A

distinct

cohort

of

the

TGFβ

superfamily

members

expressed

in

human

endometrium

regulate

decidualization

BACKGROUND

Successful

blastocyst

implantation

requires

the

differentiation

of

human

endometrial

stromal

cells

-LRB-

HESC

-RRB-

a

process

known

as

decidualization

Activin

A

a

transforming

growth

factor

β

-LRB-

TGFβ

-RRB-

superfamily

member

enhances

HESC

decidualization

and

localizes

to

decidual

cells

in

human

endometrium

Other

TGFβ

superfamily

members

including

TGFβs

and

Nodal

may

also

play

a

role

during

decidualization

This

study

aimed

to

identify

these

TGFβ

family

members

in

human

endometrium

and

to

determine

whether

they

are

involved

in

human

decidualization

IntroductionThroughout

each

menstrual

cycle

the

human

endometrium

undergoes

morphological

and

physiological

changes

in

preparation

for

pregnancy

should

the

cycle

include

conception

-LRB-

Loke

et

al.

-RRB-

An

absolute

requirement

for

successful

implantation

is

the

differentiation

and

proliferation

of

endometrial

stromal

cells

into

enlarged

phenotypically

different

decidual

cells

a

process

termed

endometrial

decidualization

-LRB-

Tang

et

al.

Salamonsen

et

al.

-RRB-

This

process

is

initiated

near

the

spiral

arterioles

-LRB-

Bell

-RRB-

and

occurs

under

the

influence

of

progesterone

-LRB-

Psychoyos

-RRB-

although

cAMP

appears

to

be

essential

for

priming

the

endometrial

stromal

cells

to

the

actions

of

progesterone

hence

both

pathways

are

absolutely

required

for

decidualization

-LRB-

Gellersen

and

Brosens

-RRB-

It

is

now

clear

that

the

decidualization

response

is

mediated

by

a

complex

array

of

bioactive

molecules

including

prostaglandin

relaxin

and

activins

-LRB-

Frank

et

al.

Robb

et

al.

Jones

et

al.

Dimitriadis

et

al.

-RRB-

The

transforming

growth

factor

β

-LRB-

TGFβ

-RRB-

superfamily

is

a

large

family

of

proteins

that

encompasses

the

sub-families

TGFβs

activins

bone

morphogenetic

proteins

-LRB-

BMPs

-RRB-

and

growth

differentiation

factors

-LRB-

GDFs

-RRB-

Overall

this

superfamily

of

proteins

exhibits

functional

diversity

with

biological

roles

in

cell

differentiation

proliferation

apoptosis

and

tissue

remodelling

all

consistent

with

various

reproductive

processes

-LRB-

Jones

et

al.

-RRB-

Activins

consist

of

two

β

subunits

βA

and

βB

that

homo/heterodimerize

to

form

activin

A

activin

B

and

activin

AB

respectively

The

activin

β

subunits

are

produced

in

the

glandular

epithelium

during

the

proliferative

and

secretory

phases

but

in

stromal

cells

only

following

decidualization

during

the

mid-late

secretory

phase

-LRB-

Otani

et

al.

Jones

et

al.

Mylonas

et

al.

-RRB-

High

levels

of

dimeric

activin

A

are

secreted

by

cAMP-treated

endometrial

stromal

cells

Addition

of

activin

A

to

decidualizing

cells

in

vitro

significantly

increased

the

secretion

of

prolactin

-LRB-

PRL

-RRB-

and

-LRB-

decidual

cell

markers

-RRB-

suggesting

activin

A

drives

decidualization

-LRB-

Jones

et

al.

Tierney

and

Giudice

-RRB-

This

decidualization

response

can

be

neutralized

by

follistatin

the

naturally

occurring

activin

antagonist

-LRB-

Jones

et

al.

Tierney

and

Giudice

-RRB-

In

addition

to

its

tight

regulation

of

activin

-LRB-

Shimonaka

et

al.

Schneyer

et

al.

