MARIA DEL MAR GIL MIRA

The Fetal Medicine Foundation

Clinical application and evidence of cfDNA testing

María del Mar Gil

No conflict of interest to disclose

The Fetal Medicine Foundation

Maternal blood

Extracellular DNA in the blood of pregnant women [Article in Russian]

Kazakov I et al. Tsitologiia 1995 Presence of fetal DNA in

maternal plasma and serum Lo D et al. THE LANCET 1997

Cell-free fetal DNA in maternal blood

The Fetal Medicine Foundation

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Cell-free fetal DNA in maternal blood

The Fetal Medicine Foundation

13 18 21

4 X Y

The cf DNA 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

Cell-free fetal DNA in maternal blood

The Fetal Medicine Foundation

In trisomy 21 cfDNA fragments from chromosome 21 are increased

13 18 21

4 X Y

Cell free DNA test

Screening for Down Syndrome

The Fetal Medicine Foundation

Chrom 21 100 units

Chrom 18 100 units

Chrom 13 100 units Chrom 21

105 units

Trisomy 21

Cell free DNA test

Screening for Down Syndrome

The Fetal Medicine Foundation

Trisomy 21 (n=8)

0 20 40 60 80 100

Risk for trisomy 21 (%)

0 20 40 60 80 100

Risk for trisomy 18 (%)

Trisomy 18 (n=2)

0 20 40 60 80 100

Risk for trisomy 21 (%)

0 20 40 60 80 100

Risk for trisomy 18 (%)

Euploid (n=1939)

0 20 40 60 80 100

Risk for trisomy 21 (%)

0 20 40 60 80 100

Risk for trisomy 18 (%)

(n=1939) (n=1937)

No result 5%

Nicolaides et al, Am J Obstet Gynecol 2012

Are the results from high-risk pregnancies applicable to routine screening?

Stored plasma (2 mL) routine combined screening at 11-13 w n=2,049

Cell free DNA test

Screening for Down Syndrome

The Fetal Medicine Foundation

Cell free DNA in maternal blood Screening for trisomies

Gil MM et al. Ultrasound Obstet Gynecol 2017; doi: uog.17484

Trisomy 21 n=1,963 Trisomy 18 n= 563 Trisomy 13 n= 119 New

Trisomy 21 n=1,051 Trisomy 18 n= 389 Trisomy 13 n= 139 Previous

Gil MM et al. Ultrasound Obstet Gynecol 2015;45:249.

The Fetal Medicine Foundation

Publication bias

0 50 100 150

Precision

-0.06 -0.03 0.00 0.03 0.06 0

500 1000 1500 2000 2500

Proportion

Precision

Trisomy 13

PrecisionPrecision

0 10 20 30

0 1000 2000 3000 4000

Proportion

-0.5 0.0 0.5 1.0 1.5 2.0

-0.050 -0.025 0.000 0.025 0.050 Proportion

1000 2000 3000 4000

0

Precision

0 20 40 60 80

Precision

0.4 0.7 1.0 1.3 1.6 Proportion

-0.050 -0.025 0.000 0.025 0.050 Proportion

Trisomy 18

0.5 0.8 1.1 1.4 1.7 Proportion

Trisomy 21

Proportion of studies with low, high or unclear risk (%)

QUADAS-2 Assessment

0% 20% 40% 60% 80% 100%

CONCERNS REGARDING APPLICABILITY

Index test

Reference standard

Patient selection

RISK OF BIAS

Patient selection Flow and timing

Index test

Reference standard

Cell free DNA test

Quality / Bias assessment

The Fetal Medicine Foundation

• Which conditions to screen for

• Interpretation of results

• Importance of fetal fraction

• Test failure

• Screening in twins

• Models of implementation

Cell free DNA test

Points of discussion

The Fetal Medicine Foundation

• Lethal Turners easily detectable by ultrasound

• Most cases are mild without intellectual disability

• 50% of sex chromosome aneuploidies are mosaics

• High incidence of maternal mosaicism

• Performance of screening is uncertain - Failure rate is higher than for T21

- Detection rate is lower (about 90%) than for T21 (>99%) - False +ve rate is higher (about 0.3%) than for T21 (<0.1%) - Positive predictive value much lower than for T21

• Management of positive result: need for amniocentesis

Cell free DNA test Which aneuploidies?

