Supachai Nathongchai, MD. Prenatal screening & Diagnosis Prenatal screening & Diagnosis Supachai Nathongchai, MD. Department of Obstetrics and Gynecology SomdejprachoaTaksinmaharaj Hospital

Population (pregnant women) Concept of Screening Population (pregnant women) Screening test Screen Positive Screen Negative Diagnostic test Amniocentesis CVS Fetal blood sampling No further test

Prenatal Screening Structural abnormalities Chromosome abnormalities

Chromosome abnormalities Trisomy 21 Trisomy 13 Trisomy 18 Monosomy X Triploidy

Chromosome abnormalities Prenatal diagnosis Chorionic villous sampling Amniocentesis Fetal blood sampling Preimplantation genetic diagnosis But ! All methods subject to risk of miscarriage

Screening for Down syndrome Maternal age Ultrasonography Maternal blood tests Combined

Incidence of Down syndrome 35 years 1/386 1/274 Maternal age LB 2nd trim. 25 years 1/1250 1/887 30 years 1/965 1/685 35 years 1/386 1/274 38 years 1/182 1/129 40 years 1/100 1/78 45 years 1/31 1/22

Maternal age for DS screening Age > = 35 yr. 10 % 20-30 % Age > 35 yr. 90 % 70-80 % Age group Pregnancy DS baby

Women < 35 years low risk 70-80% of Down syndrome Neglected

Ultrasonography First trimester screening Nuchal translucency (NT) Nasal bone

Observations on an ethnic classification of idiots Langdon Down “ ….. The nose is small. The skin has a slight dirty yellowish tinge, and is different in elasticity, giving the appearance of being too large for the body.”

Nuchal Translucency (NT)

Nuchal translucency (NT) Measurement of NT

Nuchal translucency (NT) Measurement of NT

Measurement of nuchal translucency The maximum thickness of the subcutaneous translucency between the skin and the soft tissue overlying the cervical spine should be measured by placing the callipers on the lines as shown. During the scan, more than one measurement must be taken and the maximum one should be recorded. + + + + + + + + + +

Measurement of (NT) CRL 45-84 mm GA 11-13 wk Fetus Sagittal section Neutral position Occupies 3/4 of image

Reference range of fetal nuchal translucency with CRL showing the 5th, 25th, 50th, 75th and 95th centiles The 11-14-week scan. The diagnosis of fetal abnormalities

Likelihood ratios for trisomy 21 in relation to the deviation in fetal nuchal translucency thickness The 11-14-week scan. The diagnosis of fetal abnormalities

Recent studies examining the implementation of fetal nuchal translucency screening at 10-14 weeks Authors N Risk cutoff DR(%) FPR(%) Snijders et al 96,127 1 in 300 82.2 8.3 Theodoropoulos et al 3,550 91.0 4.9 Adekunle et al 11,398 76.0 4.7 Schwarzler et al 4,523 83.0 Gasiorek-Wiens et al 23,805 87.6 13.0 Zoppi et al 12,495 90.0 9.0 Brizot et al 2,996 7.4 DR = Detection rate ; FPR = False positive rate

Observations on an ethnic classification of idiots Langdon Down “ ….. The nose is small. The skin has a slight dirty yellowish tinge, and is different in elasticity, giving the appearance of being too large for the body.”

Absence of nasal bone in fetuses with trisomy 21 at 11-14 weeks of gestation Cicero S, et al. The Lancet 2001

Estimated sensitivity and false positive rate for risk cutoffs in screening for trisomy 21 Risk NT screening NT and NB screening cutoff Sensitivity (%) FPR (%) Sensitivity (%) FPR (%) 1 in 20 57.36 0.98 81.90 0.62 1 in 35 62.88 1.35 85.89 1.02 1 in 50 65.03 1.76 86.81 1.30 1 in 100 72.09 3.01 88.65 1.64 1 in 150 74.54 4.32 89.26 1.98 1 in 200 77.30 5.71 90.18 2.37 1 in 250 80.67 7.08 91.41 2.68 1 in 300 82.21 8.28 92.02 3.02 1 in 500 85.58 14.00 92.94 4.37 1 in 1000 92.33 27.40 94.7 8.15 Cicero S, et al. The Lancet 2001

Nuchal translucency in twin pregnancies Sebire NJ,e t al. Br J Obstet Gynaecol 1996 448 twin pregnancies Fetal nuchal translucency thickness was measured by ultrasound examination at 10-14 weeks Sensitivity and false positive rates of screening for trisomy 21 were calculated The sensitivity is similar to that in singleton pregnancies The specificity is lower because translucency is also increased in chromosomally normal monochorionic twin pregnancies

Fetal karyotyping in twins In twin pregnancies, selection of the appropriate invasive technique depends on the: Accuracy of obtaining a result from both fetuses Procedure-related risk of fetal loss The risks of selective fetocide should the pregnancy be found to be discordant for an abnormality and the parents choose this option

Fetal karyotyping in twins There is an additional advantage of screening by measurement of nuchal translucency thickness; when there is discordancy for a chromosomal abnormality, the presence of a sonographically detectable marker (increased nuchal translucency) helps to ensure the correct identification of the abnormal twin should the parents choose selective termination.

