Management of High-Risk Pregnancy—A Practical Approach Manju Puri, SS Trivedi
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Prenatal Diagnosis1

Ratna Biswas,
Mandakini Pradhan
 
INTRODUCTION
Congenital disorder is one of the leading cause of mortality and morbidity in the fetus. The magnitude of problem is particularly significant in the developing world where congenital disorders are common because of consanguineous marriages and high birth rate. In India, half a million children born annually have congenital malformations and 21000 babies have Down syndrome.1 In the absence of curative treatment the financial and social burden on the family and country is enormous. Herein lies the importance of early prenatal diagnosis.
Prenatal diagnosis involves initial screening to detect at risk couples followed by definite diagnosis. The prenatal diagnostic procedures are aimed at diagnosing the disease of the fetus. It could be chromosomal abnormalities, genetic diseases or structural defects. These procedures can be invasive or noninvasive. The diagnostic tests involve chromosomal, molecular or enzyme assays on fetal samples. The aim of the diagnostic procedures is to provide an accurate and early diagnosis of the fetal disease, so as to enable the couple to make an informative choice on the management options. The modalities of management are either termination of pregnancy, fetal therapy wherever applicable or to prepare for birth of an affected child. Preimplantation diagnosis, too, is a management option whereby an unaffected embryo is chosen for implantation. Thorough genetic counseling is essential and ethical issues need's to be considered when opting for this method.2
 
Screening Methods
These are methods which can identify fetus at high risk of having malformations, genetic disorders, chromosomal abnormalities, etc. Though the screening methods widely discussed are for detection of fetal chromosomal aneuploidy, other fetal conditions which can affect the pregnancy outcome need to be kept in mind. For a screening method to be effective it needs to have a high detection rate with an acceptable false-positive rate. Following given methods can be used to detect couples at high risk of fetal disorder.
  1. History:
    • During present pregnancy
    • Previous pregnancy
    • Past medical or surgical disorder
    • Family history.
  2. Examination
  3. Investigations:
    • Serum screening
    • Ultrasound
    • Other specific tests.
 
History
  1. History during present pregnancy: Certain history during ongoing pregnancy may indicate a high-risk fetus.
    1. Exposure to teratogens like anticancer drugs, antiepileptics especially sodium valproate, anticoagulant like warfarin and radioactive iodine therapy for thyroid cancer etc.
    2. Contact with patients of infectious diseases like rubella.
    3. History of infectious diseases like chickenpox or fever with rash may indicate likelihood of fetus being infected.
    4. Anemia in the women (not due to iron deficiency or not improving after iron therapy) may need further evaluation to detect carrier of hemoglobinopathies. Such woman may be at risk of having an affected fetus.
  2. Past history: Previous history of fetus being affected with a specific disease should warrant a prenatal diagnosis in subsequent pregnancy. The method of prenatal diagnosis differs depending upon the diagnosis in the previous child. Table 1.1 delineates few of the common disorders, their risk of recurrence and method of prenatal diagnosis.
Family history: A minimum three generation pedigree should be drawn to find out any significant risk to the couple of having an affected baby. For example, a history of maternal brother being affected with Duchenne muscular dystrophy may indicate a possibility of the woman being carrier and hence having 25% risk of affected child. A history of consanguinity may indicate towards investigating parents for carrier of a common autosomal recessivedisease like β-thalassemia.3
Table 1.1   Some common disorders, their risk of recurrence and method of prenatal diagnosis
Disorder
Mode of inheritance
Recurrence risk
Method of Prenatal diagnosis
Single gene disorder
β-thalassemia
Autosomal recessive
25%
Mutation detection in both parents and subsequently in the fetal sample obtained after invasive method
Cystic fibrosis
Autosomal recessive
25%
Mutation detection in both parents and subsequently in the fetal sample obtained after invasive method
Sickle cell disease
Autosomal recessive
25%
Direct detection of mutation by molecular method in the fetal sample obtained after invasive method
Duchenne muscular dystrophy (DMD)
X-linked recessive
25% of all children or 50% of male children
Mutation detection in the previous affected child
Spinal muscular atrophy (SMA)
Autosomal recessive
25%
If diagnosis is confirmed in previous affected child, mutation detection can be done in the fetal sample obtained after invasive method
Achondroplasia
Autosomal dominant
50% if one of the parent is affected; 1% if both parents are normal as it is usually due to new mutation
Mutation detection in the fetal sample if couple is willing after counseling.
Multifactorial disease neural tube defect (NTD)
Unknown
3–5% after one affected pregnancy; 10% after two affected pregnancies
Ultrasound
Some common disorders, their risk of recurrence and method of prenatal diagnosis are listed in Table 1.1.
 
Examination
As in any clinical setting, detail examination of a pregnant woman is important. It is more so because many of them visit a doctor for the first time and asymptomatic conditions can be picked-up, e.g. this woman with history of previous two pregnancies affected with polyhydramnios and club foot, on examination was found to have myotonia and thus clinching the diagnosis of myotonic dystrophy in the family which is a triple repeat disorder (Fig. 1.1).4
zoom view
Figure 1.1: Myotonic dystrophy
 