Screening for sex chromosome aneuploidies

The Fetal Medicine Foundation

Microdeletions ^{1/ births >3Mb}

22q11.2 deletion 2-4,000 85%

Angelman / Prader Willi

(15q11.2-q13 deletion) 20,000 70%

1p36 del 5,000 85%

Cri-du-chat (5p deletion) 50,000 99%

Maternal age (yrs) Risk

T21

Di George - common features:

75% immune deficiencies 50% hypocalcemia

30% feeding problems 35% renal abnormalities 75% cardiac defects 95% intellectual deficits

25% schizophrenia in adulthood

- Detection rates *: 60-99%

- Total false positive rates: 1%

- Positive predictive value for 22q11.2 deletion: 5%

- Proportion of cfDNA panel of significant microdeletions: 10%

* quoted by companies, but do not include those of <3 Mb

Yaron Y et al. Current status of testing for microdeletion syndromes and rare autosomal trisomies using cell-free DNA technology. Obstet Gynecol 2015;126:1095 Wapner RJ et al. Expanding the scope of noninvasive prenatal testing: detection of fetal microdeletion syndromes. Am J Obstet Gynecol 2015;212:332.e1-9.

Cell free DNA test

Which aneuploidies?

The Fetal Medicine Foundation

Feta l frac tion ( % )

0 5 10 15 20 25 30 35 40

50 60 70 80 90 100 110 120

Maternal weight (kg)

Triploidy Diandric 4 Correct 4

Triploidy Digynic 4 ‘Low FF’ 3 Failed 1

Normal 48 Correct 44

Failed 4

0.5^th centile

Nicolaides KH, Syngelaki A, Gil MM, Quezada MS, Zinevich Y. Prenatal detection of fetal triploidy

from cell-free DNA testing in maternal blood. Fetal Diagn Ther 2014; 35: 212-217.

Cell free DNA test

Which aneuploidies?

The Fetal Medicine Foundation

• Trisomy 21 YES

• Trisomies 18/13 Yes

• Sex chromosomes No

• Triploidy No

• Microdeletions Not yet

Cell free DNA test

Which aneuploidies?

The Fetal Medicine Foundation

100,000 pregnancies

Trisomy 21 N=200

99,705 normal

199

99.7% 0.04% 40

Detected

DR False positive

Trisomy 18 N=67 98.2% 66 0.05% 40

Trisomy 13 N=28 99.0% 27 0.04% 40

Cell free DNA test

Interpretation of results

292 / 295 120

The Fetal Medicine Foundation

Trisomy 21

• DR 99.7%

• FPR 0.05%

• LR +ve 2506

• 1/(LR -ve) 300

a priori risk Odds of being affected

cfDNA +ve cfDNA -ve

1:100,000 1 in 42 (2.4%) 1 in 30,000,000 1:10,000 1 in 6 (20.1%) 1 in 3,000,000 1:1,000 1 in 1.4 (71.5%) 1 in 300,000 1:500 1 in 1.2 (83.4%) 1 in 150,000 1:100 1 in 1.03 (93.2%) 1 in 30,000 1:10 1 in 1.004 (99.6%) 1 in 3,000

1:2 1 in 1.001 (99.9%) 1 in 600

Cell free DNA test

Interpretation of results

The Fetal Medicine Foundation

Fetal fraction

cfDNA test Likelihood ratio FPR (%) DR (%) +ve -ve

4% 0.1 62.1 620 3

5% 0.1 87.4 870 8

6% 0.1 97.6 980 42

7% 0.1 99.8 990 410

8% 0.1 100 1,000 7,350

9% 0.1 100 1,000 >10,000

≥10% 0.1 100 1,000 >10,000

All 0.1 99.0 990 100

Wright D, Wright A, Nicolaides KH: A unified approach to risk assessment for fetal aneuploidies. Ultrasound Obstet Gynecol 2015; 45: 48-54 Fetal fraction

Chromosome 21 Z-score

-3 0 3 6 9 12 15 18 21

0 5 10 15 20 25 30 35

FF 8%

FF 2%

24

Cell free DNA test

Importance of fetal fraction

The Fetal Medicine Foundation

Management of failed result

In cases of failed result women should be offered diagnostic testing because of an increased risk of aneuploidy Septiembre 2015

Cell free DNA test

Test failure

The Fetal Medicine Foundation

Singleton pregnancies (n=10,698)

25

20

15

10

5

0 Normal

n=10,472

T21 n=160

T18 n=50

T13 n=16

Fetal fraction (%)

Failed result:

Normal 304

(2.9%)

Trisomy 21 3 (1.9%) Trisomy 18 4 (8.0%) Trisomy 13 1 (6.2%)

Revello R,Sarno L, Ispas A, Akolekar R, Nicolaides KH. Cell-free DNA testing for trisomies: management of failed result. Ultrasound Obstet Gynecol 2016; 47: 698-704.

