Cracking the Enigma of AdenomyosisAdenomyosis: An enigma • Fairly common • Presenting symptoms (in ~2/3 of patients) – Dysmenorrhea – Heavy menses – Infertility – Enlarged

Cracking the Enigma of Adenomyosis

Sun-Wei Guo Shanghai OB/GYN Hospital

Fudan University Shanghai, China

Disclosure of conflict of interest

•  Nothing to disclose

Adenomyosis: An enigma •  Fairly common •  Presenting symptoms (in ~2/3 of patients)

–  Dysmenorrhea –  Heavy menses –  Infertility –  Enlarged uterus

•  A tough disease to manage –  Hysterectomy is effective but could be traumatic to

patients –  Seemingly defies hormonal treatment –  Unclear as why there is such a refraction

•  Few, if any, novel drugs are on the horizon

Adenomyosis and endometriosis

•  Shares remarkable similarities with endometriosis –  Definition and symptomology –  Estrogen-dependency –  Inflammation –  Molecular aberrations

•  Pathogenic and pathophysiological connection with endometriosis –  DIE and focal adenomyosis (Chapron C) –  ~20% of pts with endo had adeno (Di Donato et al. 2014) –  ~48% of pts with DIE had adeno (Lazzeri et al. 2014) –  ~20% of pts undergone hysterectomy because of adeno had

endo (Li et al. 2013) –  Extensive endo in association with adeno appear to develop in

early reproductive life (Greene et al. 2009; Chapron et al. 2011) –  The TAIR theory: Leyendecker et al. 2012

What is known about its pathogenesis/pathophysiology

•  A disordered stromal differentiation (Parrott et al. 2001) – Downward extension of endometrium into the

myometrium –  Increased invasiveness of endometrial cells?

•  Estrogen-induced EMT (Chen et al. 2010) •  ↑β-catenin and markers of EMT in

adenomyotic epithelial cells (Oh et al. 2013) •  ↑HGF and markers of EMT in adenomyosis

(Khan et al. 2015)

Cautionary notes

•  EMT –  is known to be a transient state –  Involves only a small proportion of migrating

epithelial cells •  The best evidence for EMT for human

adeno invariably came from cross-sectional studies

•  Even animal data came from observation at one single time point

Cautionary notes •  EMT is known to be a transient state •  Involves only a small proportion of migrating epithelial cells •  The best evidence for EMT for human adeno invariably came from

snapshots •  Even animal data came from observation at a single time point

•  There is a lack of kinetic view of the EMT •  How does EMT occur in adenomyosis? •  What are the consequences of EMT

besides invasiveness?

What we have found so far… •  Increased platelet aggregation in endometriotic lesions (Ding et al.

2015) –  Activated platelets promote proliferation –  Activated platelets induce expression of VEGF, COX-2, MMP-9, and

Bcl-2 –  Increased thrombin concentration in peritoneal fluid from women with

endometriosis •  Activated platelets induce ER-β expression in endometriotic stromal

cells (Zhang et al. 2015) •  Recombinant P-selectin is efficacious in treating endometriosis in

mice (Guo et al. 2015) •  Evidence for a hypercoagulable state in endometriomas (Wu et al.

2015) •  Platelet-driven EMT and fibroblast-to-myofibroblast

transdifferentiation (FMT) (Zhang et al., to be submitted) •  Activated platelets are responsible for reduced NK cell cytotoxicity in

endometriosis (Du et al., in preparation)

Hypotheses

•  The progression of adenomyosis is associated with – Platelet aggregation ↑ – TGF-β/Smad3 signaling ↑ – Progressive EMT and FMT ↑ – ECM production ↑ – Fibrotic content ↑ – Hormonal response (PR-B expression) ↓

