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
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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.
Italian Society of Veterinary Physiology, 21-23 Jun e, Teramo
-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…
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-No catalytic activity due to mutation in the binding cleft
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Pregnancy - Associated Glycoproteins (PAGs)
These glycoproteins are synthesizedin the cotyledonsof ruminant species
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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) .
Italian Society of Veterinary Physiology, 21-23 Jun e, Teramo
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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)
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Few of them could be purifiedfrom placenta tissue in bovine,ovine, caprine, bison and buffalo spp. (work in progress)
Italian Society of Veterinary Physiology, 21-23 Jun e, Teramo
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
10
species
Green et al. (2000)
Italian Society of Veterinary Physiology, 21-23 Jun e, Teramo
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 !
Italian Society of Veterinary Physiology, 21-23 Jun e, Teramo
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)
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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 !
Italian Society of Veterinary Physiology, 21-23 Jun e, Teramo
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
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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
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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
(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 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
(Perenyi et al., 2002)Italian Society of Veterinary Physiology, 21-23 Jun e, Teramo
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
(Perenyi, 2002)Italian Society of Veterinary Physiology, 21-23 Jun e, Teramo
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
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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
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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
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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
(Perenyi, 2002)Italian Society of Veterinary Physiology, 21-23 Jun e, Teramo
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...
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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
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0 10 20 30 40 50 60
Days after reference estrus
0
0,5
1
1,5
0
2
4
6Day 52Day 52
EstrusEstrus
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Assay of PAG ...
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After clonage …
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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
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Assay of PAG ...
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Possible effect of under-nutrition …
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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
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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 ...
Italian Society of Veterinary Physiology, 21-23 Jun e, Teramo
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, …
Italian Society of Veterinary Physiology, 21-23 Jun e, Teramo
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
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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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