Combined risk >1 in 5 Ultrasound defects Normal

placenta Small placenta

Cell free DNA test

Test failure

The Fetal Medicine Foundation

DC twins:

-The placental products of the normal fetus may mask the abnormality of the co-twin.

-Measure fetal fraction in both.

• The test is not offered by all companies

• Only some companies measure fetal fraction

Screening in twins

Cell free DNA test

The Fetal Medicine Foundation

0

Cel l- free DN A fail ure ra te (% )

100 90 80 70 60 50 40 30 20 10

Maternal body mass index (kg/m

²

) 15 20 25 30 35 40 45 50 55 60

IVF

High failure rate in twins, compared to singletons (9% vs. 3%) mainly because many twins are IVF conceptions (56% in twins vs. 10% in singletons)

BMI (kg/m²)

Rate of failure of cell free DNA testing (%)

Singleton pregnancy Twin pregnancy

Spontaneous

IVF

Spontaneous

IVF

20 0.6 4 1 6

25 1.4 9 2 12

30 3 17 5 24

35 7 32 10 41

40 14 51 19 61

45 26 70 35 77

50 44 84 54 88

Harmony test in 10,698 singletons and 438 twins Significant predictors of fetal fraction & cfDNA test failure: increasing BMI, IVF conception and twins

Failure in singletons and twins

Sarno L, Revello R,Hanson E, Akolekar R,Nicolaides KH. Prospective screening for trisomies by cell-free DNA testing of maternal blood in first trimester twin pregnancies. Ultrasound Obstet Gynecol 2016; 47: 705-11.

Cell free DNA test

The Fetal Medicine Foundation

Stored samples from pregnancies with known outcome

Canick et al: Prenat Diagn 2012; 32: 730

Gil et al: Fetal Diagn Ther 2014; 35 : 204

Gromminger et al: J Clin Med 2014; 3: 679

Fosler et al: Ultrasound Obstet Gynecol 2016;doi10.1002/uog

Trisomy 21: DR 23/24 (96%)

Trisomy 13: DR 2/2 (100%) FPR: 0/321 (0%)

Prospective studies with complete outcome Trisomy 21: DR 28/28 (100%)

Trisomy 18: DR 4/6 (67%)

FPR: 1/1098 (0.2%)

Lau et al: J Matern Fetal Neonatal Med 2013; 26: 434

Huang et al: Prenat Diagn 2014; 34: 335

Benachi et al: Obstet Gynecol 2015; 125: 1330

Papageorghiou et al: Ultrasound Obstet Gynecol 2016; 47: 188 Sarno et al: Ultrasound Obstet Gynecol 2016; 47: 705

Tan et al: Prenat Diagn 2016; 36: 672

Le Conte et al; Ultrasound Obstet Gynecol 2017; 10.1002/uog

Trisomy 13: DR 0/1 (0%) Trisomy 18: DR 1/1 (100%)

Validation / implementation in twins

Cell free DNA test

The Fetal Medicine Foundation

Cut off

FPR (%)

DR T21 (%) 100 2.2 87.0 200 3.9 90.4 300 5.4 92.1 400 6.7 93.2 500 7.9 94.0 1000 13.0 96.1 1500 17.2 97.0 2000 20.8 97.6 2500 23.9 98.0 3000 26.6 98.3 3500 29.0 98.5 4000 31.3 98.7 5000 35.2 98.9 6000 38.7 99.1

Combined screening at 11-13 wks (age, fetal NT, serum ß-hCG & PAPP-A)

High risk

>1:10

Low risk

<1:1000

Invasive test

Intermediate risk

Nothing else cfDNA test

+ve -ve

T 21 73%

T 18 91%

T 13 85%

Turner 94%

Triploidy 86%

Other 32%

Normal 0.8%

All 1.4%

Santorum M, Wright D, Syngelaki A, Karagioti N,Nicolaides KH. Accuracy of first trimester combined test in screening for trisomies 21, 18 and 13. Ultrasound Obstet Gynecol 2016; doi 10.1002/uog.17283

Nicolaides KH, Syngelaki A, Poon LC, Gil MM, Wright D. First-trimester contingent screening for trisomies 21, 18 and 13 by biomarkers and maternal blood cell-free DNA testing. 2014; 35: 185-92.

