Biochemical Aspects and Clinical Application for Pregnancy

The Pregnancy-Associated Glycoproteins:

Biochemical Aspects and

1

Biochemical Aspects and

Clinical Application for Pregnancy Follow-up

Societá Italiana di Fisiologia Veterinaria, 21-23 giugno, Teramo

Prof. J.F. BECKERS

History of pregnancy diagnosis

- Antiquity – Interest for pregnancy diagnosis

- 600 years before Christ ... Egyptians

2

Urine of woman supposed pregnant �� Germination of grains

Fast process... �� Positive Diagnosis

Slowly process... �� Negative diagnosis

Italian Society of Veterinary Physiology, 21-23 Jun e, Teramo

The Dream of the 21st Century

Reliability ?Cost ?Precocity ?

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Precocity ?

Introduction

� In human… hCG (PU)

� In horses… PMSG (eCG)

� In ruminants… no CG !

4

� In ruminants… no CG !

But in 1982, it became evident that some « (glyco)pr oteins » could

be released in maternal blood and… used for pregnanc y

diagnosis.


-No similarity with CG structure

-Surprisingly ↑↑↑↑ 50% of similarity with pepsinogens, cathepsins D and E, …

-No catalytic activity

PAG belong to Aspartic Proteinase gene family…

5

-No catalytic activity due to mutation in the binding cleft


Pregnancy - Associated Glycoproteins (PAGs)

These glycoproteins are synthesizedin the cotyledonsof ruminant species


Trinucleate cell in the uterine epithelium

Migration of BNC till themicrovillous junction

BNC fusion with a endometrialcell (*)

More precisely in the mono or binucleate cells of t he trophoblast…

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And they are released in the maternal blood stream after migration and fusion with the maternal epithelial cells of th e endometrium…

Migration of a binucleate cell in a cow placentome. T: trophectodermal epithelium. U: uterine epithelium (Wooding & Beckers, Cell Tissue Res, 247:667) .


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Panel of cDNA expression of PAGin ruminant species

PA

G c

he z

l es

bo

v in

s , o

v in

s e t

cap

rin

s

A) B)

PA

G c

he z

l es

bo

v in

s , o

v in

s e t

cap

rin

s

A) B)

9

Few of them could be purifiedfrom placenta tissue in bovine,ovine, caprine, bison and buffalo spp. (work in progress)


PA

G c

he z

l es

bo

v in

s , o

v in

s e t

cap

rin

sP

AG

ch

e z l e

s b

ov i

ns ,

ov i

ns

e t c

apri

ns

Temporal expression of PAG in bovine and ovine species

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species

Green et al. (2000)


CHARACTERISTICS OF PAGs

Stability of PAGs

- Long half-life in vivo (≈≈≈≈ 8.5 days)

- Stables in vitro

Not degraded by red blood cells (not necessary to centrifuge just after taking blood)

boPAG-1

centrifuge just after taking blood)

In milk, PAGs are only measurable in sheep and goat …In milk, PAGs are only measurable in sheep and goat …Unfortunately not in cows !!!Unfortunately not in cows !!!

Blood samples can be kept at 4-25°C for 1-5 days be fore analysis →→→→ Tubes can be sent by post !Tubes can be sent by post !


IMPORTANCE OF PAG RIA TESTS

Measurement of PAG concentrations in maternal blood permits:

Pregnancy confirmationPregnancy confirmation

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(reproduction management)(reproduction management)

Follow up the trophoblastic function

(reproductive physiology, pathologic conditions affecting pregnancy)


Day of gestation n Se (%) Sp (%) PPV (%) NPV (%) Ref.

29-30 138 92.0 82.6 81.6 92.5 (1)

35 430 98.9 87.5 93.0 97.9 (2)

37-38 125 100 84.0 83.5 100 (1)

Diagnosis of early pregnancy in cattle

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37-38 125 100 84.0 83.5 100 (1)

(1) Szenci et al., 1998; (2) Zoli et al., 1992

Se: sensitivity; Sp: specificity; PPV: positif pred ictive value; NPV: negative predictive value

SP never reach 100% due to the embryonic mortality !


PAG RIA TESTS

Pregnancy diagnosis :

RIA: Belgium => 6,000 / year

France => 95,000 / year

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France => 95,000 / year

Spain => 1,000 / year

Scotland => 1,000 / year

Perugia => …now available

ELISA: Canada => 16,000 / year


Assay of PAG in research programs

Studies on ongoing pregnancies

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Studies on ongoing pregnancies

In the lab we try to improve the efficacy of the as say …

1st RIA : RIA 1 (AS 497) since 1992

2nd and 3 rd RIA : RIA 2 (AS 706) and RIA 3 (AS 708) since 2000


5

10

15

20PAG (ng/ml)

RIA 1

RIA 2

0 5 10 15 20 25 30 35 40 45 50 55 60

Days after AI

0

5

PAG profile of heifer n. 209 during the first 60 days after AI using 3 PAG-RIA systems. RIA 1 (AS 497) , RIA 2 (AS 708) and RIA 3 (AS 706) .

