human endometrial receptivity
TRANSCRIPT
Human endometrial receptivity duringnatural and stimulated cycles
Samir HamamahProfessor of Reproductive Medicine
Chair, ART/PGD department
ART/PGD DepartmentArnaud de Villeneuve hospital
Montpellier, FranceINSERM U 847
. in IVF/ICSI, pregnancy and birth rates remain low
. out of 3 IVF cycles fail to result in pregnancy
. more than 8 out of 10 transferred Embryo fail to implant
Rational
. a receptive endometrium
. a functionally normal blastocyst
. an adequate cross-communication between 2
Requirements for implantation
Conventional approaches used inclinical practices for assessing
endometrial receptivity
. Morphological examination
. Ultrasonography
. Cervicovaginal fluid
. Single biomarker
. Omics
Conventional approaches used in clinical practicesfor assessing endometrial receptivity
Morphological examination
. Endometrial dating by histological evaluations : Noyes’criteria
. Pinopode formation
Remains controversial as marker of endometrial receptivityInvasive method (endometrial biopsies) ?
Conventional approaches used in clinical practicesfor assessing endometrial receptivity
Ultrasonography
The 2/3D power Doppler
. Endometrial thickness
. Endometrial pattern
. Pulsatility index of uterine vessels
. Resistance index of uterine vessels
. Endometrial volume
. Vascularization index
. Flow index of endometrial & subendometrial regions
. Vascularization flow index of endometrial & subendometrial regions
Identification of patients with a poor implantation prognosis, but lowpredictive value in determining endometrial receptivity and IVF outcome
Conventional approaches used in clinical practicesfor assessing endometrial receptivity
Cervicovaginal fluid
Washing
2 approaches
Direct aspirationor
Look for cytokine, interleukin and GF secretions
Higher & variabledilution factor
Contamination bycervical mucusand/or blood
To date, supplementary studies should be performed to identify markerslinked to endometrial receptivity in endometrial secretions
Fundamental research for assessing endometrialreceptivity
Single biomarker
Numerous endometrial proteins have been suggested based on their sequentialtemporal expression with respect to the implantation window:
glycodelin A,aVb3 integrin,osteopontin,
LIF,colony stimulating factor 1 (CSF1),
mucin 1…
Further studies are needed with large patient cohorts including fertile andinfertile women in order to determine their predictive value as markers ofendometrial receptivity
. LH day +6 to +10
. Phases: signaling, apposition, attachment, invasion
. Key associated findings:luminal epithelial pinopodes;expression of adhesion molecules, andnovel cytokines profile
The implantation window
A wide range of potential candidates exists.Potential markers are in Endometrium, Embryo,Immuno system or Vascular system
Studies must show that a bio-marker is:1- measurable without disturbing implantation2- sensitive and specific
Do implantation factors explain IVFSuccess/failure ?
Carson et al., 2002;Riesewijk et al., 2003;Mirkin et al., 2005;Talbi et al.,2006
OmicsTranscriptomic approaches have been used toidentify biomarkers of the human implantationwindow.
modifications in gene expression profile associatedwith the transition of the human endometriumfrom a pre-receptive to a receptive state
Fundamental research for assessing endometrial receptivity
Proteomic approach
Study
Domínguez et al.(2009)
First sample:day of cycle
(nber of samples)
LH + 2(n=8)
Second sample:day of cycle
(nber of samples)
LH + 7(n=8)
AgePatients
(years, min-max)
Patientpopulation
Differential in-gel electrophoresis (DIGE) and mass spectometry(MS)
Oocytedonors
23-39
Foldchange
1.3
Up Down
12 23
Number of proteins
Further studies are needed to determine their predictive value as markersof endometrial receptivity
Li et al.(2006)Article in Chinese
LH + 2(n=?)
LH + 7(n=?)
?? ? 1 0 Annnexin IV(x2.12)
Candidats
Annnexin II (x2.13)
Stathmin (x-2.13)
Fundamental research for assessing endometrial receptivity‘Transcriptomic approach’
Riesewijk et al.(2003)
Study
Carson et al.(2002)
Mirkin et al.(2005)
Talbi et al.(2006)
Haouzi et al.(2009a)
First sample:day of cycle
(nber of samples)
LH + 2/4(n=3)
LH + 3(n=3)
LH + 2(n=5)
ESP(n=3)
LH + 2(n=31)
Second sample:day of cycle
(nber of samples)
LH + 7/9(n=3)
LH + 8(n=5)
LH + 7(n=5)
MSP(n=8)
LH + 7(n=31)
AgePatients
(years, min-max)
Not specified
24-32
23-39
23-50
22-36
Foldchange
2
2
3
1.5
2
Up Down
(Affymetrix Hu95A12 000 genes)
323 370
(Affymetrix Hu95A12 000 genes)
49 58
(Affymetrix Hu95A-E60 000 uniq Sequ)
153 58
1415 1463Affymetrix Hu133P(30 000 genes)
Affymetrix Hu133P(30 000 genes)
945 67
Nber of genesPatientpopulation
Fertilevolunteers
Fertilevolunteers
Oocytedonors
Normal responder
Normal responder
This is the first study analyzing by paired samples the endometrial geneexpression profiles from the same patients during the pre-receptive to thereceptive transition in a natural cycle.
