1. Prenatal diagnostic methods
Gyula Richárd Nagy MD, PhD
Semmelweis University
1st Department of Obstetrics and Gynecology

2. Diagnosis I.
 Knowledge of the disease in question is a
prerequisite of genetic counseling
 The use and interpretation of different diagnostic
procedures (e.g. sonography, karyotyping)
 Obtaining old medical records, pedigree analysis,
careful history taking, clinical examinations of
relatives, special laboratory tests should be relevant

3. Diagnosis II.
 Until the recent past, couples at high risk of
genetic disorder had the choice:
 to take the risk
 to consider other reproductive options
(long term contraception, sterilisation, TOP, or adoption)
 In 1966 the relation of advanced maternal age and
increase risk of Down syndrome was noticed
 prenatal diagnosis started to develope

4. Objectives of prenatal diagnosis
AIMS:
 to detect abnormalities and allow termination
 to allow couples at high risk to know that the disorder
could be confirmed or excluded by testing
 to provide a range of informed choice to the couples at risk
of having a child with abnormality
 to provide reassurance and reduce anxiety, especially
among high-risk groups
 to allow the couples the option of appropriate management
(psychological, postnatal)
 to enable prenatal treatment of the affected fetus

5. To make prenatal diagnosis more effective
 Less invasive techniques
 Earlier testing
 Individual diagnosis

6. Methods of prenatal diagnosis
INVASIVE
 Genetic amniocentesis
(GAC)
 Chorionic villus sampling
(CVS)
 Chordocentesis (percutan
umbilical blood sampling
– PUBS)
 (Biopsy from fetal tissues)
 (Coelocentesis)
NON-INVASIVE
 Ultrasonography (US)
 Magnetic resonance
imaging (MRI)
 (Fetal cells and) cell-free
fetal DNA in maternal
blood (NIPT)

7. Indications of prenatal diagnosis
 advanced maternal age (≥ 35 yrs)
 positive family history for
 chromosome aberration
 a monogenic disorder
 neural tube defect
 other congenital structural abnormalities
 abnormalities suspected in pregnancy
 other high risk factors (consanguinity, obstetric history,
maternal diseases)

8. Maternal age
 career or
motherhood?
 socio-economic
problem
 average age of
primiparous women
increases
 increase of incidence
of fetal aneuploidies
 increasing number
of obstetrical
complications

9. Positive family history of
chromosomal abnormality
 usually no increase in risk compared to general
population since most chromosomal disorders
arise as a result of non-disjunction
 however each situation should be confirmed by
nature of chromosome abnormality in affected
individual

10. Positive family history for a
monogenic disorder
 previous affected child
 affection of one or both parents
 positive family history
 recurrence risk 25% (AR) or 50% (AD)
 prenatal diagnosis should be offered in case of
certain conditions as many can be diagnosed by
DNA analysis or biochemical testing (cystic
fibrosis, achondroplasia, Huntington disease,
neurofibromatosis, haemophilia, muscular
dystrophy, etc.)

11. Positive family history for neural
tube defects
 Risk of recurrence: 3-5%
 Screening: MSAFP 16th
week
 Diagnosis: US
 Small closed neural tube
defects can be missed even
with the most skilled person

12. Positive history for other
congenital malformations
 Evaluation of the family history
 Calculation of the risk of recurrence
 In case of suspected disorder detailed ultrasound is
indicated (majority of the malformations can be
diagnosed by US)
 Fetal heart malformation (fetal echocardiography)

13. Other risk factors
 Parental consanguinity: increased risk of AR and
multifactorial disorders
 Poor obstetric history (recurrent miscarriages and
stillbirths) increases the risk of future pregnancies
 Maternal diseases (diabetes, etc.)

