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Cell-free DNA testing for fetal aneuploidy&First trimester combined screening for preeclampsia

Daniel L RolnikMSc, MD, PhD

MFM Fellow – Monash UniversityDiploma in Fetal Medicine (FMF, UK)

daniel.rolnik@monash.edu

Evolution of screeningCell-free DNA testing

Serum ScreeningMaternal Age Nuchal Translucency

Combined first trimester screening

Cell-free fetal DNA

1970’s 1980’s 1990’s 2011 What is next?- Chromosomal imbalances- Fetal cell microarray- Whole genome sequencing

Screening DR T21 False-positiveMaternal age > 35 years 30% 20%

2nd trimester serum screening 70-75% 5%

Nuchal translucency 80% 5%

Combined 1st trimester screening 90% 5%

Cell-free DNA 99.2% 0.09%

Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. Lancet, 1997.

TechnologyCell-free DNA testing

13 18 214 X Y

The cfDNA fragments are sequenced, their chromosome origin is established, and the quantity of each chromosome is determined

There are many millions of cf DNA fragments in maternal plasma

Screening

Cell-free DNA testing

Gil MM, Accurti V, Santacruz B, Plana MN, Nicolaides KH. Analysis of cell-free DNA in maternal blood in screening for aneuploidies: Updated meta-analysis. Ultrasound Obstet Gynecol 2017

0.04%

0.04%

FPRDR

Trisomy 21 99.7% (99.1-99.9)

Trisomy 18 97.9% (94.9-99.1)

0.04%Trisomy 13 99.0% (65.8-100)

100,000 pregnancies

T21n=200

Normaln=99,692

T18n=80

T13n=28

T21n=199 Normal

n=120

T18n=78

T13n=27

cfDNA testing

T21, T18, T13: n=304 (of 308) 0.12%Combined

ScreeningCell-free DNA testing

Population screening DR False-positive

PSA for prostate cancer 51% 12%

Breast cancer screening 79% 10%

Cervical screening 80% 5%

Combined 1st trimester screening for T21 90% 5%

Cell-free DNA for T21 99.2% 0.09%

How good is cfDNA testing for Down’s syndrome?

Contraindications for cfDNA testing• GA < 10 weeks• Vanishing twin• Miscarriage• Major fetal defects

The importance of fetal fractionCell-free DNA testing

Wright D, Wright A and Nicolaides KH. A unified approach to risk assessment for fetal aneuploidies. Ultrasound Obstet Gynecol 2015

Fetal fraction

Chro

mos

ome 2

1 Z-sc

ore

-3

0

3

6

9

12

15

18

21

0 5 10 15 20 25 30 35

FF 8%FF 2%24

Euploid T21

FF 2%

Euploid T21

FF 4%

Euploid T21

FF 10%

Euploid T21

FF 20%

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Lee TJ, Rolnik DL, Menezes MA, McLennan AC and da Silva Costa F. Cell-free fetal DNA testing in singleton IVF conceptions. Hum Reprod 2018.

Fetal fraction x BMI and IVFcfDNA and PE screening

Fetal Fraction in IVFFetal Fraction x BMI

True positiveFalse positive

%

0

10

20

30

40

50

60

70

80

90

100

110

SpontaneousT21 T18 T13 XO Other SCA

IVFT21 T18 T13 XO Other SCA

All pregnanciesT21 T18 T13 XO Other SCA

TOTALSpontaneous IVF

Performance cfDNA x IVF

RecommendationsCell-free DNA testing

• Ultrasound before cfDNA testing is advisable – finding 10% of the pregnanciesØ Miscarriage, < 10 weeks, Vanishing twin, Structural defects / increased NT

Brown I, Fernando S, Menezes M, da Silva Costa F, Ramkrishna, J, Meagher S, Rolnik DL. The importance of ultrasound preceding cell-free DNA prenatal testing for fetal chromosomal abnormalities. Submitted 2020

• 11-14 weeks morphology scan and PE screening !!!Ø A significant proportion of fetal abnormalities can be identified

Ø All: acrania, alobar holoprosencephaly, encephalocele, tricuspid or pulmonary atresia, pentalogy of Cantrell, ectopia cordis, exomphalos, gastroschisis and body stalk anomaly

Ø >50%: open spina bifida, HLHS , AVSD, complex heart defects, left atrial isomerism, lower urinary tract obstruction, amputations of extremities, arthrogryposis or fetal akinesia deformation sequence and lethal skeletal dysplasia

11-14 weeks (n=100,997) 18-24 weeks (n=99,677)

101,793 pregnancies11-14 weeks

30-37 weeks (n=71,514)

1,720 abnormalities (1.7%)796 aneuploidies

Detected 474 (27.6%) Detected 926 (53.8%) Detected 320 (18.6%)

Syngelaki A, Hammami A, Bower S, Zidere V, Akolekar R, Nicolaides K. Diagnosis of fetal non-chromosomal abnormalities on routine ultrasound examination at 11-13 weeks' gestation. Ultrasound Obstet Gynecol 2019.

RecommendationsCell-free DNA testing

• Genome-wide cfDNA analysis – NOT YETØ Clinical implications of rare autosomal trisomies?Ø Confined placental mosaicism (CPM)Ø Increase in overall false-positive rate

Ø Belgium: 3,373 tests- 17 segmental dups / dels – all normal clinical follow-up

Ø The Netherlands: 56,818 tests- 101 RATs – six confirmed and only one with abnormal phenotype

Ø DiGeorge syndrome (22.q11 microdeletion)

Do you want comprehensive screen for all 23 chromosomes and large dups / dels?

MORE IS NOT ALWAYS BETTER !