All models are wrong, but some are useful. ---- George E.P. Box

Vehicle

Tamoxifen New

born

mic

e

Birth 1 2 5 10 15 42 60

Vehicle

Dosing

Experimental design Tissue Sampling

Hotplate test

5 10 15 42 60

0.0

0.5

1.0

1.5

2.0

2.5

3.0

A: Depth of myometrial infiltration

Day of examination

Dep

th s

core

p=1.1x10^(-5)

25 30 35 40 45 50 55 60

1015

2025

B: Kinetics of hotplate latency

Day at hotplate test (since birth)

Late

ncy

(sec

onds

)

ControlAdenomyosis

***

*** ***

#NS

### #

10 20 30 40 50 60

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

D: Kinetics of platelet aggregation

Age (day)

Exte

nt

***

**

***

***ControlAdenomyosis

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14

0.00

0.01

0.02

0.03

0.04

0.05

TGF-beta1 staining levels vs. the extent of platelet aggregation

The extent of platelet aggregation

Stai

ning

leve

ls

r=0.91, p<2.2x10^(-16)

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14

0.00

0.02

0.04

0.06

0.08

0.10

0.12

p-Smad3 staining levels vs. the extent of platelet aggregation


Stai

ning

leve

ls

r=0.91, p<2.2x10^(-16)

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.140.00

00.

005

0.01

00.

015

0.02

0VEGF staining levels vs. the extent of platelet aggregation


Stai

ning

leve

ls

r=0.86, p<2.2x10^(-16)

10 20 30 40 50 60

0.00

00.

005

0.01

00.

015

0.02

0A: Kinetics of VEGF staining

Age (day)

Stai

ning

leve

ls

NS

NS

**

***


10 20 30 40 50 60

010

2030

40B: Kinetics of MVD

Age (day)

Den

sity

NS

NS

**

****Control

Adenomyosis

10 20 30 40 50 60

0.00

0.02

0.04

0.06

0.08

0.10

C: Kinetics of PCNA staining

Age (day)

Stai

ning

leve

ls

NS

****

***


10 20 30 40 50 60

0.00

0.01

0.02

0.03

0.04

0.05

0.06

E: Kinetics of TGF-beta1 staining

Age (day)

Stai

ning

leve

ls

NS**

**

***


10 20 30 40 50 60

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

F: Kinetics of p-Smad3 staining

Age (day)

Stai

ning

leve

ls

NS

****

**


10 20 30 40 50 60

0.00

0.05

0.10

0.15

0.20

G: Kinetics of E-cadherin staining

Age (day)

Stai

ning

leve

ls

NS

*

**

*** ***

ControlAdenomyosis

10 20 30 40 50 60

0.00

0.05

0.10

0.15

H: Kinetics of vimentin staining

Age (day)

Stai

ning

leve

ls

NS

*

**

***


10 20 30 40 50 60

0.00

00.

005

0.01

00.

015

0.02

0A: Kinetics of alpha-SMA staining

Age (day)

Stai

ning

leve

ls

***


10 20 30 40 50 60

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

B: Kinetics of collagen I staining

Age (day)

Stai

ning

leve

ls

NS NS NS

***

***

ControlAdenomyosis

10 20 30 40 50 60

0.00

0.01

0.02

0.03

0.04

0.05

C: Kinetics of LOX staining

Age (day)

Stai

ning

leve

ls

NS

*

**

***


10 20 30 40 50 60

0.00

0.02

0.04

0.06

0.08

0.10

D: Kinetics of PR-B staining

Age (day)

Stai

ning

leve

ls

NS NS

NS

*** ***

ControlAdenomyosis

10 20 30 40 50 60

05

1015

2025

3035

E: Proportion of fibrotic tissues (Masson)

Age (day)

Perc

enta

ge

***


10 20 30 40 50 60

010

2030

40F: Proportion of fibrotic tissues (Van Gieson)

Age (day)

Perc

enta

ge ***

***

ControlAdenomyosis

Histochemistry analysis

Picro-Sirius red staining (day 42)