-RRB-

follistatin

can

also

bind

and

regulate

other

TGFβ

members

including

the

BMPs

and

GDFs

-LRB-

Table

I

-RRB-

Table

I.Inhibitors

of

TGFβ

superfamily

ligand

receptor

ligands

and

blocks

association

with

type

II

receptorsActivin

type

II

receptor

-LRB-

ActRIIA/B

-RRB-

antagonistInhibitor

of

TGFβ

and

activin

type

I

receptorsActivin

A

-LRB-

Schneyer

et

al.

-RRB-

-LRB-

Harrison

et

al.

-RRB-

-LRB-

Inman

et

al.

-RRB-

Activin

B

-LRB-

Schneyer

et

al.

-RRB-

a

-LRB-

Inman

et

al.

-RRB-

GDF

-LRB-

Amthor

et

al.

-RRB-

a

-LRB-

Inman

et

al.

-RRB-

-LRB-

Gamer

et

al.

-RRB-

a

-LRB-

Inman

et

al.

-RRB-

-LRB-

Iemura

et

al.

-RRB-

-LRB-

Iemura

et

al.

Glister

et

al.

-RRB-

-LRB-

Iemura

et

al.

Glister

et

al.

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Nodal

-LRB-

-RRB-

-LRB-

Inman

et

al.

-RRB-

-LRB-

Inman

et

al.

-RRB-

-LRB-

Inman

et

al.

-RRB-

-LRB-

Inman

et

al.

-RRB-

aC.A

Harrison

-LRB-

unpublished

observations

-RRB-

-LRB-

-RRB-

anticipated

activity.BMPs

have

been

identified

in

the

rodent

uterus

-LRB-

Cunningham

et

al.

Ozkaynak

et

al.

Fitzpatrick

et

al.

Zhao

et

al.

Ying

and

Zhao

Erickson

et

al.

Lee

et

al.

Li

et

al.

-RRB-

In

pregnant

mice

the

temporospatial

localization

of

BMPs

and

suggested

roles

during

pregnancy

-LRB-

Ying

and

Zhao

-RRB-

is

present

within

the

decidual

area

at

implantation

sites

-LRB-

Ying

and

Zhao

Li

et

al.

-RRB-

and

plays

a

role

in

decidualization

in

vitro

-LRB-

Li

et

al.

-RRB-

Importantly

conditional

ablation

of

uterine

in

mice

resulted

in

ineffective

decidualization

and

disrupted

pregnancy

outcome

-LRB-

Lee

et

al.

-RRB-

In

a

human

model

of

endometrial

stromal

cell

-LRB-

HESC

-RRB-

decidualization

in

vitro

mRNA

increased

during

decidualization

and

addition

of

to

cultures

accelerated

PRL

mRNA

expression

-LRB-

a

measure

of

the

extent

of

decidualization

-RRB-

protein

has

not

been

examined

in

vivo

in

human

endometrial

tissue

The

only

GDFs

identified

in

the

uterus

are

and

detected

by

northern

analysis

in

both

human

and

mouse

-LRB-

Cunningham

et

al.

Fitzpatrick

et

al.

Zhao

et

al.

-RRB-

no

studies

have

shown

GDF

proteins.TGFβ

isoforms

-LRB-

-RRB-

which

can

not

bind

follistatin

have

also

been

localized

in

human

endometrial

stroma

-LRB-

Jones

et

al.

-RRB-

although

it

is

not

clear

whether

any

isoform

is

specific

to

decidualized

cells

or

increases

as

the

cells

decidualize

The

TGFβs

transmit

signal

through

the

TGFβ

type

I

II

and

III

receptors

all

of

which

are

present

on

endometrial

stromal

cells

-LRB-

Chegini

et

al.

Dumont

et

al.

-RRB-

Microarray

studies

have

demonstrated

increases

in

both

and

mRNA

with

HESC

decidualization

in

vitro

-LRB-

Popovici

et

al.

Tierney

et

al.

-RRB-

while

in

women

treated

with

medroxyprogesterone

acetate

-LRB-

MPA

-RRB-

for

days

stromal

mRNA

and

protein

increased

for

but

not

-LRB-

Reis

et

al.

