Prenatal Diagnosis for Congenital Malformations and Genetic Disorders

Updated: Feb 17, 2021
  • Author: Teresa Marino, MD; Chief Editor: Ronald M Ramus, MD  more...
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Practice Essentials

Prenatal testing and/or diagnosis is offered to all couples, who may choose prenatal serum screening, noninvasive prenatal testing, ultrasonography, or invasive procedures. Patients are initially counseled on the basis of their age and genetic and family history. The American College of Obstetricians and Gynecologists (ACOG) has recommended that serum screening, cell-free DNA screening, and diagnostic tests, such as chorionic villus sampling (CVS) and amniocentesis, be discussed with and offered to all women early in pregnancy, regardless of their age. [1]

Benefits of prenatal diagnosis

Preconception prenatal screening provides prospective parents with the option of choosing or declining to receive genetic information pertinent to their personal situation prior to planning a pregnancy. [2]

Structural abnormalities occur in approximately 3% of live births, and congenital abnormalities account for 20-25% of perinatal deaths. After conception, prenatal diagnosis can help determine the outcome of a pregnancy and identifies possible complications that can arise during pregnancy and birth. The detection of prenatal structural anomalies should lead to further genetic evaluation so that many of these conditions can be identified before birth. Prenatal diagnosis can be helpful in improving the outcome of the pregnancy, by using fetal treatment or by planning delivery in a tertiary care center. Screening may also help couples determine whether to continue the pregnancy, or it can prepare them for the birth of a child with an abnormality.

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:

  • Ultrasonography

  • Fetal echocardiography

  • Magnetic resonance imaging (MRI)

  • Radiography (post mortem)

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 pregnancy associated plasma protein (PAPP-A) and unconjugated beta-human chorionic gonadotropin (β-HCG) in the first trimester

  • Measuring maternal serum alpha-fetoprotein, unconjugated estriol, beta-HCG, and inhibin between 15 and 22 weeks (Quadruple Test)

  • Separation of fetal cells from the mother's blood, noninvasive prenatal screening using fetal cell-free DNA

  • Assessment of fetal-specific DNA methylation ratio [3]

Invasive techniques

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 fetal tissue sampling techniques include the following:

  • Amniocentesis

  • CVS

  • Percutaneous umbilical blood sampling (PUBS)

  • Percutaneous skin biopsy

  • Other organ biopsies, including muscle and liver biopsy

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)

Fetal visualization techniques that are invasive include the following:

  • Embryoscopy

  • Fetoscopy


Noninvasive Techniques

Fetal visualization - Ultrasound

Ultrasonography is a noninvasive imaging modality 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 the mother and the fetus. [4] Early first trimester ultrasonography can identify viability, gestational age, location of the pregnancy (eg, an ectopic pregnancy), abnormal gestation (eg, a molar pregnancy), and number of fetuses. In addition, the main objective of first trimester ultrasonography between 11 and 13 6/7 weeks’ gestation is to screen for aneuploidy using the nuchal translucency and other markers (eg, nasal bone, ductus venous flow, fetal heart rate, and tricuspid valve flow). With advancing ultrasound technology, transabdominal and transvaginal ultrasonography has been used for the detection of congenital abnormalities prior to 14 weeks' gestation, including cystic hygroma, abdominal wall defects, major cardiac and chest abnormalities, and skeletal defects. [5]  Accurate assessment of gestational age is important to help evaluate fetal growth abnormalities as well as to assess post-term pregnancies.

Second trimester ultrasonography should be offered to all patients, between 18 and 22 weeks' gestation, for the detection of fetal structural abnormalities. Many fetal anatomical lesions, including (not limited to) cardiac, genitourinary, gastrointestinal, skeletal, facial, and central nervous system abnormalities, can be visualized by ultrasound when performed between 16 and 20 weeks' gestation. Ultrasonography in the third trimester is also helpful to assess fetal position and presentation, placental location, fetal growth, amniotic fluid volume, and fetal well-being.

