JNH Pharmacologie de la stimulation Pharmacologie de la... · Hormonal control of ovarian function....
Transcript of JNH Pharmacologie de la stimulation Pharmacologie de la... · Hormonal control of ovarian function....
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Pharmacologie de la stimulation
DESC MDR Toulouse 2010
JN HUGUES
Hôpital Jean Verdier
& Université Paris XIII, Bondy
FRANCE
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Axe
hypothalamo -
hypophysaire
Libérines (GnRH)
StimulinesFSH et LH
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Atretic follicle
Ovulation
Dominant follicle
Developing follicles
EstradiolInhibin B ?
GnRH neurons
Corpus luteum
LHFSH
ProgesteroneEstradiol
Inhibin A ?
Hormonal control of ovarian function
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Récepteurs 7TM et couplage
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The secretion of GnRH is pulsatile and drives the secretion of LH
testis
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EFFETS HYPOPHYSAIRES DU GnRH
• Pulse de GnRH : libération des gonadotrophines (15-30 ’)
• Sécrétion pulsatile de LH : reflet de la sécrétion du GnRH
– expérience chez le mouton (Clarke) : concordance parfaite entre GnRH (système porte) et LH (veine jugulaire)
– expériences électrophysiologiques chez le singe (Knobil) :synchronisme entre fréquence des impulsions électriques hypothalamiques et décharges de LH
• Sécrétion de FSH : moins dépendante du GnRH
Pulsatilité = moins évidente car 1/2 vie plus longue.
Sécrétion pulsatile de LH et FSH
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The secretion of the pituitary gonadotropinsdepends on the hypothalamic GnRH secretion
Le phénomène de désensibilisation hypophysaire
Knobil, 1980
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EFFETS HYPOPHYSAIRES DU GnRH
Mode pulsatile de sécrétion de GnRH:indispensable pour que la cellule hypophysaire reste réactive
(libération pulsatile de FSH et LH)
Administration continue de GnRH Réduction puis disparition de la sécrétion gonadotrope
Phénomène de « Désensibilisation Hypophysaire »Internalisation et non recyclage du récepteur –
Blocage de la transmission du message intra-cellulaire
Sécrétion pulsatile de LH et FSH
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EFFETS HYPOPHYSAIRES DU GnRH
– Action génomique = augmentation de la synthèse des
ANRm des sous unités α, β de FSH et β de LH et de
l’activité transcriptionnelle.
– Augmentation de stabilité des ARNm
– Augmentation de la traduction et de la glycosylation.
Synthèse de LH et FSH
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Structure of GnRH agonists
Modifications of natural GnRH
to get GnRH agonistic properties
11 22 4433 6655 9988 101077
PyroPyro (Glu) (Glu) –– His His –– Trp Trp –– Ser Ser –– TyrTyr – Gly – Leu – Arg – Pro – Gly NHNH22
Activation of the GnRH receptor
ProteolyseRegulation ofRegulation ofbiologic activitybiologic activity
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name amino acid sequence available formu-lation/application
GnRH pGlu – His – Trp – Ser – Tyr – Gly – Leu – Arg – Pro – Gly – NH2 -
Leuprolide pGlu – His – Trp – Ser – Tyr – DLeu- Arg – Pro – NHEt s.c., depot
Buserelin pGlu – His – Trp – Ser – Tyr – DSer (O’Bu) – Leu – Arg – Pro – NHEt s.c., nasal, depot
Nafareline pGlu – His – Trp – Ser – Tyr – D2Nal – Leu – Arg – Pro – GlyNH2 s.c., nasal
Gosereline pGlu – His – Trp – Ser – Tyr – DSer (O’Bu) – Leu – Arg – Pro – AzaglyNH2 s.c., depot
Histreline pGlu – His – Trp – Ser – Tyr – DHis (Bzl) – Leu – Arg – Pro – AzaglyNH2 s.c.
