1. Genetics Series:
Prenatal Diagnosis
Dr Mathew Joseph
MBBS,MD(AIIMS),BCC(Palliative Medicine)
Assistant Professor
Department of Anatomy
Amala Institute of Medical Sciences, Thrissur

2. AN81.1 Describe various methods of prenatal diagnosis.
AN81.2 Describe indications, process, and disadvantages of amniocentesis.
AN81.3 Describe indications, process, and disadvantages of chorion villus biopsy.
OBJECTIVES - COMPETENCIES

3. • Prenatal diagnosis or screening is the way of prenatal care to
determine the well-being state of the fetus and to detect the
presence of any congenital anomaly or disorder in the fetus.
• It also includes the steps towards the decision for the treatment,
counseling of the couple and termination of pregnancy.
INTRODUCTION

4. • Maternal age : 35 years or above
• Family history : Congenital anomalies
• History of the previous child with birth defects or chromosomal abnormalities, e.g.,
X-linked diseases
• Known genetic disorders of parents: e.g. thalassemia
• History of diabetes mellitus, phenylketonuria
• Infection: During pregnancy
• Exposure to contraindicated drugs or known .
• Ultrasound examination showing fetal abnormalities
• Overanxious mothers
• Population at increased risk of genetic diseases, e.g., Indians for sickle cell
anemia
Indications for prenatal diagnosis

5. Q. List methods of prenatal diagnosis.
• Various methods available for prenatal diagnosis.
• They can be classified as Noninvasive, Minimally invasive and
Invasive methods.
• In prenatal examination, selection of the method depends on the
finding of clinical history, examination of the patient, gestational
period, and suspected disease. If possible, invasive procedures
should be avoided as they increase the chance of abortions.
METHODS OF PRENATAL DIAGNOSIS

6. Methods of prenatal diagnosis
Noninvasive methods •Prenatal checkup of mother (external examination)
•Ultrasound examination
•Fetal Doppler for heart sound
Minimally invasive
methods
•Maternal blood screening for:
–Circulating fetal cells
–Circulating cell free fetal DNA
–β-hCG (human chorionic gonadotropin)
–Alpha-fetoprotein (AFP)
–Pregnancy-associated plasma protein A
–Estriol
–Inhibin A
•Transcervical retrieval of trophoblast cells (cervical mucus, swabbing
or lavage)
Invasive methods •Amniocentesis
•Chorionic villus sampling
•Fetoscopy and fetal liver, skin, muscle biopsies
•Fetal blood sampling

7. Q. List the uses of ultrasonography in prenatal diagnosis.
• Ultrasonography can be used from the 4th week of the pregnancy till the delivery of the baby.
• It is useful for the following purposes:
– Confirmation of the pregnancy
– Confirmation of the multiple pregnancies
– Localization of the placenta and position of the fetus
– Determination of gestational age
– To monitor the fetal growth
– To detect developmental defects.
• Ultrasonography is useful for detection of the following developmental disorders:
– Polyhydramnios
– Oligohydramnios
– Anencephaly
– Hydrocephalus
– Spina bifida
– Polycystic kidney
– Limb deformities and so on.
ULTRASONOGRAPHY IN PRENATAL DIAGNOSIS

8. Nuchal Translucency
Q. Write a short note on nuchal translucency.
• Nuchal translucency is the accumulation of fluid under the skin at the back of
baby’s neck.
• Nuchal translucency is measured using ultrasonography between 11 and 13
weeks of gestational age
• Increase nuchal translucency is observed in Down syndrome.
3D and 4D Ultrasound Scan
• For detection of fetal anomalies, still picture of the baby can be generated by 3D
ultrasound scan while 4D ultrasound scan can produce a moving 3D image of
the fetus.
Nuchal translucency, 3D and 4D ultrasound scan:

