Prenatal Diagnosis for Congenital Malformations and Genetic Disorders

Updated: Jul 10, 2017
  • Author: Teresa Marino, MD; Chief Editor: Ronald M Ramus, MD  more...
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Congenital abnormalities account for 20-25% of perinatal deaths. Many genetic disorders can be detected early in pregnancy using various noninvasive and invasive techniques. These techniques are outlined below.

Noninvasive techniques

Fetal visualization includes the following noninvasive modalities:

  • Ultrasound
  • Fetal echocardiography
  • Magnetic resonance imaging (MRI)
  • Radiography

Screening for neural tube defects (NTDs) involves measuring maternal serum alpha-fetoprotein (MSAFP).

Screening for fetal Down syndrome includes the following:

  • Nuchal Translucency Measurement
  • Measuring PAPP-A and unconjugated beta-human chorionic gonadotropin (B-HCG) in the first trimester
  • Measuring maternal serum alpha-fetoprotein, unconjugated estriol, beta-human chorionic gonadotropin (HCG) and inhibin between 15-22 weeks (Quadruple Test)

Other noninvasive techniques include the following:

  • Separation of fetal cells from the mother's blood, noninvasive prenatal screening using fetal cell-free DNA
  • Assessment of fetal-specific DNA methylation ratio [1]

Invasive techniques

Fetal visualization techniques that are invasive include the following:

  • Embryoscopy
  • Fetoscopy

Invasive fetal tissue sampling techniques include the following:

  • Amniocentesis
  • Chorionic villus sampling (CVS)
  • Percutaneous umbilical blood sampling (PUBS)
  • Percutaneous skin biopsy
  • Other organ biopsies, including muscle and liver biopsy

Preimplantation biopsy of blastocysts obtained by in vitro fertilization is an invasive technique.

Cytogenetic investigations that are invasive include the following:

  • Detection of chromosomal aberrations
  • Fluorescent in situ hybridization

Invasive molecular genetic techniques include the following:

  • Linkage analysis using microsatellite markers
  • Restriction fragment length polymorphisms (RFLPs)
  • Single nucleotide polymorphisms (SNPs) - DNA chip, dynamic allele-specific hybridization (DASH)

Noninvasive Techniques

Fetal visualization - Ultrasound

Ultrasound is a noninvasive procedure for imaging that can be performed transabdominally or transvaginally. It carries almost no risk to the fetus and the mother. The objective is to obtain information that will allow optimal antenatal care and outcomes for mother and fetus. [2] When performed in early gestation, ultrasound can identify an early pregnancy and importantly help identify an ectopic pregnancy or abnormal gestation. Ultrasound can evaluate gestational age, number of fetuses, fetal position and presentation; placental location; fetal growth, and many structural birth defects. It also can assess amniotic fluid volume and fetal well-being. Accurate assessment of gestational age is important to help assess fetal growth problems as well as assessing postterm pregnancies.

Color Doppler evaluation can also be applied to the umbilical artery (UA) to assess placental function and other fetal vessels including the middle cerebral artery. In high-risk pregnancies, particularly in the setting of intrauterine growth restriction, Doppler evaluation of the UA can be used to assess vascular impedance. In certain settings, including Rh and non-Rh isoimmunization or maternal parvovirus infection, the use of Doppler evaluation of the fetal middle cerebral artery (MCA) peak systolic velocity (PSV) is now the best tool for predicting fetuses at risk for fetal anemia. Previously, amniocentesis was performed for the detection of bilirubin, with each procedure carrying a small risk of complications. When MCA-PSV is >1.5 MoM for gestational age, fetal anemia is suspected and cordocentesis to assess fetal hemoglobin level as well as possible intrauterine transfusion are recommended (see section on cordocentesis). [3]

Many fetal organ systems and anatomical lesions, including some genitourinary, gastrointestinal, skeletal, and central nervous system abnormalities and congenital cardiopathies, can be visualized by ultrasound between 16-20 weeks' gestation. Ultrasound is used to guide invasive sampling such as amniocentesis, CVS, cordocentesis, and various fetal procedures. [2] Ultrasound can also detect soft markers which are frequently found among normal fetuses and likelihood ratios have been established for many of these soft markers. [4]

These soft markers include:

●Slightly shortened humerus

●Slightly shortened femur

●Echogenic intracardiac foci

●Echogenic bowel


●Hypoplastic or absent nasal bone

●Hypoplasia of the middle phalanx of the fifth digit


●Separation of the great toe (sandal gap toe)

