Prenatal diagnosis: Basic concepts,
definitions and
methods.
.
Introduction, Definition & Purpose of
Prenatal Diagnosis
• Introduction
Prenatal diagnosis (PD) detects early in pregnancy a number of fetal anomalies and genetic
diseases.
Approximately 3% of viable fetuses would be born with a severe anomaly.
• PD allows couples at risk to envisage a pregnancy since an alternative is now offered to them.
• PD serves as a complementary/essential tool in providing anticipatory guidance/genetic counseling
for disease risk of inherited disorders, often requested by families with an affected proband.
• Definition: Prenatal diagnosis is a variety of investigations done on pregnant women at risk of
having a fetus with anomalies or genetic diseases to determine the health and condition of the
unborn fetus before birth.
• Purpose of Prenatal diagnosis
• (1) To enable timely medical or surgical treatment of a condition before or after birth.
• (2) To give the parents the chance to abort a fetus with the diagnosed condition.
• (3) To give parents the chance to prepare psychologically, socially, financially, and medically for a
baby with a health problem or disability, or for the likelihood of a stillbirth.
• (4) To plan for place and method of delivery.
Families at Risk
Families at risk
• [Link] maternal age; age 35 or above.
• 2. Recurrence of numerical and structural chromosomal anomalies
• 3. Chromosomal instability
• 4. Single gene and metabolic disorders such as SCD, Thalassemia and
other haemoglobinopathies inherited in autosomal or x-linked fashion.
• 5. Neural tube defects: Spina bifida, occulta if the skin is normal,
Anencephaly and Encephalocele.
• 6. Previous Stillbirth or Neonatal death
• 7. Exposure to teratogen: Ionizing radiation, Anticonvulsants e.g. lithium.
• 8. Infections: Rubella, Toxoplasmosis, cytomegalovirus.
• 9. Medical diseases in the mother: Dm, Phenylketonuria.
• 10. Multiple gestation: Elevated MSAFP or Reduced MSAFP.
Prenatal Screening/Diagnostic Tests
Prenatal Screening tests
• Tests used to identify pregnant women at increased risk or likelihood of a fetal abnormality in an apparently normal
pregnancy.
• They are safe, minimally invasive tests that screen for various metabolic, chromosomal and anatomic defects.
• They have relatively low sensitivity and specificity.
• It provides an assessment of the risk of having a condition.
• Ideal prenatal screening test should be easily available, affordable, should have a low rate of false positive, provides result in
a timely fashion. Should have very high sensitivity to identify patients at risk, and should allow for intervention if necessary.
• Prenatal screening covers the standard screening tests for chromosomal disorders (specifically trisomies 13, 18 & 21)
routinely offered to pregnant women during antenatal care.
• Screening tests help to reduce the number of invasive diagnostic procedures. Screening tests do not detect all cases of
aneuploidies while invasive diagnostic tests are able to identify almost all existing genetic anomalies.
The choice of screening test: Depends on the maternal GA, obstetric history, gravidity, sensitivity and availability of the test,
limitations of the test and options for termination of pregnancy, in case aneuploidy is diagnosed.
Prenatal Diagnostic tests
. They are invasive and resource-intensive but directly analyze fetal material and confirm diagnosis.
. Because the feto-maternal unit is involved, they pose risk of pain, infection, bleeding, fetal scarring and fetal loss.
First Trimester Screening Tests
1st Trimester Screening tests
• detection rates & false positive rates reported here are with respect to the detection of trisomy 21 specifically
- Maternal age
• Risk of Down’s syndrome increases with advanced maternal age.
• Ballpark risk figures of Down’s syndrome at birth:
- Maternal age of 35 years 1:350
- Maternal age of 40 years 1:100
• Risk based on maternal age is further augmented if there is a history of previous chromosomal abnormalities:
• Detection rate = 30%, false positive rate = 5%
Beta human chorionic gonadotropin (β-hCG): β-hCG is produced by the developing trophoblast in the earliest stages of pregnancy and forms
the basis for urine and serum pregnancy tests. To detect and diagnose pregnancy; in quantitative analysis, β-hCG levels lower than
expected for presumed gestation can alert the clinician to an ectopic pregnancy or threatened abortion
Pregnancy-associated plasma protein-A (PAPP-A): Is measured in maternal blood from 11-13 weeks post- menstrual age (PMA) to detect
trisomy 18 and 21 (in conjunction with β-hCG and ultrasonography for nuchal translucency) .
- Nuchal translucency (NT)
• Ultrasound measurement of subcutaneous space between fetal skin & cervical spine in the midsagittal view, It is done between 11- 13
weeks GA.