Investigations
Various screening and diagnostic procedures for prenatal diagnosis are tabulated in Table 1.2.
Table 1.2   Screening and diagnostic procedures for prenatal diagnosis
Noninvasive Screening Procedures
a. Maternal serum screening
  1. First trimester screening
  2. Triple test
  3. Quadruple test
  4. Integrated screening
  5. Sequential screening and contingent screening
b. Fetal imaging
  1. Ultrasonography – 2D, 3D, 4D
  2. Doppler sonography
  3. Magnetic resonance imaging
Noninvasive Diagnostic Procedure
Detection of fetal DNA and fetal cells in maternal circulation
Invasive Diagnostic Procedures
a. Amniocentesis
  1. Early amniocentesis
  2. Second trimester amniocentesis
b. Chorionic villous sampling (CVS)
  1. Transcervical CVS
  2. Transabdominal CVS
c. Fetal blood sampling
  1. Percutaneous umbilical blood sampling or cordocentesis
  2. Fetal intrahepatic vessel sampling
  3. Fetal cardiac sampling.
d. Fetal tissue biopsy
e. Fetoscopy
Preimplantation Diagnosis
The noninvasive procedures are generally used for screening whereas the invasive procedures give a definite diagnosis.5
Fetal aneuploidy is the commonest indication for prenatal diagnostic test. Aneuploidy is defined as an abnormal number of chromosomes in place of the usual diploid complement. Down syndrome (Trisomy 21), Edward syndrome (Trisomy 18), Patau syndrome (Trisomy 13) are the most common trisomies. Down syndrome occurs in 1: 800 to 1: 900 live births. It is compatible with life. Edward syndrome occurs in 1:3000 live births and Patau syndrome occurs in 1: 5000 live births. Both are lethal anomalies.2 The age-related risk of Down syndrome in a pregnant woman at midtrimester is, 1: 417 at 33 years, 1: 250 at 35 years, 1:149 at 37 years, 1: 69 at 40 years and 1:19 at 45 years.3
The hallmark of screening Down syndrome till few years back was Triple test which is performed in the second trimester. Now screening procedures are available in the first trimester also. Awareness amongst women regarding these procedures is lacking, hence it is important to counsel and offer screening procedures to all pregnant women.
Recent guidelines from American College of Obstetrics and Gynecology (ACOG) 2007 and American College of Medical Genetics (ACMG) recommends screening of all pregnant women regardless of maternal age.4,5
ACOG 2007 guidelines also state that integrated or sequential screening be offered to all pregnant women who seek prenatal care in the first trimester and for those who come later than 14 weeks, the second trimester serum screening should be offered.
The screening strategy chosen depends on availability of chorionic villus sampling (CVS) and expertise for nuchal translucency (NT). In the absence of expertise in NT measurement the first trimester screening is incomplete and therefore the integrated and sequential and contingent screening too, cannot be done. However the integrated serum screening is an option if expertise in NT measurement is lacking. On the other hand when CVS facilities are unavailable, the sequential and contingent screening cannot be done because in these methods if screen is positive in the first trimester then directly CVS is to be done. However integrated screening is possible even in absence of CVS facilities because here results are declared only after the second trimester tests, and an amniocentesis is performed in case of positive result.
 
Screening Methods
 
First Trimester Screening
The methods used are:
  1. Nuchal translucency (NT) sonography
  2. Nasal bone sonography
  3. Doppler sonography of ductus venosus
  4. Pregnancy associated plasma protein A (PAPP-A) in maternal serum
  5. Free β–subunit of hCG in maternal serum
6
 
Nuchal Translucency
It refers to the space between spine and overlying skin at the back of fetal neck. Measurement is done between 10 weeks + 3 days to 13 weeks + 6 days of gestation. Increased NT is a strong marker of Down syndrome.6 Possible mechanisms leading to increased nuchal translucency are fluid accumulation secondary to heart failure caused by structural anomalies of heart, abnormal extracellular matrix or abnormal lymphatic development.7
NT can be obtained by transabdominal or transvaginal route. By transabdominal route it is possible to obtain 95% of NT measurements and combining the two routes results in 100% chance of obtaining the NT measurement. Correct technique for measurement of NT is to obtain a view of the midsagittal plane with the fetal head in neutral position and fetal image occupying 75% of viewable screen. The fetal image to be obtained consists of fetal head, neck and upper part of thorax. Crown-rump length (CRL) measurement is mandatory to accurately estimate the period of gestation since NT varies with gestational age. Distinction between amnion and overlying fetal skin should be confirmed on fetal movement. Calipers are placed at inner borders of the NT space.6,8,9 Three to five NT measurements are obtained and the largest value taken.
NT measurement requires considerable skill which can be acquired with training. The accuracy of NT measurement is said to be reached when the difference in intra and interobserver variation is less than 0.5 in 95% of the measurements. Approximately 100 scans have to be performed to attain such level of perfection. A good quality ultrasound machine with video loop function and an ability to measure upto a 10th of a millimeter is needed for measuring NT.9 Adequate time should be spared for proper measurement and at least 20 minutes time should be given before labeling an attempt unsuccessful.2 In such a situation a rescan should be scheduled after a week's time or earlier depending on the period of gestation. Quality control measures need to be instituted for maintaining high standards of performance.
The rate of detection of Down syndrome based on NT in high-risk women ranges from 46–62%, whereas the detection rate in low-risk women varies from 29–91%.8 The average detection rate with NT is 77% at false-positive rate of 5%.8
Specific cutoff values (2.5 or 3 mm) are inappropriate because of gestational age dependant variation in NT. Gestational age specific thresholds are available for NT measurement which is combined with maternal age to arrive at a risk estimate.9 Alternatively it can be expressed as MoM and combined with PAPP-A and β hCG to give a risk estimate.
Detection of cystic hygroma during first trimester sonography for evaluation of NT is the most powerful predictor for trisomy 21 with 50% risk of fetal aneuploidy. This finding is an indication for a CVS.67
 
Nasal Bone Sonography
To obtain accurate measurement, the midsagittal plane is viewed with the neck in slight flexion position and the fetal profile facing upwards. In normal pregnancy two echogenic lines, one representing the fetal skin and the other the fetal nasal bone is visualized. Failure to visualize the nasal bone is an independent risk factor for Down syndrome.6 However accurate imaging of fetal nasal bone in first trimester is technically challenging with a significant intra and interobserver variation.
The ultrasound beam should strike at right angles to the nasal skin for visualization of the nasal bone. The normal ossification of nasal bone develops on both sides of the cartilaginous septum from 10 weeks onwards. New ossification appears as less echogenic lines on both sides of bone. Only the echogenic part should be measured. Difference in timing of appearance and ossification of both nasal bones may cause technical difficulty in measurement leading to intra- and interobserver variation.10,11 Hence its routine use in clinical setting is not recommended.
 
