prenatal diagnosis

Seminar and Tutorial

Prenatal Diagnosis

พญ. จนัทร์ทิพย ์ธรานนท์ท่ีปรึกษา : รศ.นพ.ถวลัยว์งค์ รัตนสิริ

Table of Contents

• Prenatal Screening and Diagnosis Of Neural Tube Defect

• Prenatal Screening and Diagnosis Of Down Syndrome and Aneuploidies

• Prenatal and preimplantation diagnosis

Prenatal Screening and DiagnosisOf Neural Tube Defect

Introduction

• Neural Tube Defect < NTD> : second most prevalent congenital anomaly, second to cardiac malformation.

• Significant Roles to prevent NTDs:

– Maternal screening programme

– Sonographic imaging with or without the AF testing

– Folic acid as the prevention of NTDs

Incidence and Epidemiology

• Highly variable as

– Ethnics and geographic data

– Folic acid supplement programme

– Prenatal screening programme

Screening Maternal AFP with folic supplement led to decreased

prevalence of NTDs

• The Unites States < By 2006>

– 3: 10000

• Englands and Wales < 1970-1997>

– 1: 10000

Higher in Young age, Hispanic and White The Highest in the United kingdom China

and Egypt

Etiology and Risk Factor

• Multifactorial**

Environmental Factors

Genetic Factors**


• Inadequate Folate*** : the nucleic formation was disturbed then the cell turnover during the neural tube closure was blocked.

• Drugs** : Disturb the folic acid metabolism : valproate* and carbamazepine* , coumadin* ,thalidomide* and efavirenz*

• Insulin dependent DM : induce hypoxia and high

oxidative stress then disturb the signaling pathways led to embryopathy and apoptosis of neural tube.


• Hyperthermia: hot tub , sauna

• Amniotic bands : physically disrupt the normal tube development

• Obesity

• Geographical—United Kingdom, India, China, Egypt, Mexico, Southern Appalachian United States

Genetic Factors

• Polymorphic variants in genes in the folate pathway

• MTHFR polymorphism : Genetic variant 677C to T which is the thermolabile enzyme which decrease the enzyme activity therefore the homocysteine levels would be higher and the DNA synthesis will be incomplete.

Genetic Factors

• Aneuploidy**—trisomy 13 and 18, triploidy

• Syndromes with autosomal recessive disorder

– Meckel Gruber, Roberts, Joubert, JarchoLevin, HARDE (hydrocephalus-agyria-retinal dysplasia-encephalocele)

• 95 percent of NTDs develop in the absence of a family history.

• Recurrence risk*****

– 3-5 percents if a couple has a previously had a child with either anencephaly or spina bifida

– 10 percents if a couple has two affected children.

– 5 percents if either parents was born with NTDs

NTDs Screening• Candidates : All should be screened since 90-95

percent of cases occur in low risk pregnancy , especially the one with risk factors< previously affected fetus , positive family history , exposure to the associated medication , GDM and obesity

NTDs Screening

• Screening Test:

– Ultrasonography < Primary screening tool>

– Maternal serum AFP < MSAFP>

– Amniotic fluid AFP < AFAFP>

– Fetal blood AFP

Ultrasound Examination• Primary screening test for NTD

• Potentially detect NTDs more than MSAFP

• 18-20 wks of GA to perform the screening since the second trimester ultrasound increase the detection of spina bifida 92-95 percent and anencephaly to 100*** percent

second-trimester fetuses with open spina bifida

• Frontal bone scalloping—the lemon sign****

• Anterior curvature of the cerebellum with effacement of the cisterna magna—the banana sign****

The lemon sign

• Transventricular plane

• Frontal bone scalloping—the lemon sign with ventriculomegaly.

The banana sign• ******

• Transcerebellar plane

• Image of the fetal head at the level of the posterior fossa,

• Anterior curvature of the cerebellum with effacement of the cisterna magna—the banana sign.