Model of clinical implementation

Cell free DNA test

The Fetal Medicine Foundation

• Which conditions to screen for: T21 and perhaps 18 / 13

• Interpretation of results: Modify prior risk with +ve and –ve LRs

• Importance of fetal fraction: Depends on the company

• Test failure: Does not increase the risk for trisomy 21

• Screening in twins: Need for more data on accuracy

• Models of implementation: Intermediate risk from combined test

Cell free DNA test

Conclusions

Thank you

The Fetal Medicine Foundation

María del Mar Gil

mgil@torrejonsalud.com

The Fetal Medicine Foundation

15 20 25 30 35 40 45 50

Maternal age (yrs)

10,000 1,000 100 10 1

Risk 1 in:

30% of fetuses with trisomy 21 in women >35 yrs

Detection rate for FPR 5%

0 10 20 30 40 50 60 70 80 90 100

%

Maternal age

Screening for Down syndrome

1970’s

The Fetal Medicine Foundation

Screening for Down syndrome

1980’s & 1990’s 2 ^nd trimester biochemical testing

Cuckle et al., 2005: Meta-analysis

hCG AFP 0

10 20 30 40 50 60 70 80 90

100 Detection rate for FPR 5%

56%

ß-hCG AFP 61%

hCG AFP E3 60%

ß-hCG AFP

E3 65%

hCG AFP E3

I

_A

67%

ß-hCG AFP

E3 I

_A

71%

Age 30%

%

Merkatz IR, Nitowsky HM, Macri JN, Johnson WE. Association between low maternal serum alpha-fetoprotein and fetal chromosomal abnormalities. Am J Obstet Gynecol 1984;

148: 886–894.

Wald NJ, Cuckle HS, Densem JW, Nanchahal K, Royston P, Chard T, Haddow JE, Knight GJ, Palomaki GE, Canick JA. Maternal serum screening for Down's syndrome in early pregnancy. BMJ 1988; 297:883-7.

Cuckle HS, Holding S, Jones R, Wallace EM, Groome NP. Maternal serum dimeric inhibin A in second-trimester Down's syndrome pregnancies. Prenat Diagn 1995; 15:385-6.

The Fetal Medicine Foundation

Crown-rump length (mm) 45 55 65 75 85 NT (mm)

0 2 4 6 8

Trisomy 21

0 4 8 n

Serum PAPP-A (M0M) 0 0.5 1.0 1.5

Euploid Trisomy 21 0

10 20 n

Serum free ß-hCG (MoM) 0 1.0 2.0 3.0 4.0 5.0

Euploid

Trisomy 21

Detection rate for FPR 5%

0 10 20 30 40 50 60 70 80 90

100 T21

Age

NT, hCG/PAPP-A T18/13

%

1990’s and beyond

•

Nicolaides KH, Azar GB, Byrne D, Mansur CA, Marks K. Nuchal translucency: ultrasound screening for chromosomal defects in the first trimester of pregnancy. BMJ 1992;

304:867

•

Snijders RJ, Noble P, Sebire N, Souka A, Nicolaides KH. UK multicentre project on assessment of risk of trisomy 21 by maternal age and fetal nuchal-translucency thickness at 10-14 weeks of gestation. Lancet 1998; 352:343

•

Spencer K, Souter V, Tul N, Snijders R, Nicolaides KH. A screening program for trisomy 21 at 10-14 weeks using fetal nuchal translucency, maternal serum free beta-human chorionic gonadotropin and pregnancy-associated plasma protein-A. Ultrasound Obstet Gynecol 1999; 13:231.

•

Kagan KO, Wright D, Valencia C, Maiz N, Nicolaides KH. Screening for trisomies 21, 18 and 13 by maternal age, fetal nuchal translucency, fetal heart rate, free ß-hCG and pregnancy-associated plasma protein-A. Hum Reprod 2008; 23:1968-75.