3-D ultrasound measurement of fetal nuchal translucency It can be used to replicated nuchal translucency measurements only when nuchal skin can also be clearly seen on 2-D ultrasound. Paul C, et al. Ultrasound Obstet Gynecol 2001 The possibility of rotating a stored volume and inspecting it in three orthogonal planes makes 3-D ultrasound a useful tool for nuchal translucency measurements, especially in doubtful cases. Clementschitsch G, et al. Ultrasound Obstet Gynecol 2001

Conditions associated with increased nuchal translucency Cardiac defects Diaphragmatic hernia Exomphalos Achondrogenesis type II Achondroplasia Asphyxiating thoracic dystrophy Beckwith-Wiedemann syndrome Blomstrand osteochondrodysplasia Body stalk anomaly Campomelic dysplasia EEC syndrome Fetal akinesia- deformation sequence Fryn syndrome GM1-gangliosidosis Hydrolethalus syndrome

Ultrasound screening of Down syndrome The second trimester Ultrasound screening of Down syndrome

Genetic Sonogram: Ultrasound indicators for Down 1. Structural anomalies : Cardiac anomaly, DU atresia 2. Soft marker : Echogenic intracardiac foci Pyelectasis, Femur length Humerus Length

Structural anomalies Trisomy 13 Trisomy 18 Trisomy 21 90% 70-80% 20%

Soft ultrasound makers Nuchal skin fold thickness Choroid plexus cyst (CPC) Mild ventriculomegaly Nasal bone Echogenic intracardiac foci (EIF) Echogenic bowel Renal pyelectasia Hypoplastic middle phalanx of the fifth digit

Nuchal skin fold thickness Langdon Down (1866) =  thickness of skin of the back Benacerraf et al (1985) = Using as an U/S marker for Down

Fig NT

Cut off threshold of nuchal thickness > 6 mm. 16-20 wks gestation Down abnormal nuchal thickness 40% LR: 17 (CI 8-35) => Expert review is recommended, and karyotyping should be offered Sensitivity 34% Specificity 99.5%

Nuchal thickness : Efficacy Associated with - Congenital heart disease - Genetic syndromes (rare) => Expert review is recommended

Choroid plexus cyst (CPC) Cyst > 3 mm 14 to 24 weeks’ gestation Locate in lateral ventricle Related more to Trisomy 18 Not associated with other fetal abnormalities or abnormal postnatal development.

CPC and Trisomy 18 Snijders et al (1994) Trisomy 18 (58 cases) CPC 50% Normal fetusesCPC 1% Isolated CPCminimal increase risk

Isolated CPC Trisomy 18 Trisomy 21 Size, Disappearance and Laterality LR (trisomy 18) = 7 (CI 4-12) Trisomy 21 Likelyhood ratio = 1.87 (CI 0.78-4.46) Size, Disappearance and Laterality => not increase or reduce risk Gross et al 1995, Gupta et al 1997 , Yoder et al 1999

Recommendation Fetal karyotyping should only be offered if isolated CPCs are found in women 35 years or older or if the maternal serum screen is positive for either trisomy 18 or 21 All women with fetal CPCs and additional malformation should be offered referral and karyotyping All women with CPCs and additional soft markers should be offered additional counselling and further ultrasound review

Mild ventricular dilatation Lateral ventricle : constant along gestation 6.1 + 1.3 mm. Male : female = 6.4 :5.8 mm Definition 10-15 mm. Dangling choroid plexus > 3 mm.from medial wall

Isolate mild ventriculomegaly  abnormal fetal karyotype ~ 3.8% (0-28.6%)  0.15% of chromosomally-normal fetuses  1.4% of trisomy 21 fetuses in the second trimester  LR 9

Recommendations Neonatal assessment and follow-up are important to rule out associated abnormalities and are important because of the potential for subsequent abnormal neurodevelopment Cerebral ventricles greater than or equal to 10 mm are associated with chromosomal and central nervous system pathology. Expert review should be initiated to obtain the following: a detailed anatomic evaluation looking for additional malformations or soft markers laboratory investigation for the presence of congenital infection or fetal aneuploidy MRI as a potential additional imaging technique

Echogenic intracardiac focus (EIF) A bright focus in papillary muscle Attach to cordae tendinae Move with valvar movement Calcium deposit and surrounded by fibrous tissue Bettelheim et al 1999 Lt ventricle 96% Both ventricle 4.3% Rt ventricle 4.7%

