the receptive endometrium: providing the essential ... ppt/salamonsen… · the receptive...

31
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

Upload: others

Post on 16-Jan-2020

7 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 2: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 3: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 4: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

The peri-implantation microenvironment in women

Salamonsen

apposition

changed

adhesion

attachment UTERINE CAVITY invasion

hCG+

Page 5: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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)

Page 6: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 7: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 8: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 9: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 10: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 11: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 12: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

***

Page 13: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 14: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 15: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 16: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 17: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 18: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 19: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

1 5 10 15 20 25 28

IVF cycles

Days

Receptive

OPU

ET

Various protocols gonadotrophins

GnRH agonists/antagonists

hCG/LH

Page 20: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 21: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 22: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 23: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

This laboratory-based data supports a strategy of freezing all embryos and

replacing in a natural cycle

Page 24: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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)

Page 25: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 26: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 27: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 28: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

Prediction of outcome of fresh embryo

transfer: individual analytes Lavage at hCG+2

Pilot study

Factor 1 Factor 2

Page 29: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 30: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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

Page 31: The receptive endometrium: providing the essential ... PPT/Salamonsen… · The receptive endometrium: providing the essential microenvironment for embryo implantation Ovarian Club

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