Vehicle TAM

VEGF

Vimen

tin

PCNA LO

X

CD41

p-Sm

ad3

MVD

aSMA

Colla

gen I

Fibro

sis_V

TGFb

1

Fibro

sis_M

E-ca

d

PR-B

C60C10C15C60A5C10C15C60C60C15A5C42C5A5C10C42A10A10A10C10C5C42C60C42C42C60C42C60C42A5C5C10C42C60C5C42C15C5A5C15A10A10A15A15A15A15A15A42A42A42A42A42A42A60A60A60A60A60A60A60

Summary •  Our data are consistent with

–  Increased platelet aggregation –  Increased activation of TGF-β/Smad3 signaling

pathway – Progressive EMT – Progressive activation of myofibroblasts –  Increased production of extracellular matrix –  Increased fibrosis – Reduced hormonal receptor PR-B expression

•  Responsible for increased progestin-resistance?

Validation in human adenomyosis

Variable name Controls (n=20)

Adenomyosis (n=34)

P-value

Age (year) Mean=38.5 (SD=7.3) Median=40.5 range=25--48

Mean=44.6 (SD=5.4) median=45.0 Range=29--52

0.003

Menstrual phase Proliferative Secretory

10 (50.0%) 10 (50.0%)

12 (35.3%) 22 (64.7%)

0.39

Parity 0 1 ≧2

5 (25.0%) 12 (60.0%) 3 (15.0%)

4 (11.8%) 22 (64.7%) 8 (23.5%)

0.47

Visual analog scale on the severity of dysmenorrhea

Mean=0.5 (SD=1.3) Median=0.0 range=0--5

Mean=436 (SD=2.9) median=5.0 Range=0--8

1.4x10-5

Uterus size (cm3)

Median=70.4 Range=31.5—113.9 Missing: n=1

Median=229.6 Range=78.5—618.2

3.0x10-8

Co-occurrence of Endometriosis No Yes

20 (100.0%) 0 (0.0%)

25 (73.5%) 9 (26.5%)

0.019

Co-occurrence of Uterine fibroids No Yes

19 (95.0%) 1 (5.0%)

33 (97.1%) 1 (2.9%)

1.0

CD42b: Marker for platelets Vimentin: stromal cells DAPI: neulei

Control Adenomyosis0.00000

0.00004

0.00008

0.00012

Extent of platelet aggregation

Group

Density

p=0.017

Density of CD42b+ platelets

Representative IHC results

Control Adenomyosis

0.00

0.02

0.04

0.06

A: Platelet aggregation

Group

Ext

ent o

f agg

rega

tion

p=1.9x10 (̂-9)

Control Adenomyosis

0.00

0.04

0.08

B: VEGF

Group

Sta

inin

g le

vel

p=1.0x10 (̂-7)

Control Adenomyosis

1030

50

C: MVD-CD31

Group

# of

ves

sels p=9.7x10 (̂-8)

Control Adenomyosis

0.00

0.10

0.20

D: PCNA

Group

Sta

inin

g le

vel p=2.9x10 (̂-11)

Control Adenomyosis

0.00

0.04

0.08

E: TGF-beta1

Group

Sta

inin

g le

vel p=6.2x10 (̂-15)

Control Adenomyosis

0.00

0.04

0.08

F: p-SMAD3

Group

Sta

inin

g le

vel

p=1.2x10 (̂-9)

Control Adenomyosis

0.0

0.4

0.8

G: E-cadherin

Group

Sta

inin

g le

vel

p=6.2x10 (̂-15)

Control Adenomyosis

0.00

0.10

H: Vimentin

Group

Sta

inin

g le

vel p=5.9x10 (̂-4)

Control Adenomyosis

0.00

0.10

I: alpha-SMA

Group

Sta

inin

g le

vel

p=1.7x10 (̂-9)

Control Adenomyosis

0.00

0.10

J: Collagen I

Group

Sta

inin

g le

vel

p=8.7x10 (̂-13)

Control Adenomyosis

0.00

0.06

0.12

K: LOX

Group

Sta

inin

g le

vel

p=1.0x10 (̂-7)

Control Adenomyosis

0.00

0.04

0.08

L: PR-B

Group

Sta

inin

g le

vel

p=5.5x10 (̂-10)

Control Adenomyosis

010

2030

4050

60Extent of fibrosis

Group

Perc

enta

ge o

f fib

rosi

s

p=5.1x10^(-5)

Based on Masson trichrome staining

Does anti-platelet treatment have any therapeutic value?

