Prenatal diagnostic methods

Gyula Richárd Nagy MD, PhD

Semmelweis University

1st Department of Obstetrics and Gynecology

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

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

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

To make prenatal diagnosis more effective

Less invasive techniques

Earlier testing

Individual diagnosis

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)

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)

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

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

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.)

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

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)

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.)

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)

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)

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.

Complications:

Fetal loss: 0.5-1%

Fetal injury: very rare because of ultrasound

guidance

Leakage of amniotic fluid, bleeding, uterine

irritability

Genetic amniocentesis III.

Chorionic villus sampling (CVS)

The most common first trimester invasive prenatal diagnosis technique: chromosomal abnormalities, monogenic disorders

Ultrasound-guided procedure between the 10-12thgestational weeks

Initially transcervical, nowadays transabdominal

CVS obtains chorionic tissue from the developing placenta

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.

Amniocentesis vs. CVS

AMNIOCENTESIS CVS

Procedure

AF removed by needleCV removed by catheter

(TC) or needle (TA)

Timing 16-20th week 10-12th week

Fetal malform. risk-

1:3000 vascular limb

malformation

Pregnancy loss0.5-1% 2-3%

Time required for

cytogenetic dg.2-3 weeks 1 week

Accuracy Highly accurate Highly accurate

Risk of placental

mosaicism

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)

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)

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.

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

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.

Cell-free fetal DNA in maternal

blood

Non-invasive prenatal testing

Hungarian research (2003-2015)

Cell-free fetal DNA in maternal blood

Both the mother and fetus produce cell-free DNA fromapoptotic cells– maternal DNA originates in bone marrow

– fetal DNA originates in placenta• 150-200bp

• Adequate amounts for clinical testing after 10 weeks (detected after32 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

Euploid fetusFetus 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

Improvements in DNA sequencing

gel-based systems

capillary sequencing

massively parallel

sequencing

kb

/day

/mach

ine

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

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)

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

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)

Screening vs. diagnosis

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)

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.

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.

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)

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.

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

Thank you for your attention!