Human endometrial receptivity during

natural and stimulated cycles

Samir Hamamah
Professor of Reproductive Medicine
Chair, ART/PGD department

ART/PGD Department
Arnaud de Villeneuve hospital
Montpellier, France
INSERM U 847

Rational

. 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

Requirements for implantation

. a receptive endometrium
. a functionally normal blastocyst
. an adequate cross-communication between 2

Conventional approaches used in

clinical practices for assessing
endometrial receptivity
. Morphological examination
. Ultrasonography
. Cervicovaginal fluid
. Single biomarker
. Omics

Conventional approaches used in clinical practices

for assessing endometrial receptivity

Morphological examination
. Endometrial dating by histological evaluations : Noyescriteria

. Pinopode formation

Remains controversial as marker of endometrial receptivity

Invasive method (endometrial biopsies) ?

Conventional approaches used in clinical practices

for 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 low

predictive value in determining endometrial receptivity and IVF outcome

Conventional approaches used in clinical practices

for assessing endometrial receptivity

Cervicovaginal fluid

Look for cytokine, interleukin and GF secretions

2 approaches

Washing

Higher & variable

dilution factor

or

Direct aspiration

Contamination by
cervical mucus
and/or blood

To date, supplementary studies should be performed to identify markers

linked to endometrial receptivity in endometrial secretions

Fundamental research for assessing endometrial

receptivity

Single biomarker
Numerous endometrial proteins have been suggested based on their sequential
temporal 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 and
infertile women in order to determine their predictive value as markers of
endometrial receptivity

The implantation window

. LH day +6 to +10
. Phases: signaling, apposition, attachment, invasion
. Key associated findings:
luminal epithelial pinopodes;
expression of adhesion molecules, and
novel cytokines profile

Do implantation factors explain IVF

Success/failure ?

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 implantation
2- sensitive and specific

Omics

Transcriptomic approaches have been used to

identify biomarkers of the human implantation
window.

modifications in gene expression profile associated

with the transition of the human endometrium
from a pre-receptive to a receptive state

Carson et al., 2002;

Riesewijk et al., 2003;
Mirkin et al., 2005;
Talbi et al.,2006

Fundamental research for assessing endometrial receptivity

Proteomic approach

Study

Patient
First sample:
Second sample:
Age
Fold
Number of proteins
population
day
of
cycle
day
of
cycle
Patients
change
(nber of samples) (nber of samples)
(years, min-max)
Up Down

Li et al.
(2006)

Candidats

LH + 2
(n=?)

LH + 7
(n=?)

Annnexin IV

LH + 2
(n=8)

LH + 7
(n=8)

1.3

12

23

Annnexin II (x2.13)
Stathmin (x-2.13)

(x2.12)

Article in Chinese

Domnguez et al. 23-39

(2009)

Oocyte
donors

Differential in-gel electrophoresis (DIGE) and mass spectometry

(MS)

Further studies are needed to determine their predictive value as markers

of endometrial receptivity

Fundamental research for assessing endometrial receptivity

Transcriptomic approach
Study

Age
Patients
(years, min-max)

Carson et al. Not specified

(2002)
Mirkin et al.
(2005)

24-32

Riesewijk et al. 23-39

(2003)

Patient
population

First sample:
day of cycle

Second sample: Fold

Nber of genes
day of cycle
change
(nber of samples) (nber of samples)
Up Down

Fertile
volunteers

LH + 2/4
(n=3)

LH + 7/9
(n=3)

Oocyte
donors

LH + 3
(n=3)

LH + 8
(n=5)

Fertile
volunteers

LH + 2
(n=5)

LH + 7
(n=5)

Talbi et al.
(2006)

23-50

Normal responder

ESP
(n=3)

MSP
(n=8)

1.5

Haouzi et al.
(2009a)

22-36

Normal responder

LH + 2
(n=31)

LH + 7
(n=31)

323

370

(Affymetrix Hu95A
12 000 genes)

49

58

(Affymetrix Hu95A
12 000 genes)

153

58

(Affymetrix Hu95A-E
60 000 uniq Sequ)

1415 1463

Affymetrix Hu133P
(30 000 genes)

945

67

Affymetrix Hu133P
(30 000 genes)

This is the first study analyzing by paired samples the endometrial gene
expression profiles from the same patients during the pre-receptive to the
receptive transition in a natural cycle.

we revisited the global gene expression

profile of human endometrial biopsies of a
natural and stimulated cycles
during :

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)

Experimental design

Endometrium cells

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RNA extraction

Amplification & labelling

Hybridization on chip

Affymetrix GeneChip
Operating Software (GCOS)

Expression
profile of 30 000
genes

Haouzi .Hamamah, HR 2009a

Haouzi .Hamamah, HR 2009a

Haouzi .Hamamah, HR 2009a

Validation of selected genes

identified by the microarray
data from natural cycles in the
pre-receptive (LH + 2) and
receptive (LH + 7)
endometrium.