Triptoreline pGlu – His – Trp – Ser – Tyr – DTrp - Leu – Arg – Pro – GlyNH2 s.c., depot
Structure of GnRH agonists
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Chronic administration of GnRH agonist
Testostérone
LH
« flare-up effect »
suivi de
« désensibilisation »
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Action of GnRH agonists
postpost--receptorreceptor--cascadecascade
GnRH GnRH -- receptorreceptor
GnRH
GnRH GnRH -- agonistagonist
flare up effect
Down-regulation
pituitary suppression
refractory phaseRelease and synthesis
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Structure of GnRH antagonists
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PyroPyro (Glu) (Glu) –– His His –– Trp Trp –– Ser Ser –– TyrTyr – Gly – Leu – Arg – Pro – Gly NHNH22
Activation of the GnRH receptor
ProteolyseRegulation ofRegulation ofbiologic activitybiologic activity
To achieve antagonistic properties of natural GnRH moremodifications than only in position 6 and 10 are necessary
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1 2 3 4 5 6 7 8 9 10
pyroGlu
pyroGlu HisHis TrpTrp SerSer TyrTyr GlyGly LeuLeu ArgArg ProPro Gly
NH2
GlyNH2
AcD-Nal
AcD-Nal
D-4FPhe
D-4FPhe D-TrpD-Trp SerSer TyrTyr D-ArgD-Arg LeuLeu ArgArg ProPro D-AlaD-Ala
AcD-Nal
AcD-Nal
D-4CiPhe
D-4CiPhe D-PalD-Pal SerSer ArgArg D-Glu
(AA)D-Glu(AA) LeuLeu ArgArg ProPro D-AlaD-Ala
AcD-Nal
AcD-Nal
D-4CiPhe
D-4CiPhe D-PalD-Pal SerSer TyrTyr D-4Aph
(Et2)
D-4Aph(Et2)
LeuLeu L-4Aph(Et2)
L-4Aph(Et2) ProPro D-AlaD-Ala
AcD-Nal
AcD-Nal
D-4CiPhe
D-4CiPhe D-PalD-Pal SerSer TyrTyr D-CitD-Cit LeuLeu ArgArg ProPro D-AlaD-Ala
GnRH
Nal-Arg
Nal-Glu
GanirelixGanirelix
CetrorelixCetrorelix
First-Generation
Second-Generation
ThirdThird--GenerationGeneration
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Action of GnRH antagonists
postpost--receptorreceptor--cascadecascade
GnRH GnRH -- receptorreceptor
GnRH
GnRH GnRH -- antagonistantagonist
No flare up effect
Immediate effect
No Down-regulation
Reversible effectRelease and synthesis
Competition atthe pituitary
level
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1 2 3 4 5 6 7 8 9 10pyroGlu
pyroGlu HisHis TrpTrp SerSer TyrTyr GlyGly LeuLeu ArgArg ProPro Gly
NH2
GlyNH2
AcD-Nal
AcD-Nal
D-4CiPhe
D-4CiPhe D-PalD-Pal D4Aph
(Et2)D4Aph(Et2)
L4Aph(Et2)
L4Aph(Et2)
D-AlaD-Ala
AcD-Nal
AcD-Nal
D-4CiPhe
D-4CiPhe D-PalD-Pal D-CitD-Cit D-AlaD-Ala
Native Native GnRHGnRH
GanirelixGanirelix
CetrotideCetrotide
Third-Generation D-LeuD-Leu NHEtNHEtLeuprolide
D-SerD-Ser NHEtNHEtBuserelin
activation of the GnRH receptor
regulation ofreceptor affinity
regulation ofbiologic activity
GnRH Antagonists
GnRH AgonistsKarten MJ & Rivier JE, 1986
DTrpDTrp Triptorelin GlyNH2
GlyNH2
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How do GnRH analogs work ?