The value of ultrasonography in the third trimester has been questioned. In a prospective study of over 52,000 women who had first trimester and second trimester ultrasound examinations and returned at 35-37 weeks' gestation, the incidence of detection of a new abnormality was 0.5%. Some abnormalities may not be present at the time of a second trimester ultrasound examination, including some cardiac lesions, duodenal atresia, and achondroplasia. [6]

Ultrasound can also detect soft markers, which are frequently found in normal fetuses but may increase the risk of aneuploidy. Likelihood ratios have been established for many of these soft markers. [7]

These soft markers include the following:

  • Slightly shortened humerus
  • Slightly shortened femur
  • Echogenic intracardiac foci
  • Echogenic bowel
  • Pyelectasis
  • Hypoplastic or absent nasal bone
  • Hypoplasia of the middle phalanx of the fifth digit
  • Clinodactyly
  • Separation of the great toe (sandal gap toe)
  • Choroid plexus cysts

Color Doppler evaluation can 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 fetal growth restriction, Doppler evaluation of the UA can be used to assess vascular impedance and guide clinical management. 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 the best tool for predicting the risk of fetal anemia. Previously, amniocentesis was performed for the detection of bilirubin, with each procedure carrying a small risk of complications. When the MCA-PSV is >1.5 MoM for gestational age, fetal anemia is suspected and cordocentesis to assess fetal hemoglobin level and possibly perform an intrauterine transfusion is recommended (see section on cordocentesis). [8]

Ultrasound is used to guide invasive sampling techniques such as amniocentesis, CVS, cordocentesis, and various fetal procedures. [4]

Transvaginal ultrasonography is the gold standard for evaluating placenta location when placenta previa is suspected. When combined with color Doppler evaluation, transvaginal ultrasonography can establish the diagnosis of vasa previa prior to the onset of labor or the rupture of membranes. Evaluation of the cervical length by transvaginal evaluation, until 24 weeks' gestation, 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

Structural congenital abnormalities remain a leading cause of infant mortality. Optimal timing of fetal echocardiography appears to be between 18 and 22 weeks; however, it can be performed at an earlier gestational age. When this technique is used with duplex or color flow Doppler, it can identify a number of major structural cardiac defects and rhythm disturbances. [9]  Identification of fetal arrhythmias may help identify high-risk pregnancies, as transplacental medical therapy can improve the prognosis of some fetal arrhythmias. [10] Finally, identification of congenital heart abnormalities may be associated with reduced fetal morbidity and allow for delivery in a medical center capable of managing these congenital lesions. Not all cardiac defects, however, are detected at this gestational age, as some may appear later in gestation.

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

  • Enlarged nuchal translucency

  • Conception through in vitro fertilization

  • Identification of an extracardiac abnormality detected on routine ultrasound

  • Monochorionic diamniotic twins

  • Suspected genetic disease or fetal chromosome abnormality detected by cell-free DNA screening or invasive genetic testing

  • Exposure to potentially teratogenic agents (eg, paroxetine)

  • Family history of congenital heart defects, particularly in the parent or a previous child

  • Maternal diseases, such as pregestational diabetes, phenylketonuria, lupus, or Sjogren syndrome, in which the presence of antibodies such as SSA and SSB can lead to heart block

  • Maternal rubella infection during early 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 on the pregnant woman or fetus. [11]

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 an increased risk of stillbirths and neonatal death, as well as a wide range of rheumatologic, inflammatory, and infiltrative skin conditions. [11]  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. The added value of MRI in the diagnosis of placenta accrete spectrum remains controversial.

Fetal visualization - Radiography

Prenatal radiography has a very limited role. Owing 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 3-dimensional computed tomography can provide more detailed images of the spine and pelvic bones than ultrasonography, the fetal radiation exposure is in the 3 mGy range and thus is not used. 

Screening for neural tube defects

The prevalence of neural tube defects (NTDs) varies worldwide, which reflects differences in genetic and environmental factors. ACOG recommends screening for open NTDs by ultrasound, with or without maternal serum alpha-fetoprotein (MSAFP) measurement. [12, 13] NTDs 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, especially a mother with NTDs

  • Type 1 diabetes mellitus during pregnancy

  • Maternal exposure to drugs, such as valproic acid

  • Elevated level of MSAFP

  • Race: MSAFP levels are 10-20% 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.

MSAFP levels increase with gestational age; as such, incorrect dating or fetal demise may result in an elevated MSAFP level. 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. Other causes of elevated MSAFP include maternal diabetes, multiple gestations, pregnancies complicated by bleeding, abnormal placentation or function (placenta accrete spectrum or fetal growth restriction), as well as other fetal malformations (fetal sacral teratoma) and rarely maternal liver tumors.