●Widened iliac angle

●Short ear length

●Short frontal lobe

One of the largest evaluations of routine ultrasonographic examination was the Eurofetus study that analyzed the accuracy of a sonogram by a trained ultrasonographer on pregnant women at 19 to 22 weeks in unselected populations. This study was performed in 61 large European centers and found that the sensitivity for the detection of all anomalies was 56.2 %. Major anomalies were detected more frequently than minor abnormalities, (73.7 vs 45.7%) with detection of central nervous system abnormalities highest at 88.3%. Only 38.8 % of major cardiac abnormalities were detected. They concluded that overall, 44% of abnormalities (55% of severe abnormalities), were detected before 24 weeks gestation. [5]

Evaluation of the cervical length by transvaginal evaluation may help identify women at high risk for preterm delivery and allow for possible intervention including cerclage placement or supplemental progesterone. 

Fetal visualization - Fetal echocardiography

Fetal echocardiography can be performed at 15 weeks' gestation, however, optimal timing appears to be between 18-22 weeks. Despite this, some cardiac lesions are detected later in gestation.  When this technique is used with duplex or color flow Doppler, it can identify a number of major structural cardiac defects and rhythm disturbances. [6]  Structural congenital abnormalities remain a leading cause of infant mortality, and up to 50% of these are due to cardiac lesions. Identification of fetal arrhythmias may help identify high-risk pregnancies as transplacental medical therapy can improve the prognosis of some fetal arrhythmias. [7] Finally, identification of congenital heart abnormalities may be associated with reduced fetal morbidity, and delivery in a center that is capable of managing these congenital lesions may be arranged prior to delivery. Generally, mode of delivery is dependent on the usual obstetrical indications. 

Fetal echocardiography is recommended in cases where cardiac defects are suspected, including the following:

  • Identification of an extracardiac malformation/abnormality of another major organ system on routine ultrasound
  • Monochorionic diamniotic twins
  • Suspected genetic disease or fetal chromosome abnormality associated with heart defects
  • Exposure to potentially teratogenic agents
  • Family history of congenital heart defects, particularly in a parent or sibling
  • Maternal diseases, such as diabetes or phenylketonuria associated with fetal structural heart defects, in particular heart blocks, such as lupus or other immune disorders
  • Alcohol or drug consumption by mother during pregnancy
  • Maternal rubella infection during pregnancy

Fetal visualization - Magnetic Resonance Imaging

Magnetic resonance imaging (MRI) uses electromagnetic radio waves to generate detailed computer images. The possible direct biologic effects of MRI include induction of local electric fields and currents as well as radiofrequency radiation resulting in heating of tissue. There are however no reported harmful effects from MRI of the pregnant woman or fetus. [8]  Gadolinium is the contrast agent most commonly used for MRI. It crosses the placenta and is excreted by the fetus into the amniotic fluid, then swallowed such that it can be reabsorbed into the fetal circulation. Gadolinium potentially has a long half-life in the fetus and is associated with increased risk of still births and neonatal death as well as a wide-range of rheumatological, inflammatory, or infiltrative skin conditions. [8]  It is not recommended for use in the pregnant patient unless the benefit outweighs the potential risk to the fetus. After 20 weeks of gestation, MRI may play a complementary role to ultrasound. MRI can better identify and delineate some abnormalities, particularly abnormalities of the brain.  There are currently no known deleterious effects of fetal MRI on the developing fetus.

Fetal visualization - Radiography

Prenatal radiography has a very limited role. Due to the dangers of radiography to the fetus and the availability of other imaging options, this technique rarely is used. Superimposition of maternal and fetal bones also makes interpretation difficult. There remains a role postnatally in the setting of skeletal dysplasia where it can define characteristics of the skeleton. Although prenatal 3D-CT can provide more detailed images of spine and pelvic bone than ultrasound; the fetal radiation exposure is in the 3 mGy range and thus not used. 

Screening for Neural Tube Defects

The prevalence of neural tube defects varies worldwide which reflects the differences in genetic and environmental factors. ACOG recommends screening for open neural tube defects by ultrasound, maternal serum alpha-fetal protein or both. [9] Neural tube defects can also be associated with genetic syndromes (eg Meckel-Gruber syndrome).