Sonographic Findings in NTDs

AFP– Synthesized by fetal yolk sac and later by GI tract

and liver

– Function: immunoregulation and intravascular transport protein : analogous of albumin

– normal concentration gradient between fetal plasma and maternal serum 50,000:1.

– neural-tube and ventral wall defects, permit AFP to leak into the amnionic fluid, resulting in increased maternal serum AFP levels

AFP

• Maternal serum AFP Peaks at 28-32 wks inconsequence of the high fetoplacental permeability

• AFAFP < Amniotic fluid AFP > secreted from kidney to AF and peaks at GA 12-14 wks and declines until undetectable level at term

• Fetal plasma AFP peaks at 10-13 wks but no clinical roles

MSAFP testing

• MSAFP could be used at GA of 15 -20 wks

• Result was expressed as multiple of median for each gestational age which can be compared with different lab and populations

• The value above 2-2.5 of MOMs , designated for abnormal result.

• If the value elevation persist after a repetition then a specialised ultrasound examination is needed for assessment

MSAFP

• 2009 meta-analysis

• The positive predictive value is only 2 to 6 percent

• Sensitivity :

– 90 percents in anencephaly

– 80 percent for spina bifida

• The positive predictive value of MSAFP level between 2.5-2.9 MOMs for NTDs is 1.45 percents

• MSAFP level greater than 7 MOMs ,the positive predictive value of for NTDs is 13.5 percents

• false-positive rate of up to 5 percent (black hatched area)

• false-negative rates of up to 20 percent for spina bifida (tan hatched area) and 10 percent for anencephaly (red hatched area).

Factors increasing MSAFP***

• GA**: increasing by 15 percents per week during second trimester , if BPD differs more than 1 week comparing to the stated age then recalculated MSAFP must be done.

• Maternal weight **: vary in each women in term of volume distribution

• Diabetes mellitus**: lower the threshold to 1.5 MOMs

Factors increasing MSAFP

• Fetal anomalies** : abdominal wall defect, 89 % in omphalocele and 100% in gastroschisis

• Multiple gestation** : the concentration is proportional to the number of fetuses : the upper limit for twins pregnancy is 4-5 MOMs

• Race: Black women

Factors increasing MSAFP

• Fetal viability : fetal death** raise the MSAFP

• Preeclampsia **

• Fetal-growth restriction**

• Maternal hepatoma or teratoma

Fetal anomalies increasing AFP***

• Gastroschisis, Omphalocele **

• Cystic hygroma

• Esophageal or intestinal obstruction**

• Liver necrosis **

• Renal anomalies—polycystic kidneys, renal agenesis, congenital nephrosis, urinary tract**

• Sacrococcygeal teratoma**

Fetal anomalies increasing AFP

• Pilonidal cyst**

• Chorioangioma of placenta **

• Placenta intervillous thrombosis

• Placental abruption

• Oligohydramnios

AFAFP and AFAChE

• AFAFP : Amniotic fluid AFP

• AFAChE : Amniotic fluid acetylcholine esterase

• A primary biochemical test performed to diagnose open NTDs when the MSAFP is positive and ultrasound is equivocal or normal.

• An elevation of both AFP and AChE values in amniotic fluid suggest an open fetal NTDs with 98 percents accuracy and a false positive rate of 0.4 percents

• Fetal genetic study if amniocentesis is performed .

Fetal MRI

• Ultrasounds with limitations including the reverberation causing suboptimal visualization of the brain , maternal obesity and oligohydramnios can hamper the accurate diagnosis

• Further studies are needed to evaluate the diagnostic capability of MRI ,considered when the ultrasound is not optimal

Prenatal diagnostic study

• Targeted Ultrasound imaging

• If ultrasound is equivocal, AFAFP and AFAChE is performed as the diagnostic test

• Amniocentesis for genetic study and AFAFP and AFAChE

Additional Diagnostic study

• Ultrasound Survey the other malformation which is associated with NTD namely oral cleft ,musculoskeletal renal cardiovascular were the most commonly found as the associated anomaly.