Ductus Venosus Sonography
The blood flow in the fetal ductus venosus in first trimester by Doppler ultrasound shows triphasic flow pattern with forward flow reaching peak during ventricular systole, early ventricular diastole and during atrial contraction. Absence or reversal of flow at atrial systole is a marker for trisomy 21.6 Difficulty in measuring flow through ductus venosus limits its use in routine clinical practice.10
 
Biochemical Markers in Maternal Serum
PAPP-A is 50% lower in trisomy 21 as compared to normal pregnancy when measured between 10–13 weeks of gestation, whereas maternal serum levels of free β hCG is twice as high in trisomy 21 as compared to normal pregnancy.6
 
Combined First Trimester Screening
Combined NT, PAPP-A, β hCG in association with maternal age detects 85% of trisomy 21 with the false-positive rate of 5%. The earlier in first trimester the measurements are taken the higher is the detection rate, 87% at 11 weeks as compared to 82% at 13 weeks.6
A National Institute of Health (NIH) sponsored study called the Blood, Ultrasound and Nuchal Translucency (BUN) trial evaluating the results of first trimester screening in 8500 high-risk cases detected 79% of all Down syndrome cases with 5% false-positive rate.12 The FASTER study with 36000 pregnancies detected 85% of Down syndrome with 5% of false-positive.13 8The one stop clinic to assess risk (OSCAR) study involving 12000 pregnancies had a 90% detection rate at a false-positive rate of 5%.14 The Serum, Urine and Ultrasound (SURUSS) study involving 48000 pregnancies had a 86% detection rate for a 5% false-positive rate.15 The average derived from these 4 large trials is 85% which is better than quadruple screening in second trimester. Both BUN and FASTER study support the efficacy of free β hCG over total β hCG. However the recent guidelines from the American College of Medical Genetics state that free β hCG, total hCG or hyperglycosoylated hCG should be interchangeable.5 As individual marker NT is most informative followed by PAPP-A.
 
Second Trimester Screening
It is indicated in pregnant women who present for the first time in second trimester or when expertise in first trimester screening, like measurement of NT is not available. Methods of second trimester screening are:
  1. Biochemical serum markers
  2. Sonographic detection of major structural malformation
  3. Sonographic evaluation of minor markers.
 
Biochemical Serum Markers
  1. Maternal serum alpha fetoprotein (MSAFP)
  2. β hCG
  3. Unconjugated estriol (uE3)
  4. Inhibin A.
 
Maternal Serum Assay in Second Trimester
MSAFP screening is most accurate when performed between 16–18 weeks of gestation.14 Low MSAFP has been associated with Down syndrome. One-third to one-fifth of Down syndrome pregnancies present with low MSAFP levels with the median MSAFP level of 0.7.16 MSAFP values need correction for weight, race, diabetes and multiple gestation.3 In insulin dependent diabetics MSAFP levels are 60% of nondiabetics.16 In twin pregnancy the median MSAFP levels from 16–20 weeks of gestation is 2.5 MoM for single pregnancy. Combined biochemical screening procedures are:
 
Triple Test
MSAFP, unconjugated estriol and hCG are the components of triple test. Maternal serum levels of AFP and unconjugated estriol are 25% lower in Down syndrome as compared to normal pregnancy. Serum hCG is twice as high in Down syndrome as compared to normal pregnancy. It is usually performed between 15–18 weeks of gestation. The serum levels of each of the proteins is 9expressed as multiple of median (MoM) for women with gestational age same as patients. The composite estimate of the risk of trisomy 21 is calculated. Standard cut off is 1/270 which is equal to the second trimester risk of Down syndrome in a 35-year-old woman.3,12 Triple test identifies around 60% of the pregnancies at risk of Down syndrome with 5% screen positive rate in women younger than 35 years. In women older than 35 years it detects 75% or more pregnancies affected with Down syndrome or other aneuploidies.17
 
Quadruple Test
Maternal serum inhibin A levels are increased to 2.1 times the median value of control in Down syndrome. Quadruple test is done between 14–21 weeks. It combines measurement of MSAFP, serum estriol, free β hCG and inhibin A together with maternal age.3,18 It has a 67 to 76% detection rate of trisomy 21 in women younger than 35 years.17 Some studies have quoted a detection rate of 80%.
 
Combined First and Second Trimester Screening
It maximizes the performance of these screening modalities. It is of two types—Integrated screening and sequential or contingent screening.
Integrated screening: It is a two step approach with the results announced at the time of the second test. In the first trimester, between 10–14 weeks of gestation NT and PAPP-A are measured. In the second trimester, between 15–16 weeks of gestation, MSAFP, hCG, estriol and inhibin A levels are measured. The results are analysed only at the end of second test and the probability of trisomy 21 is calculated using a computerized algorithm. The detection rate is 95% with a false-positive of 5%.2,19
Sequential and contingent screening: In sequential screening NT and PAPP-A or NT, PAPP-A and β-hCG are analysed to detect an interim risk in the first trimester. If interim risk is sufficiently high (e.g. >1 in 25 or > 1 in 50) then the pregnant women are informed of their screen positive result and offered early diagnostic intervention. All women who are below high-risk level subsequently go for second trimester screening with either a screen positive or negative result and advised for a diagnostic test according to the results of the second trimester screening. In the contingent screening the first trimester screening with NT and PAPP-A or NT, PAPP-A and β hCG is analysed to assess an interim risk. But instead of just a high interim risk group, a low interim risk group is also identified for e.g. < 1 in 2000. The high interim risk group is informed of their screen positive status like in sequential screening and advised diagnostic test while the low interim risk group do not need any further testing thereby reducing the cost incurred. The intermediate risk group which fall between the high- and low-risk group are advised for a second trimester screening 10with the four biochemical markers to receive a fully integrated report and accordingly advised diagnostic invasive testing based on the report.2,19 The advantage of this screening is that if the first trimester screening is positive then chorionic villus sampling is offered to the pregnant women leading to early diagnosis and an early termination of pregnancy if needed.
The first trimester serum markers are also abnormal in trisomy 18. Levels of PAPP-A and βhCG are low and NT is increased. In the second trimester, alfa-fetoprotein, βhCG and estriol are all low in trisomy 18. Algorithms are available to detect risk of trisomy 18 based on first trimester and integrated screening.
 