11. Q. Write a short note on Amniocentesis.
• Amniocentesis is a prenatal diagnostic invasive procedure to collect amniotic fluid for
analysis.
• Amniocentesis was first introduced by Fritz Friedrich Fuchs and Polv Riis in 1956.
• Ultrasound-guided amniocentesis was first reported by Jens Bang and Allen
Northeved in 1972.
Suitable gestational age: 14–20 weeks.
Procedure
• With the aid of the ultrasound guidance, amniotic sac is tapped to collect 10–20 mL of
amniotic fluid.
• Fluid is centrifuged to isolate the amniotic cells (cells of the fetal origin). These cells
are grown in the culture medium and examined for chromosomal abnormalities by
karyotyping.
• Supernatant amniotic fluid is screened for the alpha-fetoprotein (AFP) and other
biochemical parameters.
AMNIOCENTESIS

12. Risk
• The risk of miscarriage is low (0.5–1%).
• Other risks include injuries to the fetus, umbilical cord and placenta.
Screening Benefits
It is useful for detection of the following conditions.
• Chromosomal disorders such as Down syndrome, Turner syndrome and so on.
• Genetic disorders such as cystic fibrosis, sickle cell anemia, Tay-Sachs disease.
• Neural tube defects such as spina bifida and anencephaly (increased AFP).
• Quantitative fluorescent PCR (QF-PCR) is performed for rapid detection of
aneuploidies of chromosome 13, 18, 21, X, and Y.
Limitations
Amniocentesis cannot detect structural birth defects such as cleft lip, cleft palate,
heart malformations and so on. These can be detected by ultrasound.
AMNIOCENTESIS

13. Procedure for amniocentesis.

14. Q. Write a short note on chorionic villus sampling (CVS).
• Chorionic villus sampling is a prenatal test in that a sample of chorionic villi is
aspirated from the placenta for testing.
• It was first time performed by Giuseppe Simon in 1983.
Suitable gestational age: 10–12 weeks
Procedure
• Chorionic villus sampling is done under ultrasound guidance.
• There are two approaches for CVS:
1. In this method, a thin catheter is passed through the cervix
into the uterus to collect cells of chorionic villi from the placenta
2. Transabdominal approach: In this method, a needle is inserted through the anterior
abdominal wall into the placenta to remove chorionic villi cells.
• Placental biopsy: It is the term used for CVS when the procedure is carried out in later
stage of pregnancy.
CHORIONIC VILLUS SAMPLING

15. Risk
• The risk of miscarriage is about 1–2%.
• Other risks include infection, amniotic fluid leakage and limb defects.
Merits
• Chorionic villus sampling can be done much earlier (in 10–12 weeks) as compared to amniocentesis
(in 14–20 weeks). Hence, CVS can give early diagnostic results than amniocentesis.
• As CVS contains enough cells for genetic analysis, it provides rapid diagnosis than amniocentesis.
• If the fetus is suffering from major genetic abnormality, due to early diagnosis by CVS, termination of
the pregnancy may be planned safely in the first trimester itself.
Demerits and Limitations
• The risk of miscarriage is slightly higher in CVS than that in amniocentesis.
• In CVS, alpha-fetoprotein levels cannot be measured for the diagnosis of neural tube defects.
• Cells of CVS may show mosaicism (1–2% cases), but it may not be there in the fetus.
• Maternal cell contamination: Placental blood may contain maternal cells.
• Chorionic villus sampling does not rule out structural abnormalities of the fetus.
CHORIONIC VILLUS SAMPLING

18. Q. Write a short note on maternal blood screening test.
•Maternal blood/serum screening tests are performed for confirmation of
pregnancy and to detect birth defects.
•Maternal blood screening is done for the detection of following substances:
– Circulating fetal cells
– Circulating cell free fetal DNA
– β-hCG (human chorionic gonadotropin)
– Alpha-fetoprotein
– Pregnancy-associated plasma protein A
– Estriol
– Inhibin A.
MATERNAL BLOOD SCREENING TEST

19. Triple Screening Test
Q. List the components of triple screening test.
• It is also known as multiple marker screening test, AFP plus, Kettering test or Bort’s test.NEXT
• It is a maternal blood screening test.
• Purpose: To screen the cases for chromosomal abnormalities such as Down syndrome and
neural tube defects such as spina bifida.
• Suitable gestational age: 15–18 weeks.
• Sensitivity: 70% for detection of Down syndrome.
Components of triple screening test.
Component Source
•Maternal serum alpha-fetoprotein (AFP) Fetus
•Human chorionic gonadotropin (hCG) Placenta
•Unconjugated estriol (uE) Fetus and placenta
MATERNAL BLOOD SCREENING TEST