GnRH-AntagonistBlockade of GnRH
receptor
GnRH- Agonist
0
5
10
15
20
25
30
-6 0 6 12 18 24 30 36 42 48
Hours
LH (
IU/L
) Agonist
Antagonist
LH FSH
binding to GnRH receptor 1. Release of FSH & LH
2. Ptituitary desensitization
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GnRH Agonist vs Antagonists
Effect on LH levels
0
5
10
15
20
25
30
-6 0 6 12 18 24 30 36 42 48
Hours
LH
(IU
/L)
AgonistAntagonist
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Les anti – oestrogènes
et
Les inhibiteurs de l’aromatase
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Hypothalamus
Hypophyse
Effet
Ovaires
Glaire
Endomètre
Cible Conséquences
GnRH
LH
action FSH / aromatase
volume, filance
épaisseur
altération vascularisation
Citrate de Clomiphène
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ERER
ERER
E2
FSH
Day 5Day 5
Clomiphene Citrate Treatment
ERER
ERER
Day 10Day 10
FSH
E2
CC CC
ERER
ERER
ERER
ERER
• Long half live: persistent effects by late FP
• No negative feed back on FSH secretion
Risk of multifollicular development
Blockade of estrogen receptors
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Inducteur d’ovulation le plus prescrit
1.873.000 cp / an
• Utilisé > 30ans• Simple
• Peu coûteux (4.54ε 5 cp)• Peu risqué, bien que !
• Effets anti-oestrogéniques• Mauvaises indications• Prescriptions stéréotypées• Risque KC ovarien ?
Citrate de Clomifène
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ERERE2
FSH
Day 5Day 5
Aromatase Inhibitor Treatment
ERER
ERER
Day 10Day 10
FSH
E2
AI
ERER
ERER
ERER
ERER
• short half live: no persistent effects by late FP
• effective negative feed back on FSH secretion
Lower risk of multifollicular development
Blockade of androgen to estrogen conversion
AA…………..E2..E2
AI
AA…………..E2..E2
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Stimulation par injectionsde gonadotrophines
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La structure des gonadotrophines
FSH, LH et hCG : protéines composées de deux sous-unités
alpha commune et beta spécifique
Produite par technologie d’ADN recombinant
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Molécule de FSH
J Xiang 2002 SRBI
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FSH-FSHR interaction
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FSH-FSHR interaction
extracellular domain
transmembrane domain
intracellular domain – G protein
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α
βα
calcium channel
Ca 2+
FSH
γβ
GTPcAMP
PKA
protein phosphorylation
CRE mRNA transcription
transcription factors
nucleus
Signal transduction pathway of the FSH receptor
FSHR
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FSH isoforms
Hypophyseal secretion
~ 12 isoforms with different sialic acid content
resulting in
• different half-lives
• different bio-activities
Crucial role of the isoform profile.
The final step of glycosylation is critical
in the manufacturing process.
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Molécule de FSH hétérogèneité : le degré de glycosylation
Mannose
Galactose
Sialic Acid
Protein
GlcNAc
Acide sialique
Acide sialique
Différents profils de glycosylation
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Contenu en acide sialiqueet isoformes de FSH
Howles CM. Hum Reprod Update. 1996;2:172.