Screening at risk patients for NTDs is recommended if the following are present:

  • Ultrasound findings suspicious for NTD
  • A previous child with NTDs 
  • A family history of NTDs exists, especially a mother with NTDs.
  • Type 1 diabetes mellitus during pregnancy.
  • Maternal exposure to drugs, such as valproic acid
  • Elevated level of MSAFP is present.
  • Obesity 
  • Race: MSAFP level is 10 to 20 percent higher in black women​

Measuring Maternal Serum Alpha-Fetoprotein

The developing fetus has 2 major blood proteins, albumin and alpha-fetoprotein (AFP), while adults have only albumin in their blood. The MSAFP level can be used to determine the AFP levels from the fetus. AFP is produced by the yolk sac and later by the liver; it enters the amniotic fluid and then the maternal serum via fetal urine.

In conditions such as open NTD (eg, anencephaly, spina bifida) and abdominal wall defects in the fetus, AFP diffuses rapidly from exposed fetal tissues into amniotic fluid, and the MSAFP level rises. However, the MSAFP levels also increase with gestational age, as such, incorrect dating or fetal demise are a cause of elevated MSAFP. Other causes of elevated MSAFP include maternal diabetes, multiple gestations, pregnancies complicated by bleeding, abnormal placentation or function (accreta or intrauterine growth restriction) as well as other fetal malformations (fetal sacral teratoma) and rarely maternal liver tumors.

The MSAFP test can be performed between 15-22 weeks' gestation. A combination of the MSAFP test and ultrasonography detects almost all cases of anencephaly and most cases of spina bifida. When 2.0 or 2.5 MoM is used, the American College of Medical Genetics and Genomics reported the detection rate for anencephaly is ≥95%. NTD can also be distinguished from other fetal defects, such as abdominal wall defects, by the use of an acetylcholinesterase test carried out on amniotic fluid obtained by amniocentesis. AFAFP and amniotic fluid acetylcholinesterase (AChE) are the primary biochemical tests performed on amniotic fluid for detection of NTDs. AChE is an enzyme contained in blood cells, muscle, and nerve tissue. An elevation of both AFAFP and AChE values suggests a fetal NTD with 96% accuracy; [10]  

An unexplained elevated MSAFP, with negative targeted ultrasound, may be a marker for pregnancies at increased risk of some complications, including fetal growth restriction, preeclampsia and demise. However, as the predictive value is low, there have not been recommendations regarding increased surveillance in pregnancy or improved outcomes secondary to increased surveillance. [11]

In cases where a low level of MSAFP is reported, it may be associated with Down syndrome, other chromosomal aneuploidy or failing pregnancies. [12, 13]

Screening for Fetal Down Syndrome

The quadruple test is usually performed at 15 to 18 weeks of gestation but can be done as late as 22 weeks. In 2012, the quadruple test was the most common Down syndrome screening test performed in the United States. However, there are several advantages to earlier assessment including maximum time for decision making and safer methods of termination. Early risk assessment includes 3 markers: one ultrasound and 2 biochemical:

The nuchal translucency measurement  

Maternal serum pregnancy associated plasma protein (PAPP-A)  

Maternal serum beta human chorionic gonadotropin (beta hCG)

The nuchal translucency (NT) is the normal fluid filled space behind the fetal neck. Fetal NT thickness is measured between 10 3/7 and 13 6/7 weeks gestation and is increased in fetuses with Down syndrome. [14] As a screening test, it is weaker as gestational age increases from 10 to 13 weeks and the cut-off for abnormal values are in the 95-99th percentile based on gestational age. Without biochemical markers, the sensitivity of NT is lower.  

PAPP-A is a complex, high molecular weight glycoprotein with levels that are lower in pregnancies affected with fetal Down syndrome and as a marker, decreases with increasing gestational age between 9 and 13 weeks. In contrast, B-hCG levels on average will double in pregnancies at risk for Down syndrome. Two other markers have been studied including placental growth factor (PlGF) and alpha-fetoprotein (AFP).  PIGF and AFP levels are lower in pregnancies affected with fetal Down syndrome. These are currently not clinically used the addition of these 2 markers may increase detection rate to 90% however the false positive rate is also increased to 20%. [15, 16, 17]

There have been several ways to offer screening including the full integrated test which consists of nuchal translucency and PAPP-A measured at 10 to 13 weeks followed by measurement of AFP, uE3, hCG, and inhibin at 15 to 18 weeks as well as serum integrated test which includes all of the tests of the full integrated test (PAPP-A, AFP, uE3, beta-hCG, inhibin), but no ultrasound marker (ie, no nuchal translucency). There are also different ways to offer testing including contingency testing, where if the first trimester screen is positive, women are offered invasive testing whereas if the first trimester test is negative, they can opt to complete second trimester screening or not have any other testing.