Screening for Down syndrome

1 ^st trimester combined test

The Fetal Medicine Foundation

DR for FPR 3%

0 10 20 30 40 50 60 70 80 90

100

97%

%

Screening for Down syndrome 1 ^st trimester combined test PLUS

11 12 13 14 Gestation (wks)

0.6 Trisomy 21

0.0 0.5 1.0 1.5 2.0

Maternal serum PLGF (MoMs)

2000-10

•

Cicero S, Curcio P, Papageorghiou A, Sonek J, Nicolaides K. Absence of nasal bone in fetuses with trisomy 21 at 11-14 weeks of gestation: an observational study. Lancet 2001;358:1665-1667.

•

Kagan KO, Cicero S, Staboulidou I, Wright D, Nicolaides KH. Fetal nasal bone in screening for trisomies 21, 18 and 13 and Turner syndrome at 11-13 weeks of gestation. Ultrasound Obstet Gynecol 2009; 33:259-64.

•

Kagan KO, Valencia C, Livanos P, Wright D, Nicolaides KH. Tricuspid regurgitation in screening for trisomies 21, 18 and 13 and Turner syndrome at 11 + 0 to 13 + 6 weeks of gestation. Ultrasound Obstet Gynecol 2009; 33:18-22.

•

Maiz N, Valencia C, Kagan KO, Wright D, Nicolaides KH. Ductus venosus Doppler in screening for trisomies 21, 18 and 13 and Turner syndrome at 11-13 weeks of gestation. Ultrasound Obstet Gynecol 2009; 33:512-7.

The Fetal Medicine Foundation

Prospective validation of 1

^st

trimester combined screening for trisomy 21 (n=108,982); FPR 5.3%

Dete c tion ra te a t ris k c ut -of f 1 i n 1 0 0 (% )

T18 T13

T 21 45,XO Triploidy

0 10 20 30 40 50 60 70 80 90 100

96% 93%

92% 98% 97%

Other 46%?

Screening for Down syndrome 1 ^st trimester combined test

A beneficial side-effect of screening for trisomy 21 is detection of many other aneuploidies

Santorum M, Wright D, Syngelaki A, Karagioti N,Nicolaides KH. Accuracy of first trimester combined test in screening for trisomies 21, 18 and 13. Ultrasound Obstet Gynecol 2016; doi 10.1002/uog.17283

The Fetal Medicine Foundation

Holoprosencephaly, Exomphalos, Megacystis, NT >3.5 mm

Screen +ve rate 1.1%

This group contained 57% of all aneuploidies

Trisomy 21 53%

Trisomy 18 72%

Trisomy 13 88%

Triploidy 34%

Monosomy X 94%

Other 23%

50% T21 80% T13 20% all 45% T18

1 ^st trimester combined test – validation n = 108,982

Syngelaki A,Guerra L, Ceccacci I,Efeturk T,Nicolaides KH. Impact of holoprosencephaly, exomphalos, megacystis and high NT in first trimester screening for chromosomal abnormalities. Ultrasound Obstet Gynecol 2016; doi 10.1002/uog.17283

The Fetal Medicine Foundation

Screen positive rate

T ris omy 1 8 dete c tion % (n)

0 10 20 30 40 50 60 70 80 90 100 0

10 20 30 40 50 60 70 80 90 100

1 in 10 1 in 100 1 in 1000 (DR 99%)

T ris omy 1 3 dete c tion % (n)

0 10 20 30 40 50 60 70 80 90 100 0

10 20 30 40 50 60 70 80 90 100

1 in 10 1 in 100 1 in 1000 (DR 96%)

FHR

T ris omy 2 1 dete c tion % (n )

0 10 20 30 40 50 60 70 80 90 100 0

10 20 30 40 50 60 70 80 90 100

1 in 1000 (DR 98%)

1 in 100

1 in 10

1 ^st trimester combined test

Prospective validation n=108,982

Santorum M, Wright D, Syngelaki A, Karagioti N,Nicolaides KH. Accuracy of first trimester combined test in screening for trisomies 21, 18 and 13. Ultrasound Obstet Gynecol 2016; doi 10.1002/uog.17283