• Detection rate = 80%, false positive rate = 5%
Absence of Nasal Bone (NB)
Ultrasound measurement done between 11-14 weeks of pregnancy to demonstrate absence of nasal bone which is a marker for aneuploidy.
Nasal bone is absent in 68.5% of down syndrome fetuses and 32.2% of fetuses with other chromosomal abnormalities.
First Trimester Screening Tests Contd
- Combined test
• NT + HCG + PAPPA (pregnancy-associated plasma protein A ) (w.r.t. maternal age).
Detection rate = 90%, false positive rate = 5%
• Best screening test offered locally, thus routinely recommended for all patients eligible for it.
• Nuchal translucency (NT) can be measured as early as the 6th week of gestation. Increased NT has been found to
be associated with trisomies and congenital cardiac defects.
Ultrasonography and fetal echocardiography can be offered to pregnant women if fetal NT is found to be at least 3.5
mm and aneuploidy screen is negative and no chromosomal abnormalities have been detected) as there is still a
high risk of congenital cardiac/abdominal wall defects and other genetic syndromes. It can detect the risk of Down
syndrome and is recommended from 11 to 14 weeks of pregnancy.
Cell-free fetal DNA test[cffDNA] This blood test determines the risk of down syndrome and other chromosomal
abnormalities in pregnant women who have a high risk of having a baby with a chromosomal abnormality. DNA of
fetus is analyzed from the pregnant mothers’ blood as it is present in her blood in trace amounts. This test is
recommended from the 10th week of pregnancy. According to recent research, cell-free fetal (cff) DNA in maternal
plasma can specify common aneuploidies (trisomies 21, 18, 13) in high-risk pregnant women.
• It is a non-invasive prenatal screening test. Relatively new technology.
• It is primarily placental in origin and is released from apoptotic trophoblast.
• First-trimester screening (FTS) is helpful, women diagnosed to have a malformed fetus or aneuploidy can be
offered the options in the early pregnancy such as CVS, genetic counseling as well as second-trimester
amniocentesis, especially if she is at high risk.
2nd Trimester Screening Tests
2nd Trimester Screening tests
- Triple test
• Alpha fetoprotein + hCG + unconjugated estriol (w.r.t. maternal age)
Detection rate = 70%, false positive rate = 5%
• Not as good as combined test, but the only alternative that can be offered to patients presenting in the 2nd trimester.
- Quadruple test
• Alpha fetoprotein + hCG + unconjugated estriol + inhibin A (w.r.t. maternal age)
Detection rate = 81%, false positive rate = 5%
• Better than triple test, but not offered locally as assay for inhibin A is not available locally.
• The high value of alpha-fetoprotein level in the blood determines the high risk of Neural tube defects, birth defects of the abdominal
wall and complications in pregnancy such as, miscarriage, IUGR or death of the fetus.
Penta test
• It is carried out at the same time with triple and quad screen. It involves addition of a 5th marker to the quad screen which is
hyperglycosylated hCG or Invasive trophoblastic antigen (ITA) produced by cytotrophoblast during embryonic implantation and
trophoblast invasion of the uterine wall.
• Levels tend to increase in down syndrome affected pregnancies.
• The essence of this test is to increase the sensitivity of the test result for aneuploidy while reducing the false positive rate.
Fetal Ultrasonography
• US is the single most valuable modality in the identification of fetal and/or placental structural anomalies. It is also useful in the
detection of abnormal growth patterns in the fetus, in estimating gestational age, and in assessing fetal well-being . It is important in
guiding the operator during procedures such as amniocentesis and cordocentesis.
• US is widely available and has no known adverse effects.
• US makes use of ultrasounds to study tissues and organs. It is applied from the first trimester but it is only during the second trimester
that one can best evaluate fetal morphology and preferably from 18th week of gestation.
• Cardiac ultrasonography, Allows examination of great vessels and heart chambers, it is done from 20th -23rd week of pregnancy.
US Markers Suggesting Fetal Anomaly
Markers suggesting the presence of a birth defect.
Ultrasonographic markers are variations observed during the ultrasound session that will alert the examiner to the possibility of an abnormal
fetal development or a genetic disease. These markers can reveal the possibility of a chromosomal anomaly such as trisomy 21, 13, 18,
or chondrodysplasia.
Ultrasound markers suggesting the presence of a fetal anomaly.
