Prof.Prof. Giuseppe SIMONIGiuseppe SIMONITOMA, Advanced Biomedical Assays, S.p.A.TOMA, Advanced Biomedical Assays, S.p.A.

VILLOCENTESI O AMNIOCENTESI?VILLOCENTESI O AMNIOCENTESI?

Vantaggi e svantaggi da una Vantaggi e svantaggi da una

esperienza di 115esperienza di 115’’000 analisi 000 analisi

cromosomiche prenatalicromosomiche prenatali

Bologna, 5 Aprile 2008Bologna, 5 Aprile 2008

Controversie e nuove tecnologie nella diagnosi prenatale del priControversie e nuove tecnologie nella diagnosi prenatale del primo mo

trimestre: dal laboratorio alle procedure strumentalitrimestre: dal laboratorio alle procedure strumentali

FREQUENCIES OF UNBALANCED FETAL KARYOTYPES ATFREQUENCIES OF UNBALANCED FETAL KARYOTYPES AT

AMNIOCENTESISAMNIOCENTESIS ANDAND CVSCVS

1994- January 15th 2008: 115’576 AF AND CVS

N° CASES %

70,860 1.38%

742 0.94%

252 6.35%

8,042 2.42%

4,951 8.44%

84,847

16

195

418

TOTAL 1612 (1.9%)

AF

Balanced chr abnorm. In parents

Increased risk at screening test

Other

Maternal age

Previous child/fetus with chr abnorm.

UNBALANCED ABNORMALITIES*

976

7

INDICATION

N° CASES %

25997 2.24%

972 2.06%

209 10.05%

445 6.07%

3106 21.25%

30729

CVSUNBALANCED ABNORMALITIES*

Maternal age 582

INDICATION

Previous child/fetus with chr abnorm. 20

Balanced chr abnorm. In parents 21

Increased risk at screening test 27

Other 660

TOTAL 1310 (4.25%)

D EL/ D UP

OT H ER

T R IS 21

T R IS 18

T R IS 13

*UN B A L.A B N OR M .

SEX C H R .A N EUP L.

M OSA IC S

SUP ER N .M A R KER S

D E N OVO R EA R R .

TYPES OF UNBALANCED ABNORMALITIES FOUND ATTYPES OF UNBALANCED ABNORMALITIES FOUND AT

AMNIOCENTESISAMNIOCENTESISN° CASES

TRIS 21 697

TRIS 18 174

TRIS 13 72

SEX CHR.ANEUPL. 218

45,X 53

OT H ER SEX C H R A B N 165

MOSAICS 219

SUPERN.MARKERS 47

TRIPLOIDS 27

OTHERS 158

TOTAL 1612

9.80%

13.52%

13.59%

2.92%

1.67%

10.24%

3.29%

REL. PROPORTION (%)

43.24%

10.79%

4.47%

FREQUENCIES OF UNBALANCED FETAL KARYOTYPES FREQUENCIES OF UNBALANCED FETAL KARYOTYPES

ATAT AMNIOCENTESIS AMNIOCENTESIS ACCORDING TO MATERNAL AGEACCORDING TO MATERNAL AGE

N° OF CASES %

AGE ≥ 35 years 48446 1.65%

< 35 years 22414 0.78%

N° OF UNBALANCED ABNORMALITIES

801

175

FREQUENCIES OF THE DIFFERENT TYPES OF FREQUENCIES OF THE DIFFERENT TYPES OF

UNBALANCED FETAL KARYOTYPES ATUNBALANCED FETAL KARYOTYPES AT

AMNIOCENTESISAMNIOCENTESIS ACCORDING TO MATERNAL AGEACCORDING TO MATERNAL AGE

TRIS 21

TRIS 18

TRIS 13

SEX CHR.ANEUPL.

45,X

OT H ER SEX C H R

MOSAICS

SUPERN.MARKERS

TRIPLOIDS

OTHERS

SUBTOTAL

TOTAL

5 (2,9) 25 (3,1)

27 (3,4)

40 (22,9) 109 (13,6)

32 (18,3) 106 (13,2)

< 35Y N° CASE (%) ≥ 35Y N° CASE (%)

51 (29,1) 398 (49,7)

5 (2,9) 73 (9,1)

175 (17.9) 801 (82.1)

976

37 (21,1) 60 (7,5)

7 (4,0)

33 (18,9)

0

8 (1,0)

101 (12,6)

3 (0,4)

5 (2,9)

TYPES OF UNBALANCED ABNORMALITIES FOUND ATTYPES OF UNBALANCED ABNORMALITIES FOUND AT CVSCVS

N° CASES

TRIS 21 623

TRIS 18 230

TRIS 13 84

SEX CHR.ANEUPL. 176

45,X 102

OT H ER SEX C H R A B N 74

MOSAICS confirmed at AF 88

SUPERN.MARKERS 16

TRIPLOIDS 40

OTHERS 53

TOTAL 1310

REL. PROPORTION (%)