Contenu en Ac. Sialique
Demi-vieIn Vivo
Elevé Longue
Type d’isoformes
Acide
Basique Faible Courte
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Analyse de la structure des chaînes glycosylées des isoformes de la FSH
Neutre Mono Di Tri Tetra
Cartographie glycoprotéique selon teneur en acide sialique (charge)
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ETAPES CLES DANS LA TECHNOLOGIE D’ADN POUR LA PRODUCTION DE hFSH
Recombinant human FSH
1975 : sequence de la sous-unité α de FSH humaine
1976: sequence de la sous-unité β de FSH
1985 : Le gene de ß FSH exprimé dans les fibroblastes
1988: FSH humaine exprimée dans les cellules CHO
1992: 1ere grossesse avec r- hFSH
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ACTIVE SUBSTANCESUBSTANCE ACTIVE
Procédés de production
COLLECT URINECOLLECTE D’URINE
PURIFICATIONPURIFICATION
CELL CULTURE CULTURE DE TISSU
TRADITIONALTRADITIONEL BIOTECHNOLOGYBIOTECHNOLOGYBIOTECHNOLOGIE
AMPOULEFORMULATION
Deux types de production de gonadotrophines
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Biotechnology in the early 90 ’s
Manufacturing process
• Over 70 human proteins are clinically available
• More than 30 complex glycosylated-proteins manufactured using mammalian cells (final step of
glycosylation) : FSH belongs to this family
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NoyauNoyau
MaturationMaturation
SecretionSecretionTranslationTranslation GolgiGolgi
ERER
mRNAmRNA
TranscriptionTranscription
IncorporationIncorporation
Expression
Molécule mature
TransfectionTransfection
Production par génie génétique
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FSH / LH Nom activité spécifique /mg de prot
HMG HP 75 / 75 Ménopur 2000 SCFSH u HP 75 / <0,001 Fostimon 4200 SC
hCG u 5.000 Gonado Endo IM
FSH rec 75 / 0 Puregon 10 000 SC300/600/900
75 / 0 Gonal F 13.450 SC300/450/900
LH rec 0 / 75 Luveris 10 000 SC
hCG rec 6.500 Ovitrel SC
Gonadotrophines
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Biotechnology in the early 2000 ’s
Improved manufacturing process (Gonal F)
Highly consistent isoform profile
Specific Activity : 13.745 IU / mg
Calibration of the final product
Was still dependent on the bio-assay described by
Steelman-Pohley Ovarian in 1953
Weight Augmentation Assay for FSH
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Steelman-Pohley OvarianWeight Augmentation Assay for FSH
Sacrifice day 4and collect ovaries
Ovaries are weighedand data processed
FSH injected sc 1 x 3 days
21-22 day old female ratsRandomized + hCG primed
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Rat in vivo bioassay - limitations in precision
CV: 80 CV: 80 -- 125 %125 %
Ovaries stimulated by 4 different doses of r-hFSH
European Pharmacopoeia
Dose 1
Dose 2
Dose 3
Dose 4
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In Vivo BioassayPharmacoepial Release Specification applied to all FSH
products is 80-125% of labelled value
60
75
94
50
55
60
65
70
75
80
85
90
95
Minimum Labelled Value Maximum
US and European Pharmacopeia
Inte
rnat
iona
l Uni
ts (
IU)
75
60
94
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150 225 300 37575
150
225
300
375
FSH (IU)actual
content
FSH Batches
450
188
120
240
375
300
468
FSH content overlap between the highest value of one batch and the lowest value of the upper batch greatly increases with FSH requirement
Major drawback of the rat bio-assayVariability in the FSH content of IU batches
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Biochemical Characteristics of Pergonal
1 2 3 4 5 6 7 8 9Bio LH
(IU/amp) 6.1 17.1 14 6.4 9.6 6.2 6 6.3 6.2
Bio FSH(IU/amp) 298 354 246 614 362 224 273 825 332
Bio FSH :LHratio 49 20 17 95 37 36 45 131 53
Follicles atOPU (SEM)