RIA 2

RIA 3

(Perenyi et al., 2002)Italian Society of Veterinary Physiology, 21-23 Jun e, Teramo

5

10

15

20PAG (ng/ml)

RIA 1RIA 2

PAG profile of heifer nº 1654 during the first 60 days after AI using 3 PAG-RIA systems. RIA 1 (AS 497) , RIA 2 (AS 708) and RIA 3 (AS 706) .

0 5 10 15 20 25 30 35 40 45 50 55 60

Days after AI

0

5RIA 2RIA 3


5

10

15

20PAG (ng/ml)

RIA 1RIA 2

PAG profile of heifer n. 9447 during thePAG profile of heifer n. 9447 during the first 60 daysfirst 60 days after AI using after AI using 3 PAG3 PAG--RIA systems. RIA systems. RIA 1 (AS 497) , RIA 2 (AS 708) and RIA 3 (AS 706) .

0 10 20 30 40 50 60

Days after AI

0

5RIA 2RIA 3

0.8 ng/ml0.8 ng/ml

pregnantpregnant

nonpregnantnonpregnant


100

150

200

250

300

350

RIA 1

60 80 100 120 140 160 180 200 220 2400

50

100

PAG profiles of heifer n. 209 during 60-250 days of pregnancy using 3 PAG-RIA systems. RIA 1 (AS 497) , RIA 2 (AS 708) and RIA 3 (AS 706) .

RIA 2RIA 3

(Perenyi, 2002)Italian Society of Veterinary Physiology, 21-23 Jun e, Teramo

RIA 11000

1500

2000

2500

3000

3500

1000

1500

2000

2500

3000

3500Calving

RIA 1RIA 2

Calving

RIA 1RIA 2RIA 3

250 270 290 3100

500

1000

250 270 290 3100

500

1000

PAG profiles of heifers n. 209 and 579 after day 250 using 3 PAG-RIA systems. RIA 1 (AS 497) , RIA 2 (AS 708) and RIA 3 (AS 706) .

RIA 2RIA 3


PAG on ongoing pregnancies

21Zoli et al., 1991Italian Society of Veterinary Physiology, 21-23 Jun e, Teramo

Assay of PAG …

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Studies on embryonic or fetal mortalities


Embryonic or fetal mortalities

Cow 3129 – Fetal mortality with a functionalcorpus luteum ( ↑↑↑↑ P4 conc.)

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Plasmatic profiles of PAG and P4associated to positive (P) or negative (N)ultrasound pregnancy diagnosis

Szenci et al. 1998

Cow 3570 – Early embryonic mortality with↓↓↓↓ in concentrations of P4


Cow 3369 – Embryonic mortality after asecond AI

Embryonic or fetal mortalities

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Plasmatic profiles of PAG and P4associated to positive (P) or negative (N)ultrasound pregnancy diagnosis

Szenci et al. 1998

Cow 3004 – Early embryonic mortalityduring the postpartum period


PAG concentrations in animals with spontaneous preg nancy failure

10

15

20PAG (ng/ml)

RIA 1Day 70Day 70Rectal Rectal

palpation:palpation:

Day 49Day 49Rectal Rectal

palpation:palpation:pregnantpregnant

80

Days after AI

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 750

5

RIA 1RIA 2RIA 3

palpation:palpation:nonpregnantnonpregnant


Day Day 144144

40

60

80

RIA 1

PAG concentrations after fetal mortality due to a Neospora caninuminfection (Heifer 8368)

++--++------

0 20 40 60 80 100 120 140 160 180 2000

20

RIA 1

RIA 2

RIA 3

--(Zs. Perenyi in cooperation

with Ch. Rettigner; 2002)Neosporadiagnosis

Assay of PAG ...

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Embryo transfer during anoestrus...


PAG concentrations after embryo transfer during ano estrus

1,5

2

2,5PAG (ng/ml)

6

8

10P4 (ng/ml)

P4Day 52Day 52

RIA 1RIA 2RIA 3

28

0 10 20 30 40 50 60

Days after reference estrus

0

0,5

1

1,5

0

2

4

6Day 52Day 52

EstrusEstrus


Assay of PAG ...

