoral length, thus helping to identify a growth-restricted fetus.
  • In the third trimester, ratios of morphometric measures such as abdominal circumference and femoral length are used to diagnose growth retardation.

  • Oligohydramnios and a poor biophysical score support the diagnosis of growth retardation secondary to uteroplacental insufficiency. Oligohydramnios is defined as the absence of amniotic fluid pockets or the presence of an amniotic fluid index (AFI; sum of the vertical distance of the largest pocket in each of 4 equal uterine quadrants) of less than 5.

  • Estimated fetal weights, derived by combining several parameters (usually head, abdominal, and femur measurements), are useful. However, they are inaccurate at the extremes of birth weight. Symmetric growth retardation begins earlier in gestation, affects both head and abdominal measurements, and is caused by chromosomal or genetic anomalies or intrauterine infections.

• Macrosomia: Serial ultrasonography can be used to measure the ratio of abdominal circumference to head circumference to detect macrosomia.

Detection of Fetal Anomalies

Fetal CNS anomalies

• Ultrasound is 95% sensitive in the diagnosis of hydrocephalus and myelomeningocele.

• Ventriculomegaly has been defined in some studies by a measurement at the atrium of the lateral ventricle of more than 10 mm at any time during pregnancy.

• In myelomeningocele, diagnosis is made by noting a divergence of the pedicles of the vertebrae or the presence of a fluid-filled sac. Some intracranial signs are associated, such as ventriculomegaly, small BPD, biconcave frontal bones at 18-24 weeks, a distorted position of the cerebellum, and obliteration of the cisterna magna, especially if associated with Chiari II malformation.

• Diagnosis of anencephaly, encephalocele, craniosynostosis, and brain malformations, such as porencephaly, can be made based on ultrasound findings. Fetuses with hydrocephalus or meningomyelocele should be evaluated for chromosomal abnormalities or anatomic defects in the cardiac, renal, and skeletal systems. Associated defects are present in 90-95% of cases.

Fetal chest abnormalities

• Pulmonary hypoplasia, pleural effusions, cystic adenomatoid malformations, sequestration, and bronchogenic cysts are all pulmonary lesions that can be diagnosed based on ultrasound findings.

• Congenital diaphragmatic hernia (CDH): Diagnosis of CDH is made based on the presence of bowel or liver in the thorax with the accompanying blood supply, mediastinal shift, and pulmonary hypoplasia. Polyhydramnios may be present. After the diagnosis, serial ultrasonography should be performed to monitor fetal growth and hydramnios and to evaluate for cardiac anomalies. Workup may include ultrafast MRI, echocardiography, and karyotyping with amniocentesis to exclude associated anomalies. Hydrops is a predictor of a poor outcome.

Fetal cardiac abnormalities

• Detailed cardiac ultrasonography is indicated in fetuses with the following:
  • Chromosomal anomalies

  • Hydrops

  • Oligohydramnios or polyhydramnios

  • Exomphalos

  • Diaphragmatic hernia

  • Defects in other systems with known cardiac associations.

• Other indications for a cardiac echo antenatally include the following:
  • Family history of congenital heart defect

  • Maternal diabetes or systemic lupus erythematosus (SLE)

  • Maternal lithium, alcohol, or progesterone intake

  • Fetal arrhythmias

• M-mode echocardiography is used to measure chamber size, cardiac rhythm, pericardial effusions, and wall thickness and motion. Cross-sectional echocardiograms show the heart position and situs and the atrioventricular (AV) connections. Doppler echocardiograms show the direction and pattern of blood flow, and they can depict valvular regurgitation or stenotic lesions.

• Prenatally diagnosed heart disease has been associated with reduced early neurologic morbidity in certain lesions, such as a hypoplastic left heart. Conversely, whereas a poorer prognosis was reported earlier in prenatal cohorts because more severe lesions are more likely to be detected, especially when they are associated with a structural or chromosomal defect.