• · Abdominal calcifications (meconial peritonitis)
• · Bladder hypertrophy (urethral valve)
• · Bone hypodensity (hypophosphatasia)
• · Cerebral ventricles increased (hydrocephaly)
• · Cono-truncal defect or defect of the heart common trunk, manifested as a tetralogy of Fallot or a vascular defect (Di George, and velo-
cardio-facial syndromes secondary to a deletion — del 22q11-)
• · Cranial vault ossification defect (anencephaly)
• · Cystic hygroma (Turner syndrome, 45X)
• · Double buble in the gastric region (duodenal atresia)
• · Endocardial cushion defect, or of the primary cardiac septum, described as an atrium septal defect (trisomy 21)
• · Facial hypoplasia and cleft lip ( trisomy 13 - holoprosencephaly)
• · Fractures (osteogenesis imperfecta)
• · Increased number of choroidal cysts ( trisomy)
• · Increased volume of cerebral ventricles (hydrocephaly)
• · Lemon sign, lemon shape head (spina bifida)
• · Long bones shortening (bone dysplasia)
• · Nuchal skin folds increased (trisomy 21)
• · Persistant flexed fingers (trisomy 18 - arthrogryposis)
• · Polydactyly (trisomy 13; Ellis Van-Creveld syndrome)
• · Pterygium colli (Turner syndrome; pterygium multiple)
• · Stomach unseen (oesophageal atresia)
• · Thoracic deformity (skeletal dysplasia)
Non-Invasive Prenatal Screening Tests
Non-Invasive Prenatal Screening Tests
• Cell-free fetal DNA (cffDNA Test) in maternal circulation
• A new genetic test that analyzes the DNA of the fetus from maternal
blood. It can be performed from the 10th -22nd GA and results are available
in 10-15 days.. A positive test is indicative of birth defects and further
invasive tests may be needed to confirm the diagnosis.
• NIPS detects trisomies 21, 18 and 13, cystic fibrosis, haemophilia, etc. It
can also reveal the gender of the fetus. This test has higher sensitivity
compared to nuchal translucency as well as first-trimester screening tests
and quad test. Currently, ACOG Committee on Genetics recommends NIPS
for high –risk women with
• Age above 35 years during pregnancy
• Positive first-trimester screening tests, e.g., triple or quadruple screen
• Ultrasound finding of an anatomical abnormality
• History of previous trisomy
• History of a balanced translocation in a parent or partner’s parent
Invasive Prenatal Diagnostic Tests
Invasive Prenatal Diagnostic Tests
- Indications for invasive prenatal diagnostic tests are:
• Advanced maternal age
• Ultrasound screening indicative of fetal abnormalities
• Previous history of fetal abnormality
• Abnormal triple or quadruple marker tests (hCG, unconjugated oestriol
and alfa-fetoprotein)
• Maternal anxiety due to a history or family history of fetal malformations
• Invasive prenatal diagnostic tests include (3 main modalities) chorionic
villus sampling (CVS), amniocentesis and fetal blood sampling (FBS).
• These tests are associated with an elevated risk of complications, e.g.,
limb injuries have been reported after CVS; miscarriages have been
reported after amniocentesis and fetal blood sampling.
Chorionic Villus Sampling (CVS)
CVS is done in 1st trimester by transabdominal or transcervical routes via Ultrasound-guided sampling.
• The approach is based on the placental location. A transabdominal approach is preferred for anterior and fundal placentas
and in active vaginal and cervical infections. The sample is smaller than that obtained with the transcervical method.
• The transcervical approach is indicated in cases with interposed bowel loops or uterine retroversion and a posterior or low-
lying placenta. The transvaginal approach has limited application and is used when the placenta is posteriorly, the uterus is
retroverted and retroflexed, and the cervical canal points toward the abdomen.
• Tissue culture yields cytogenetic results in 6-8 days
• Direct visualization of dividing villi cells allows for detection of chromosomal abnormalities in 3 days
• CVS is performed from 9th to 12th weeks of gestation.
• CVS is the technique of choice for prenatal diagnosis prior to 12 weeks' gestation for detection of a chromosomal anomaly,
DNA molecular diagnosis of classic genetic disorders, and the detection of defects in lysosomal enzymes or
mucopolysaccharidoses.
• A diagnosis of enzymatic defects, such as 21-hydroxylase deficiency, which causes congenital adrenal hyperplasia (CAH), can
be made with an allele-specific amplification analysis technique of DNA obtained with CVS.
• For technical reasons, it is difficult to perform on multiple gestations. This test cannot be used to diagnose anatomic
abnormalities, such as NTDs and abdominal-wall defects.
• - Complications: Fetal loss rate = 1%
• Culture failure rate = 0.5% (i.e. no results can be determined after procedure is performed)
• Mosaicism rate = 1% (i.e. >1 cell population which differ in genetic makeup present in chorionic villi, resulting in sampling
error; however, the presence of mosaicism is almost always detectable during analysis of the sample.
• -Reduction limb deformities. Risk of transplacental hemorrhage (need to give anti-D to rhesus-ve mothers after procedure).