Fig

EIF : Rt ventricle : Both ventricle Bromley 1998 EIF : Rt ventricle : Both ventricle Twice the risk Wax + Philput 1998 EIF : Multiple : Dense echo Increase risk

Location of EIF The evidence is best for left or biventricular EICF LR 2.8 (95% CI 1.5-5.5) =>high-risk women. Low-risk => LR 0–1.8 Consensus of the SOGC Imaging and Genetics Committees suggests an LR of 2

Recommendations EICF should be evaluated as part of the 4-chamber cardiac review during the 16- to 20- week ultrasound Isolated EICF with a fetal aneuploidy risk less than 1/600 by maternal age (31 years) or maternal serum screen requires no further investigations Women with an isolated EICF and a fetal aneuploidy risk greater than 1/600 by maternal age (31 years) or maternal serum screening should be offered counselling regarding fetal karyotyping Women with right-sided, biventricular, multiple, particularly conspicuous, or nonisolated EICF should be offered referral for expert review and possible karyotyping

Echogenic bowel echogenicity of abdominal content Compare to liver and bone echo Associated with - Trisomy 21 - Congenital infection - Meconeum ileus (cystic fibrosis) - Bowel atresia - FGR, FDIU

Echogenic bowel Nyberg et al : Grading system Grade 1 : mild echogenic ; diffuse Grade 2 : mod echogenic ; focal Grade 3 : Very echogenic ; bone echo

Echogenic bowel Echogenic bowel seem to increase risk 0.6-2.4% of all 2nd trimester fetuses Increased risk for fetal aneuploidy =>LR 6 (CI 2.7 - 6.8) Sensitivity 14.7% Specificity 98% Echogenic bowel seem to increase risk The more the echo, The higher the risk Deren et al 1998

Recommendations 1. Evaluation of the fetal bowel should be done routinely during 16-20 week obstetric ultrasound. 2. Echogenic bowel should be identified by comparison with the echogenicity of surrounding bone using an appropriate transducer and gain setting. Bowel echogenicity equal to or greater than bone is significant (grade 2 or 3). 3. No further investigation are required for grade 1 echogenic bowel

4. Grade 2 and 3 echogenic bowel is associated with both chromosomal and nonchromosomal abnormalities. Expert review is recommended to initiate the following: a. detailed ultrasound evaluation looking for additional structural anomalies or other soft markers of aneuploidy b. detailed evaluation of the fetal abdomen looking for signs of bowel obstruction or perforation c. detailed evaluation of placental characteristics (echogenicity, thickness, position, and placental cord insertion site) d. genetic counselling e. laboratory investigations that should be offered, including fetal karyotype, maternal serum screening, DNA testing for cystic fibrosis (if appropriate), and testing for congenital infection

MILD PYELECTASIS 5 mm - 10 mm

MILD PYELECTASIS Isolated pyelectasis 0.7% Isolated finding in fetal Down syndrome ~ 2% LR for Down syndrome ~ 1.9 (95% CI 0.7–5.1)

Recommendations 1. Evaluation of fetal kidneys is a part of the screening ultrasound at 16 to 20 weeks,’ and if pyelectasis is visualized, the renal pelvis should be measured in the anterior/posterior diameter 2. All fetuses with renal pelvic measurements > 5 mm should have a neonatal ultrasound, and those having measurements > 10 mm should be considered for a third trimester scan 3. Isolated mild pyelectasis does not require fetal karyotyping 4. Referral for pyelectasis should be considered with additional ultrasound findings and (or) in women at increased risk for fetal aneuploidy owing to maternal age or maternal serum screen results

SHORT FEMUR LENGTH Below 2.5th percentile or Less than 0.9 of that predicted by the measured BPD Sensitivity 16% FPR 4% LR 2.7 (95% CI 2.1-6.0)

SHORT HUMERUS LENGTH Below 2.5th percentile or Less than 0.9 of that predicted by the measured BPD Sensitivity 9% FPR 3% LR 7.5 (95% CI 4.5-12)

FIFTH FINGER CLINODACTYLY 3.4% of normal fetuses 18.8% of fetuses with Down syndrome. LR 5.6 (95% CI 2.5–11.9).