Fetal gastrointestinal anomalies

Gastroschisis and omphalocele are easily detected on ultrasound. Ultrasonography has low sensitivity in the diagnosis of obstruction, which is indirectly indicated by the presence of polyhydramnios, poorly visualized gut distal to the obstruction, and a fluid-filled portion proximal to it. An echogenic bowel, meconium peritonitis, and pseudocyst formation are suggestive of cystic fibrosis. All of these findings indicate the need for further cytogenetic evaluation of the fetus.

Fetal genitourinary tract anomalies

Ultrasound can detect renal agenesis, cystic disease, obstructive lesions (uteropelvic junction obstruction and posterior urethral valves), and renal tumors. Renal dimensions, parenchymal thinning and cysts, ratios of renal circumference to abdominal circumference, pelvic diameters, and urinary ascites can be assessed, along with urethral and bladder anatomy. The amniotic fluid volume provides an indication of renal function. Oligohydramnios is associated with a poor prognosis.

Fetal skeletal anomalies

A detailed fetal ultrasound attempts to rule out skeletal dysplasias, achondroplasia, osteogenesis imperfecta, polydactyly, and absence of a bone. Long bones are evaluated for size, shape, symmetry, and proportions of the different segments, and the skull is evaluated for shape and deformity. Examination of the spine and ribs helps in the delineation of the disorder. Identification of a skeletal dysplasia and prognosis are relatively accurate; however, in one study, a specific antenatal diagnosis was made in 60% of cases, but these were incorrect in 19% postnatally. According to some reports, 3-dimensional ultrasonography seems to provide additional visualization of skeletal deformities and abnormal spatial relationships, such as short ribs and absent bones, and to enable specific diagnosis.

Fetal chromosomal anomalies

In a recent meta-analysis, nuchal thickening was found to be the most accurate marker in the second trimester. It was associated with a 17-fold increased risk of Down syndrome.

Nuchal thickening in the first trimester has a sensitivity of 60-70% for the detection of Down syndrome, with a 5% false-positive rate, whereas ultrasonography and biochemical screening, in combination, improve the sensitivity to 80%. Other single subtle markers (eg, choroid plexus cysts, shortened long bones, echogenic bowel) are not sensitive.

MRI Examination

MRI is an important adjunct to ultrasonography. It is used mainly in the assessment of cases with equivocal ultrasonographic findings or when prenatal ultrasound is not reliable in the identification of fetal anomalies, such as in the setting of maternal obesity or oligohydramnios. The advantage of the newer fast and ultrafast sequence MRI is that they have minimized motion artifacts; thus, sedation is not needed. A variety of sequences have been used, including echoplanar, half-Fourier single-shot turbo spin-echo (HASTE), and fast spin-echo sequences. Of these, HASTE has proven to be an excellent method of fetal imaging. A recent meta-analysis showed that ultrafast MRI in the third trimester provided additional information compared to ultrasound in fetal diagnosis in 23-100% of cases, particularly those involving the posterior fossa of the head.

Advantages of MRI include the following:

• Absence of ionizing radiation

• Multiplanar capability

• Large field of view

• Superior soft tissue contrast enhancement

• Good image quality in oligohydramnios

• More precise volumetric measurement

• Better intracranial delineation

Limitations of MRI include the following:

• Spatial resolution inferior to that of ultrasonography

• Poor depiction prior to 20 weeks' gestation

• Safety question - not approved by the Food and Drug Administration (FDA), but no known significant risk beyond the first trimester

MRI may be used if other nonionizing imaging modalities are inadequate or if ionizing radiation would otherwise be required for further evaluation.

Indications for MRI evaluation include the following:

• Fetal cerebral anomalies: MRI has been most successful in the identification of posterior fossa abnormalities, migrational anomalies (eg, lissencephaly, polymicrogyrias, schizencephaly) at 30-32 weeks' gestation, agenesis of the corpus callosum, white matter disease, hydrocephalus, and ischemic or hemorrhagic lesions.