Incidence PPV FPR RecommendationGeneral population 0.1% 3.8% 0.24% No Screening

Aortic arch abnormalities 20% 89% 10% Invasive testing

Gross SJ, Stosic M, McDonald-McGinn DM, Bassett AS, et al. Clinical experience with single-nucleotide polymorphism-based non-invasive prenatal screening for 22q11.2 deletion syndrome. Ultrasound Obstet Gynecol 2016.

First trimester combined screening for preeclampsia

Why?Screening for PE

Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. Lancet, 1997.

2 - 3% of all pregnancies

6.6 million cases per year worldwide

US - 18% of maternal deaths

1 maternal death every 12 minutes

15% of premature deliveries

Future cardiovascular risk

Risk scoring / guidelines

Screening for PE

High risk factors• Previous pre-eclampsia• Chronic renal disease• Chronic hypertension• Diabetes mellitus• SLE or APS

Moderate risk factors• First pregnancy•Age > 40 yrs• Body mass index > 35 kg/m2

• Inter-pregnancy interval > 10 yrs• Family history of pre-eclampsia

NICE guidelines 2010

High risk factors

• Previous pre-eclampsia• Chronic renal disease• Chronic hypertension• Diabetes mellitus• SLE or APS

Moderate risk factors

• First pregnancy•Age > 35 yrs• Body mass index > 30 kg/m2

• Inter-pregnancy interval > 10 yrs• Family history of pre-eclampsia• Black or poor

ACOG guidelines 2018SMFM

Simple, but:

- No personalized risk- And…

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Risk scoring / guidelinesScreening for PE

Tan MY et al. Comparison of diagnostic accuracy of early screening for pre-eclampsia by NICE guidelines and a method combining maternal factors and biomarkers: results of SPREE. Ultrasound Obstet Gynecol 2018

23%

High-risk x prevention – NICE, UK

010

20304050

60

80

90100

70

Det

ectio

n ra

te (%

)

<37w >37w Total

SPR 10.2%

41%

26%30%

Combined screening 11-14 weeksScreening for PE

0

10

20

30

40

50

60

80

90

100

70

Det

ectio

n r

ate

(%)

History, MAP, UT PI, PLGF

PE <37w

75%

PE <34w

90%

PE >37w

45%

SPR 10%

Low risk

24 28 32 36 40 44 48 52 56 60 64 68 72 76 80Gestational age at delivery with preeclampsia (w)

0.5%

24 28 32 36 40 44 48 52 56 60 64 68 72 76 80Gestational age at delivery with preeclampsia (w)

High risk30%

O'Gorman N, Wright D, Syngelaki A, Akolekar R, Wright A, Poon LC and Nicolaides KH. Competing risks model in screening for preeclampsia by maternal factors and biomarkers at 11-13 weeks gestation. Am J Obstet Gynecol 2016.

The Fetal Medicine Foundation

Rolnik DL, Wright D, Poon L, et al. Aspirin versus placebo in pregnancies at high risk of preterm preeclampsia. N Engl J Med 2017.

0

10

20

30

40

50

60

80

90

100

70

Pre

ve

nti

on

ra

te (

%)

<34w

18%

38%

<37w

95%

>37w

82% 62%

<32w

11%

89%

High-risk group agreeing to RCT

Screening at 11-13wN = 26,941

Assigned to aspirinN = 798

Assigned to placeboN = 822

150 mg/day12 to 36 w Spain: Murcia, Granada, Tenerife

Belgium: Brussels Greece: AthensItaly: MilanEngland: 6 NHS hospitals

Aspirin 11-14 weeks

PE prevention

Rolnik DL, da Silva Costa F, Lee TJ, Schmid M and McLennan AC. Association between fetal fraction on cell-free DNA testing and first-trimester markers for pre-eclampsia. Ultrasound Obstet Gynecol 2018.

Fetal fraction x pregnancy complicationscfDNA and PE screening

Will fetal fraction become a marker of placentation and adverse pregnancy outcome?

Cell-free DNA test is very good for T21Cell-free DNA test is good for T18 and 13 – Ultrasound Cell-free DNA test is really bad for everything else

Fetal fraction is important and closely related to test accuracy

Regardless, 11-14 weeks ultrasound is essential

Maternal history alone is poorly predictive of PE

Combined screening for pre-eclampsia at 11-14 weeks Aspirin high-risk groups reduces early-onset PE by 90%

Fetal fraction is likely to become a good marker of placentation (PE, FGR)

ConclusionscfDNA and PE screening

Thank you

The Fetal Medicine Foundation

Transabdominal ultrasound• Identify uterine arteries:

- Sagittal section of the cervix- Color flow mapping- Move transducer from side to side- Arteries are at the level of the internal os

• Sampling gate: 2 mm to cover whole vessel• Angle of insonation: less than 30º• Peak systolic velocity: more than 60 cm/s

• Mean PI: average PI (left + right / 2)

Uterines 11-14 weeksScreening PE

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The Fetal Medicine Foundation

• Device: Automated (3BTO-A2, Microlife, Taipei, Taiwan),calibrated at regular intervals.

•Method: Women rested for 5 minutes, arms supportedat the level of the heart.

• Cuff size: Small (<22 cm), normal (22-32 cm) orlarge (33-42 cm), depending on the mid-armcircumference.

• Both arms: Take average of two measurements in each arm.

MAP 11-14 weeks

Screening PE The Fetal Medicine Foundation Guidelines – ISUOG

Screening PE

The Fetal Medicine Foundation Guidelines – ISSHP

Screening PE The Fetal Medicine Foundation Guidelines – FIGO

Screening PE