-RRB-

Whether

the

TGFβs

play

a

functional

role

during

human

decidualization

remains

unknown.Given

that

the

TGFβ

superfamily

of

proteins

are

such

common

and

crucial

differentiative

and

proliferative

factors

it

is

surprising

that

very

few

members

of

the

family

including

the

GDFs

and

BMPs

have

been

examined

in

the

human

endometrium

particularly

in

the

mid-secretory

phase

when

differentiation

of

stromal

cells

is

initiated

The

high

level

of

redundancy

between

TGFβ

family

members

and

the

promiscuity

of

receptor

ligand

interactions

would

suggest

that

it

is

likely

that

a

number

of

other

family

members

are

involved

in

decidualization

and

in

the

other

processes

occurring

in

this

highly

dynamic

tissue.In

this

study

the

presence

of

BMPs

-LRB-

-RRB-

GDFs

-LRB-

-RRB-

Nodal

and

TGFβs

were

examined

in

vivo

by

immunohistochemistry

in

secretory

phase

endometrial

tissue

and

in

vitro

for

mRNA

expression

in

both

non-decidualized

and

decidualized

HESC

Inhibitors

with

different

specificities

were

administered

to

HESC

to

elucidate

whether

activin

is

the

major

family

member

driving

decidualization

or

whether

other

family

groupings

-LRB-

BMPs

GDFs

TGFβs

-RRB-

might

equally

contribute

to

the

process

As

these

are

secreted

factors

individual

ligands

were

measured

in

conditioned

medium

from

non-decidualized

and

decidualized

HESC

to

assess

whether

they

increased

with

decidualization

The

effect

of

the

secreted

ligands

on

decidualization

was

also

examined

It

is

important

to

identify

which

TGFβ

members

are

important

during

decidualization

since

this

process

is

critical

for

implantation

and

the

establishment

of

pregnancy

Materials

and

MethodsTissue

collectionsEndometrial

biopsies

-LRB-

n

-RRB-

were

collected

by

dilatation

and

curettage

from

fertile

women

who

were

scheduled

for

tubal

ligation

or

were

undergoing

testing

for

tubal

patency

Tissues

were

assessed

by

a

pathologist

and

had

no

obvious

endometrial

pathology

The

women

had

no

steroid

treatment

or

other

medication

for

at

least

months

before

the

collection

of

tissue

Written

and

informed

consent

was

obtained

from

all

women

participating

in

the

study

and

the

protocols

were

approved

by

Monash

Medical

Centre

Human

Ethics

Committee.ImmunohistochemistryImmunohistochemical

analysis

was

performed

using

a

total

of

nine

endometrial

tissue

biopsies

from

fertile

women

confirmed

by

Noyes

criteria

-LRB-

Noyes

et

al.

-RRB-

as

mid-late

secretory

phase

tissue

-LRB-

POD

-RRB-

For

each

of

the

antibodies

activin

βA

and

βB

Nodal

and

n

different

tissue

biopsies

were

used

For

immunolocalization

in

first

trimester

placental

tissue

-LRB-

kindly

provided

by

Professor

Euan

Wallace

Obstetrics

and

Gynaecology

Monash

University

Melbourne

Australia

-RRB-

n

different

biopsies

were

used

per

antibody

Briefly

µm

sections

of

formalin-fixed

paraffin-embedded

tissues

were

dewaxed

and

rehydrated

For

each

antibody

an

antigen

retrieval

step

was

required

which

involved

microwave

exposure

or

trypsin

digestion

-LRB-

in

min

at

°C

-RRB-

Endogenous

hydrogen

peroxidase

activity

was

quenched

using

in

for

min

at

room

temperature

Non-specific

binding

was

prevented

by

pre-incubation

of

tissue

sections

with

a

non-immune

block

-LSB-

fetal

calf

serum

-LRB-

FCS

-RRB-

normal

human

serum

in

Tween/Tris-buffered

saline

-LRB-

TBS

-RRB-

with

addition

of

normal

horse

serum

for

normal

swine

serum

for

Nodal

normal

goat

serum

for

activin

βA

and

βB

-RSB-

Primary

antibodies

were

against

activin

βA

and

activin

βB

-LRB-

and

µg/ml

respectively

both

gifts

provided

by

W.

Vale

Salk

Institute

La

Jolla

CA

USA

-RRB-

Nodal

-LRB-

µg/ml

Santa

Cruz

Biotechnology

Inc.