RISK ASSESSMENT

The Scoring Index Major anomaly 2 Nuchal fold > 6 mm 2 Short femur 1 Short humerus 1 Pyelectasis > 4 mm 1 Hyperechoic bowels 1 Echogenic intracardiac focus 1 J Ultrasound Med 11:449-458, 1992

Modified scoring index Sensitivity 86.8%; False positive rate 27.1% Maternal age 35-39 1 Maternal age > 40 2 Scoring index > 2 Sensitivity 86.8%; False positive rate 27.1%

Abnormal genetic sonogram one or more abnormal USG markers sensitivity 87% specificity 91% positive predictive values 11% negative predictive values 99.8% Yeo L, Vintzileos AM: The use of genetic sonography to reduce the need for amniocentesis in women at high risk for Down syndrome. Semin Perinatol 27:152-159, 2003

Likelihood Ratios for Down Major anomaly 25.0 Nuchal fold 18.6 Hyperechoic bowels 5.5 Short humerus 2.5 Short femur 2.2 Echogenic intracardiac focus 2.0 Pyelectasis 1.6 No markers 0.4 Ultrasound Obstet Gynecol 1998;12:8-14.

The Extended Genetic Sonogram

Screening Modalities Maternal age Ultrasonography Biochemical markers Combined screening

Maternal age + free bhCG + PAPP-A Detection rate for Down 60%

“Combined testing” Nuchal translucency and free bhCG and PAPP-A Brizot et al 1994/1995 Orlandi et al 1997 de Graaf et al 1999 De Biasio et al 1999 Spencer et al 2000 Studty Trisomy 21 GA FPR(%) DR(%) 80 11 37 13 210 10-14 5.0 3.3 89 87 85

Second Trimester Biochemical Screening Triple Screening Alpha-fetoprotein Human chorionic gonadotropin Unconjugated estriol Other markers Dimeric inhibin A Dried blood, urine markers

Objective : To increase prenatal detection of Down syndrome (DS) Neural tube defects (NTD) Trisomy 18 Timing ONLY between 14-21 weeks

AFP hCG uE3 Trisomy 21 Open NTD Trisomy 18

Factors affecting triple analytes levels Gestational age Maternal weight Ethnicity Multiple pregnancy Smoking Type I diabetes mellitus

Screen positive for NTD (AFP > 2.5 MoM) USG Confirm GA R/O twins, gross anomalies Repeat AFP test Targeted USG Brain (Lemon and Banana signs) Spine

Screen positive for DS (DS risk) > 1:270 USG Confirm GA R/O twins, gross anomalies Genetic counselling risk of DS based on age and TS results risk of amniocentesis (1:200)

Screen positive for trisomy 18 (Trisomy 18 risk > 1:100) USG Confirm GA R/O gross anomalies Genetic counselling risk based on age and TS results risk of amniocentesis (1:200)

Triple screen Negative DS screening efficacy - 70% for women under 35 years - 90% for women 35 years and over Open NTD Screening efficacy - 95% for anencephaly - 80% for spinal bifida

Second trimester comparable US screening vs Biochemical screening Sensitivity False positive Biochemical Reduce risk : more No need for highly skilled physician Combine test : most benefit comparable

Diagnostic tests (Gold standard) Karyotyping Down syndrome Trisomy 18 Targeted ultrasonography Open neural tube defects

Prenatal diagnosis Prenatal diagnosis has been called “one of the most powerful advances in medical technology.”

Amniocentesis Technique Blindly Static ultrasound (late 1970s and early 1980s) Real-time ultrasound - Marking - Ultrasound guidance

Amniocentesis Technique 16-20 weeks’ gestation 20-22 guage spinal needle Approximately 20 ml of amniotic fluid is collected The first 1-2 ml of amniotic fluid is discarded to minimise the chances of maternal cell contamination

Amniocentesis Safety The Canadian Collaboration Trial 1020 pregnancies No difference in loss rates from that of controls Correlation between fetal losses and > 2 insertions per procedures and the needles of 19-guage or higher

CVS Technique 9-12 weeks’ gestation Transcervical or transabdominal (Equally safe and efficacious ) The chorion frondosum, which contains the most mitotically active cells, is the area of the placenta that is sampled

Fetal blood sampling Technique Cordocentesis or Percutaneous umbilical blood sampling (PUBS) 2. Intrahepatic vein sampling 3. Cardiocentesis * 2 & 3 are associated with increased risks

PUBS Technique 20-22 guage needle. The most commonly used location is within 2 cm. of the placental insertion. Fetal blood have been obtained from umbilical artery, but generally the umbilical vein is preferred because it is larger and less likely to be associated with a fetal bradycardia.

Diagnostic embryofetoscopy History Embryoscopy was first performed via the transcervical approach early in gestation, previous to fusion of the chorion and amnion, to make a phenotypic diagnosis. Percutaneous techniques were then developed to introduce the fetoscope directly into the amniotic cavity transabdominally.

7 weeks’ gestation

14 weeks’ gestation

16 weeks’ gestation

Recommendation Offer amniocentesis or CVS to Offer screening to Women age 35 years and over at EDC Previous aneuploidy Offer screening to Women age 34 years or under at EDC Women age 35 years and over who are reluctant to accept amniocentesis