Fetal Imaging
 
Sonographic Detection of Major Fetal Structural Malformation
This is done between 17–18 weeks of gestation to maximize fetal anatomic evaluation. The major structural malformations associated with aneuploidy are:3,6,10
  1. Cardiac defects: Atrial septal defect, ventricular septal defects, teratology of Fallot, double outlet right ventricle, hypoplastic left heart, septal and endocardial cushion defects, valvular defects, coarctation of aorta.
  2. Gastrointestinal malformations: Duodenal atresia, esophageal atresia, omphalocele, tracheoesophageal fistula, annular pancreas, imperforate anus, diaphragmatic hernia.
  3. Central nervous system malformations: Meningomyelocele, holoprosencephaly, agenesis of corpus callosum, ventriculomegaly (Fig. 1.2), microcephaly, posterior fossa defects, etc.
    zoom view
    Figure 1.2: Venticulomegaly
    11
  4. Skeletal malformations: Club feet, rocker bottom feet, polydactyly.
  5. Renal malformations: Horseshoe kidney, polycystic kidney, hydronephrosis.
  6. Facial anomalies: Cleft lipand palate, microphthalmia, anophthalmia, macroglossia, cyclopia.
 
Sonographic Evaluation of Minor Markers
These are specific ultrasound findings that have been associated with increased risk of fetal aneuploidy. The minor markers are:
  1. Nuchal fold: This measures the area between the outer aspect of occipital bone and outer aspect of skin in the axial plane passing through the posterior fossa. It is measured between 15–21 weeks. In normal pregnancy it is less than 5 mm, if it is more than 5 mm then the risk of Down syndrome increases by 11 fold.6 It is also increased in Noonan's syndrome and in congenital cardiac defects. It is considered to be the single most sensitive and specific marker for aneuploidies in the second trimester sonography.10
  2. Echogenic bowel: A very bright bowel in the fetal abdomen is associated with a 6.7 fold increased risk of Down syndrome. While performing ultrasound it is important to check the gain settings as false-positive results will occur if the gain settings are high. The echogenicity of bowel should be comparable to that of fetal bone.6 It is also seen in association with disorders like cystic fibrosis, cytomegalovirus infection and other aneuploidies. Structural malformations like small bowel obstruction, malrotation of gut, meconium ileus and peritonitis are also associated with this observation.10 In it's presence a maternal screening for cystic fibrosis, CMV infection and aneuploidy should be done. On the other hand amniocentesis can be done for fetal karyotyping, detection of cystic fibrosis mutation, and DNA analysis of CMV infection.
  3. Echogenic intracardiac focus: This is caused by calcification of papillary muscles of ventricle. It is mainly seen in the left ventricle. To prevent overdiagnosis multiple angles are visualized to rule out specular reflections. The presence of echogenic intracardiac focus increases the risk of Down syndrome by 1.8 fold. However the point to be noted is that it may be present in 30% of normal Asian fetuses and hence it is not a sensitive or specific marker for aneuploidies. Therefore, other ultrasound findings or positive serum screening should be present to justify invasive testing.6,10
  4. Short humerus and femur: In normal fetus the ratio of expected to observed length of these two bones should not be less than 0.90. The presence of short humerus increases the risk of Down syndrome by five fold, whereas short femur increases the risk by 1.5 fold. However racial difference in biometry exists and Asian fetuses have shorter biometric measurements. Hence normogram for individual population should be available to prevent overdiagnosis. The sensitivity and specificity of this marker is not high and hence it's isolated presence does not justify an invasive diagnosis.612
  5. Pyelectasis: A renal pelvis measurement in the anteroposterior diameter of more than 4 mm is abnormal when measured between 14–20 weeks. Bilateral pyelectasis is associated with 1.5 fold increased risk of Down syndrome.6
  6. Nasal bones: Hypoplastic or absent nasal bones on second trimester sonography is associated with increased risk of Down syndrome. The data available till now is limited to assess the strength of this association.6
  7. Choroid plexus cyst: The presence of cyst in the choroid plexus in an axial view through the upper portion of fetal head has been correlated with the increased risk of trisomy 18. However isolated choroid plexus cyst in low risk woman is not clinically relevant6,10 (Fig. 1.3). It disappears in 2% fetuses by 20 weeks and resolves in more than 90% by 26 weeks. It is a soft marker of aneuploidy.
zoom view
Figure 1.3: Choroid plexus cyst
Nicolaides and colleagues observed that detection of aneuploidy on sonography was related to the number of defects identified.10,20 They assigned points to specific markers. Structural malformation and thickened nuchal fold were assigned two points each and shortened femur, shortened humerus, echogenic intracardiac focus, hyperechoic bowel and pylectasis were given one point each. An invasive test is recommended in high-risk women with one or more points or in low-risk women with two or more points based on his scoring system.13
 
Genetic Sonography
It is a targeted sonography which is done for fetal aneuploidy. It is done between 18 to 20 weeks of gestation and special attention is given to detect abnormal fetal biometry, fetal structural anomalies and minor (soft) markers of fetal aneuploidy.17
In Down syndrome the following sonographic findings should be looked for:
Skeletal abnormalities like sandal gap toes, 11 pairs of ribs, short fingers, brachymesophalangia, etc. Facial abnormalities like flattened face and occiput, oblique palpebral fissure, high arch palate, low nasal bridge, low set ears, nasal hypoplasia, macroglossia, epicanthic fold. CNS abnormalities which are frequently associated with Down syndrome are ventriculomegaly, microcephaly and brachycephaly. Cardiovascular defects include ASD, VSD, TOF, endocardial cushion defects, echogenic cardiac focus and pericardial effusion. Gastrointestinal and genitourinary abnormalities like duodenal atresia, tracheoesophageal fistula, omphalocele, annular pancreas, imperforate anus, hyperechogenic bowel and mild pylectasis. Hypotonia and goiter are other findings seen in trisomy 21. IUGR may or may not be an associated finding.10
In trisomy 18, growth is usually restricted. CNS abnormalities associated with Edward syndrome are choroid plexus cyst, abnormal cisterna magna, absent corpus callosum, cerebellar hypoplasia, ventriculomegaly, strawberry-shaped calvarium, and neural tube defects. Skeletal abnormalities include, limb reduction defects, overlapping index finger, clubbed feet, rocker bottom feet and polydactyly. Facial defects seen are, cleft lip and palate, micrognathia, low set ears, and microphthalmos. Cardiovascular abnormalities like septal and valvular defects may be present. Gastrointestinal and genitourinary anomalies associated with this syndrome are omphalocele, diaphragmatic hernia, hydronephrosis and horseshoe kidney. Umbilical cord cysts and double vessel umbilical cord may also be observed.10
Trisomy 13 is associated with growth restriction. CNS malformations like alobar holoprosencephaly, posterior fossa abnormalities, corpus callosum agenesis, ventriculomegaly and neural tube defects may be seen. Skeletal abnormalities like polydactyly and facial malformations like cyclopia, midline facial clefts, anophthalmia, hypoplastic nose are associated with Patau syndrome. CVS abnormalities like septal defects, tetralogy of Fallot, hypoplastic left ventricle, coarctation of aorta, echogenic intracardiac focus, gastrointestinal malformations like omphalocele and genitourinary abnormalities like polycystic kidneys, horseshoe kidney, enlarged echogenic kidney and nuchal thickening may be observed.1014
 