Measuring maternal unconjugated estriol

The amount of estriol in maternal serum depends upon viable fetus, a properly functioning placenta, and on maternal well-being. Fetal adrenal glands produce dehydroepiandrosterone (DHEA) that gets metabolized to estriol in the placenta. Estriol crosses to the maternal circulation and is excreted either by maternal kidney in urine or by maternal liver in the bile. A low level of estriol can be an indicator of Down syndrome, adrenal hyperplasia with anencephaly, [18, 19]  as well as Smith-Lemli-Opitz syndrome (SLOS) which is an autosomal recessive defect in a cholesterol biosynthetic enzyme, C7-reductase. SLOS is manifested by intellectual disability, poor growth, and phenotypic abnormalities. A very low uE3 level (median 0.21 MoM) is noted because the steroid precursors required for estriol synthesis in the fetus are defective. [20]

Measuring maternal serum beta-human chorionic gonadotropin

Following conception and implantation of the developing embryo into the uterus, the trophoblasts produce enough beta-HCG, which is an indication for pregnancy. In the middle to late second trimester, the level of beta-HCG also can be used in conjunction with other biomarker level to screen for chromosomal abnormalities. An increased beta-HCG level coupled with a decreased MSAFP level suggests Down syndrome. [13, 21] The beta-HCG level also can be quantified in serum from maternal blood, and, if its amount is found to be lower than expected, it indicates abortion or ectopic pregnancy. If the level of HCG is estimated to be considerably high, then it indicates the possibility of trophoblastic diseases.

Measuring maternal inhibin-A levels

The hormone inhibin is secreted by the placenta and the corpus luteum. Inhibin-A can be measured in maternal serum. An increased level of inhibin-A is linked with an increased risk for trisomy 21. A high inhibin-A level may also be associated with a risk for adverse perinatal outcome including preterm delivery and fetal growth restriction.

Screening for fetal Down syndrome - Cell-free fetal DNA

Non-invasive prenatal screening uses next-generation sequencing of cell-free DNA (cfDNA) in the maternal circulation. Circulating cfDNA is derived from both the mother and the fetal-placenta unit and is highly fragmented. Cell-free fetal DNA and RNA can be extracted from maternal blood around 7 weeks’ gestation, which can be used to screen for Down syndrome, as well as other trisomies (18 and 13), and common sex chromosome aneuploidies (45,X; 47,XXX; 47,XXY; 47,XYY). The test can identify 98-99% of affected pregnancies. [22, 23] Sex determination for families with inherited sex-linked diseases, diagnosis of certain single gene disorders, and blood Rhesus factor status (in the case of Rhesus D-negative mothers) can also be performed using cell-free fetal nucleic acids from the placenta. [3]  

The test is best drawn after 10 weeks to allow the cell fraction to increase to at least 4% of the total fetal cell fraction. As such in obesity, as maternal weight increases, the fetal fraction may be low leading to a non-result. Factors that may increase the risk of false positive include; demise of one fetus, confined placental mosaicism, maternal mosaicism, or maternal cancer. If results are not reported, indeterminate, or uninterpretable from cell-free DNA screening, women should receive further genetic counseling, ultrasound evaluation and diagnostic testing. There appears to be an increased risk of aneuploidy. This test does not replace invasive testing like CVS or amniocentesis as it is limited in its ability to identify all chromosome abnormalities. Women should also be counseled that cell free DNA testing does not eliminate the risk of a structural congenital abnormality and these patients should still be offered ultrasound and MSAFP. [24]  Use of cfDNA to screen for other aneuploidies and sex chromosomes is technically possible, but not recommended.


Invasive Techniques

Fetal visualization - Fetoscopy

Fetoscopy can be performed during the second trimester. In this technique, a fine-caliber endoscope is inserted into the amniotic cavity through a small maternal abdominal incision, under sterile conditions and ultrasound guidance, for the visualization of the embryo to detect the presence of subtle structural abnormalities. It also is used for fetal blood and tissue sampling. Fetoscopy is associated with a 3-5% risk of miscarriage. In modern obstetrics, it is used in the treatment of twin to twin transfusion syndrome where laser is used to coagulate anastomotic vessels. Twin to twin transfusion syndrome is divided into stages. [25] For Quintero stages II to IV, fetoscopic laser ablation of placental anastomoses is the preferred procedure for definitive treatment between 16 and 26 weeks of gestation. 