• Volumetric analysis: The size of the fetus or individual organs, such as the liver, can be determined. In CDH, volumetric measurements of the right fetal lung and the position of the left hepatic lobe appear to be good prognostic indicators and help in planning therapeutic interventions.

• Congenital high airway obstruction syndrome: MRI can be used to confirm upper airway obstruction by demonstrating hyperinflation of both lungs and dilated fluid-filled airways below the level of obstruction. Fetal neck masses such as cystic hygromas and teratomas can be identified, allowing early intervention when indicated.

• Congenital hemochromatosis: T2-weighted MRI can be used to confirm the diagnosis.

• Amniotic band syndrome: MRI provides an accurate diagnosis.

CT Scanning

CT scanning has limited applications in prenatal diagnosis. It is used mainly when MRI is contraindicated in the mother (eg, if she has a pacemaker, an intraocular metallic foreign body, or intracranial ferromagnetic surgical clips).

The advantage of CT scanning is that it better delineates fetal bony anatomy than other imaging modalities.

The limitations of CT scanning include possible teratogenesis due to ionizing radiation if it is performed in the first trimester and a risk of cancer induction. In children, a risk of mortality from cancer of 1 per 220-440 cases has been reported.

Indications for CT scanning include pelvimetry and CT amniography to confirm monoamnionicity if ultrasonography provides inconclusive data.

Fetal Magnetocardiography

Treatments for fetal disorders

• Neural tube defects
  • Perhaps the best known and most extensively studied pharmacologic intervention is folic acid. It has been proven to reduce the incidence of neural tube defects in women with one or more previously affected children and in women who have no risk factors.

  • All women are advised to take folic acid prior to conception (0.4 mg/d PO for 3 mo), and 4 mg/d is recommended for women with a previously affected child, beginning at least 1 month prior to conception through 3 months of pregnancy.

• Congenital adrenal hyperplasia
  • Since the differentiation of external genitalia begins at 7 weeks' gestation, the mothers of all fetuses at risk (those with a previously affected child) are given dexamethasone (0.25 mg PO qid) at 7-9 weeks.

  • Direct studies using probes or linkage DNA studies are performed with CVS. Affected female fetuses are identified at a rate of 1 case per 8 carrier parents.

  • Treatment is continued until term only in affected fetuses. Stress-dose glucocorticoids are administered at delivery and gradually tapered in the mother after delivery.

  • The long-term outcome of patients treated in utero is still being assessed. The treatment has proven effective in preventing masculinization.

• Thyrotoxicosis
  • Fetal thyrotoxicosis is usually seen in infants of mothers with Grave disease or autoimmune thyroiditis. The diagnosis is made with cordocentesis.

  • Maternal treatment with propylthiouracil (initial dose 300 mg/d PO, then titrate according to effect) or methimazole is associated with a good fetal outcome.

• Hypothyroidism
  • Fetal hypothyroidism is linked to maternal hyperthyroidism, use of radioactive iodine, drugs, and excessive maternal iodine intake.

  • Fetal status is evaluated at ultrasonography and by direct cordocentesis.

  • Intra-amniotic L-thyroxine (500 mcg q2wk initiated at 34 wk of gestation) has been shown to cause regression of fetal goiters and normalization of hormone levels.

• Methylmalonic acidemia
  • This is caused be a deficiency of methylmalonyl CoA mutase or its coenzyme adenosylcobalamin, which results in an accumulation of methylmalonic acid and its precursors in body fluids. Clinically, patients present in the first few weeks of life with poor feeding, vomiting, hypotonia, lethargy, dehydration, ketosis and acidosis.

  • Prenatal cyanocobalamin has been empirically administered orally to the mother at a dose titrated to achieve high maternal plasma B12 levels and normal maternal urinary methylmalonic acid excretion.

  • The long-term effects on fetal development have not been studied extensively.

