Post on 16-Jan-2020
The receptive endometrium: providing the essential
microenvironment for embryo implantation
Ovarian Club
Paris, 2014
Lois Salamonsen MIMR-PHI Institute Melbourne, AUSTRALIA
Photo: G Nikas
Wellcome Library
Progesterone
Oestrogen Ovarian hormone cycle
Uterine menstrual cycle
O v u l a
t i o
n
0 5 1 4 19 24 28
Secretory phase
Cycle day
Menses Proliferative phase
follicular development corpus luteum present/regresses
Uterine fluid: important for implantation
Gray et al Biol.Reprod. 2001
Filant et al Biol.Reprod. 2013
Retarded conceptus development
No implantation
Due to lack of
uterine secretory products
In mice, no implantation, no decidualization
Sheep without
uterine glands
Contains
glycoproteins
glycogen, glycodelin A
EGF, LIF, VEGF
Burton et al, JCEM 2002
Hempstock et al, RBE 2004
Human: histotroph
important throughout 1st
trimester
The peri-implantation microenvironment in women
Salamonsen
apposition
changed
adhesion
attachment UTERINE CAVITY invasion
hCG+
Some molecular changes associated with receptivity
in the uterine cavity
glandular secretions – nutrients, enzymes, cytokines, antiproteases, transport proteins etc
exosomes released from endometrial (? And trophectodermal) surface
at the luminal epithelial surface (mucins, integrins, processing proteases etc)
within the luminal epithelial cells (changes in junctional complexes, Ca++ regulators, growth factors, cytokines, chemokines etc)
Cyclical changes in epithelial proteins suggest a role in embryo-maternal interactions
Vogiagis et al. 1996; Nie et al. 2006; Dimitriadis et al, 2000, Hannan, JCEM 2004
O v u l a t i o n
Secretory Menstrual Proliferative
IL11 LIF
PC6
FKN
LIF
D24
D2
3
D1
9
IL-11
PC6
D19 D2
6
CX3CL1 (FKN)
ES MS
Some proven actions of endometrial secreted proteins
Improve blastocyst development
Act as chemoattractants to blastocyst / trophoblast
Alter adhesive capacity and outgrowth of blastocysts
changed
adhesion
attachment
apposition
DISCOVERY STUDIES
Lavage fluid
Specific groups
Functional studies: Actions on blastocyst,
attachment etc
Build and test multiplex assays:
multivariate analysis
Unknown proteins: MS technology
Known proteins: Multiplex analysis
Serpin C1; F vs IF
Validation: IHC in tissues, secretions from primary hEEC
Endometrial secreted proteins: discovery 2009-2011
Hannan et al, J Prot Res 2009, 2010; Endocrinol 2011, Paiva et al, Hum Rep 2011
MP vs. MS (fertile
Fertile vs. Infertile(MS)
MP vs. MS Fertile vs. MS Infertile
Changed between
2D-PAGE + LC-MS
>400
proteins
LuminexTM multiplex analysis
• 33/42 growth factors, cytokines, chemokines
• range of concentrations
• some altered with cycle phase and fertility status
• similar profile in endometrial epithelial cell culture
Actions of uterine fluid on mouse embryo outgrowth
+ MP uterine fluid
control
+ MS uterine fluid
MS
0
20
40
60
80
100
120
140
160
66 74 90 98
Hours post hatching
Are
a o
f bla
sto
cyst
outg
row
th
**
** *
*
*
Cont
MP
Growth on FN, Pooled UF, 8 women 15 blastocysts / treatment
Hannan et al, 2011
Uterine fluid components enhance epithelial cell adhesive capacity to embryonic FN
EEC a
dhesi
on t
o F
N:
fold
change
*
0
1
2
3
4
5
6
C UF C VEGF
*
FN+
Shimomura et al Mol HR 2006
Hannan et al, 2010, 2011
Also other factors: FKN, HCC1 Meduri BOR,2000
Flt-1
Endometrial chemokines differently regulate trophoblast adhesion to fibronectin
0
1
2
3
4
5
Con FKN HCC1
Adh
esio
n to f
ibro
nectin fo
ld
cha
ng
e fro
m c
on
trol *
*
Hannan & Salamonsen Biol.Reprod. 2008
Regulated by FKN
CTNNA1 ↑
ECM1 ↑
SPP1
ITGA6
ITG5 ↑
Inte
grin-β
5 m
RN
A
0
1
2
3
4
CONT FKN HCC-1
*
*
ECM
-1 m
RN
A
0
2
4
6
8
CONT FKN HCC-1
***
The blastocyst also signals to the endometrium to enhance receptivity
In vivo infusion of hCG into the uterine cavity, LIF, VEGF secretion (Licht et al
Reprod Biol 2001)
In vitro treatment of endometrial epithelial cells enhanced implantation-related cytokines (Paiva et al
Human Reprod 2011)
+hCG
6/43 factors by hCG
Receptivity enhanced
by embryo
Exosomes 50-100/150nm vesicles
of endocytic origin
Released into the extracellular space
Mediate extracellular communication by transfer of genetic material, proteins etc
Alter phenotype of recipient cells
Théry, The Scientist, 2011
Li et al, 2012
Hum Mol Genetics
Exosomes can be retrieved from human uterine fluid and associated mucus
(in vivo production)
mucus associated
fluid
CD63 CD81 merged
600
Particle Diameter (nm) 0 100 200 400 500 300
Uterine Fluid
Size analysis
ECC1 exosomes contain specific miRNA vs parent cells
217 13 5
hsa-let-7e*, hsa-let-7f-2*
hsa-miR-122,
hsa-miR-124, hsa-miR-1248,
hsa-miR-129*,
hsa-miR-142-3p, hsa-miR-222*
hsa-miR-376c
hsa-miR-409-3p,
hsa-miR-432, hsa-miR-451
hsa-miR-520h
hsa-miR-149*
hsa-miR-187
hsa-miR-202,
hsa-miR-25*
hsa-miR-767-5p
exosomes cells
Ng, Rome et al, Plos One, 2013
Endometrial exosome miRNAs potentially target pathways important for implantation
Adherens junctions
Tight junctions
ECM-receptor interactions
VEGF signalling
Jak-stat signalling
Amino acid metabolism
and others
What about the endometrium in IVF cycles?