The Fetal Medicine Foundation

100,000 pregnancies

Trisomy 21 N=200 99,800 normal

Serum biochemistry at 16 wks 70% 140 5% 4990

Combined test at 12 wks 90% 180 3% 2994

Cell-free DNA >99% 199 0.04% 40

Maternal age 30% 60 5% 4990

METHOD OF SCREENING DR Detected False positive

Cell-free DNA in maternal blood

Screening for Down syndrome

The Fetal Medicine Foundation

Source:

MEDLINE, EMBASE

Key words:

Non invasive/Non-invasive/Noninvasive prenatal detection/diagnosis, Cell free DNA prenatal

Inclusion criteria:

• Peer-reviewed

• Year of publication >2011

• Language English

• Follow-up rate >85%

• Analysis performed blindly

Exclusion criteria:

• Proof of concept articles

• Case-control studies

Bivariate analysis performed to calculate random-effect estimate of pooled weighted for:

• Singletons: Trisomies 21, 18 and 13

Univariate analysis performed to calculate random-effect estimate of pooled weighted for:

• Singletons: Monosomy X and SCA; Twins: Trisomy 21

Records screened n = 3,353

Included in systematic review n = 35

Non-relevant articles n = 2,001

Proof of concept articles n = 29

Records identified n = 7,759

Duplicates n= 4,406

Case-control studies n = 35

Conference abstracts n = 1,070

Review / opinion articles n = 154

Outcome in < 85% cases n = 29

Cell free DNA test

Systematic review 31/12/16

The Fetal Medicine Foundation

Cell free DNA test

Importance of fetal fraction

Company Result

A No aneuploidy, female fetus B No aneuploidy, female fetus C No aneuploidy, female fetus

FF on request: 4.3% and 3.9%

D No result due to FF of 0.6%

E No result due to low FF

Takoudes T, Hamar B. Performance of non-invasive prenatal testing when fetal cell-free DNA is absent. Ultrasound Obstet Gynecol 2015; 45: 112.

The Fetal Medicine Foundation

Miscarriage from amnio / CVS

Cell free DNA test

RCOG: Amniocentesis and Chorionic Villus Sampling. Green Top Guideline No.8. London: RCOG, 2010.

ACOG: Invasive prenatal testing for aneuploidy. ACOG Practice Bulletin No. 88. Obstet Gynecol 2007; 110: 1459-1467.

SOGC: Mid-trimester amniocentesis fetal loss rate. J Obstet Gynaecol Can 2007; 29: 586-595.

SEGO: Diagnóstico prenatal de los defectos congénitos. Cribado de las anomalías cromosómicas. 2010.

Amnio 1%

BC 1-2%

Amnio 0.3-0.5%

BC 0.3-0.5%

Amnio 0.2-1.5%

BC 0.2-1.5%

Sociedad Española de Ginecología y

Obstetricia

Amnio 0.2-1.5%

BC 0.7-1%

The Fetal Medicine Foundation

King’s College hospital study

Singleton pregnancies with combined screening at 11-13 w

• Expectant management

• Livebirth n = 33,310; Miscarriage n = 404 (1.2%)

• Regression model to predict miscarriage

Variable OR 95% CI

Age (per year) 0.870 0.766-0.988

Delta nuchal translucency 1.778 1.496-2.114 Ductus venosus: reversed a-wave 2.208 1.508-3.232

Log

₁₀

PAPP-A MoM 0.356 0.233-0.543

Akolekar R, Bower S, Flack N, Bilardo K, Nicolaides KH: Prediction of miscarriage and stillbirth at 11-13 weeks and the contribution of chorionic villus sampling. Prenat Diagn 2011:31:38.

CVS n = 2,396

Miscarriage

Observed: 44 (1.8%)

Expected: 45 (95% CI 32-58)

Miscarriage from amnio / CVS

Cell free DNA test

The Fetal Medicine Foundation

Wulff CB, Gerds TA, Rode L, Ekelund CK, Petersen OB, Tabor A: Risk of fetal loss with invasive testing following first trimester screening: national cohort of 147,987 singleton pregnancies. Ultrasound Obstet Gynecol 2016; 47: 38-44.

Danish study

• 147,987 singleton pregnancies

• All had first trimester combined screening

• Propensity score stratification

Miscarriage risk difference: CVS -0.2%

Amniocentesis 0.6%

Stillbirth risk difference: CVS -0.3%

Amniocentesis 0.1%

Miscarriage from amnio / CVS

Cell free DNA test