•  Two kinds of intervention – Ozagrel (a thromboxane synthase inhibitor)

•  Low dose (12.5 mg/kg) (Lo Oz) •  High dose (25 mg/kg) (Hi Oz)

– Platelet depletion using anti-GPIbα antibody •  For controls, isotope-matched non-immune anti-

IgG antibody – Versus vehicle control

Experimental Design ne

wbo

rn fe

mal

e m

ice

Randomization

d 2-5

Wk 4 Wk 19

Uterine contractility Plasma CORT IHC for Trpv1, OTR,F4/80 NF-κB,COX-2, PR-B GAD65

Tam

oxife

n do

sed

BLK n=12

Blank

Lo Oz

Hi Oz

IgG

Wk 8 Wk 12 Wk 16 Birth

Intervention

Hotplate test

Lo Plt Dptn

Hi Plt Dptn

Untreated

Dosing Treatment

CTL UNT HO LO HPD LPD IgG

0.00

050.

0015

0.00

250.

0035

Uterine weight/bodyweight ratio among different groups

Group

Ute

rine

wei

ght v

s. b

odyw

eigh

t rat

io

UNT HO LO HPD LPD IgG

01

23

4Treatment effect on depth of myometrial infiltration

Group

Dep

th o

f myo

met

rial i

nfilt

ratio

n ******

******

C CC

C C

0 5 10 15 20

05

1015

Time course of mean hotplate latency

Time of test (in weeks)

Hot

plat

e at

ency

(in

sec) U

U

U

U

U

O

O

O

O

O

o

o

oo

oD

D

D

D

D

d

d

d

d

dG

G

G

G

G

C: Blank controlU: UntreatedO: hi-dose Ozagrelr: lo-dose OzagrelD: hi-dose platelet depletiond: lo-dose platelet depletionG: IgG control

Tamoxifen fed Test 1Test 2

Test 3Test 4

Tx beganTest 5

Exp’t ended


0.0

0.5

1.0

1.5

2.0

2.5

A: Amplitude of uterine contraction

Group

Ampl

itude

of u

terin

e co

ntra

ctio

n

*** ****** ***

***


1015

2025

3035

40C: Frequency of uterine contraction

Group

Frequency

** ***

NS

NS


02

46

810

A: Plasma corticosterone levels

Group

Plas

ma

corti

cost

eron

e le

vel *** **

*** ******

4 6 8 10 12 14 16

02

46

810

B: Plasma corticosterone levels vs. hoplate latency

Hotplate latency after treatment

Cor

ticos

tero

ne le

vels

CC

CC

C

CCCC

CCC

U

U

U

U

UU

U

U

U

OO

O

O

O O

O

O

O

O

o

o

o

o

o

o

o

o

o

oD

D

D

D D

D

D

DD

dd

d

d

dd

dd

dd

G

GG

G

G

G

G

G

r=-0.90, ****

Immunofluorescent staining of GAD65+ neurons in the nucleus raphe magnus GAD65 in the

Immunofluorescent staining of GAD65+ neurons in the nucleus raphe magnus GAD65 in the