The MSAFP test can be performed between 15 and 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. [14]  

An unexplained elevated MSAFP, with negative targeted ultrasound, may be a marker for pregnancies at increased risk for perinatal morbidity, including fetal growth restriction, preeclampsia, and fetal demise. As the predictive value is low, there have not been recommendations regarding increased surveillance in pregnancy or improved outcomes secondary to increased surveillance. [15]

A low MSAFP may be associated with Down syndrome, other chromosomal aneuploidy, or failing pregnancies. [16, 17]

Screening for fetal chromosomal abnormalities

In 2012, the quadruple test was the most common Down syndrome screening test performed in the United States. The quadruple test is usually performed at 15-18 weeks of gestation but can be done as late as 22 weeks. There are several advantages to earlier assessment, including maximum time for decision making, obtaining genetic results, 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, when the crown-rump length is between 38 and 84 mm. An enlarged NT is defined as a space that is greater than the 99th percentile for gestational age. An enlarged NT, as a screening test, is associated with an increased risk of aneuploidy and structural abnormalities, including congenital heart defects. [18] When the NT is enlarged, genetic counseling and diagnostic testing should be offered. Regardless of the choice of testing, a second trimester ultrasound examination to rule out fetal structural abnormalities should be offered, as well as a fetal echocardiogram. [1]   

When used alone to modify the risk of age-related trisomy 21, the NT detection rate is approximately 70%. PAPP-A is a complex, high molecular weight glycoprotein with levels that are lower in pregnancies affected with fetal trisomy 21. As a marker, PAPP-A decreases with increasing gestational age between 9 and 13 weeks. Low PAPP-A in the first trimester has been associated with fetal loss, abruption, preeclampsia, and fetal growth restriction. In contrast, beta-hCG levels on average will double in pregnancies at risk for trisomy 21.

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 the quadruple test (AFP, uE3, hCG, and inhibin) at 15 to 18 weeks and one result given. The serum integrated test includes all of the tests of the full integrated test (PAPP-A, AFP, uE3, beta-hCG, inhibin) but no nuchal translucency. Sequential screening involves receiving the first trimester results, then opting for second trimester testing and obtaining the results separately. Finally, there is 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. [1]

Measuring maternal unconjugated estriol

The amount of estriol in maternal serum depends upon a viable fetus, a properly functioning placenta, and maternal well-being. Fetal adrenal glands produce dehydroepiandrosterone (DHEA) that is metabolized to estriol in the placenta. Estriol crosses to the maternal circulation and is excreted either by the maternal kidney in urine or by the maternal liver in the bile. A low level of estriol can be an indicator of aneuploidy, adrenal hyperplasia with anencephaly, [19, 20]  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. [21]

Measuring maternal serum beta-human chorionic gonadotropin

Following conception and implantation of the developing embryo into the uterus, the trophoblasts produce beta-HCG. The level of beta-HCG can be followed in maternal blood. Early in gestation, the level will double in 48 hours in 66% of patients. An abnormal rise may indicate an ectopic pregnancy or a nonviable gestation. An excessively high level may indicate trophoblastic disease. In the middle to late second trimester, the level of beta-HCG also can be used in conjunction with other biomarker levels to screen for chromosomal abnormalities. An increased beta-HCG level coupled with a decreased MSAFP level increases the risk of trisomy 21. [17, 22]

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, and an elevated level of inhibin-A is linked with an increased risk of trisomy 21. A high inhibin-A level may also be associated with a risk of adverse perinatal outcome, including preterm delivery and fetal growth restriction.

Screening for chromosomal abnormalities - Cell-free fetal DNA

Noninvasive prenatal screening uses next-generation sequencing of cell-free DNA in the maternal circulation. This technology has changed prenatal screening for aneuploidy. Placental trophoblasts are released into the maternal circulation, and when these cells die, they release the fetal portion of cell-free fetal DNA. Cell-free fetal DNA can be extracted from maternal blood starting at 9-10 weeks' gestation.

Cell-free fetal DNA can be used to detect trisomy 21,18, and 13 and common sex chromosome aneuploidies (45,X; 47,XXX; 47,XXY; 47,XYY). In a singleton pregnancy, cell-free DNA can detect 99% of fetuses with trisomy 21, 98% of fetuses with trisomy 18, and 99% of fetuses with trisomy 13. The number of cases of sex chromosome abnormalities remains small for accurate estimation. [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. [8]  Use of cell-free DNA to screen for other aneuploidies (trisomy 16 and 22), microdeletions, and expanded cell-free DNA genome-wide screening is technically possible, but not recommended owing to lack of clinical validation. [1]

Maternal blood is best drawn after 10 weeks to allow the cell fraction to increase to at least 4% of the total fetal cell fraction. The quantity of cell fraction increases with increasing gestational age and for quality purposes, it is important to use a laboratory that reports fetal fraction. Factors that may affect the fetal fraction include maternal obesity and race, medication exposure, and presence of an aneuploidy. The fetal fraction may be low, leading to a non-result. Factors that may increase the risk of false-positive results include the demise of one fetus in a twin gestation, confined placental mosaicism, maternal mosaicism, or maternal cancer. If the results are not reported, indeterminate, or uninterpretable from cell-free DNA screening, women should be offered genetic counseling, ultrasound evaluation, and diagnostic testing.