Other Imaging Studies
  1. 3D Ultrasound: Other than the standard 2D ultrasound, 3D ultrasound allows multiplanar imaging and is an adjunct to the 2D ultrasound. In special situations such as fetal echocardiography, detection of cleft lip and cleft palate and in skeletal abnormalities, it's role is more certain.2 Because of multiplanar imaging and rapid acquisition of volume data, more accurate information is possible like for example, in the evaluation of the extent and size of anomaly.17 It provides better comprehension of fetal anatomy specially for the patient. It is semimanual and dependent on operator skill partly, hence adequate training is required.
  2. Magnetic resonance imaging (MRI): It was first used in obstetrics in 1983. It's advantage lies in it's ability to use multiple planes for reconstruction and a large field of view making the visualization of complicated anomalies easier. The development of single shot rapid acquisition sequence with refocused echoes and high quality T2 weighted image provides excellent fetal imaging. This has obviated the need to sedate the mother or fetus. It is specially useful when ultrasound findings are inconclusive as in oligohydramnios, maternal obesity or when posterior cranial fossa is to be visualized where bone calcification hampers the ultrasonographic study. MRI has been used extensively in the diagnosis of ventriculomegaly, corpus callosum agenesis, posterior fossa abnormalities, cortical gyral malformations, hemorrhage, holoprosencephaly, arachnoid cyst, neural tube defects and vascular malformations.2
 
Summary of Recommendations and Conclusions of ACOG Practice Bulletin No. 77 (2007)
 
Screening for Fetal Chromosomal Abnormalities
The following recommendations are based on good and consistent scientific evidence (Level A):
  • First trimester screening using both nuchal translucency measurement and biochemical markers is an effective screening test for Down syndrome in the general population. At the same false-positive rates, this screening strategy results in a higher Down syndrome detection rate than does the second trimester maternal serum triple screen and is comparable to the quadruple screen.
  • Measurement of nuchal translucency alone is less effective for first trimester screening than is the combined test (nuchal translucency measurement and biochemical markers).
  • Women found to have increased risk of aneuploidy with first trimester screening should be offered genetic counseling and the option of CVS or second trimester amniocentesis.15
  • Specific training, standardization, use of appropriate ultrasound equipment, and ongoing quality assessment are important to achieve optimal nuchal translucency measurement for Down syndrome risk assessment and this procedure should be limited to centers and individuals meeting these criteria.
  • Neural tube defect screening should be offered in the second trimester to women who elect only first-trimester screening for aneuploidy.4
    The following recommendations are based on limited or inconsistent scientific evidence (Level B):
  • Screening and invasive diagnostic testing for aneuploidy should be available to all women who present for prenatal care before 20 weeks of gestation regardless of maternal age. Women should be counseled regarding the differences between screening and invasive diagnostic testing.
  • Integrated first and second trimester screening is more sensitive with lower false-positive rate than first trimester screening alone.
  • Serum integrated screening is a useful option in pregnancies where nuchal translucency measurement is not available or cannot be obtained.
  • An abnormal finding on second trimester ultrasound examination identifying a major congenital anomaly significantly increases the risk of aneuploidy and warrants further counseling and the offer of a diagnostic procedure.
  • Patient who have a fetal nuchal translucency measurement of 3.5 mm or higher in the first trimester, despite a negative aneuploidy screen or normal fetal chromosomes, should be offered a targeted ultrasound examination, fetal echocardiogram or both.
  • Down syndrome risk assessment in multiple gestation using first or second trimester serum analysis is less accurate than in singleton pregnancies.4
    The following recommendations are based primarily on consensus and expert opinion (Level C).
  • After first trimester screening, subsequent second trimester Down syndrome screening is not indicated unless it is being performed as a component of the integrated test, stepwise sequential, or contingent sequential test.
  • Subtle second trimester ultrasonographic markers should be interpreted in the context of a patient's age, history and serum screening results.4
 
Non Invasive Diagnostic Method
 
Detection of Fetal DNA and Fetal Cells in Maternal Circulation
Presence of cell free fetal DNA in maternal plasma was first discovered in 1997. At present the circulating fetal DNA detected in maternal plasma can be analysed for noninvasive diagnosis of RhD status of the fetus, sex-linked disorders, single gene disorder like congenital adrenal hyperplasia, beta- thalassemia and achondroplasia.22 Karyotyping is also possible.16
The different fetal cells which can be isolated are nucleated erythrocytes, CD-34 positive hematogenic progenitors and syncytiotrophoblast. These cells can be detected as early as 6 weeks of gestation.14 The fetal cells are differentiated from maternal cells by various cell sorting techniques like density gradient or protein separation, fluorescence activated cell sorting and magnetic activated cell sorting.3 But there are certain limitations of fetal cell detection. These are—nucleated RBC cannot be expanded in cultures hence metaphase chromosomes are not available for prenatal diagnosis, CD-34 cells from previous pregnancies may persist and cause error in diagnosis and trophoblasts are not always present in maternal circulation. Hence none of these cells are ideal for analysis of fetal disease. Also the sorting methods are not consistently reliable for fetal cell detection.23
Fetal DNA is detected by real time PCR techniques. Recently plasma epigenetic markers have been used for differentiating maternal and fetal DNA. DNA methylation techniques is one such epigenetic method. Search is on for techniques which are superior to the methylation specific PCR assays like the mass spectrometric-based methods.24,25
 
Invasive Prenatal Diagnostic Procedures
These are techniques to obtain sample which are of fetal origin and can be used for analysis to make a definite fetal diagnosis.
In presence of indications patients have to be counseled for an invasive test such as amniocentesis. Counseling has to encompass all aspects of the procedure like the technique, the rate of complications especially that of pregnancy loss, the timing of results and the management options available. The decision of going for the procedure is absolutely voluntary and written consent mandatory. All the forms, i.e. Form D,E,F and G need to be filled up duely as per PNTD act.
 