• 6.5 percents of NTD with anomalies associated with chromosome and genetic abnormality

• < Trisomy 18 is the most common>***

Unexplained MSAFP Elevation

• Normal specialized sonographic evaluation, with or without amniocentesis

• Increased risk for various adverse pregnancy outcomes. < Not used as a screening test >

– fetal-growth restriction

– Oligohydramnios

– placental abruption

– preterm membrane rupture

– preterm birth– fetal death.

Pregnancy management

• ACOG recommends the route of delivery for the fetus with spina bifida should be individualized.

– cesarean section to reduce the chance of mechanical trauma and spinal infection

– Fetal surgery to improve the motor outcomes but increasing the maternal and fetal risk.

• Pregnancy termination

– In 83 percents of anenchephaly

– In 63 percents of spina bifida

Prevention

• 0.4 mg of folic acid orally every day before conception and through the first trimester, to reduce the NTD risk by 80 percents in low risk women****

• 4 mg folic acid taken daily

1 month before ******conception and through

the first trimester in women increased risk for NTDs

• Increased risk for NTDs:

– one or more prior affected children

– either parents has such a defect

• Folic acid supplementation may not decrease the risk for NTDs

• valproic acid exposure

• pregestational diabetes

• first-trimester fever or hot tub exposure

• defects associated with a genetic syndrome

Prenatal diagnosis of Down Syndrome and other aneuploidies

Introduction

• Down syndrome is the most common chromosome abnormality

• Moderate to severe learning disability, heart defect, intestinal malformation, vision and hearing loss

• High financial and psychological support to the affected family

• Aneuploid conceptuses

– 50 percent of first-trimester abortions

– 5 to 7 percent of all stillbirths and neonatal deaths.

Prenatal diagnosis and screening test of aneuploidies.

Screening test< noninvasive>

• First trimester– Ultrasound < nuchal

translucency and CRL>– Maternal serum marker

< BhcG, PAPP-A>• Integrated test < Full>• Sequential test• Contingent test

• Second trimester– Triple test– Quad test– Maternal serum free cell fetal

DNA

Diagnostic test

• Invasive procedure – Chorionic villi sampling

– Amniocentesis

– Fetal blood sampling

• Preimplantation diagnostic testing

Candidates for invasive prenatal diagnosis

• Maternal age : risk of having down syndrome and any aneuploidies increased with age from 35 years to 40 years by four folds comparing to the young age and 10 fold increased risk at the age of 40- 45 .

BUT 70 percent of Down syndrome pregnancies are in women younger than

35 years.

Candidates for prenatal diagnosis

• Dizygotic twins and maternal age older than 31 years at delivery

• Previous autosomal trisomy , triploidy or other chromosome abnormality birth

• Patient or partner is a carrier of chromosome translocation or inversion

• Some cases of repetitive early pregnancy loss

• Major structural defect by sonography

First trimester Screening

• Most common used protocol

• GA 11-14wks

– Nuchal translucency alone

– Combined test involoves

• Nuchal translucency and gestational age combines with serum marker PAPP-A and free or total BhCG< double test>

Nuchal translucency

• This is the maximum thickness of the subcutaneous translucent area between the skin and soft tissue overlying the fetal spine at the back of the neck

• It is measured in the sagittal plane, when the crown-rump length measures between 38 and 84 mm.

Nuchal translucency

• The NT measurement is expressed as a multiple of the gestational age specific median.

• NT ≥ 3.5 mm , offered targeted sonography, in addition to fetal karyotyping

• One third of fetuses with increased nuchaltranslucency thickness will have a chromosome abnormality

• isolated marker, NT detects 70 percent of fetuses with Down syndrome at a false-positive rate of 5 percent

• maximal sensitivity at 11 weeks

• NT is used as an isolated marker in multifetal gestations as serum screening is not as accurate or not available.

Serum analytes in first trimester

• free β-hCG and PAPP-A <pregnancy-associated plasma protein A >

• fetal Down syndrome :

– free β-hCG level ≥ 2.0 MoM

– PAPP-A level ≤ 0.5 MoM.