6.8(1.7)
7.2(0.6)
7.3(0.5)
9.0(0.7)
8.5(0.7)
6.4(0.8)
7.8(0.8)
10.4(1.4)
8.5(0.7)
Biochemical characteristics of HMG Batches
(Stone et al. 1989)
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Patient Response to Different hMG Lots
Rector NA, et al. Fertil Steril 60:1082, 1993
0
0.1
0.2
0.3
0.4
0.5
0.6
Lot 1 Lot 2 Lot 3 Lot 4
# oocytes/amp# fertilized oocytes/amp
*p<0.05
†p<0.05
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Corrélation : activité biologique FSH et nombre of follicules matures
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0
10
20
30
40
50
60B
FD
A98
501
BF
DA
9850
4
BF
DA
9850
7
BF
DA
9851
0
BF
DA
9851
3
BF
DA
9851
6
BF
DA
9851
9
BF
DA
9852
2
BF
DA
9852
5
BF
DA
9852
8
BF
DA
9853
1
BF
DA
9853
4
BF
DA
9853
7
BF
DA
9854
0
BF
DA
9854
3
BF
DA
9854
6
BF
DA
9855
0
BF
DA
9855
3
BF
DA
9855
6
BF
DA
9855
9
BF
DA
9950
1
BF
DA
9950
4
BF
DA
9950
7
BF
DA
9951
0
BF
DA
9951
3
BF
DA
9951
6
BF
DA
9951
9
Batch No
Neutrals
Mono
Di
Tri
Tetra
FSH Isoform ‘Families’ in Follitropin Alpha Highly Batch to Batch Consistency
Per
cent
of t
otal
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r-hFSH (follitropin alfa) a une grande reproductibilitéinter-lots: activité spécifique
. Bassett et al. RBM Online 2004; 10: 169–177
La variabilité inter-lots n’est que de 8.5% durant les 7 années de production
Batches (1997–2003)
13.645 IU / mg protein
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A Validated Physico-chemical Method (SE-HPLC) Allows Follitropin Alpha to be Measured in
Mass Units
14.0012.0010.008.006.004.002.000.00-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
6.59
7
7.71
0
Minutes
AU
Follitropin Alfa
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Follitropine alpha en 2004 : Fbm
Mode de Production : Biotechnologie
Activité Spécifique : 13.745 IU / mg
Profile d ’isoformes très constant
Calibration du produit final
Steelman - PohleyBioassay
75 IU
60 IU 94 IU(80%) (125%)
HPLCHPLCFbMFbM
5.5 5.5 µµgg
5.4 5.4 µµg 5.6 g 5.6 µµgg(2%) (2%)(2%) (2%)
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Utilisation des GonadotrophinesAugmentation de la r-hFSH
-
5,000
10,000
15,000
20,000
25,000
30,000
35,000
40,000
45,000
1996 1997 1998 1999 2000 2001 2002 2003
Year
75iu
Equ
iv. A
mps
(in
thou
sand
s)
u-hMG u-FSH r-hFSH
61.5%3%
60%
37%
5%
33.5%
IMS data, 2003
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Calculation of costs in € when looking for the number of ampoules (75 IU)
r-hFSH hMG HP Differencer-hFSH vs. hMG HP
€ € € %
Ø AVP per 75 IE * 42.17 33.70 8.47 20.1
per cycle 1.307.27 1.270.49 36.78 2.8
per pregnancy 4.925.46 5.058.37 -132.91 - 2.7
per life birth 7.413.49 8.266.61 - 852.61 - 11.5
• Calculation of costs for r-hFSH: average price of all GONAL-f and Puregon doses and packages for 75 IU ampoules;
• Calculation of costs for hMG: average of Menopur with 5 and 10 ampoules with 75 IU
Source of costs: Lauer-Taxe from December 15th, 2005
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Reference
� Barri PN, et al. Revista Iberoamericana de Fertilidad 2002;19(3):195-202.� Daya S, et al. Hum Reprod 2001;16:2563-9.
� Felberbaum R, et al. Geburtsh Frauenheilk 2002;62:668-76� Romeu A, et al. J Assist Reprod Gen 2003;20:294-300
� Silverberg K, et al. Fertil Steril 2002;77:107-13.
Felberbaum€21 686€22 188GermanSilverberg$40 688$47 096Per pregnancy (societal)
Daya€8 876€9 108 UK
Silverberg$28 481$32 967Per pregnancy (insurer)
Battaglia€28 827€29 860Italy
Romeu€12 791€13 007Spanish public healthBarri€19 739€20 467Spanish private clinics
Referencer-hFSHu-hFSHSetting
Coût total de traitement par grossesse dans les différents pays
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Coût-bénéfices ....