Laser energy (20 to 40 watts from a diode or YAG laser) is applied through a 400 to 600-micron quartz fiber. This is sleeved through an operating channel in the fetoscope and a second channel is inserted for continuous irrigation.  The anastomotic vessels are then coagulated in a method called sequential selective laser photocoagulation which starts with Arteriovenous (AV, donor artery to recipient vein), then venous-arterial (VA, donor vein to recipient artery), then arterial-arterial (AA) and venous-venous (VV) anastomoses. This sequential selective procedure has been associated with a 40-50% decrease in intrauterine fetal demise of the donor twin than previous non-sequential procedure. [26] Complications of the procedure include preterm labor, rupture of the membrane between fetuses creating a monochorionic-monoamniotic gestation, premature rupture of membranes, placental abruption and twin anemia polycythemia sequence. Fetoscopic laser therapy can also have implications in the management of other fetal pathologies such as chorioangiomas, amniotic band syndrome and sacrococcygeal teratoma. [27]

Fetal tissue sampling - Amniocentesis

Amniocentesis is an invasive, well-established, safe, reliable, and accurate procedure usually performed at 15-18 weeks, but can be performed any time in gestation after 15 weeks. Prior to 15 weeks, there has been an increased risk of loss and fetal clubbed foot. It is performed under ultrasound guidance. A 22-gauge needle is passed through the mother's abdomen through the uterus, into the amniotic cavity.  About 10-20 mL of amniotic fluid that contains cells from amnion, fetal skin, fetal lungs, and urinary tract epithelium are collected. These cells are grown in culture for chromosomal, biochemical, and molecular biologic analyses. Supernatant amniotic fluid is used for the measurement of substances, such as amniotic fluid AFP, hormones, and enzymes. Karyotype can detect chromosomal changes as small as 5-10 Mb as well as balanced translocations or inversions. The use of chromosomal microarray (CMA) has increased as it can detect smaller (10-100 Kb) gains and losses of genetic material that would not be detected by traditional karyotype. Another advantage of CMA is that it does not require cell culture, allowing availability of results in shorter time frames.

The results of cytogenetic and biochemical studies on amniotic cell cultures are more than 90% accurate. In the third trimester of pregnancy, the amniotic fluid can be analyzed for determination of fetal lung maturity. Risks with amniocentesis are rare but include 0.5% fetal loss and maternal Rh sensitization, as such women with Rh negative blood type should receive RhoGAM post procedure.  


Fetal tissue sampling - Chorionic villus sampling

The choice of chorionic villi sampling versus amniocentesis is personal as they essentially provide the same genetic information. CVS is performed very early in gestation between 9-12 weeks, ideally at 10 weeks' gestation. A catheter is passed through the cervix or through the abdominal wall into the uterus under ultrasound guidance, usually at a tertiary care facility and a sample of chorionic villi surrounding the sac is obtained. The number of procedures that should be carried out annually to maintain competency is unclear. The villi are dissected from the decidual tissue, and chromosome analysis is carried out on these cells to determine the karyotype of the fetus (see image below).

Prenatal diagnosis for congenital malformations an Prenatal diagnosis for congenital malformations and genetic disorders. Karyotype showing normal male chromosomal constitution (46, XY).

DNA can be extracted from these cells for molecular analysis. DNA analysis of CVS specimens is helpful for early diagnosis of diseases such as hemoglobinopathies. [28] In addition, tissue culture can be initiated on these cells for further studies. Fetal DNA from both villi or amniocytes can also be tested for specific genetic conditions. Single gene testing and other genetic conditions in the prenatal period often relies on a positive family history or a previously identified mutation, thus parental blood samples are often required for confirmatory testing.

The major advantage of CVS over amniocentesis is its use in earlier in pregnancy. Abnormalities can be identified at an early stage, and more acceptable decisions about termination of the pregnancy can be taken. Abortion is also much safer at this early stage. A disadvantage of CVS as compared to amniocentesis is a 1-2% risk of miscarriage, and, rarely, CVS can result with limb defects in the fetus. [29] Maternal sensitization is possible, and known maternal alloimmunization is a relative contraindication as it may result in more severe disease. A higher rate of maternal cell contamination and confined placental mosaicism with CVS may result in diagnostic ambiguity, leading to the need for additional invasive diagnostic tests. [30]   

Fetal tissue sampling - Percutaneous umbilical blood sampling

Cordocentesis (PUBS) is a method for fetal blood sampling. [31] A needle is inserted into the umbilical cord under ultrasound guidance, and fetal blood is collected from the umbilical vein for chromosome analysis , genetic diagnosis. infection and fetal blood cell counts.  An advantage of PUBS is the rapid rate at which lymphocytes grow, allowing prompt genetic diagnosis. The disadvantage of the procedure is the higher fetal loss rate and need to experienced operator. Other possible complications include hemorrhage from puncture site, cord hematoma, fetomaternal hemorrhage, transient bradycardia and possible vertical transmission of maternal infections such as hepatitis C and HIV. The vertical transmission risk is likely low and related to maternal viral load. 