47.56%

17.56%

6.41%

13.44%

6.72%

1.22%

4.05%

7.79%

5.65%

3.05%

FREQUENCIES OF UNBALANCED FETAL KARYOTYPES FREQUENCIES OF UNBALANCED FETAL KARYOTYPES

ATAT CHORIONIC VILLI SAMPLINGCHORIONIC VILLI SAMPLING ACCORDING TO ACCORDING TO

MATERNAL AGEMATERNAL AGE

N° OF CASES %

AGE ≥ 35 years 20,596 2.49%

< 35 years 5,401 1.28%

N° OF UNBALANCED ABNORMALITIES

513

69

FREQUENCIES OF THE DIFFERENT TYPES OF FREQUENCIES OF THE DIFFERENT TYPES OF

UNBALANCED FETAL KARYOTYPES ATUNBALANCED FETAL KARYOTYPES AT CVSCVS

ACCORDING TO MATERNAL AGEACCORDING TO MATERNAL AGE

TRIS 21

TRIS 18

TRIS 13

SEX CHR.ANEUPL.

45,X

OT H ER SEX C H R A B N

MOSAICS

SUPERN.MARKERS

TRIPLOIDS

OTHERS

SUBTOTAL

TOTAL

12 (2,3)

8 (11,6)

9 (1,8)

55 (10,7)

39 (7,6)

5 (7,2)

5 (7,2)

13 (18,8)

16 (23,2)

1 (1,4)

7 (10,1)

< 35Y N° CASE (%) ≥ 35Y N° CASE (%)

582

20 (29,0)

20 (3,9)

513 (88.1)69 (11.9)

276 (53,8)

69 (13,5)

24 (4,7)

64 (12,5)

2 (2,9) 11 (2,1)

5 (7,2)

SUMMARY OF PRENATAL DIAGNOSIS SUMMARY OF PRENATAL DIAGNOSIS

OFOF MOSAICISM AT AMNIOCENTESESMOSAICISM AT AMNIOCENTESESTYPE OF MOSAICISM

Autosomes

Sex chromosome

Marker chromosome

TOTAL

% on total cases 219/84847=0,26%*

219

63

117

39

N° CASES

*Frequencies*Frequencies of of TrueTrue Fetal Fetal MosaicismsMosaicisms at CVS: at CVS:

10,53% X 2.05% /100= 0.23%10,53% X 2.05% /100= 0.23%

VANTAGGI DELLVANTAGGI DELL’’ANALISI CITOGENETICA SUI VILLI CORIALIANALISI CITOGENETICA SUI VILLI CORIALI

⇒⇒⇒⇒⇒⇒⇒⇒ IDENTIFICAZIONE DI MOSAICISMO SU VILLI CORIALIIDENTIFICAZIONE DI MOSAICISMO SU VILLI CORIALI

⇒⇒⇒⇒⇒⇒⇒⇒ CONFERMA DEL MOSAICO NEGLI AMNIOCITI (TFM)CONFERMA DEL MOSAICO NEGLI AMNIOCITI (TFM)

⇒⇒⇒⇒⇒⇒⇒⇒ ASSOCIAZIONE DEL RITARDO DI CRESCITA FETALE ASSOCIAZIONE DEL RITARDO DI CRESCITA FETALE

IDIOPATICO CON CPMIDIOPATICO CON CPM

⇒⇒⇒⇒⇒⇒⇒⇒ ESCLUSIONE DI UPD FETALE NEIESCLUSIONE DI UPD FETALE NEI CASI DI CPM CASI DI CPM

⇒⇒⇒⇒⇒⇒⇒⇒ MARGINI DI TEMPO SUFFICIENTI PER INDAGINI MARGINI DI TEMPO SUFFICIENTI PER INDAGINI

SUPPLEMENTARI (FISH, ANALISI MOLECOLARI, SUPPLEMENTARI (FISH, ANALISI MOLECOLARI, MLPAMLPA……) )

Confined placental Confined placental mosaicismsmosaicisms

Kalousek D., 1984

TIPO I TIPO II TIPO III

MOSAICISM MOSAICISM

IN IN

CHORIONIC CHORIONIC

VILLIVILLI

CONFIRMATORY CONFIRMATORY

AMNIOCENTESISAMNIOCENTESIS

MOSAICISM MOSAICISM

IN IN

AMNIOCYTESAMNIOCYTES

CONFIRMATORY CONFIRMATORY

FETAL BLOOD FETAL BLOOD

SAMPLINGSAMPLING

Table 1: Incidences of the different types of mosaicisms (CPM and TFM) found after chrionic villous and

amniocytes karyotyping

TROPHOBLAST MESENCHYME

(direct) (culture)