Cell-free fetal DNA testing performs better than serum analyte screening, with a lower false-positive rate. This test does not, however, replace invasive testing such as CVS or amniocentesis, as it is limited in its ability to identify all chromosome abnormalities. Women should also be counseled that cell-free fetal DNA testing does not eliminate the risk of a structural congenital abnormality or a genetic condition. [24]  Cell-free fetal DNA remains a non-diagnostic test, and if an abnormal result is obtained, this should be confirmed with CVS or amniocentesis. Alternatively, if a fetal structural abnormality is detected on prenatal ultrasound evaluation, the indication for amniocentesis (with or without chromosomal microarray) should not be modified by a negative cell-free fetal DNA result.


Invasive Techniques

Chorionic villus sampling

The choice of CVS versus amniocentesis is personal, as these procedures essentially provide the same genetic information. CVS is performed early in gestation, between 10 and 13 weeks. This procedure can be performed transabdominally or transcervically. A catheter is passed through the maternal abdomen or cervix into the uterus, under ultrasound guidance, and into the placenta. A sample is obtained using a syringe with negative pressure to aspirate from the placenta. 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. [25] 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 testing for other genetic conditions in the prenatal period often rely 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 earlier in pregnancy. Abnormalities can be identified at an early gestational age.

Vaginal bleeding may occur after CVS. The risk of fetal loss after CVS has decreased over time and is now estimated at 0.22%. Other potential complications, including leaking amniotic fluid, culture failure, or infection, occur in less than 0.5% of patients. [26]  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. [27]  Reports that CVS can result in limb defects in the fetus can be addressed by performing CVS after 10 weeks’ gestation. [28] Maternal sensitization is possible, and known maternal alloimmunization is a relative contraindication, as it may result in more severe disease.


Amniocentesis is a safe, reliable, and accurate procedure that is usually performed at 15-20 weeks' gestation but can be performed any time in gestation after 15 weeks. An increased risk of loss and fetal clubbed foot has been reported when amniocentesis is performed prior to 15 weeks. It is performed with ultrasound guidance. A 22-gauge needle is passed through the mother's abdomen, through the uterus, and into the amniotic cavity.

About 20-30 mL of amniotic fluid that contains cells from amnion, fetal skin, fetal lungs, and urinary tract epithelium is collected. These cells are grown in culture for chromosomal, biochemical, and molecular biologic analyses. Single gene disorders can be detected. Supernatant amniotic fluid is used for the measurement of substances such as amniotic fluid AFP, hormones, and enzymes. Karyotype analysis can detect chromosomal abnormalities in number and structure as small as 5-10 Mb, as well as balanced translocations or large rearrangements.

The use of chromosomal microarray (CMA), which can be performed on cells from CVS or amniocentesis, has increased. CMA can detect major chromosome abnormalities but also submicroscopic abnormalities, gains and losses of genetic material that would not be detected by traditional karyotype analysis. Another advantage of CMA is that it does not require cell culture, allowing the availability of results in shorter time frames. CMA cannot detect balanced translocations or triploidy.

The risk of loss after amniocentesis is 0.1-0.3%. Other potential complications, including vaginal bleeding and amniotic fluid leakage, occur in 1-2% of cases. [26]  Women with Rh negative blood type should receive RhoGAM post procedure (unless there is confirmation that the father of the baby is also Rh negative).