Evaluation of amniocytes, chorionic villi, or maternal blood can often provide similar information as fetal blood, as such fetal blood sampling should be limited to clinical scenarios where amniocentesis or CVS do not provide the information or are not timely enough. One of the most common indications for PUBS is evaluation of fetal anemia secondary to isoimmunization or parvovirus infection.  Fetal blood is obtained for hemoglobin determination and intrauterine fetal transfusion performed only if fetal hemoglobin is more than two standard deviations below the mean value for gestational age (reference values available). The procedure s generally limited to pregnancies between 18 and 35 weeks of gestation. Prior to 18 weeks the small size of the umbilical cord makes the procedure technically challenging and after 35 weeks, it is considered riskier than delivery. [32]


Fetal tissue sampling - Percutaneous skin biopsy

To prenatally diagnose a number of serious skin disorders, such as anhidrotic ectodermal dysplasia, epidermolysis bullosa letalis, epidermolysis bullosa dystrophica, hypohidrotic ectodermal dysplasia, oculocutaneous albinism, and genetic forms of ichthyosis, percutaneous fetal skin biopsies are taken under ultrasonic guidance between 17-20 weeks' gestation.

Fetal tissue sampling - Other organ biopsies, including liver and muscle biopsy

Case reports have described fetal liver biopsy to diagnose an inborn error of metabolism, such as ornithine transcarbamylase deficiency, [33] glucose-6-phosphatase deficiency, [34] glycogen storage disease type IA, nonketotic hyperglycemia, [35] and carbamoyl-phosphate synthetase deficiency. [36]  Fetal muscle biopsy has also been described to analyze the muscle fibers histochemically for prenatal diagnosis of Becker-Duchenne muscular dystrophy. [37] . These are not routinely used nor readily available outside specialized centers. 

Fetal tissue sampling - Preimplantation biopsy of blastocysts obtained by in vitro fertilization

Techniques are being developed to test cells obtained from biopsy of early cleavage stages or blastocysts of pregnancies conceived through in vitro fertilization. [38] These techniques will be helpful for selective transfer and implantation of those pregnancies into the uterus that are not affected by a specific genetic disorder. 


Cytogenetic Investigations

Detection of chromosomal aberrations

Chromosomal aberrations, such as deletions, duplications, translocations, and inversions diagnosed in affected parents or siblings, can be detected prenatally in a fetus by chromosomal analysis (see image below).

Prenatal diagnosis for congenital malformations an Prenatal diagnosis for congenital malformations and genetic disorders. Karyotype showing trisomy 21 (47, XY, +21) in a male.

This analysis can be undertaken on fetal cells obtained through such techniques as amniocentesis and CVS.

Fluorescent in situ hybridization(FISH)

FISH uses different fluorescent-labeled probes, which are single-stranded DNA conjugated with fluorescent dyes and are specific to regions of individual chromosomes. These probes hybridize with complementary target DNA sequences [39] in the genome and can detect chromosomal abnormalities, such as trisomies, [40] monosomies, and duplications. This technique allows counting of the number and location of large pieces of chromosomes and increased the sensitivity, specificity, and resolution of chromosome analyses. FISH can be performed on metaphase chromosomes or interphase nuclei and is technically straightforward. [41]

Three types of DNA probes are used in FISH analysis. Whole chromosome probes are specific to a whole chromosome or a chromosome segment and are applied to metaphase spread for the identification of translocations or aneuploidy. Repetitive probes, such as alpha satellite sequences located in the centromeric regions of human chromosomes, are used in the identification of marker chromosomes and aneuploidy. Unique sequence probes are single clones or a series of overlapping clones corresponding to a specific gene or a confined region of a chromosome that do not contain major repetitive sequences and are used for the identification of specific translocation events in cancer [42] and for the detection of submicroscopic deletions. [43]  Some of the advantages of FISH include that its resolution is much better than traditional chromosome banding ( 2 megabases (Mb) in length vs 6 Mb for chromosomal banding), it can be applied to both dividing (metaphase) and non-dividing (interphase) cells and FISH can identify many structural abnormalities including deletions, duplications, aneuploidy and the structurally rearranged chromosomes. However, disadvantages do exist in that small mutations, such as small deletions, insertions as well as point mutations, cannot be identified. Uniparental disomy (inheritance of both copies of a chromosome from the same parent) will also be missed. Chromosomal inversions will not be detected as the probe can only detect the presence of a specific sequence not its precise location within the chromosome. 