Success rates of IVF have remained ~30% /cycle in spite of improvements in embryo culture and selection
The endometrium has been largely ignored by IVF clinics
Attention to endometrial receptivity – likely to improve IVF success rates
1 5 10 15 20 25 28
IVF cycles
Days
Receptive
OPU
ET
Various protocols gonadotrophins
GnRH agonists/antagonists
hCG/LH
Substantial morphological disturbance following COH: LH /hCG +2
Fertile Infertile antagonist Donor agonist
Infertile agonist, NPr Infertile agonist, Pr
Evans, Hannan et al, PLOS ONE 2012
Endometrial abnormalities at hCG+2 combined 8 parameters
Glands Stroma BV
* *
*
Fertile Agonist NPR
CD34
Elastase (NP)
ER, PR, oedema,
Stromal decidualization Epithelial transformation
Leukocyte numbers Glandular secretions
Also
Endometrial response to hCG is lost following chronic exposure
Cell culture models: HCG, acute 20 IU for 24h OR chronic 5 IU for 3-7 days, then acute 20 IU for 24h
Evans & Salamonsen, Hum Reprod 2013
*
0
50
100
150
200
D3 2.5 D3 5 20IU short
Adhesion to Fibronectin
Shimomura et al Mol HR 2006
50
60
70
80
90
100
110
120
day 1 day 2 day 3 day 4 day 5 day 7 day 8
control
short
5IU
acute chronic/acute
This laboratory-based data supports a strategy of freezing all embryos and
replacing in a natural cycle
Strong clinical evidence also supports freeze-all with replacement in natural
cycles
Other outcomes also improved. ↑ birth wt, ↓ risk of low bw, preterm birth, small for gestational age, ectopics, preeclampsia (Shapiro et al, 2002-2013)
Biomarkers for endometrial receptivity are needed
What will be best for clinical use?
What to sample?
blood, tissue, uterine fluid, urine
When to sample?
mid-secretory, at oocyte pickup
What to measure?
mRNA, protein, lipids
single or multiple markers proliferative secretory
Multivariate modelling of 10 analytes in uterine fluid
Dating by Noyes criteria
Exclusion: tubal, ovarian, metaplastic pathologies
Lavage fluid, multiplex ELISA
Women <43y, fertile or infertile, non-stimulated cycles
subjects n F/IF
10X cross validation
ES F vs IF 20/18 70.27%
MS F vs IF 17/19 77.78%
ES vs MS (Pre-R vs R)
20/17 72.22%
Representative significant data
0
2000
4000
6000
8000
10000
12000
Fertility code
g-c
sf
0 10
10
20
30
40
50
Fertility code
gcsf/p
rote
in
0 1
1
10
100
1000
10000
100000
Fertility code
CF
H
0 11
10
100
1000
10000
Fertility code
Il8
0 1
10
100
1000
10000
100000
Fertility code
VE
GF
R1
0 1
10
100
1000
10000
100000
1000000
Fertility code
sG
P1
30
0 1
0.1
1
10
100
1000
Fertility code
PlG
F
0 1
0.0001
0.001
0.01
0.1
1
10
100
1000
Fertility code
fgf2
_cfh
0 1
0.01
0.1
1
10
100
1000
10000
Fertility code
fgf-
2/p
lgf
0 1
0.001
0.01
0.1
1
10
100
1000
10000
Fertility code
il8
_fg
f2
0 1
AUC 0.848
Criterion Value
0.685
Sensitivity 78.95%
Specificity 88.24%
Significance 0.0001
6 analytes
ES F v IF
AUC 0.784
Criterion Value
0.280
Sensitivity 88.89%
Specificity 63.16%
Significance 0.0003
AUC 0.932
Criterion Value
0.108
Sensitivity 84.21%
Specificity 94.12%
Significance 0.0001
MS F vs IF
7 analytes
ES v MS Non-rec vs rec
8 analytes
Prediction of outcome of fresh embryo
transfer: individual analytes Lavage at hCG+2
Pilot study
Factor 1 Factor 2
Optimising the intrauterine environment The next leap forward for IVF
Need an optimal intrauterine environment within the uterine cavity, to prepare the blastocyst and the
endometrial surface for implantation
Biomarkers in uterine fluid will provide:
clear information on inadequate uterine receptivity as a primary cause of infertility
a guide for changes in IVF protocols
information on likely outcome of transfer in any treatment / non-treatment cycle
potential targets for treatment of some female infertility without IVF
Endometrial Remodelling Team 2014
Clinical
Monash IVF
Gab Kovacs
Luk Rombauts
Bev Vollenhoven
Judi Hocking
Elise Forbes
Collaborators
Natalie Hannan
Richard Simpson
David Greening
Sophy Rome
Salamonsen Lab Tracey Edgell
Jemma Evans
Hong Nguyen
Angela Morgan
Patrick Martin
Harriet Fitzgerald
Ann Winter
Adriana Krysta
Long term collaborators
Guiying Nie
Evdokia Dimitriadis
Funding: NHMRC, Merck-Serono GFI, Monash IVF