Amniocentesis
It is an invasive procedure where a needle is introduced into the amniotic cavity under a sonographic guidance and amniotic fluid is withdrawn for analysis. The indications for amniocentesis are:3
  1. Mother at high risk of fetal aneuploidy:
    1. Maternal age 35 years or more at the time of delivery in singleton pregnancy
    2. Maternal age 31 years or more at the time of delivery in twin gestation
    3. Increased risk on first or second trimester screening for Down syndrome
    4. Ultrasound finding suggestive of major structural malformation.3
    5. One of the parents is a carrier of balanced chromosomal translocation.
  2. Fetus at high risk of metabolic disorder:
    1. A definite diagnosis was made in previous affected child.
    2. Previous affected child may have a typical clinical presentation/investigations without a definite diagnosis.17
  3. Fetus at risk of single gene disorder (autosomal recessive, autosomal dominant and X-linked recessive disorder) where amniotic fluid can also be used for extracting DNA for diagnosis.
  4. RhD isoimmunized pregnancy management by spectrophotometric analysis of bilirubin in amniotic fluid and plotting of Liley's curve.
Amniocentesis is done between 15–20 weeks of gestation. Technically the procedure is easier to perform at this period of gestation since the uterus is accessible per abdomen and sufficient amount of amniotic fluid is available for withdrawing.
It is performed under aseptic precaution using a 22 G needle. A preliminary sonography is performed to document the number of fetuses, the viability of the fetus, gestational age, placental localization and liquor volume. A site which has sufficient liquor volume and which is free of placenta or cord is chosen for needle insertion. Local anesthesia at puncture site is optional. A sterile glove is put over the transducer and under sonographic vision, 22G needle is inserted into the amniotic cavity. The stilette is removed and fluid is aspirated (Fig. 1.4).3,18
zoom view
Figure 1.4: Amniocentesis
Dry tap during amniocentesis is due to tenting of membranes. In such a situation needle should be withdrawn into the myometrium and reinserted with a forceful thrust to penetrate the membranes. Another method for successful entry is to penetrate the needle into the posterior myometrium under sonographic guidance and then gradually withdraw the needle into the amniotic fluid pocket. The use of stylet with needle also helps to overcome the problem of tenting of membranes. The stylet is longer than the needle and by advancing the stylet 10 mm beyond the needle tip the membranes can 18be entered.26 Initial 3–4 ml of fluid is discarded to prevent contamination of maternal cells. Approximately 20 ml of fluid is aspirated and transferred into centrifugation tubes. Following the procedure the fetal heart rate is checked and anti-D is given to Rh negativemothers. Postprocedure amniotic fluid leakage occurs in 2% cases but is usually self-limiting. Chorioamnionitis occurs in 1 in 1000 procedures. The rate of pregnancy loss is as low as 0.3% in experienced hands.16
Early amniocentesis is performed between 11–14 weeks of gestation. The incidence of limb deformities, like talipes equinovarus is significantly high in early amniocentesis. It is seen in 0.75–1.7% cases. The miscarriage rate is 1.5%.3 Hence this procedure is not preferred. The Canadian Early and Midtrimester Amniocentesis Trial (CEMAT) randomized 4000 pregnant women for early and midtrimester amniocentesis. The success rate of culture was 97.7% with accuracy of 99.8% for early amniocentesis but there was higher rate of complications in form of fetal loss, increased incidence of talipes equinovarus and postprocedural amniotic fluid leakage.2729
Amniotic fluid is analysed routinely for alfa fetoprotein levels. Fetal cells obtained from amniotic fluid or chorionic villus sampling are cultured for karyotype determination and DNA study. In recent times rapid diagnosis of common fetal aneuploidies like trisomy 13, 18, 21 and sex chromosomes is possible. The techniques of rapid diagnosis is fluorescent in situ hybridization(FISH) and quantitative fluorescent-PCR(QF-PCR). Specific probes for fluorescent in situ hybridization technique has been developed to detect numerical aberrations of metaphase chromosomes and interphase nuclei of nonmitotic cell.18,30
The limitations of rapid diagnostic techniques are that they fail to detect structural chromosomal aberrations like translocation and inversion and in addition they miss out nearly half (47.4%) of the aneuploidies when compared to karyotyping.31 However in more recent studies involving more than 2000 women both the sensitivity and specificity of FISH on uncultured amniocytes for detecting these chromosomal abnormalities was > 99%. But there were both false-positive and negative reports. Further maternal cell contamination on uncultured cells remains a problem. Mosaicism and confined placental mosaicism(CPM) in CVS samples is not identified accurately by FISH. The American College of Medical Genetics does not recommend management on basis of result of FISH alone.
QF-PCR amplifies highly polymorphic short tandem repeats on chromosomes 13, 18, 21, X and Y using fluorescent primers and PCR. This method is comparable to FISH in sensitivity but maternal cell contamination is more easily identified. Hence it has replaced FISH for rapid aneuploidy screening in European countries.30
Newer technologies are being developed like the array-based comparative genome hybridization (aCGH). It has a high resolution, genome wide screening strategy for obtaining DNA copy number information in a single 19measurement. It is readily amendable to automation and is less labor intensive. But it's role in detection of submicroscopic chromosome imbalances is unclear. It fails to detect balanced chromosome rearrangements, alteration in ploidy levels like triploidy and tetraploidy, and mosaicism necessitating further diagnostic tests like karyotyping.30 Role of aCGH in prenatal diagnosis needs further evaluation.
 