1020

3040

5060

70# of GAD65+ neurons in the NRM

Group

# of

GAD

65+

neur

ons

***

***** ***

***


0.00

0.02

0.04

0.06

0.08

A: Platelet aggregation

Group

CD

41 s

tain

ing

leve

l


05

1015

20

B: # of F4/80+ macrophages

Group

# of

F4/

80+

mac

roph

ages


0.0

0.1

0.2

0.3

0.4

C: OTR

Group

OTR

sta

inin

g le

vel


050

100

200

300

D: Myometrial OTR

Group

Myo

met

rial O

TR s

tain

ing

leve

l


0.00

0.10

0.20

0.30

p-p65

Group

p-p6

5 st

aini

ng le

vel


0.00

0.05

0.10

0.15

0.20

COX-2

Group

CO

X-2

sta

inin

g le

vel


0.00

0.05

0.10

0.15

Collagen I

Group

Col

lage

n I s

tain

ing

leve

l


0.00

0.05

0.10

0.15

0.20

0.25

PR-B

Group

PR

-B s

tain

ing

leve

l

Summary

•  Both Orzagrel treatment and platelet depletion – Reduce myometrial infiltration –  Improve adenomyosis-associated

hyperalgesia – Reduce uterine hyperactivity – Reduce stress levels –  Improve central inhibition of pain

Are there any human data?

Valproic acid

•  Valproic acid can relieve dysmenorrhea and reduce uterus size – A pilot study (n=3) (Liu and Guo 2008) – A second study (n=12) (Liu et al. 2010)

Liu et al. Gyn Minim Inv Thera 2015

Consistent and significant reduction of dysmenorrhea after treatment with andrographolide

*** ***

0 1 2 3 4 5 6

02

46

8

A: Kinetics of change in VAS score

Time at evaluation (month)

Dys

men

orrh

ea V

AS s

core

Pre-Tx 3 months post-Tx 6 months post-Tx

02

46

810

B: Boxplot of VAS scores at different time point

Time at evaluation

VAS

scor

e *** ***

16 patients (66.7%) reported some relief of pain by the end of 6th month Liu et al. Gyn Minim Inv Thera 2015

Change in uterus size

Caution: n=11 at Month 3

*

0.0 0.5 1.0 1.5 2.0 2.5 3.0

100

200

300

400

500

A: Kinetics of change in uterus size

Time at evaluation (month)

Ute

rus

size

(cm

^3)

Pre-Tx 3 months post-Tx

100

200

300

400

500

B: Boxplot of uterus size before/after treatment

Time at evaluation

Ute

rus

size

(cm

^3)

*

(n=9)

Liu et al. Gyn Minim Inv Thera 2015

EGCG

Curcumin Quercetin

Anti-platelet

The commonality of promising drugs

Conclusions •  Platelet-driven activation of TGF-β/Smad3

pathway results in – Progressive EMT and FMT in the development of

adenomyosis –  Increased proliferation, angiogenesis, ECM

production –  Leading to eventual fibrotic nodules

•  This explains as why adenomyosis is difficult to manage by medication

•  Anti-platelet therapy appears to be a promising therapeutics

Anti-platelet

PR-B

NF-κB

COX-2

OTR TRPV1

EMT (Zeb1, Snail1,

Slug)

Fibroblast activation

MΦ infiltration

PGE2

Fibrosis

Collagens

# GAD65+ neurons in the NRM

Uterine hyperactivity

Pain

Platelet activation

Pain

Schematic diagram showing possible mode of actions for anti-platelet therapy

Acknowledgment •  Minghong Shen, M.D. •  Bo Zhu, M.D., M.S. •  Qi Zhang, M.D., M.S. •  Xishi Liu, M.D., Ph.D. •  Hongqi Zhang, M.D. •  Yanbo Du, M.D., M.S. •  Jie Duan, M.D., M.S. •  Qiumin Qi, M.D. •  Ding Ding, M.D., Ph.D. •  National Science Foundation of China

Documents

Cracking the Enigma of AdenomyosisAdenomyosis: An enigma • Fairly common • Presenting symptoms (in ~2/3 of patients) – Dysmenorrhea – Heavy menses – Infertility – Enlarged