Microarray comparative genomic hybridization

Recently, array-CGH (microarray comparative genomic hybridization) also referred to as chromosomal microarray analysis (CMA), is considered to be useful in detecting genomic imbalance in the fetus (duplications/deletions, see above in amniocentesis). A 2013 meta-analysis showed that in fetuses presenting with structural abnormality (referral for structural abnormality) noted on ultrasound and a normal G-banded karyotype, CMA was abnormal in 7.2%. [44]

CMA is also useful in evaluation of stillbirth (pregnancy loss ≥20 weeks of gestation) because both chromosomal abnormalities and culture failure are common in these cases. Culture failure is common when the fetus has died, and thus prevents the accurate diagnosis of a karyotypic abnormality in these cases.

Possible prenatal CMA indications after a normal G-band karyotype or instead of conventional karyotyping include cases of intrauterine growth restriction (IUGR), increased nuchal translucency, stillbirth and positive cell-free DNA screen for a microdeletion. Non-invasive prenatal screening with cell-free DNA can identify fetal genomic microdeletions; however, sensitivity and specificity remains unknown. Expanded panels, including screening for common microdeletion syndromes such as 22q11.2 deletion (DiGeorge syndrome) are available. [45]


Molecular Genetic Techniques


Molecular genetic techniques are being used for prenatal diagnosis. [46] These techniques are based upon the fact that DNA complement is generally identical in every cell of the body; therefore, any hereditary defect diagnosed at the DNA level will be present in nucleated cells from that individual. For molecular analysis, DNA is extracted from amniocytes, chorionic villi, or fetal blood cells. Then, it is amplified by PCR and is used for the diagnosis of genetic mutations or deletions within a gene that causes a specific genetic disease. The following molecular biologic techniques can be used for prenatal diagnosis of different diseases.

Linkage analysis by microsatellite markers

Microsatellites are short tandem repeats of 2-6 base pairs that are highly polymorphic and are distributed throughout the genome. This form of polymorphism is inherited in a mendelian codominant manner. For linkage analysis, primers for regions flanking the repeat sequences are designed and used to amplify these microsatellites by PCR, initially for candidate gene regions and on their exclusion for whole genome analysis.

On gel electrophoresis, the genotype of different individuals in the family indicating 2 alleles for each microsatellite marker is established, and haplotypes are constructed with the analyzed markers. Cosegregation of a particular allele of any of these analyzed markers with the disease phenotype, in all the affected but in none of the unaffected individuals, indicates the probability of linkage with that marker at that particular locus, which is confirmed statistically by calculating the LOD scores. A LOD score value of greater than 3 indicates linkage of that particular marker with the disease locus in that family. In informative families affected with a disease, linkage can be confirmed by LOD score and haplotype analysis. Segregation of a particular allele linked with disease phenotype also can be tested in the fetus by haplotype analysis (see image below).

Prenatal diagnosis for congenital malformations an Prenatal diagnosis for congenital malformations and genetic disorders. Segregation of haplotypes for 10 markers (M1-M10) in a family. Diseased haplotype, as indicated by red bars, is shared by all of the affected individuals (filled circles and squares) and by none of the unaffected individuals (unfilled circles and squares).

Carter et al [47] identified an intragenic polymorphic marker linked with human CP49 gene (that codes for intermediate filament protein in lens fiber cells) on chromosome 3 at band 3q21-22 for the genetic linkage analysis of autosomal dominant congenital cataract. Toudjarska et al [48] demonstrated molecular diagnosis of Marfan syndrome by linkage analysis.

Restriction fragment length polymorphism

In the human genome, variations are common and reportedly occur approximately once every 200 base pairs. These single base pair differences in DNA nucleotide sequences are inherited in a mendelian codominant manner. Restriction endonucleases are the enzymes that recognize and cut DNA within a specific base sequence recognition site. If a difference occurs in the DNA sequence within the recognition sequence of a restriction enzyme, it results in fragments of different size by that restriction enzyme. This difference is recognized by the altered mobility of the restriction fragments on gel electrophoresis, which is known as RFLP (see image below). This technique is used to detect deletions within the gene and DNA polymorphisms and to identify mutant genes and mutations at hot spots.