14. Methods of prenatal diagnosis
INVASIVE
 Genetic amniocentesis
(GAC)
 Chorionic villus sampling
(CVS)
 Chordocentesis (percutan
umbilical blood sampling
– PUBS)
 (Biopsy from fetal tissues)
 (Coelocentesis)
NON-INVASIVE
 Ultrasonography (US)
 Magnetic resonance
imaging (MRI)
 (Fetal cells and) cell-free
fetal DNA in maternal
blood (NIPT)

15. Genetic amniocentesis
 Ultrasound-guided intervention between 16-20
gestational week
 Fetal karyotype analysis: from fetal cells (cell
culture). Result of karyotyping from cell culture
takes ~3 weeks
 Ability of QF-PCR for most common trisomies
 Material for diagnosing monogenic disorders
 Measurement of the amniotic fluid alpha-
fetoprotein (AFAFP)

16.  Ultrasound prior to amniocentesis: to determine
fetal cardiac activity, estimated gestational age,
location of placenta, amniotic fluid volume,
number and position of fetuses
 Avoidance of the placenta is recommended
 Sterile technique
 Volume of amniotic fluid removed: 8-15 ml
 After the examination: 2 days bed rest
Genetic amniocentesis II.

17. Complications:
 Fetal loss: 0.5-1%
 Fetal injury: very rare because of ultrasound
guidance
 Leakage of amniotic fluid, bleeding, uterine
irritability
Genetic amniocentesis III.

18. Chorionic villus sampling (CVS)
 The most common first trimester invasive prenatal
diagnosis technique: chromosomal abnormalities,
monogenic disorders
 Ultrasound-guided procedure between the 10-12th
gestational weeks
 Initially transcervical, nowadays transabdominal
 CVS obtains chorionic tissue from the developing
placenta

19.  Ultrasound is performed prior to CVS: fetal cardiac
activity, gestational age, number of fetuses, uterine fibroid
 Sterile technique
 Ultrasound-guided intervention
 Pregnancy loss: 2-3%
 Placental mosaicism can cause diagnostic difficulties
Chorionic villus sampling (CVS) II.

20. Amniocentesis vs. CVS
AMNIOCENTESIS CVS
Procedure
AF removed by needle
CV removed by catheter
(TC) or needle (TA)
Timing 16-20th week 10-12th week
Fetal malform. risk
-
1:3000 vascular limb
malformation
Pregnancy loss
0.5-1% 2-3%
Time required for
cytogenetic dg.
2-3 weeks 1 week
Accuracy Highly accurate Highly accurate
Risk of placental
mosaicism

21. Chordocentesis (PUBS)
 Ultrasound guided puncture of the umbilical cord
 „pure” fetal blood can be sampled
 The sample is rich in cells
 Can be used in cases of Rh isoimmunisation:
diagnosis, fetal therapy (tranfusion)

22. Ultrasound
 Screening (chromosome aberrations) and
diagnostics (e.g. spina bifida) in one
 Five US scan during pregnancy
 0. US: to diagnose pregnancy (embryonal heart
function)
 1st US scan: 12th week (NT<3 mm!)
 2nd US scan: 17-21st week (detailed examination:
diagnosis of congenital malformations)

23.  3rd US scan: 28-32nd week (IUGR, flowmetry:
placental circulation)
 4th US scan: 36-38th week: fetal position,
estimated weight, placental position, width of the
scar)
 Fetal echocardiography: 18-22nd week: fetal heart
anatomy and function
Ultrasound II.

24. Fetal MRI I.
 Fetal MRI: an adjunct tool to US in fetal screening
 Images to be obtained from any directions
 Excellent soft tissue contrast
 Substituting US in case of oligohydramnios or
obesity

25. INDICATIONS
 Central nervous system malformations (NTD,
holoprosencephaly, hydranencephaly, AV
malformations, intracranial haemorrhage)
 Cervical teratoma (relationship to vessels and
airways)
 Chest masses (diaphragmatic hernia, CAM)
 Adrenal neuroblastoma
Fetal MRI II.

26. Cell-free fetal DNA in maternal
blood
 Non-invasive prenatal testing

27. Hungarian research (2003-2015)

28. Cell-free fetal DNA in maternal blood
 Both the mother and fetus produce cell-free DNA from
apoptotic cells
– maternal DNA originates in bone marrow
– fetal DNA originates in placenta
• 150-200bp
• Adequate amounts for clinical testing after 10 weeks (detected after
32 days gestation)
• Can also be used in the third trimester
• Undetectable after 2 hours postpartum
 An average maternal plasma sample contains
– ~90% maternal cell-free DNA
– ~10% fetal cell-free DNA
isolate cell-free fetal DNANot to