Chorionic Villus Sampling
This procedure is carried out between 10–13 weeks of gestation. It can be performed by transabdominal or transvaginal route. The indication of CVS is essentially the same as amniocentesis except for a few conditions which require either amniotic fluid or placental tissue for analysis.3 The advantage of CVS is that the results are available earlier hence providing enough time for medical termination of pregnancy if needed. The complications of these two procedures are similar.
 
Transcervical Chorionic Villus Sampling
This procedure is performed using a polyethylene catheter of 5.7 F diameter and 27 cm length. The cervix and placenta are visualized by transabdominal sonography and the catheter is introduced through the cervix along the uterine wall to reach the placental tissue till it's distal end. The stylet is removed and 20 cc syringe containing 5 ml tissue culture fluid is attached to the catheter. Suction is applied as the catheter is gradually withdrawn.32 This allows placental tissue aspiration into the syringe. If material obtained is inadequate then another two attempts are permitted with fresh catheters.33 This route is rarely used because of higher miscarriage rate of 3.7% compared to midtrimester amniocentesis.18
 
Transabdominal Chorionic Villus Sampling
There are two techniques of transabdominal CVS. The two needle method recommended by Smidt Jensen and Hahnemann utilizes an outer needle as trocar to pierce the maternal skin, uterine myometrium and the placenta. A thinner needle is passed through it into the placental tissue. This technique allows reinsertion in case the tissue sample is inadequate and also prevents maternal contamination.34
The single needle technique proposed by Brambati35 is the commonly preferred method for chorionic villus sampling. A 20 G spinal needle is inserted percutaneously into the placenta under sonographic vision. The stylet is removed and contents are aspirated into a 20 ml syringe containing 5 ml of media. The needle is moved backwards and forwards in the placental tissue for a few times to obtain adequate amount of tissue(Fig. 1.5).20
zoom view
Figure 1.5: Chorionic villus sampling
The cytogenetic analysis is similar to amniocentesis. Chorionic villus has four different type of tissues—cytotrophoblast, mesenchyme, fetal blood vessels and fetal blood. Cytotrophoblast can be analysed by “rapid prep” between 24–48 hours because of presence of spontaneous mitosis in these cells. Mesenchymal cells need culturing and hence tissue analysis takes five to ten days time. But the result is more reliable. Sometimes there is a discrepancy between the placental and fetal tissue which could be either due to contamination by maternal cells or due to confined placental mosaicism. In confined placental mosaicism, the normal euploid chromosome is found along with abnormal cell line in the placental tissue, although the fetus may have normal karyotype. In such a situation amniocentesis is done to confirm fetal karyotype. Mosaicism is identified in 1% of CVS samples and the abnormal cell line is confirmed in 10–40% of these fetuses.32 The complication of these procedures is vaginal spotting, bleeding or leakage of fluid which occurs in 7–10% of cases performed transvaginally and 1% of cases performed transabdominally. Miscarriage rate is 2.5%. Procedure related fetal malformations like limb reduction defects and oromandibular hypoplasia is seen if CVS is performed below 9 weeks hence early CVS has been abandoned now.32
 
Fetal Blood Sampling
  1. Percutaneous umbilical blood sampling or cordocentesis.
  2. Intrahepatic vessel sampling.
  3. Fetal cardiac sampling.
21
 
Percutaneous Umbilical Blood Sampling or Cordocentesis
This procedure is done under real time ultrasonographic guidance. Pre- procedure antibiotics prophylaxis may be given and antenatal corticosteroid is administered in preterm fetuses 24 hours prior to the procedure. The free-hand technique of sampling uses a 20–22 G spinal needle of length around 15 cm to pierce the umbilical vein at relatively fixed segment of the cord near its insertion into the placenta. In this method the needle is free to move in all directions and hence the chances of injury to cord and other structures are more. In the other technique a fixed needle guide is attached to the ultrasound transducer through which a 22 to 25G needle is introduced. The predicted path of needle appears on screen and allows prior selection of sample site. Lateral movement of the needle is not possible.36,37 The most accessible cord loop is selected for puncture. In both methods umbilical vein is punctured and the blood obtained is tested for its fetal origin. The acid dilution test of Londersloot distinguishes maternal from fetal blood. When fetal blood is added to 0.1 M potassium hydroxide it turns pink, while if maternal blood is added it turns brown. In addition the mean corpuscular volume of fetal blood can be measured which is larger than maternal RBC.36 After confirming the fetal origin of blood the sample are put in tubes containing EDTA or heparin and sent for karyotyping. Postprocedure the puncture site is monitored for bleeding.
 
Fetal Intrahepatic Vessel Sampling
In this procedure intrahepatic vessels are sampled. This procedure is opted when cordocentesis is difficult to perform or is unsuccessful. It is rarely used nowadays. Fetal hepatic necrosis is a known complication.38
 
Fetal Cardiac Sampling
Fetal blood sampling by cardiac puncture is used when other methods fail to provide a sample. It is an easy procedure and is quite safe. This route can also be utilized for emergency fetal blood transfusion like in case of post procedural fetal bleed or for performing feticide.38
The results of karyotype analysis is available within a few days. Blood obtained from cordocentesis can also be analysed for fetal infection, fetal Rh D analysis, for assessment of fetal anemia and need for transfusion in Rh incompatibility, immunologic thrombocytopenias and coagulopathies.37,38 The complications are cord vessel bleeding (50%), cord hematoma (17%), feto-maternal hemorrhage (66%), intrauterine death (1.4%).3,37,38 Fetal loss is more when concurrent fetal pathology like growth restriction and hydrop fetalis is present.
 