With trisomy 18 and trisomy 13, levels of both analytes are lower

Combined first trimester testing• NT measurement with serum hCG and PAPP-A.

• Down syndrome detection rates is 79 to 87 percent, a false-positive rate of 5 percent

• trisomies 18 and 13 detection rates is 90 percent, 2-percent false-positive rate

Full Integrated test

• Ultrasound measurement of nuchal translucency and serum analytes at the age of 10 -13 then

• At GA of 15-18 wks AFP,uE3, hCG, and inhibin A

• Detection rate 85-95 percents

• Result withheld until quad test completed

• Lowest false positive test in down syndrome

Sequential and contingent testing

• The disadvantages of integrated test is patients have to wait until the second trimester to perform the screening test thereby the sequential and contingent testing develops.

Sequential testing• Performing at first trimester

• first trimester screening test and then quad test :

– 1 % offer diagnostic test after screening

– 99% procede to quad test ,result withheld until quad test completed

• Using first trimester screening and then offering CVS only to the high risk one.

• If not a very high risk ,go on to the quad test

Contingent testing

• First trimester screen and quad test

• Three risk cut-offs

– 1. women at a very high risk< 1%>, > 1 in 50 , after 1 st trimester testing , CVS should be performed.

– 2. women at a very low risk< 84%> ,< 1 in 2000 , no additional testing after the first trimester screening

– 3. women at intermediate risk < 15%>, > 1 in 2000, but < 1 in 50, receiving second trimester test.

Quadruplet test

• Best available screening test for women who present for prenatal care in second trimester

• GA 15-18 wks

• Maternal serum for AFP uE3 hCG and inhibin A

Abnormal Serum analyte in Down syndromes

Serum analyte Result 1.Total or Free BhCG ≥ 2

2. PAPPA ≤ 0.5

3. AFP ≤ 0.74

4. Unconjugated estriol ≤ 0.8

5. Inhibin alpha ≥ 1.7

Cell free fetal DNA in maternal blood

• Next generation genomic sequencing

• 99.5% detection of trisomy 13 18 and 21 and possibly sex aneuploid

• False positive rate of 0.5% or less

• Perform 12-20 wks of gestation.

Free cell fetal DNA in maternal serum

• Candidates for using cell free fetal DNA testing as a primary screening in singleton

– maternal age of 35 years and older

– Presence of sonographic finding associated with fetal aneuploidy

– History of previous pregnancy with fetal trisomy

– Screening positive test

– Patients carry a balanced robertsoniantranslocation

Free cell fetal DNA in maternal serum

• Limitation : uninformative test, expensive

• High sensitivity and specificity : selecting the one who will be beneficial for the invasive prenatal diagnosis

Soft sign

• Soft signs: Normal variants

• Used in 15-22 weeks of gestation

• Risk increase steeply with the number of markers identified.

Sonographic markers

Likelihood ratio of markers in down syndrome

• 10 percents will have this soft signs in unaffected pregnancy

• ACOG recommends the risk adjustment based on second trimester sonographic markers is concerned because more fetal losses would result than case of ofdown syndrome identified.

Bernaceraf scoring index

Minor sonographic finding that associated with Down syndrome• 1. nuchal skinfold thickening

• 2. echogenic intracardiac focus

• 3. mild renal pelvis dilatation< pyelectasis>

• 4.echogenic bowel

• 5.clinodactyly

• 6. sandal gap

Minor sonographic finding that associated with Down syndrome

• Nuchal skinfold

– Transcerebellar view of fetal head from the outer edge of skull to the outer skin

– A measurement of 6 and greater is considered abnormal.

– More than 10 fold risk of down syndrome

– Amniocentesis should be performed even found as an isolated finding.