Prenatal diagnosis for congenital malformations an Prenatal diagnosis for congenital malformations and genetic disorders. Pedigree (A) with RFLP analysis (B) with restriction enzyme BfaI. Due to sequence alteration, on restriction analysis affected individuals (4, 10, 14, 21) show 2 bands, whereas unaffected individuals (1, 2, 3, 9, 22) have only 1 undigested fragment.

Churchill et al [49] performed prenatal diagnosis in a familial case of aniridia by extracting DNA from cultured fibroblasts obtained through amniocentesis, RFLP with restriction enzyme Ava1, and electrophoresis by single-strand confirmation polymorphism to screen the PAX6 gene.

Single nucleotide polymorphisms

SNPs are single base differences in the genome of an individual, which occur about every 1000 bases. A single-nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide adenine (A), thymine (T), cytosine (C), or guanine (G]) in the genome differs between paired chromosomes in an individual. Each SNP has 2 alleles; they can be used for linkage analysis to carry out fine mapping of regions on the chromosomes and to study mutations in the genes. The advantages of SNPs are their abundant numbers, and they can be typed by oligonucleotide hybridization assay, without gel electrophoresis. Two methods are available for oligonucleotide hybridization assay, DNA chip and DASH.

DNA chip

A DNA chip is a wafer of silicon, usually 2 cm3 or less in area, and carries many different oligonucleotides (short single-stranded DNA molecule less than 50 nucleotides in length synthesized artificially in a test tube) in a high-density array. The DNA to be analyzed is labeled with a fluorescent marker and is pipetted onto the surface of the chip. Hybridization of labeled DNA is detected by examining the chip with a fluorescent microscope. The position where the hybridization signal is emitted indicates which oligonucleotide has hybridized with the test DNA. If there is a single mismatch at a single position within the oligonucleotide, that mismatch does not form a base pair, and hybridization does not occur. In this way, oligonucleotide hybridization discriminates between the 2 alleles of a SNP.

Dynamic allele-specific hybridization

In this technique, hybridization takes place in solution, in 1 of the 96 well microtiter tray. Hybridization is detected by a fluorescent marker that binds only to double-stranded DNA and emits a signal on hybridization. Initially, hybridization is carried out under conditions that allow mismatched hybrids to form, and, at this stage, oligonucleotides and the test DNA hybridize regardless of which SNP allele is contained by the DNA. By raising the temperature, the mismatched hybrids, which are less stable as compared to complete hybrids, break down. Detecting which allele is present in the test DNA can be determined from the temperature at which the hybridization-dependent fluorescent signal disappears.

Currently, SNPs can be used for the molecular genetic analysis of many eye disorders, such as congenital cataract, myopia, Marfan syndrome, and glaucoma.


Prerequisites of Prenatal Diagnosis

Prenatal testing and/or diagnosis is offered to all couples, whether it involves prenatal serum screening, cf-DNA, ultrasound or invasive procedures. Some common indications include:

  • Advanced maternal age defined as age 35 at time of delivery
  • A previous child with a chromosomal abnormality.
  • The couple is known to be carriers of a chromosomal translocation.
  • The pregnant woman is affected with type 1 diabetes mellitus, epilepsy, or myotonic dystrophy.
  • Exposure to viral infections, such as rubella or cytomegalovirus.
  • The mother is exposed to excessive medication or to environmental hazards.
  • In her or her spouse's family, a history of Down syndrome or some other chromosomal abnormality is present.
  • A history of single gene disorder is present in her or her spouse's family.
  • Her male relatives have Duchenne muscular dystrophy, severe hemophilia or intellectual deficiency (Fragile X).
  • She is suspected of an X-linked chromosomal abnormality
  • The fetus is diagnosed in utero to have some hereditary error of metabolism.
  • The fetus is detected to be at increased risk for a NTD or other structural or multiple structural abnormalities.

Benefits of Prenatal Diagnosis

An offer of prenatal screening or diagnosis provides prospective mothers and couples the option of choosing or declining to receive genetic information pertinent to their personal situation prior to conception. [50]

After conception, prenatal diagnosis provides various benefits. Prenatal diagnosis determines the outcome of pregnancy and identifies possible complications that can arise during pregnancy and birth. It can be helpful in improving the outcome of pregnancy using fetal treatment. Screening can help couples determine whether to continue the pregnancy and prepares couples for the birth of a child with an abnormality.