Fetal Tissue Biopsy
Biopsy of fetal skin, liver and muscle is performed under sonographic or fetoscopic visualization. Tissue is analysed by electron microscopy or for 22biochemical analysis. Condition like muscular dystrophy, mitochondrial myopathy and skin disorders like epidermolysis bullosa can be diagnosed by this procedure.3,38
 
Fetoscopy
It is done through an endoscope which allows fetal inspection for structural anomalies and allows fetal blood sampling and tissue biopsy.
It is a technique of direct visualization of the fetus using a small bore fiberoptic endoscope. The trocar of the fetoscope measures 2.2 mm in diameter and the scope measures 1.7 mm in diameter.16 In the first trimester embryofetoscopy, a 0.8 mm fiberoptic endoscope and a 27G needle are passed through a 16G double barrel instrument sheath. It is passed transabdominally into the extracelomic or amniotic cavity. Study of morphology of the embryo-fetus and fetal blood sampling are it's major clinical applications.39 The drawback of fetoscopy is narrow field of vision and limited view of the small parts of the fetus because of short focal length. Scopes small enough to go through the shaft of a 20G needle have been developed but vision is hampered because of diminished intensity of light.16
 
Summary of Recommendations and Conclusions of ACOG Practice Bulletin No. 88 (2007)40
 
Invasive prenatal testing for aneuploidy
The following recommendation is based on good and consistent scientific evidence (Level A):
  • Early amniocentesis (at less than 15 weeks of gestation) should not be performed because of the higher risk of pregnancy loss and complications compared with traditional amniocentesis (15 weeks of gestation or later).
The following conclusions are based on limited or inconsistent scientific evidence (Level B):
  • Amniocentesis at 15 weeeks of gestation or later is safe procedure. The procedure-related loss rate after midtrimester amniocentesis is less than 1 in 300–500.
  • In experienced individuals and centers, CVS procedure-related loss rates may be the same as those for amniocentesis.
The following recommendations and conclusions are based primarily on consensus and expert opinion (Level C):
  • Invasive diagnostic testing for aneuploidy should be available to all women, regardless of maternal age.
  • Patients with an increased risk of fetal aneuploidy include women with a previous fetus or child with an autosomal trisomy or sex chromosome abnormality, one major or at least two minor fetal structural defects 23identified by ultrasonography, either parent with a chromosomal translocation or chromosomal inversion or parental aneuploidy.
  • Nondirective counseling before prenatal diagnostic testing does not require a patient to commit to pregnancy termination if the result is abnormal.39
 
Preimplantation Diagnosis
It is a technique of diagnosing genetic abnormalities before the pregnancy is formally established. It has been used in couples who have an age-related risk of aneuploidies or who are carriers of balanced translocations. It has also been used for diagnosis of X-linked disorder or single gene disorder.2,23
Preimplantation diagnosis is done during in vitro fertilization procedures wherein the gametes or the cells from an embryo are biopsied for genetic analysis. The oocyte is tested indirectly by analyzing the first polar body. It is removed by micromanipulation technique and subjected to DNA analysis or chromosomal study. If the first polar body is tested abnormal then the oocyte is presumed to be normal and vice versa. The disadvantage of this procedure is that it is an indirect analysis and it can be used only for maternal carriers. Furthermore recombination will occur if defective gene lies away from the centromere making result indeterminate. Contamination with the cumulus cells can lead to error in diagnosis.
Sperm sorting techniques using molecular probes tagged with laser activated dye has been used followed by separation techniques like flow cytometry. But this method can cause considerable damage to the sperm.16
The most successful method of preimplantation diagnosis is biopsy of single cell from embryo at the eight cell stage or blastocyst stage. The eight cell stage embryo have independent cells and a single cell can be removed without damage to the neighboring cells because at this stage, cells do not develop gap junctions and are less adherent. But the drawback of this technique is that the cells are large hence difficult to remove, contamination can occur by sperm or cumulus cells and there is short time for analysis of results since successful implantation depends on early transfer of embryo.16
Blastomere biopsy is performed 5 days following fertilization at the 120 cell stage. It contains mainly trophoblastic cells but the inner cell mass that forms the embryo is clearly demarcated at this stage. The cells are biopsied after mechanical disruption of the zona or after spontaneous hatching. The advantages are that more number of cells are available for analysis and they are well-differentiated. The disadvantage is that there is increased risk of damage to the inner cell mass due to greater adherence of the cells to each other and there is limited incubation time. The trophoectoderm may not always be similar to the fetus in karyotype and biochemical status. The role of pre-implantation diagnosis at present is limited.1624
 
KEY POINTS
  • Prenatal screening procedures are non-invasive methods whereas the diagnostic procedures are usually invasive, except for the detection of fetal DNA and fetal cells which is a noninvasive diagnostic procedure. The ACOG 2007 and ACMG 2007 guidelines recommends screening for all pregnant women.
  • The noninvasive prenatal screening procedures are a combination of maternal serum assays and fetal sonographic evaluation.
  • The first trimester combined screening measures fetal nuchal translucency and maternal serum markers PAPP-A and β hCG and in association with maternal age provides a risk estimate for trisomy 18 and 21.
  • The second trimester screening traditionally called the triple screening measures maternal serum alfa-fetoprotein, hCG, and unconjugated estriol and predicts relative risk of trisomy 18 and 21 in relation to the maternal age. Quadruple screening incorporates serum inhibin A levels in addition to the above three serum markers and has a better detection rate than triple screening.
  • Combined first and second trimester screening has higher detection rate than individual screening. A positive result has to be confirmed by a diagnostic invasive procedure. If a pregnant woman presents early in pregnancy for antenatal care, that is before 14 weeks of gestation than integrated or sequential or contingent screening should be offered while if she presents after 14 weeks than a second trimester screening procedures should be recommended.
  • Detection of fetal cells or DNA in maternal circulation can revolutionize the noninvasive methods of prenatal diagnosis but is still in the infancy stage.
  • Invasive methods of prenatal diagnosis like chorionic villus sampling, amniocentesis, percutaneous umbilical blood sampling, etc. provide embryonic or extraembryonic tissue for chromosomal, genetic, or biochemical analysis. The techniques used for analysis of fetal material are PCR, FISH, aCGH or karyotyping of cultured fetal cells. Mutation study on DNA and biochemical analysis is also possible. While rapid aneuploidy tests like FISH and QF-PCR provide an early report, they have higher rate of false-positive and negative results. Traditional karyotyping gives more accurate results.
  • Preimplantation diagnosis is an invasive method of diagnosis of fetal disease before the formal establishment of pregnancy. Hence it offers a choice to differentiate the healthy embryos from unhealthy ones and pick up the healthy embryos for embryo transfer.
25
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