Haouzi .Hamamah, HR 2009a

Number of genes significantly modulated in 6 microarray analyses

between natural and stimulated cycles
Study

Mirkin et al.
(2004)

Number
of
samples

Natural cycle
(nber of samples)

13

LH+8

hCG+9 Atg

LH +8

hCG+9 Ag

LH +7

hCG +7

LH +7

hCG +7 Atg standard dose

(n=14)

(n=4)

LH +7

19

49

1.2

No

281

277

No

22

69

hCG +7 Atg high dose

88

24

LH +7

hCG +7 Ag

(n=14)

(n=5)

22

100

LH+2

hCG+2

(n=5)

(n=5)

LH+7

hCG+7

69

73

LH+7

hCG+7 high serum E2 levels

244

159

LH+7

hCG+7 low serum E2 levels

(n=5)

Liu et al.
(2008)

13

(n=5)
(n=5)

Haouzi et al.
(2009b)

84

Number of genes

1.19

(n=5)

(n=14)

Horcajadas et al.
(2008)

Fold
change

Down

(n=14)

28

Paired
analysis

Up

(n=5)

Horcajadas et al.
(2005)
Simon et al.
(2005)

Stimulated cycle
(nber of samples)

(n=5)
(n=3)

(n=5)

(n=5)

(n=4)

No

(n=4)

hCG+2

LH+7

hCG+5

(n=21)

No

(n=5)

LH+2
(n=21)

No

(n=21)

Yes

321

657

(n=21)

This is the first study analyzing by paired samples the endometrial gene
expression profiles from the same patients during the pre-receptive to the
receptive transition both in a natural and in a subsequent stimulated cycle.

Comparison of gene expression profiling across the

WI in natural Vs stimulated cycles

Elucidate the genomic impact of COS on endometrial

development and
Search for novel gene targets to improve endometrial
receptivity in IVF

LH+2 versus hCG+2

LH+7 versus hCG+5

Haouzi .Hamamah, HR 2009b

Haouzi .Hamamah, HR 2009b

Haouzi .Hamamah, HR 2009b

15% Atypical profiles

Molecular signature
of COS cycles
during the WI
differs from natural
cycles

EFR
ER after freezigthawing on NC

PP 27
27

PP 22
22

P 25

P 42

Haouzi .Hamamah, HR 2009b

Haouzi .Hamamah, HR 2009b

Haouzi .Hamamah, HR 2009b

GnRH agonist long protocol affects more the

local microenvironment of the human
endometrial receptivity than antagonist
protocol

% of genes in
common between
stimulated and
natural cycles
during the
receptive
endometrium.

Major differences of chemokines, and growth factors involved during

endometrial receptivity between natural, GnRH antagonist and agonist
long protocols.
Chemokines (A), and growth factors (B) up-regulated during the
endometrial receptivity under natural, GnRH agonist long and
antagonist protocols.
Red, not present in natural cycles

Unsupervised classification with PCA of the hCG+2 and

hCG+5 samples under GnRH agonist long and antagonist
protocols with the predictor list.

CONCLUSIONS

The transcriptomic pattern of endometrial cells in N and S

cycles in the same patients reveals either moderate or strong
alterations of endometrial receptivity under COS protocols.
This information open new perspectives, particularly in patients
with multiple implantation failures.
In this case, analysis of the endometrial profile could reveal a
strongly altered profile during COS protocols, prompting the
clinician to either adapt the IVF stimulation protocol or to
perform embryo transfer later during a N cycle.

CONCLUSIONS

Both protocols affect endometrial receptivity in comparison

with their N cycle.
Major differences in endometrial chemokines and growth
factors in S cycles in comparison with N cycles were observed,
associated with gene expression alterations of endometrial
receptivity genes.
The endometrial receptivity under GnRH antagonist was closer
to the N cycle receptivity than under GnRH agonist protocols.

CONCLUSIONS

GnRH antagonist mimics more closely the natural

endometrial receptivity than GnRH agonist long
protocols.
The use as a first choice of GnRH agonist long
protocol for normal responder patients should be
reconsidered.

Aknowledgements
ART/PGD Department

INSERM U 847

Prof. Samir Hamamah

Pr. Herv Dechaud
Pr. Bernard Hedon
Dr. Lionel Reyftman
Dr. Tal Anahory
Dr. Alice Ferriere
Dr. Vanessa Loup

Dr. Meryline Dijon

Dr. John De Vos
Dr. Franck Pellestor
Dr. Dephine Haouzi
Dr. Said Assou
Mis. Cecile Donzo