Nuchal skinfold

Echogenic intracardiac focus

• Echogenic intracardiac focus < EIF> is focal intracardiacpapillary calcification

• Not a cardiac abnormality

• Usually left sided

• Double the risk of down syndrome if found isolated

• Common in trisomy -13

Echogenic intracardiac focus

Pyelectasis

• Transient or physiological finding in normal fetus

• Measured on transverse image of the pelvis

• 4mm or greater is found about 2 percents of of fetuses and double the risk of down syndrome

• Likelyhood of having renal abnormality and further additional testing should perform at GA34wks

Pyelectasis

Echogenic fetal bowel

• Six fold increased risk of down syndrome

• Appears as bright as bones

• Represents :

– Small amounts of swallowed blood

– Fetal CMV infection

– Cystic fibrosis in fetus

Echogenic fetal bowel

Femur and Humeral length

• Short femur and humerus

• Short femur: ≤ 90 percents of expected which correlated with the BPD measurement

• Short humerus: ≤ 89 percents of expected which correlated with the BPD measurement

Clinodactyly, Hypoplasia of the fifth finger middle phalanx

Sandal Gap

First trimester sonographic finding in Down syndrome• Absent of fetal nasal bone

• Nuchal Translucency

• Wider frontomaxillary facial angle : flat facial profile

• Tricuspid Regurgitation

• Abnormal ductus venosus flow

– also associated with trisomy 18 and 13

Not routinely used in the US

Fetal nasal bone

• Two thirds of fetuses with down syndrome, the nasal bone is not visible in GA of 11-14 wks

• adequate assessment : 45-degree angle of insonationwith the fetal profile; in the midsagittal plane, with the tip of the nose and the third and fourth ventricles visible; and that the nasal bone brightness be greater than or equal to that of the overlying skin

Fetal nasal bone

Prenatal and preimplantation diagnosis

Prenatal and preimplantation diagnosis

• Invasive procedure

– Chorionic villi sampling

– Amniocentesis

– Fetal blood sampling

• Preimplantation diagnostic

Amniocentesis

• Transabdominal withdrawal of amniotic fluid

• Most common prodedure

• Used to diagnose the fetal genetic conditions

• GA 15-20 wks to be performed

• Assess fetal karyotype

• Takes 7 to 10 days to culture the amniocyte and the assessment of the karyotype

Technique

• Sonographic guidance

• Using 20 to 22 gauze spinal needle

• Initially 1 -2 ml of fluid is contaminated

• 20 ml of AF was collected

• After removing of the needle , puncture site is observed to check bleeding and fetal cardiac motion is documented

• If maternal Rh-D negative ,RhoGram is given after the procudure

Typical volume required for selected test.

• AF should be clear

• If blood tinged fluid is detected , transplacental passage is suspected but not associated with pregnancy loss

• In Multifetal pregnancy : indigo carmine dye is often injected before removing the first needle as the second sac entrance with clear fluid would verify the needle in the second sac.

complication

• Pregnancy loss : 1 in 300-500

• Double the pregnancy loss if BMI ≥40 kg/m2

• Twins : 1.8 percents

• Amniocentesis leakage < usually within 48 hrs after the procedure>

• Chorioamnionitis

Early amniocentesis

• Performing at the age of 11 -14 wks

• Less fluid is withdrawn : I ml per each GA

• Higher rate of complication : AF leakage, talipesequinovarus ****and fetal loss

• ACOG not recommend

CVS

• Between 10 -13 wks

• Karyotype and specialized genetic test

• Results are earlier ,allowing safer pregnancy termination

• Karyotype available in 7 -10 days

CVS Technique

• Transabdominal

• Transcervical, both are equally safe

• Transcervical villus sampling is performed using a specifically designed catheter made from flexible polyethylene that contains a blunt-tipped, malleable stylet.

CVS Technique

• Transabdominal sampling is performed using an 18- or 20-gauge spinal needle.

• With either technique, transabdominal sonography is used to guide the catheter or needle into the early placenta —chorion frondosum, followed by aspiration of villi into a syringe conta

CVS Technique

• Relative contraindications:

– vaginal bleeding or spotting

– active genital tract infection,

– extreme uterine ante- or retroflexion

. If the patient is Rh D-negative and unsensitized, anti-D immune globulin is administered following the procedure

• fetal loss rate following CVS was 2 percent compared with less than 1 percent following amniocentesis.