• Multiple carboxylase deficiency
  • This disorder is caused by a deficiency of holocarboxylase synthetase or biotinidase, two enzymes essential to rendering the carboxylases functional. The carboxylase enzymes are involved in the metabolic pathways of isoleucine, leucine, and valine.

  • Clinically, patients present in the first few weeks of life or later in childhood with hypotonia, seizures, vomiting, failure to thrive, dermatitis, developmental delay, hearing loss, and acidosis.

  • Maternal biotin supplementation may prevent neonatal complications.

• Lung maturity induction
  • In multiple studies, maternal corticosteroid therapy, used to induce lung maturity and surfactant synthesis in the fetus, has been proven effective in significantly reducing respiratory distress syndrome (RDS) in the neonatal period. Controlled studies have shown a reduction from 20.2% to 11.2%.

  • Betamethasone (12 mg IM q24h for 2 doses) or dexamethasone (6 mg IM q12h for 4 doses) is recommended for fetuses at 24-34 weeks' gestation who are at risk of preterm delivery.

• Maternal HIV infection: Maternal administration of zidovudine (AZT), started at 14 weeks' gestation, continued throughout pregnancy, and given intravenously during labor, followed by treatment of the neonate for the first 6 weeks, has been documented to decrease the rate of vertical transmission from 25% to 8%.

• Immune hydrops
  • Hemolytic disease of the fetus is a condition of fetal anemia caused by Rh isoimmunization. An antibody screen, performed periodically in a sensitized mother, can detect the presence and titer of maternal antibodies.

  • If the paternal screen for the antigen is positive, PCR can determine fetal blood group from samples obtained at amniocentesis or cordocentesis. Reverse transcriptase-polymerase chain reaction (RT-PCR) of fetal cells in maternal blood is also being used for the same purpose.

  • The fetuses at risk are then monitored with serial ultrasounds (for evidence of hydrops) and Doppler assessment of the velocity of blood flow in the middle cerebral artery (higher in anemic fetuses), starting at 16-18 weeks' gestation and repeated every 1-2 weeks until 35 weeks' gestation.

  • Alternatively, one can perform amniocentesis serially (10 day to 2 week intervals) with measurement of bilirubin, beginning at 18 weeks, and determine when the result is abnormal from the Queenan and Liley charts.

  • Serial cordocentesis is indicated for severely affected fetuses for direct measurement of hematocrit (Hct), reticulocyte count, and bilirubin.

  • Intrauterine transfusions can be performed as indicated based on the results of the diagnostic tests. Direct intravascular transfusions through umbilical vein puncture or a combination of intraperitoneal and intravascular transfusions can be used. The combination achieves a more stable Hct and delays the time to the next transfusion.

  • The volume in milliliters of intraperitoneal transfusion can be calculated as the (gestational age in weeks minus 20) X 10 and repeated at 2-week intervals until fetal erythropoiesis decreases.

  • Monitor this with Kleihauer Betke stains and size of fetal liver and spleen on ultrasound as indicators of extramedullary hematopoiesis. Repeat at 3- to 4-week intervals, with monitoring of the fetal Hct level.

  • The intravascular transfusion alone aims to achieve a fetal Hct of 35- 40%. The intraperitoneal transfusion provides a reservoir of blood and achieves a final Hct of 50-60%.

  • A fetus with hydrops requires careful transfusion until the Hct level reaches 25%. Transfusion is repeated in 2 days and then weekly to achieve the final Hct level.

  • O-negative, CMV-negative allogenic or maternal blood is tested for infection, washed, packed (Hct level, 75-85%), filtered, irradiated with 2500 Gy, and then transfused. Repeat transfusions are indicated until pulmonary maturity or a gestational age of 35 weeks is reached.

  • Postnatal "top-up" transfusions and erythropoietin, along with folic acid, may have a role in the first postnatal week.

• Fetal thrombocytopenia
  • Maternal thrombocytopenia has many causes, many of which do not place the fetus at risk of bleeding.

