10th Postgraduate course in Reproductive Medecine andReproductive Biology

Metalloproteinases as a diagnostic tool of Premature Rupture of Membranes?

Claudia Carrillo MSc

Tutor: Professor P. Bischof

12,October 2000

MMPs and TIMPs

• Definition• Function and activation• TIMPs

Structures of the MMPs

PRCG[V/N]PP

Zn

HEXGHX[L/M ]G[L/M ]XH

cataly tic dom ain

propeptide

C-term inal domain "hem opex in"

fibronec tin type II dom ain

collagen like dom ain

MMP-2

MMP-9

Classification

• Gelatinases (MMP-2, MMP-9)

• Collagenases (MMP-1, MMP-8, MMP-13, MMP-18)

• Stromelysines (MMP-3, MMP-7, MMP-10, MMP-11)

• MemType MMPs(MMP-14, MMP15, MMP-16, MMP-17)

• Others (MMP-12, MMP-19)

ClassificationMMPs Others names Substrates aminoacids M.W ( Kd) M.W ( Kd)

pro-enzyme activeMMP-1 Intest.collase Col I,II,III,VII,& X, 469 54 42

Fibro.collase entactinMMP-2 Gelatinase A Col I,IV,V,VII,X,&XI 660 72 66

Intest.collase fibronectin,elastinMMP-3 Stromelysin-1 Col III,IV,IX,X,gelatin 477 57 48

Transin-1 laminin,fibron,elas,casMMP-7 PUMP -1 casein,fbronectin,gelatin 267 28 19

MatrilysinMMP-8 Neutrop.collase Col I, III 467 53 65

PMNL collaseMMP-9 Gelatinase B Col IV,V,XIV,gelatin 707 92 88

92Kd gelatinaseMMP-10 Stromelysin-2 Col II,IV,V,VII,gelatin,fibron. 476 53 47

Transin-2MMP-11 Stromelysin-3 Col IV,fibronectin,laminine 488 54

aggrecan, gelatinesMMP-13 Collagenase-3 Col I,II,III 471 53MMP-14 MT1-MMP Col I,II,III active pro- MMP-2 582 65

MMP-XI et MMP-13MMP-15 MT2-MMP MMP-2 669 75MMP-16 MT3-MMP active la pro-MMP-2 604 69

MMP-X2

Clinical relevance

• Cancer• Pregnancy• Preeclamsia• PRM

MMP-2 is over-expressed in Hydatiforme Mole

MMP-9 in first and second trimester of pregnancy

100

150

200

250

300

350

MM

P-9

(ng/

ml)

03-0

4

04-0

5

05-0

6

06-0

7

07-0

8

08-0

9

09-1

0

10-1

1

11-1

2

12-1

3

13-1

4

14-1

5

15-1

6

16-1

7

17-1

8

18-1

9

w eeks

MMP-9 in first and second trim ester of pregnancy

AIMS of the project

• To see if MMP-2 & MMP-9 can be usedas a diagnostic marker for RPM.

• We therefore compared MMPs with thePROM test (IGFBP-1), Bromo andclinical evaluation

• Development of a biochemical test based on MMPs levels as an indicatorfor RPM

Materials and methods

• Vaginal secretion of pregnant women• IGFBP-1,Bromo,clinical evaluation• ELISA

The principle of ELISA1 plate

2. wash

3. add test antibody

4. wash

5. add ligand

6. wash

7. add chromogen

8. develop plated

chromogen

ligand

Patients

• 39 Pregnant women (March - July 2000)

• MMP-2 (39) and MMP-9 (29)

• Age: 41-21 years old, mean (31,7)

• Gestational age: 28- 41 2/7, mean (37 2/7)

Ruptured, doubtful, unruptured

-1

4

9

14

19

24

29

34

39

ruptured doubtfu l unrupture

MMP-2 and MMP-9 comparedto the PROM test

MMP-2 compared to the PROM-test

0

20

40

60

80

100

1 2

(+) (-)

MMP-9 compared to the PROM-test

0

50

100

150

200

250

1 2

(+)

(-)

MMP-9 and MMP-2 compared to BROMO

MMP-2 in BROMO(+) and (-)

0

20

40

60

80

100

1 2

(+)(-)

MMP-9 in BROMO(+) and (-)

0

50

100

150

200

250

1 2

(+)(-)

MMP-2 and MMP-9 compared to clinical evaluation

MMP-2 compared to clinical evaluation

020406080

100120

1 2

(+) (-)

MMP-9 compared to clinical evaluation

0

50

100

150

200

1 2

(+)(-)

Conclusions• MMP-2 and MMP-9 immunoreactivity

correlate well with the results fromBROMO and PROM tests but not withthe clinical evaluation.

• Further studies can be done to assaythe activity of the MMP-2 and MMP-9.

ZymogrammsZymogramms of MMPs in of MMPs in cytotrophoblasticcytotrophoblastic cellscells

kDa

212

170116

76

53

MMPMMP--99

MMPMMP--22

53