20. Quad Screening Test
Q. List the components of quad screening test.
• It is a maternal blood screening test.
• Purpose and time: Similar to triple test.
Components of quad screening test.
Component Source
•Maternal serum alpha-fetoprotein (AFP) Fetus
•Human chorionic gonadotropin (hCG) Placenta
•Unconjugated estriol (uE) Fetus and placenta
•Inhibin A (IA) Placenta and ovary
MATERNAL BLOOD SCREENING TEST

21. Q. Write a short note on percutaneous umbilical blood sampling.
• Percutaneous umbilical blood sampling (PUBS) is also known as fetal blood
sampling, cordocentesis, or umbilical vein sampling.
• Under ultrasound guidance, PUBS was first performed by Daffos et al. in 1983.
Procedure
Under ultrasound guidance, a thin needle is inserted into the umbilical cord
through the anterior abdominal wall of mother to collect fetal blood.
Purpose
• To detect chromosomal, genetic and hematological abnormalities rapidly. It gives
results within 2–3 days.
• To rule out mosaicism that is detected by amniocentesis or CVS.
Risk
• The risk of miscarriage is about 1–2% cases.
• Other risks include infection and blood loss from the site of the puncture.
PERCUTANEOUS UMBILICAL BLOOD SAMPLING
(CORDOCENTESIS)

22. Fetoscopy
• Fetoscopy is the endoscopic visualization of the fetus, amniotic cavity, umbilical
cord and the fetal surface of the placenta.
• Under ultrasound guidance, fetoscope is introduced through the abdominal wall
of the mother.
Indications
1. To take skin biopsy in case of epidermolysis bullosa, muscle biopsy.
2. For surgical interventions such as insertion of catheter drain in urinary tract.
–Note: Fetoscopy use is decreased due to the availability of ultrasound scan and
genetic methods for prenatal diagnosis.
–Risk: The risk of miscarriage is about 3–5% cases.

23. Acid elution test or Kleihauer-Betke test (1957)
This test is performed to detect the amount of fetal hemoglobin transferred from the fetus to the maternal blood.NEXT
Purpose
•To determine the required dose of RhO (D) immunoglobulin that inhibits the formation of Rh antibodies in Rh-
negative mother and to prevent Rh disease in future in the Rh-positive fetus.
Lecithin-sphingomyelin Ratio
•Lecithin-sphingomyelin ratio (L/S ratio) in amniotic fluid is performed to assess the fetal lung maturity.
•Surfactant (contains lecithin, sphingomyelin and other glycoproteins) is required for viability of the newborn in the
external environment.
Urine Strip Test for Confirmation of the Pregnancy
•It is most widely used test for confirmation of pregnancy. This test depends on detection of β-hCG in maternal
urine.
•It gives positive results after 10 days of first missed period. β-hCG can be detected after implantation.
Preimplantation Genetic Diagnosis
•Preimplantation genetic diagnosis (PGD) has been used to detect genetic defects before the implantation for in
vitro fertilization (IVF) cases.
•In PGD, one or two cells are collected from fertilized ovum and will be screened for genetic disorders.
SOME INTERESTING FACTS

24. Lecithin-sphingomyelin ratio.NEXT
Lecithin-sphingomyelin ratio Indicates risk of fetal respiratory
distress syndrome
More than 2 No risk, matured fetal lung
1.5–2 Mild–moderate
Less than 1.5 High risk

25. • The amniotic fluid index represents the amount of amniotic fluid observed on
ultrasonography.Viva
• Amniotic fluid index is expressed in centimeters
Amniotic fluid index.
Amniotic fluid index.NEXT
Amniotic fluid index Indicated quantity of amniotic fluid
8–11 cm Normal
Less than 5 cm Oligohydramnios
More than 20 cm Polyhydramnios