• An early CVS was its association with limb-reduction**** defects and oromandibular limb hypogenesis performed at 7 weeks’ gestation

• When performed at ≥ 10 weeks’ gestation, as is commonly done today, the incidence of limb defects does not exceed the background rate of 1 per 1000

• Vaginal spotting is common following transcervicalsampling, but it is self-limited and not associated with pregnancy loss.

• The incidence of infection is less than 0.5 percent

limitation of CVS

• chromosomal mosaicism is identified in up to 2 percent of specimens.

• In most cases, the mosaicism reflects confined placental mosaicism rather than a true second cell line within the fetus.

• Amniocentesis should be offered, and if the result is normal, the mosaicism is presumed to be confined to the placenta.

• Confined placental mosaicism has been associated with fetal-growth impairment and stillbirth.

Fetal Blood Sampling

• cordocentesis or percutaneous umbilical blood sampling (PUBS).

• It was initially described for fetal transfusion of red blood cells in the setting of anemia from alloimmunization

• fetal anemia assessment remains the most common indication.

Fetal Blood Sampling

• Fetal blood sampling is also performed for fetal karyotype determination, particularly in cases of mosaicism identified following amniocentesis or CVS.

• Fetal blood karyotyping can be accomplished within 24 to 48 hours.

• Although fetal blood can be analyzed for virtually any test performed on neonatal blood

Fetal Blood Sampling Technique

• Under direct sonographic guidance,

• using a 22- or 23-gauge spinal needle into the umbilical vein, and blood is slowly withdrawn into a heparinizedsyringe.

• Adequate visualization of the needle is essential.

• Fetal blood sampling is often performed near the placental


• cord insertion site, where it may be easier to enter the cord if the placenta is anterior (Fig. 14-9). Alternatively, a free loop of cord may be punctured.

• a local anesthetic may be administered. Prophylactic antibiotics are used at some centers, although there are no trials to support this policy.


• Arterial puncture is avoided, because it may result in vasospasm and fetal bradycardia.

• After the needle is removed, fetal cardiac motion is documented, and the site is observed for bleeding.

• fetal loss rate is approximately 1.4 percent

• Other complications

– cord vessel bleeding in 20 to 30 percent of cases,

– fetal-maternal bleeding in 40 percent of cases in which the placenta is traversed

– fetal bradycardia in 5 to 10 percent

Most complications are transitory, with complete recovery, but some result in fetal loss.

Preimplantation Genetic Testing

• Genetic testing performed on oocytes or embryos before implantation ,in vitro fertilization (IVF), may provide valuable information regarding the chromosomal complement and single-gene disorders

Preimplantation Genetic Testing

• Preimplantation genetic diagnosis

• Preimplantation genetic screening

– 1. polar body analysis

– 2. blastomere biopsy

– 3. trophectoderm biopsy

Polar body analysis

• Maternally inherited genetic disorder.

• The first and second polar bodies are extruded from the developing oocyte.

• Sampling should not affect fetal development

• Disadvantages : paternal genetic contribution is not evaluated.

Blastomere biopsy

• Done at the 6- to 8-cell (cleavage) 3 days old, then one cell is removed.

• limitation : mosaicism of the blastomeres may not reflect the chromosomal complement of the developing embryo

• The technique is associated with a 10-percent reduction in the pregnancy rate

Blastomere biopsy

Trophectoderm biopsy

• 5 to 7 cells from a 5- to 6-day blastocyst

• Advantage :no embryal cells are removed as trophectoderm cells give rise to the trophoblast .

• Disadvantageously :performed later in development

Trophectoderm biopsy

Preimplantation Genetic diagnosis

• PCR amplified genome region

• FISH : chromosome rearrangement

• CVS or AC should be done to confirm the PGD

prenatal diagnosis

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