Coût par grossesse évolutive:
• r- hFSH £8 992• uFSH – HP £10 834• uhMG £9 472
Sykes et al 2001
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La structure des gonadotrophines
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• La première préparation de LH recombinante
�Composée d’une sous-unité alpha commune à la FSH et àl’hCG et d’une sous-unité beta spécifique
�Bénéficie de la technologie de l’ADN recombinant, procédéde fabrication identique à celui de la FSH recombinante et
de l’hCG recombinante
Luveris : LU VERITAS
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Production de r.hLH par recombinaison génétique
α
β
2 gènes humains
hLH
secretion
CHO: cellule de hamster
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Signal transduction pathway of the LH receptor
regulation of cholesterol transport to the inner
mitochondrial membrane
PKA
LH
GTPcAMP
Cl-Ca 2+
Ca 2+
Ca/CaM kinase
PLA2
arachidonic acid
leukotrienes
PKC
+
+
+LHR
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Pourquoi utilise-t-on l’hCG dans le déclenchement ?
Sous unité alpha commune : 92 AA
Sous unité bêta spécifique 121 AA 111 AA 145 AA
Similitude entre les 121 premiers AA de la chaîne bêta de l' hCG et les 121
AA de la chaîne bêta de la LH. Fixation sur le même récepteur LH/hCG
⇒ l' hCG est utilisée dans le déclenchement de l’ovulation
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CaractCaractCaractCaractééééristiques du pic de LH ristiques du pic de LH ristiques du pic de LH ristiques du pic de LH
Paramètres
Signal
Durée du pic(ttes phases)
Phase ascendante
Plateau (h)
Phase descendante
Pic de LH physiologique
Déclenché par un taux seuil d’E2
50 heures
14 heures
14 heures
20 heures
Pic de « LH »induit par
5 000 UI hCG IM
Aucun
> 96 heures
20 heures
0
72 heures
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Rôle des facteurs paracrines et autocrines
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Secretory activity of different follicular cell types
Theca cells
Oocyte
Cumulus GC
Mural GC
Theca capillaries
Intra-follicule concentration
gradients
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TGF-ββββ superfamily
Granulosa cell Differentiation Theca cell Theca cellAndrogen Production Progesterone Production
TGF Stimulation of FSH Inhibitory Stimulatory (CYP 17)rats (Rosairo) rats (Hernandez; Fournet) rats
GDF9 Inhibition of FSH Inhibitory Inhibitory (LHR)rats (Vitt) humans (Yamamoto) rats; bovine (Spicer) humans (Yamamoto)
BMP Inhibition of FSH Inhibitory Stimulatoryrats (Otsuka) HOTT (Dooley) HOTT (Dooley)
Inhibin Inhibition (?) Stimulatory Inhibitoryhumans (Hillier) HOTT (Sawetawan)
Activin Stimulation of FSH (primates Hillier) Inhibitory StableInhibition of hCG (Myers) humans (Hillier) HOTT (Sawetawan)
AMH Inhibition of FSH
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Molecular markers of follicular development
INHA
INHBA
INHIBIN A
INHA
�����
INHIBIN B
Tertiary
AMH
αµηαµηαµηαµη
αµηαµηαµηαµη
PrimaryPrimordial Secondary
INHBA
INHBB
ACTIVIN
Development-related transition from Activin to Inhibin formation during folliculogenesis
Primary to pre-antral stageβ subunit formation: Homodimers
antral stageα subunit formation: Heterodimers
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Inhibins and activins
Early follicular phase (low E2)
Activin • enhances the inductive actionof FSH on aromatase activity
• suppresses theca cell androgen synthesis
Late follicular phase (high E2)
• FSH and LH increase Inhibin and aromatase activity
• inhibin promotes LH / IGF1androgen synthesis by theca cell
Theca cell Androgen synthesis is amplified to sustain oestrogen synthesis
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A.Themmen
Anti-Müllerian Hormone (AMH)•Inhibitory effect on primordial follicular recruitment
• Inhibits pre-antral and small antral follicle growth by fine tuning the sensitivity of growing follicles to FSH
AMHKO female mice : more growing follicles and follicles more sensitive to FSH
AMH : one of the factors involved in the FSH threshold
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Members of the TGF- ββββ superfamily implicated in the bidirectional communication between theca - granulos a
cells and granulosa cells - oocyte
Autocrine (thick grey arrows)and paracrine (thick black arrows)signalling events depend on the expression of type-I and type-II receptors
on the cell surface