Human Endometrial Receptivity

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