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After clonage …


PAG concentrations after clonage

Schistosomusreflexus

Placental abnormality

6000

8000

10000

12000

14000PAG (ng/ml)

Cow presenting aplacental abnormality

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Comparison of PAG concentrations in cows presenting a normal gestation and in one cow presenting a placental abnormality in addition to a normal fetus.

-11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1

Semaines avant la parturition

0

2000

4000

Weeks before parturition


Assay of PAG ...

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Possible effect of under-nutrition …


Animaux ...

- 12 Azawak zebu cows- BCS 4-5

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Blood samplimg from the 8 th week

of pregnancy till the 10 th week

postpartum Burkina-Faso


Cow with ↓↓↓↓ in BCS

Mean concentration inAzawak zebu cows

600

800

1000

1200

PA

G (

ng/m

l)

33PAG concentrations in a Azawak zebu cow with a degrad ation of BCS.

0 5 10 15 20 25 30 35 40 5pp 10pp 15pp

Semaines de gestation et post-partum

0

200

400PA

G (

ng/m

l)

Weeks before and after parturition

New investigations in Buffalo…

Collaboration with Perugia (Prof. A. Debenedetti and Dr. O. Barbato)…

Preliminary studies:

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Preliminary studies: - PAG concentrations higher than in cattle (9.0

±±±± 4.0 ng/ml) at Day 30 of pregnancy - PAG RIA: suitable for pregnancy diagnosis in

this species and other ruminant species

In summary ...

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In summary ...


With PAG RIA …

The concentrations of Pregnancy -Associated Glycoproteins can help…

Farmers … - For pregnancy diagnosis in cow, sheep, goat and bu ffalo …- Isolated or in conjunction with other sero-diagnos is:brucellosis, leucosis, IBR, BVD, Visna Maedi, CAEV.

Veterinarians … - When developing rectal palpation or ultrasound

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Veterinarians … - When developing rectal palpation or ultrasoundinvestigations:

- in order to confirm a diagnosis…- in pregnancy with mummified fetus…

Researchers … - When studying the embryonic or fetal mortality,the consequence of diseases, of nutrition, …


Fecundation

Parturition

Pregnancydiagnosis

Clinical or RIA

Clinicalor RIA

Clinicalor RIA

Clinicalor RIA

Clinicalor RIA

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AI AI ET

EEM LEM

Newborns with excessive or insufficient weights

Abortion - Mummification - Maceration

Gestation


Staff (Faculty of Veterinary Medicine, ULg): Collaborators :

Dr. J. Sulon (Post-Doc) Prof. F. Ectors (University of Liège, Belgium)

Dr. N.M. Sousa (Post-Doc) Prof. A. Debenedetti (University of Perugia, Italy)

Dr. B. Remy (DMV) Dr. O. Barbato (University of Perugia, Italy)

Mrs R. Fares Prof. F. Lopez-Gatius (University of Lleida, Spain)

Dr. C. Gatez (DMV) Prof. F. Gonzalez (Canary Islands University, Spain )

Mrs Ch. Poullet Prof. B. Szafranska (Univ. Warmia and Mazury, Polan d)

Mr. J.A. Michiels (Ing.) Drs. Y. Heyman and P. Chavatte -Palmer (INRA -Tours)

Acknowledgements ...

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Mr. J.A. Michiels (Ing.) Drs. Y. Heyman and P. Chavatte -Palmer (INRA -Tours)

Mr. B. Glaude (Ing.) Prof. O. Szenci (University of Budapest, Hungary)

Mr. M. Machado Prof. M.A.M. Taverne (University of Utrecht)

Researchers : Dr. B. El Amiri (INRA-Settat, Marocco)

Mr. A. Ayad (DVM) Dr. M. Zongo (Univ. of Ouagadougou, Burkina-Faso)

Mrs. A. Alvarez-Oxiley (Ing.) Grants :

Ms. A. Bella (DVM) DGA-Ministère de la Region Wallonne

Mr. N. van Hahn (Ing.) Ministère de l’Agriculture

FNRS

University of Liège

More information about PAG -RIA:

In Belgium: Professor Jean-François BECKERSPhysiology of Animal ReproductionFaculty of Veterinary Medicine - ULgBd de Colonster n. 20, B41, Parking 72Sart-Tilman, B-4000, LiègeTel.: 04 366-4161 ; Fax: 04 366-4165 ; GSM: 04 77 2 9 7018

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Email: [email protected]

In Italy: Dr. Olimpia BARBATODip. di Scienze Biopatologiche ed Igiene delle Produ zioni animali ed alimentariUniversità di Perugiavia San Costanzo, 4, 06126, PerugiaTel.: 0039 075 5857640Fax 0039 075 5857654email: [email protected]

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Biochemical Aspects and Clinical Application for Pregnancy