  • In idiopathic thrombocytopenic purpura, the fetus has a low risk for intracranial hemorrhage. Scalp electrodes, forceps, and vacuum are not used at the time of delivery. In early labor, a fetal platelet count may be obtained via cordocentesis or a scalp blood smear. If the count is less than 20,000, a cesarean delivery may be preferred.

  • Alloimmune thrombocytopenia is the most common type of platelet isoimmunization that occurs in a PLA1 Ag–negative mother, with an incidence of 1 per 5000. If a history of an affected sibling exists, the direct fetal platelet count is measured with blood sampling, and maternal platelets can be transfused with cordocentesis. Disadvantages are the short life span of the platelets, which makes frequent transfusions necessary, and the possibility of further sensitization. Maternal intravenous immunoglobulin G (IVIG; 1 g/kg/wk) coadministered with corticosteroids has been tried with some good results.

• Fetal hematopoietic stem cell transplantation
  • Hematopoietic stem cell (HSC) transplantation in utero is an attractive theoretical option for the treatment of congenital disease that can be diagnosed antenatally and improved by engraftment of HSCs.

  • Prior to 14 weeks' gestation, the fetal bone marrow has not yet developed sites for hematopoiesis and is receptive to the engraftment of circulating hematopoietic stem cells. Thymic processing of self-antigens has not yet started, and differentiated T lymphocytes have not yet been released into the circulation.

  • At this stage, theoretically, foreign HSCs should engraft without inducing an immune rejection or graft-versus-host disease and without needing myeloablation. HLA matching is not required. Additionally, specific tolerance for donor antigen is induced, allowing additional cells or bone marrow to be transplanted postnatally from the same donor.

  • In utero treatment may also preempt many clinical manifestations of the condition, such as recurrent infections, failure to thrive, and neurologic damage.

  • The disadvantages of the modality are the maternal and fetal risk from the procedures used to diagnose the disease and to perform the actual HSC transplantation, the technical expertise needed for the procedure, and the expense.

  • Diseases theoretically amenable to HSC transplantation are hemoglobinopathies such as sickle cell disease and thalassemias, immune deficiency diseases, and inborn errors of metabolism.

• Congenital heart disease
  • The precise diagnosis of congenital heart lesions with the aid of newer echocardiographic techniques has created the potential for prenatal surgery or interventional catheterization.

  • In the treatment of hypoplastic left heart syndrome, umbilical vessel catheterization and balloon valvuloplasty in utero for aortic stenosis are being attempted, with equivocal results.

  • In critical pulmonary stenosis, experimental valvotomy in utero may prevent right ventricular hypoplasia.

  • At present, the major goal of prenatal diagnosis of congenital heart lesions is genetic counseling and delivery at a tertiary center, where early and optimal management is possible in the neonatal period.

• Fetal arrhythmias
  • Most fetal arrhythmias are benign, and 90% are atrial extrasystoles. These should be observed twice weekly to exclude sustained supraventricular extrasystoles or atrial flutter. In ventricular extrasystoles, myocardial ischemia and tumors (eg, rhabdomyomas) must be excluded.

  • Prerequisites prior to starting antiarrhythmic therapy include the following:
    • An understanding of the electrophysiologic basis of the abnormal rhythm and its natural history

    • Good understanding of the pharmacokinetics of the agent in the mother, fetus, and placenta

    • Maternal consent for treatment

    • Presence of hydrops fetalis with a sustained supraventricular arrhythmia

    • Early gestation with a sustained arrhythmia in which the risk for development of hydrops is perceived to be high

  • The disadvantages include early and late mortality in the mother and fetus.

• Supraventricular tachycardias
  • These must be treated if they are sustained and associated with hydrops or upon evidence of left atrial preexcitation and a small foramen ovale.

  • Inpatient maternal treatment is started after 12-24 hours of fetal cardiac monitoring.

  • Maternal workup includes ECG to exclude a maternal Wolff-Parkinson-White syndrome and a determination of electrolyte, blood urea nitrogen (BUN), and creatinine levels prior to digoxin loading.