26. It is the screening test for the cells from preimplantation embryo for the detection of
metabolic, genetic or chromosomal abnormality before embryo transfer.
Indications
• Advanced maternal age 35 years and above
• Recurrent pregnancy loss (2 or more consecutive loss)
• Repeated IVF cycles failures (3 or more IVF failures)
• Unexplained infertility
• History of chromosomally abnormal previous child
• Family history of autosomal or X-linked diseases or metabolic disorders.
PREIMPLANTATION DIAGNOSIS

27. Procedure
• After fertilization, the first and second polar bodies biopsy or blastomere biopsy
cells are collected and processed.
Disadvantages
• Invasive procedure.
• Risks of ovarian hyperstimulation syndrome, multiple pregnancy and birth
defects due to IVF procedure but not related to the genetic test.
• Does not screen for all genetic diseases.
• Does not detect phenotypic disorders.
PREIMPLANTATION DIAGNOSIS

28. • AFP is a protein synthesized by immature fetal liver cells.
• Healthy, nonpregnant adults have very low levels of AFP.
• In pregnant women, with advancing gestational age, levels of AFP increase in
the maternal serum, urine, and in the amniotic fluid.
• In the following conditions, AFP levels are increased than expected during
pregnancy:
– Neural tube defects: Anencephaly, open spina bifida
– Incorrect gestational age
– Multiple pregnancy
– Sacrococcygeal teratoma
– Renal anomalies
– Cystic hygroma, liver necrosis
• In Down syndrome, the level of AFP is found to be lower than the expected
Alpha-fetoprotein (AFP).

29. • Cell-free fetal DNA (cffDNA) is derived from placental trophoblast tissue.
• This cffDNA circulates in the maternal blood and detectable as early as 6–7 weeks of
pregnancy.NEXT
• Suitable timings for NIPT: 11th week of gestation and onward.NEXT
Advantages
• No need of fetal invasive procedures. No risk of miscarriage.
• NIPT was developed in 2012 for detection of Down syndrome, Edward syndrome and Patau
syndromes (La et al., 1997, showed circulating DNA in maternal blood).
Disadvantage
• Maternal blood contains 80–90% of maternal cell-free DNA and only 10–20% cffDNA.
Indications
• Increased maternal age
• Anxiety for invasive procedures.
NON-INVASIVE PRENATAL TESTING (NIPT)

30. Procedure
Under all aseptic precautions, 5 mL maternal blood is collected. Cell-free DNA is isolated. Based on
the suspected anomaly, this DNA is analyzed using various genetic tests such as real time PCR,
next-generation sequencing, and so on.
Uses
• Detection of X-linked recessive disorders
• Detection of SNPs
• Fetal sex detection by presence of Y chromosome (banned in India).
Limitations
• Fetal or maternal mosaic, multiple pregnancies (triplet)
• Cannot detect phenotypic findings
• Very less quantity of cffDNA is main constrains
• Cannot detect fetal anomalies (USG is useful).
NON-INVASIVE PRENATAL TESTING (NIPT)

31. This Act prohibit sex selection (before or after conception), and regulate prenatal
diagnostic techniques for the detection of abnormalities, metabolic disorders,
chromosomal aberrations, congenital malformations or sex-linked disorders and
preventions of misuse for sex determination leading to female feticide.
• This law defines requirements for:
– Legal genetic counseling centers
– Prenatal diagnostic procedures
– Prenatal diagnostic tests
– Sex selection
– Qualified persons
– Procedures for registrations, and record keeping
– Offences and penalties for violation of Act.
Pre-Conception and Pre-Natal Diagnostic Techniques Act (PC-PNDT Act)
1994.

32. • This Act prevents misuse of technology for:
– Sex selection before conception
– Prenatal sex determination
– Female feticide
– Communication of sex.
• According to the amendment 2003, ultrasonography clinics are also incorporated under the control of
this law.
• Violation of law
– Provision of imprisonment up to 3 years, and up to Rs. 10,000 fine.
– On repeat offence, up to 5 years imprisonment, and up to 50,000 fine. The name of
practitioner to be removed from the council for 5 years if guilty and permanently if repeat
offence is committed under section 23 of the Act.
• Amendment 2018 proposed six months training in ultrasonography for quality to conduct prenatal
diagnostics only.
Pre-Conception and Pre-Natal Diagnostic Techniques Act (PC-PNDT Act)
1994.

33. Thank you…