29. Euploid fetus
Fetus with Down
syndrome
Maternal plasma
Maternal cell-free DNA
from chromosome 21
Fetal cell-free DNA
from chromosome 21
10 11
Fetus with trisomy 21 releases an an extra amount of cell-free fetal
DNA representing chromosome 21 into the maternal circulation

30. Improvements in DNA sequencing
gel-based systems
capillary sequencing
massively parallel
sequencing
kb/day/machine
1 000 000 000
100 000 000
10 000 000
1 000 000
100 000
10 000
1 000
100
10
1980 1990 2000 2010
year

31. Massively parallel sequencing
 Both maternal and fetal DNA are examined from a
peripherial maternal blood sample
– Fetal DNA is NOT isolated from maternal blood, BOTH maternal and
fetal DNA are sequenced
• library preparation
• cluster generation
• sequencing
 MPS is used as a molecular counting system rather than as a
sequencer
– 25-36 base pair reads
 Alignment of reads
– Measure counts relative to a reference value (from a normal genome)

32. Overall performance of NIPT for
aneuploidy
 Sensitivity for detection of trisomy 21 is >99%
 False positive rates are extremely low (~0,5%)
– May have a biological basis
 False negative rates are even lower (but not 0%)
– Mostly due to low fetal cell-free DNA fraction

33. Screening for a genetic disease
 Screening program’s aim:
 the prevention of a certain disease
 the early diagnosis of a certain disease
 Effective screening programs need general
agreement, in the society or population
 There may be opportunities for prenatal prevention:
primary (e.g. folic acid supplementation vs. NTD),
secondary (induced abortion)

34. Screening vs. diagnosis

35. Biochemical screening tests
II. trimester
Triple test:
AFP, hCG, unconjugated estriol
Quadruple test:
AFP, hCG, unconjugated estriol, inhibin-A
I. trimester
Serum screening alone:
free beta-hCG, PAPP-A
Combined test:
free beta-hCG, PAPP-A + NT
Nondisclosure sequential
screening (results not
reported until all are
complete)
Integrated serum test
Integrated test
Stepwise sequential
screening (intermediate
results reported as they are
ready)
Contingent screening (only
those with borderline risks
have second-trimester tests)

36.  Detection rate
– Maternal age 30%
– Quadruple test (FPR 5%) 60-65%
– NT alone 70-79%
– Combined test (NT + free beta-hCG and PAPP-A)
(FPR 5%) 87%
– NT + nasal bone
(FPR 1-2%) 90%
– Combined test + nasal bone
(FPR 1%) 95%
– Integrated test, stepwise sequential screening
(FPR 4-5%) 95%
Biochemical screening tests II.

37. Decision making, parental rights and
responsibilities
 The couple decide whether or not to undertake
pregnancy (before 12th week)
 The couple decide whether or not to accept the offer of
prenatal diagnosis
 Genetic counselor: gives the adequate information
 Parents: make the decision
 Factors influencing the decision: optimistic/pessimistic
attitude, ethical or religious principles etc.

38. Termination of pregnancy (TOP) I.
 TOP may be permitted at any time when serious disease
threatens the mother’s life (e.g. heart failure, obstetric
complication)
 TOP may be permitted at any time when fetal disease is
incompatible with postpartum life (e.g. anencephaly)
 TOP up to the 12th gestational week is permitted, when
the risk of genetic disorder or teratogenic damage to the
fetus exceeds 10%
 TOP is possible until the 24th gestational week, when the
risk of a severe, difficultly curable or incurable fetal
disease is 50-100% (e.g. Down syndrome)

39. Fetal indications, e.g.
 The probability of a severe AD disorder is 50%
(Huntington disease)
 Mother carrying an XR-gene is pregnant with a male
fetus
 Severe CNS malformation
 Severe bilateral kidney disease
 Severe chromosome aberration
Termination of pregnancy (TOP) II.

40. Malformations with better prognosis
E.g. Cleft lip, cleft palate, mild ventriculomegaly,
multicystic kidney etc.
 Follow-up during the pregnany (US)
 Exclude associating malformation, chromosome
aberrations
 The optimal hospital or clinic for the delivery
 Caesarean section vs. vaginal delivery?
 Detailed neonatological examinations
 Postnatal medical treatment
 Postnatal surgery

41. Thank you for your attention!