  • Digoxin is the first-line drug. Propranolol, procainamide, and quinidine have also been used. All fetal antiarrhythmic medications are associated with risks of proarrhythmia and mortality in both mother and fetus. Carefully select patients for treatment and monitor drug levels and toxicity. Structural defects, such as Ebstein anomaly and mitral insufficiency, must be excluded.

• Congenital complete heart block
  • This is associated with major congenital heart disease in approximately 50% of cases.

  • Diagnoses have included left atrial isomerism, physiologically corrected transposition, atrioventricular canal defects, and ventricular septal defects. This group has a high incidence of congestive heart failure or cyanosis and requires postnatal permanent pacemakers.

  • The remaining cases (50%) are associated with maternal autoimmune diseases (eg, systemic lupus erythematosus, Sjögren disease). Experimental protocols have used immunosuppressives, b-mimetics, and inotropes, with variable results.

	FETAL THERAPY: SURGERY	Section 8 of 9
Author Information Fetus As A Patient Prenatal Diagnosis Prenatal Screening Tests Prenatal Diagnostic Tests Imaging Modalities In Prenatal Diagnosis Fetal Therapy: Options And Medical Treatment Fetal Therapy: Surgery Bibliography

Anesthesia

As new intrauterine surgical techniques have been developed, anesthesia for the procedures has also evolved. The major objectives are to ensure maternal and fetal safety. Specific goals are the prevention of maternal hypoxia and hypotension, together with the maintenance of optimal uterine blood flow. Lower doses of epidural and spinal anesthetic agents are needed in pregnant women because of increased epidural pressure and a lower volume of cerebrospinal fluid in the vertebral space.

• To promote fetal safety, procedures are generally performed in the second trimester, if possible, to avoid potential teratogenicity from the anesthetic agents.

• Fetal asphyxia: Normal maternal PaO₂ should be maintained, and blood pressure should be maintained (with intravenous fluids and, if necessary, ephedrine, a vasopressor with central adrenergic stimulant action).

• Tocolysis: Uterine contractions are stimulated with the uterine incision and need to be stopped before preterm labor sets in. The agents used for this purpose include indomethacin, magnesium sulphate, and terbutaline.

• Indomethacin is administered preoperatively and continued postoperatively for 3-5 days. Fetal adverse effects include premature closure of the ductus arteriosus.

• Anesthetic agents commonly used are isoflurane inhalation with 100% oxygen along with muscle relaxants. For surgeries involving direct fetal manipulation, direct intramuscular fentanyl and pancuronium (a muscle relaxant and vagolytic) administered to the fetus have been tried prior to hysterotomy under ultrasound guidance.

Monitoring During Surgery

The parameters monitored during and after surgery include the following:

• Myometrial contractions and intrauterine pressures

• Maternal blood pressure, ECG, and pulse oximetric and blood gas levels

• Fetal pulse oximetric measurement (50-60% saturation), heart rate, blood gases, and ECG

• Ultrasonographic findings in cases of fetoscopic surgery

• Fetal temperature (Maintain temperature with continuous warm sodium chloride irrigation, minimized exposure, and increased ambient temperature.)

Surgical Interventions

Three approaches are currently used for invasive fetal therapy.

1. Ultrasonography-guided vesicoamniotic and, less commonly, thoracoamniotic shunt placement, is used in a fetus from 16 weeks' gestation to when lung maturity makes postnatal treatment the best option. Complications are inadequate function, migration, and iatrogenic gastroschisis.

2. Fetoscopic techniques now have a clinical application in the ligation of umbilical cords in acardiac twins, in selective laser photocoagulation of communicating vessels in twin-to-twin transfusions, and in the ablation of posterior urethral valves.
   • The procedure is performed inside the uterus using endoscopes, with a much smaller hysterotomy than that needed for open procedures. This lessens the risks of preterm labor and fetal hypothermia and improves fetal hemostasis during the procedure.