Percutaneous umbilical blood sampling

Cordocentesis (PUBS) is a method for fetal blood sampling. [29] A needle is inserted into the umbilical cord under ultrasound guidance near the cord insertion or free loop, and fetal blood is collected from the umbilical vein for chromosome analysis, genetic diagnosis, detection of 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 the need for an experienced operator. Other possible complications include hemorrhage from the puncture site, cord hematoma, feto-maternal 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 is performed only if the fetal hemoglobin level is more than two standard deviations below the mean value for gestational age (reference values available). The procedure is 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. [30]


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, fetoscopy is used in the treatment of twin-to-twin transfusion syndrome, in which a laser is used to coagulate anastomotic vessels. Twin-to-twin transfusion syndrome is divided into stages. [31] 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 compared with the previous non-sequential procedure. [32] 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. [33]

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

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, can be diagnosed with percutaneous fetal skin biopsies that are obtained with ultrasonic guidance between 17 and 20 weeks' gestation.

Case reports have described fetal liver biopsy to diagnose an inborn error of metabolism, such as ornithine transcarbamylase deficiency, [34] glucose-6-phosphatase deficiency, [35] glycogen storage disease type IA, nonketotic hyperglycemia, [36] and carbamoyl-phosphate synthetase deficiency. [37]  Fetal muscle biopsy has also been described to analyze the muscle fibers histochemically for prenatal diagnosis of Becker-Duchenne muscular dystrophy. [38] .

These procedures are rarely performed nor are they readily available outside specialized centers. 

A genetic diagnosis can assist not only in determining the fetal prognosis, but in helping with prenatal care, including decisions on reproductive choice, in utero therapy, delivery planning, and neonatal management. It can also report the recurrence risk, leading to informed genetic counseling about reproductive options, including preimplantation genetic testing, diagnostic prenatal testing, and consideration of donor gametes or adoption.


Cytogenetic Investigations

Preimplantation biopsy of blastocysts obtained by in vitro fertilization

Techniques have been developed to test cells obtained from biopsy of early cleavage stages or blastocysts of pregnancies conceived through in vitro fertilization. [39] These techniques are helpful for the selective transfer and implantation of those pregnancies that are not affected by aneuploidy or a specific genetic disorder. Usually one or a few cells are examined. Even in the setting of preimplantation genetic screening, confirmatory testing with CVS or amniocentesis is recommended.

Fluorescent in situ hybridization

Fluorescent in situ hybridization (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 [40] in the genome and can detect chromosomal abnormalities, such as trisomies, [41] monosomies, microdeletions and duplications. This technique allows counting of the number and location of large pieces of chromosomes and increases the sensitivity, specificity, and resolution of chromosome analyses. FISH can be performed on metaphase chromosomes or interphase nuclei and is technically straightforward. [42] This is a rapid and accurate test for aneuploidies, including trisomy 21,13, and 18; monosomy X; and triploidy.

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 [43] and for the detection of submicroscopic deletions. [44]  These probes must be requested, and one of the most common deletions is 22q11.2.

Some of the advantages of FISH include that its resolution is much better than traditional chromosome banding, it can be applied to both dividing (metaphase) and non-dividing (interphase) cells, and it can identify many structural abnormalities, including deletions, duplications, aneuploidy, and structurally rearranged chromosomes. However, disadvantages do exist in that small mutations, such as deletions, insertions, and 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.

Although the positive predictive value for trisomy 21,18, and 13 is reportedly 100%, confirmatory testing with CVS or amniocentesis is still recommended.

Microarray comparative genomic hybridization

Microarray comparative genomic hybridization, also referred to as chromosomal microarray analysis (CMA), is considered to be useful in detecting genomic imbalance in the fetus. CMA can detect major chromosomal abnormalities as well as submicroscopic changes that are too small to be detected by routine karyotype. A meta-analysis showed that in fetuses presenting with structural abnormality (referral for structural abnormality) noted on ultrasound and normal karyotype, CMA could identify a significant chromosomal abnormality in 6% of fetuses. [45] Pathogenic copy variants are present in 1.7% of fetuses with a normal ultrasound examination and normal karyotype. ACOG recommends that CMA be made available to any patient who desires invasive prenatal diagnosis. [1]

CMA is also useful in the evaluation of stillbirth (pregnancy loss at ≥20 weeks of gestation) because CMA can be performed on cultured, uncultured, and non-viable cells.  


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 polymerase chain reaction (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.

Exome sequencing

Exome sequencing may be considered when a fetus has structural abnormalities detected by ultrasound evaluation and karyotype analysis and CMA has failed to yield a diagnosis.

The exome is a region of the gene known to contain proteins. It contains 1% of the genome but 85% of disease-causing mutations. It interrogates the genome at the nucleic level. Prenatal exome sequencing could make a definitive diagnosis; however, it may increase the likelihood of identifying variants of uncertain significance (VUS).

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.