I CPM Abnormal Normal Normal 36,58% (139/380)

II CPM Normal Abnormal Normal 44,7% (170/380)

III CPM Abnormal Abnormal Normal 8,1% (31/380)

IV TFM Abnormal Normal Abnormal 1,58% (6/380)

V TFM Normal Abnormal Abnormal 4.7% (18/380)

VI TFM Abnormal Abnormal Abnormal 4,2% (16/380)

TYPE NATURE AMNIOCYTES RELATIVE FREQUENCIES

RISULTATIRISULTATI

Frequenze dei differenti tipi di mosaicoFrequenze dei differenti tipi di mosaico

10.53%* (40/380)

= 0.23% CVS SAMPLES

10.53%* (40/380)

= 0.23% CVS SAMPLES

24237 24237 mosaicismomosaicismo CV: 498 (2,05%) CV: 498 (2,05%) 380 AF380 AF

*Summary*Summary of of prenatalprenatal diagnosisdiagnosis of of MosaicismsMosaicisms at at amniocentesisamniocentesis: :

219/84847= 0.26%219/84847= 0.26%

Table 2: Probabilities of confirmation on amniocytes of Mosaic or Non Mosaic abnormal cell

line considering the different combinations of the affected placental tissues

TROPHOBLAST MESENCHYME

(direct) (culture)

A N 4,14% (6/145)

MA N Type IV/Type I+IV=3/112+3= 2,6%

NMA N 3/27+3= 10%

N A 9,6% (18/188)

N MA Type V/Type II+V= 6/150+6= 3,8%

N NMA 12/20+12= 37,5%

A A 34% (16/47)

MA MA Type VI/Type III+VI= 9/19+9= 28,5%

NMA MA 3/9+3= 25%

MA NMA 4/0+4= 100%

NMA* NMA* 0/3+0= 0%

A=Abnormal; N=Normal; MA=Mosaic Abn; NMA=Non Mosaic Abn;*exhotic chr

CONFIRMATION

RISULTATIRISULTATI

ProbabilitProbabilitàà di conferma nel fetodi conferma nel feto

203 TOTAL CASES OF WHICH 51 INVOLVED AN IMPRINTED CHROMOSOME203 TOTAL CASES OF WHICH 51 INVOLVED AN IMPRINTED CHROMOSOME

VANTAGGI DELLVANTAGGI DELL’’ANALISI CITOGENETICA SU ANALISI CITOGENETICA SU

AMNIOCITIAMNIOCITI

⇒⇒⇒⇒⇒⇒⇒⇒ MIGLIORE MIGLIORE RISOLUZIONE DEL BANDEGGIO RISOLUZIONE DEL BANDEGGIO

CROMOSOMICOCROMOSOMICO

⇒⇒⇒⇒⇒⇒⇒⇒ CELLULE DERIVATE DA DIVERSI TESSUTI E PARTI CELLULE DERIVATE DA DIVERSI TESSUTI E PARTI

FETALEFETALE

REPORTED DETECTION RATES (REPORTED DETECTION RATES (d.rd.r.).)

REPORTED DETECTION RATESREPORTED DETECTION RATES

SURUSS (U.K.) (n=47'507)

I TRIMESTER (9-13wg) MESUREMENTS (+MAT. AGE) TRIS 21* TRIS 21* TRIS 18 & OTHER°

Combined-1 NT+f-β-hCG 65% / /

Combined-2 Combined-1+PAPP-A 83% 85% 78%

Combined-3 Combined-2+AFP 84% / /

Combined-4 Combined-3+uE3 86% / /

°False Positive Rate: 6%

FASTER (U.S.A.) (n=38'033)

Detection Rates

*False Positive Rate: 5%

SURUSS (U.K.) (n=47'507)

II TRIMESTER (14-22wg) MESUREMENTS (+MAT. AGE) TRIS 21* TRIS 21* TRIS 18 & OTHER§

DOUBLE AFP+f-β-hCG 71% / /

TRIPLE DOUBLE+uE3 77% 70% /

QUADRUPLE TRIPLE+inhA 83% 81% 69%

§False Positive Rate: 8,9%

FASTER (U.S.A.) (n=36'171)

Detection Rates

*False Positive Rate: 5%

SURUSS (U.K.) (n=47'507)

I+II TRIMESTER MESUREMENTS (+MAT. AGE) TRIS 21* TRIS 21* TRIS 18 & OTHER

Integrated (NT+PAPP-A) + QUADRUPLE 93% 86% /

*False Positive Rate: 5%

FASTER (U.S.A.) (n=36'171)

Detection Rates

RELATIVE INCIDENCE OF +21,+18,+13,X0,TRIPLOIDS ON RELATIVE INCIDENCE OF +21,+18,+13,X0,TRIPLOIDS ON

THE TOTAL FETAL CHROMOSOMOPATHY IN OUR SURVEYTHE TOTAL FETAL CHROMOSOMOPATHY IN OUR SURVEY

CV

(582 cases with a

significant

chromosomopathy:

XXX and XYY

excluded)

Chr. Abnormality N° Cases Relative %

TRIS 21 20 29%

TRIS 18 5 7.2%

TRIS 13 5 7.2%

X0 5 7.2%

TRIPLOIDY 2 2.9%

37/69 53.6%

TRIS 21 276 53.8%

TRIS 18 69 13.5%

TRIS 13 24 4.7%

X0 9 1.8%

TRIPLOIDY 11 2.1%

389/513 75.8%

<35

(69 abn. cases)

≥35

(513 abn. cases)

AF

(976 cases with a

significant

chromosomopathy:

XXX and XYY

excluded)

Chr. Abnormality N° Cases Relative %

TRIS 21 51 29.1%

TRIS 18 5 2.9%

TRIS 13 5 2.9%

X0 7 4.0%

TRIPLOIDY 0 -

68/175 38.9%

TRIS 21 398 49.7%

TRIS 18 73 9.1%

TRIS 13 27 3.4%

X0 8 1.0%

TRIPLOIDY 3 0.4%

509/801 63.5%

≥35

(801 abn. cases)

<35

(175 abn. cases)

PERCENTAGES OF CHROMOSOMAL ABNORMALITIES PERCENTAGES OF CHROMOSOMAL ABNORMALITIES

ACTUALLYACTUALLY DETECTED BY PRENATAL SCREENING ON DETECTED BY PRENATAL SCREENING ON

THE TOTAL FETAL CHROMOSOMOPATY (1)THE TOTAL FETAL CHROMOSOMOPATY (1)

1° TRIMESTER age OBSERVED % a

<35 29.0%

≥35 53.8%COMBINED-2

d.r. b

DETECTABLE % a

∆% c

24.6% -4.4%

45.7% -8.1%85%

TRISOMY 21

OBSERVED % a

24.6% (17/69)

22.0% (113/513)

+18,+13,X0,TRIPLOIDS

d.r. b DETECTABLE % a ∆%

c

19.2% -5.5%

17.2% -4.8%78%

Σ% OBSERVED

53.6%

75.8%

Σ% DETECTABLE

43.8%

62.9%

1° TRIMESTER age

<35

≥35COMBINED-2

∆ % d

-9.80%

-12.90%

1° TRIMESTER

aON TOTAL FETAL CHROMOSOMOPATHY

bBest Reported Detection Rate

cpartial (DETECTABLE%-OBSERVED%)

dtotal (DETECTABLE%-OBSERVED%)

2° TRIMESTER age OBSERVED % a

DOUBLE

TRIPLE

QUADRUPLE

DOUBLE

TRIPLE

QUADRUPLE

<35 29.1%

≥35 49.7%

PERCENTAGES OF CHROMOSOMAL ABNORMALITIES PERCENTAGES OF CHROMOSOMAL ABNORMALITIES

ACTUALLYACTUALLY DETECTED BY PRENATAL SCREENING ON DETECTED BY PRENATAL SCREENING ON

THE TOTAL FETAL CHROMOSOMOPATY (2)THE TOTAL FETAL CHROMOSOMOPATY (2)

1I° TRIMESTER

d.r. b

DETECTABLE % a

∆% c

71% 20.7% -8.4%

77% 22.4% -6.7%

83% 24.2% -4.9%

71% 35.3% -14.4%

77% 38.3% -11.4%

83% 41.2% -4.5%

TRISOMY 21 +18,+13,X0,TRIPLOIDS

OBSERVED % a

9.7% (17/175)

13.9% (111/801)

d.r. b

DETECTABLE % a

∆% c

N.R. / /

N.R. / /

69% 6.7% -3.0%

N.R. / /

N.R. / /

69% 9.6% -4.3%

Σ% OBSERVED

38.90%

63.50%

Σ% DETECTABLE

/

/

30.9% /

/

50.8%

∆ % d

-8.00%

-12.70%

aON TOTAL FETAL CHROMOSOMOPATHY

bBest Reported Detection Rate

cpartial (DETECTABLE%-OBSERVED%)

dtotal (DETECTABLE%-OBSERVED%)

2° TRIMESTER age

DOUBLE

TRIPLE

QUADRUPLE

DOUBLE

TRIPLE

QUADRUPLE

≥35

<35

IN SUMMARYIN SUMMARY

56%

37%

69%

49%

43.8%

62.9%

30.9%

50.8%

0%

50%

100%

<35 ≥35 <35 ≥35

1st TRIM-COMBINED 2nd TRIM-QUADRUPLE

TO

TA

L F

ET

AL

CH

RO

MO

SO

MO

PA

TH

Y

DETECTABLE

NOT DETECTED