   • The success of the procedure depends on the use of both a transabdominal ultrasound intraoperative view and a simultaneous endoscopic view to guide placement of the trocars and cannulae.

   • The drawbacks of fetoscopic surgery are the risk of bleeding (avoiding the transplacental route decreases this risk), rupture of membranes, and chorioamnionitis. Fetoscopy may also be difficult because of poor access to the fetus due to fetal position or polyhydramnios.

3. Open fetal surgery is currently performed at select centers in instances in which the risk of the procedure to the mother and fetus is overridden by a diagnosis with a known poor outcome. Complications such as chorioamnionitis, preterm labor, bleeding, and direct trauma to the fetus are risks in most of these procedures.

These surgical techniques are considered appropriate for 9 lesions.

1. Obstructive uropathy

   Patients with severe obstructive uropathy with bilateral hydronephrosis and oligohydramnios revealed with ultrasonography should be evaluated for possible fetal therapy.

   • Prior to intervention, a cordocentesis is performed to document a normal karyotype and to exclude other major fetal anomalies.

   • This is followed by serial fetal bladder aspirations of urine under ultrasonographic guidance, which can help in the diagnosis of progressive renal damage (tonicity and electrolyte levels in the urine) and can relieve pressure if performed prior to 20 weeks' gestation.

   • A vesicoamniotic shunt is indicated in persistent megacystis with adequate ultrasonographic and biochemical renal function to reduce pressure in the urinary tract and to improve pulmonary development and decrease uterine compression.

   • Fetoscopic techniques can be used for fulguration of posterior urethral valves, placement of vesicoamniotic shunts, and vesicostomy. If all of these procedures fail, open vesicostomy with marsupialization of the bladder wall to the abdomen may be attempted.

   • Open surgery has a high fetal mortality rate (45%). In a study evaluating long-term postnatal outcomes after fetal surgery for posterior urethral valves in 14 patients, 8 patients lived to a follow-up period of 11.6 years. Chronic renal failure was present in 5 of them. This study emphasized that fetal intervention may assist in prolonging gestation to term, but the sequelae of the lesion on renal function may not be preventable. Fetuses with urethral atresia, despite vesicoamniotic shunts, have a poor prognosis, probably due to the severity and timing of the lesion.

2. Hydrocephalus

   Ventriculoamniotic shunts used for the decompression of obstructive hydrocephalus have had poor results and have caused procedure-related complications. Thus, their use is not indicated.

   • Fetal surgical procedures, both open and endoscopic, have been performed to repair myelomeningocele in utero. The open procedure is performed at 24-30 weeks' gestation and is shown to reduce both hindbrain herniation and the number of patients requiring shunts for hydrocephalus postnatally.

   • An endoscopic procedure has been performed by the Vanderbilt group, which consists of maternal laparotomy, followed by placement of a split-thickness maternal skin graft over the exposed spinal cord and neural elements of the fetus. The skin graft is attached with fibrin glue prepared from autologous maternal cryoprecipitate. The procedure has been performed at 22-24 weeks' gestation, with the rationale that neurologic injury is partly acquired through exposure of neural elements to amniotic fluid and the uterine wall.

3. Pleural effusion

   The use of thoracoamniotic shunts is indicated in a fetus with pleural effusion that reaccumulates after thoracocentesis and causes mediastinal shift. The aim of the shunt is to decompress the chest, promote pulmonary development, and treat the hydrops.

4. Twin-to-twin transfusion syndrome

   Umbilical cord ligation may be indicated in some cases of twin-to-twin transfusion syndrome. In acardiac twins, twin reverse arterial perfusion (TRAP) is characterized by artery-to-artery and vein-to-vein communications between twins in a monozygotic placenta. The donor twin is at risk for congestive failure, and the recipient is acardiac and inadequately perfused. Umbilical cord ligation is indicated in the acardiac twin or a nonviable twin involved in twin-to-twin transfusion after 21 weeks' gestation. Selective laser photocoagulation of the cord circulation, using YAG laser, can be performed prior to 21 weeks. In this procedure, an endoscope is introduced intra-amniotically through a port with ultrasonographic guidance.