Illustration of the CVS
Amniocentesis
Amniocentesis
• - Ultrasound-guided aspiration of amniotic fluid sample using a 22G needle transabdominally.
• Amniocytes in sample are cultured, requiring 1-2 weeks for final chromosomal analysis.
• Fluorescent in-situ hybridization (FISH) can be used using chromosome-specific probes (e.g. for trisomy 13,
18 & 21) can give preliminary results in 3 days.
• Amniotic fluid itself can be tested for various substances depending on the indication for the test.
• Performed in the 2nd trimester from 16th - 20th GA. Risks of talipes and fetal loss if done earlier.
• - Utility of test:
• Karyotype for chromosomal abnormalities
• Identify single gene mutations (e.g. thalassemia)
• Test for perinatal infections:
• - Detect intrauterine infections (TORCH, VZV, parvovirus B19) if maternal exposure has occurred
with ultrasonographic features suggestive of fetal infection (fetal hydrops, echogenic bowel,
ventriculomegaly, intracranial calcification)
• - Analysis of amniotic fluid to diagnose preterm chorioamnionitis (gram stain, white cell count,
glucose level, culture).
Amniocentesis Contd
• Biochemical tests for fetal structural abnormalities:
• - AFP: elevated levels in neural tube (e.g. spina bifida) & ventral abdominal wall defects (e.g. gastrochisis, omphalocele)
• - Acetylcholinesterase: presence indicates open neural tube defect
• Test for fetal pulmonary maturity:
• - Test for amniotic phospholipids to calculate
• lecithin:sphingomyelin (L/S) ratio; L/S ratio >2
is associated with low risk for respiratory distress in the neonate (not done anymore with advent of
routine corticosteroid injections in threatened preterms to mature fetal lungs)
• Therapeutic amniocentesis for:
• - Polyhydramnios
• - Twin-twin transfusion syndrome (uses serial amniocentesis to remove excessive amniotic fluid from sac of recipient twin).
Complications:
• Uterine bleeding (1.9%)
• Uterine cramping
• Leakage of amniotic fluid (1%)
• Pregnancy loss (0.5%)
• Increased risk of clubfoot when performed prior to 12 weeks' gestation
• Procedural failure due to tenting of the membranes ahead of the needle
• Culture failure rate of 1%
• Mosaicism rate = almost zero
• Transplacental hemorrhage (hence need to give anti-D to rhesus-negative mothers after procedure)
Illustration of the amniocentesis procedure
Fetal Blood Sampling
Fetal Blood Sampling
• US-guided sample of fetal blood via umbilical vessel with a 20-27 guage needle, transplacental in an anterior placenta or transamniotic in a posterior
placenta.
• Sources of fetal blood (umbilical vein e.g, cordocentesis, intrahepatic blood sampling, cardiocentesis):
• Fetal leukocytes are cultured, requiring 3 days for chromosomal analysis results
• Hence main indication is for rapid karyotyping (as fetal leukocytes can be cultured more rapidly than amniocytes)
• The greatest advantage of this technique is that it provides a direct fetal sample and access to the fetus for treatment in utero.
• - Performed from 20-23 weeks onwards
• - Utility of test:
• Karyotype for chromosomal abnormalities
• Identify single gene mutations (e.g. thalassemia)
• Assessment of fetal anemia (historically in the setting of rhesus isoimmunization; not necessary anymore with the advent of middle cerebral artery
Doppler as an effective indicator of fetal anemia)
• Therapeutic in-utero blood transfusion (e.g. for parvovirus B19 infection, Bart’s hydrops)
• - Complications:
• Fetal loss rate = 2-5%
• Cord hematoma, haemorrhage
Cordocentesis:
• Complications associated with cordocentesis are more common in posterior placentae and when the procedure is performed prior to 19 weeks‘GA.
These include the following:
• Fetal loss (1-2.3%)
• Preterm labor (5-9%)
• Hematoma of the umbilical cord and placental abruption
• Chorioamnionitis (0.6%)
• Fetal exsanguination from the procedure site
• Rh isoimmunization - Rh immunoprophylaxis is mandatory in all Rh-negative nonsensitized women after the procedure
• Placenta penetration during cordocentesis has been associated with a higher risk for fetal loss, preterm birth, and low birth weight.
Risk for this procedure is higher than Aminocentesis, CVS or Placental biopsy. Anti D globulin must be given to rhesus negative women.
Future perspectives or trends
• Future perspectives or trends
1. In-utero treatment.
2. Pre-implantation diagnosis.
3. Preconception screening .
4. Fetal nucleated cells in maternal circulation
5. Three dimensional ultrasound
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