Natural cycle- early secretory phase, 2 days after the LH surge (LH +2)- mid-secretory phase (LH +7)
Stimulated cycle- Egg collection day (hCG +2)- Transfert dat (hCG +5)
we revisited the global gene expressionprofile of human endometrial biopsies of anatural and stimulated cycles
during :
Experimental designExperimental design
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Amplification & labelling
Hybridization on chip
Endometrium cells
RNA extraction
Affymetrix GeneChip®Operating Software (GCOS)
Expressionprofile of 30 000
genes
Haouzi ….Hamamah, HR 2009a
Haouzi ….Hamamah, HR 2009a
Haouzi ….Hamamah, HR 2009a
Validation of selected genesidentified by the microarraydata from natural cycles in thepre-receptive (LH + 2) andreceptive (LH + 7)endometrium.
Haouzi ….Hamamah, HR 2009a
Horcajadas et al.(2005)
Study
Mirkin et al.(2004)
Horcajadas et al.(2008)
Haouzi et al.(2009b)
Natural cycle(nber of samples)
LH+8(n=5)
LH +8(n=5)
LH +7(n=14)
LH+2(n=21)
Stimulated cycle(nber of samples)
hCG+9 Atg(n=5)
hCG+9 Ag(n=3)
hCG +7(n=5)
hCG+2(n=21)
Numberof
samples
13
19
49
84
Foldchange
1.19
1.2
3
-
2
Up Down
6 6
5 1
281 277
321 4
Number of genes
LH+7(n=21)
hCG+5(n=21)
Simon et al.(2005)
LH +7(n=14)
hCG +7 Atg standard dose(n=4)
28 2 22 69
LH +7(n=14)
hCG +7 Atg high dose(n=5)
LH +7(n=14)
hCG +7 Ag(n=5)
88 24
22 100
LH+2(n=5)
hCG+2(n=5)
LH+7(n=5)
hCG+7(n=5)
657 0
Pairedanalysis
No
No
No
Yes
No
0 0
69 73
Liu et al.(2008)
13 LH+7(n=5)
hCG+7 high serum E2 levels(n=4)
LH+7(n=5)
hCG+7 low serum E2 levels(n=4)
No 2 244 159
5 2
This is the first study analyzing by paired samples the endometrial geneexpression profiles from the same patients during the pre-receptive to thereceptive transition both in a natural and in a subsequent stimulated cycle.
Number of genes significantly modulated in 6 microarray analysesbetween natural and stimulated cycles
Comparison of gene expression profiling across theWI in natural Vs stimulated cycles
Elucidate the genomic impact of COS on endometrialdevelopment and
Search for novel gene targets to improve endometrialreceptivity in IVF
LH+2 versus hCG+2LH+7 versus hCG+5
Haouzi ….Hamamah, HR 2009b
Haouzi ….Hamamah, HR 2009b
Haouzi ….Hamamah, HR 2009b
P 27 P 22 P 25 P 42
15% Atypical profiles
Molecular signatureof COS cyclesduring the WI
differs from naturalcycles
EFR
ER after freezig-thawing on NC
P 27 P 22 P 25Haouzi ….Hamamah, HR 2009b
Haouzi ….Hamamah, HR 2009b
Haouzi ….Hamamah, HR 2009b
GnRH agonist long protocol affects more thelocal microenvironment of the human
endometrial receptivity than antagonistprotocol
% of genes incommon between
stimulated andnatural cycles
during thereceptive
endometrium.
Major differences of chemokines, and growth factors involved duringendometrial receptivity between natural, GnRH antagonist and agonistlong protocols.Chemokines (A), and growth factors (B) up-regulated during theendometrial receptivity under natural, GnRH agonist long andantagonist protocols.
Red, not present in natural cycles
Unsupervised classification with PCA of the hCG+2 andhCG+5 samples under GnRH agonist long and antagonist
protocols with the predictor list.
The transcriptomic pattern of endometrial cells in N and Scycles in the same patients reveals either moderate or strongalterations of endometrial receptivity under COS protocols.
This information open new perspectives, particularly in patientswith multiple implantation failures.
In this case, analysis of the endometrial profile could reveal astrongly altered profile during COS protocols, prompting theclinician to either adapt the IVF stimulation protocol or to
perform embryo transfer later during a N cycle.
CONCLUSIONS
Both protocols affect endometrial receptivity in comparisonwith their N cycle.
Major differences in endometrial chemokines and growthfactors in S cycles in comparison with N cycles were observed,
associated with gene expression alterations of endometrialreceptivity genes.
The endometrial receptivity under GnRH antagonist was closerto the N cycle receptivity than under GnRH agonist protocols.
CONCLUSIONS
GnRH antagonist mimics more closely the naturalendometrial receptivity than GnRH agonist long
protocols.
The use as a first choice of GnRH agonist longprotocol for normal responder patients should be
reconsidered.
CONCLUSIONS
ART/PGD Department INSERM U 847
Pr. Hervé DechaudPr. Bernard HedonDr. Lionel ReyftmanDr. Tal AnahoryDr. Alice FerriereDr. Vanessa Loup
Dr. Meryline DijonDr. John De VosDr. Franck PellestorDr. Dephine HaouziDr. Said AssouMis. Cecile Donzo
Prof. Samir Hamamah
Aknowledgements