5. Amniotic band syndrome

   In amniotic band syndrome, recent attempts have been made to lyse amniotic bands using fetoscopic techniques when a high risk of limb amputation is present.

6. Congenital diaphragmatic hernia

   Many investigators believe that intrauterine therapy is indicated in fetuses with CDH who have a poor prognosis.

   • These patients have been defined as those with the liver in the chest and those with a low lung-to-head ratio (<1.0) on ultrasound.

   • Additional criteria for intervention include a singleton fetus, normal karyotype, diagnosis made prior to 25 weeks' gestation, and absence of associated anomalies.

   • The procedures that have been attempted since the early 1990s involved definitive repair by reduction of viscera from the chest, patch placement over the diaphragm, and abdominal silo construction to reduce intra-abdominal pressure. These carried a high mortality rate in patients with a poor prognosis and have since been abandoned.

   • The current fetal surgery for CDH is tracheal occlusion.
     • This causes enlargement and real growth of the lungs, often pushing the abdominal viscera back into the abdomen. The trachea is occluded by external metal clips placed either fetoscopically or in an open fashion, delivering the head and neck through a hysterotomy.

     • Both fetoscopic and open methods have had comparable outcomes. Survival rates in these high-risk patients have been approximately 33%, compared to 10% with conventional postnatal therapy. Significant morbidity related to prematurity, atrial perforation, pulmonary insufficiency, and neurologic complications have been observed.

     • An ex utero intrapartum (EXIT) procedure to remove the clips, aspirate lung fluid, administer surfactant, and intubate the trachea is then performed while the fetus is still on placental support, followed by delivery of the baby. The EXIT procedure is performed at 36 weeks' gestation or earlier if fetal hydrops or impending preterm labor is present.

     • Recent small trials of internal tracheal occlusion by a detachable balloon placed through a single uterine port using fetal bronchoscopy and ultrasound have yielded good results. The advantage of the technique is that it is technically less demanding and has a lower risk of recurrent laryngeal nerve and tracheal injury.

7. Congenital high airway obstruction syndrome

   When congenital high airway obstruction syndrome (CHAOS) is complicated by hydrops, an EXIT procedure to place a tracheostomy may be of use. Earlier, fetoscopic, intervention may also be reasonable. The usual causes are laryngeal or tracheal stenosis.

8. Sacrococcygeal teratoma

   Fetuses with sacrococcygeal teratoma may develop hydrops from high output failure. Early attempts at open resection of the teratoma or radiofrequency ablation of the tumor proved to have a high rate of fetal mortality and maternal morbidity.

   • Coagulation or ligation of the feeding vessels at the base of the tumor directly at fetoscopy by laser is now possible at an early gestation. This treatment slows the vascular steal and reverses the high-output failure.

   • Targeted radiofrequency ablation of the feeding vessels via a percutaneous probe under ultrasonic guidance is also effective. The potential risks include gas emboli, hyperkalemia, thrombosis, hemorrhage, hemolysis, burn injury to the adjacent tissue, and premature rupture of membranes.

   • Fetal hemodynamic status requires monitoring during and immediately following the ligation because of an increase in afterload after ligation of the previously low-resistance tumor circuit.

9. Congenital cystic adenomatoid malformations

   Of fetuses with congenital cystic adenomatoid malformations, 10% develop hydrops, and these have a mortality rate approaching 100%. They can be treated at open fetal surgery with resection of the cystic lobe prior to 32 weeks' gestation. In some instances, this improves lung growth and allows the hydrops to resolve. The macrocystic form of cystic adenomatoid malformation may be drained with pleuroamniotic shunts, thus ameliorating the space-occupying effects and improving lung growth.