Santa

Cruz

CA

USA

-RRB-

-LRB-

mg/ml

Orbigen

San

Diego

CA

USA

-RRB-

-LRB-

μg/ml

Abcam

Cambridge

UK

-RRB-

Specificity

for

these

antibodies

has

been

previously

published

-LRB-

Nadiri

et

al.

Kiyono

and

Shibuya

Mendler

et

al.

McPherron

et

al.

Garba

and

Relinger

-RRB-

For

and

Nodal

the

specificity

has

been

shown

by

western

blot

analysis

-LRB-

unpublished

observation

-RRB-

Antibodies

were

diluted

to

µg/ml

in

the

non-immune

block

and

applied

overnight

-LRB-

h

-RRB-

at

°C

A

non-immune

goat/rabbit/mouse

IgG

-LRB-

R&D

Systems

Incorporated

Minneapolis

MN

USA

-RRB-

was

used

at

a

matching

concentration

to

the

primary

antibody

and

was

included

for

each

tissue

After

stringent

washing

with

high

salt

TBS

and

Tween

-LRB-

BioRad

Laboratories

Hercules

CA

USA

-RRB-

detection

of

positive

binding

was

performed

by

the

sequential

application

of

the

appropriate

secondary

antibody

diluted

in

non-immune

block

and

avidin

biotin

peroxidase

conjugate

-LRB-

Dako

Glostrup

Denmark

-RRB-

This

was

followed

by

the

substrate

diaminobenzidine

-LRB-

Dako

-RRB-

for

between

and

min

Sections

were

counterstained

with

Harris

hematoxylin

-LRB-

Sigma

Chemical

Company

St

Louis

MO

USA

-RRB-

dehydrated

and

mounted

from

Histosol

with

DPX

mounting

medium

-LRB-

BDH

Laboratory

Supplies

Poole

UK

-RRB-

Immunostaining

was

analysed

by

two

independent

observers

for

staining

intensity

and

heterogeneity

in

each

of

the

endometrial

compartments

-LRB-

glandular

and

luminal

epithelium

stroma

including

decidualized

stromal

cells

vasculature

-RRB-

Stromal

cell

isolation

and

cultureTissue

biopsies

ranging

from

Days

were

used

for

in

vitro

decidualization

studies

HESC

were

isolated

from

tissue

by

enzymatic

digestion

and

filtration

as

described

previously

-LRB-

Dimitriadis

et

al.

-RRB-

Briefly

tissue

was

finely

minced

with

scissors

and

digested

by

bacterial

collagenase

type

III

-LRB-

Worthington

Biochemical

Corporation

Freehold

NJ

USA

-RRB-

at

a

concentration

of

IU/ml

in

the

presence

of

µg/ml

deoxyribonuclease

-LRB-

DNase

Boehringer

Mannheim

Biochemica

Mannheim

Germany

-RRB-

After

min

of

agitation

at

°C

the

digested

tissue

was

filtered

sequentially

through

and

µm

nylon

filters

to

remove

glands

Cell

suspensions

were

centrifuged

at

rpm

for

min

Cells

were

plated

at

a

density

of

in

a

flask

and

grown

to

confluency

in

DMEM

and

Ham

's

medium

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

Trace

Biosciences

Sydney

Australia

-RRB-

supplemented

with

PSF

-LRB-

penicillin

streptomycin

and

fungizone

-RRB-

-LRB-

Commonwealth

Serum

Laboratories

Melbourne

Australia

-RRB-

and

charcoal

stripped

FCS

-LRB-

csFCS

-RRB-

-LRB-

Thermo

Scientific

Maple

Plain

USA

-RRB-

Approximately

min

after

plating

medium

containing

non-attached

epithelial

cells

was

removed

and

replaced

with

fresh

medium

This

results

in

a

pure

stromal

cell

culture

-LRB-

Dimitriadis

et

al.

-RRB-

In

vitro

decidualizationConfluent

HESC

were

rinsed

with

phosphate-buffered

saline

trypsinized

and

replated

into

plates

using

and

csFCS

Once

confluent

HESC

were

washed

with

and

the

medium

replaced

with

a

serum-free

medium

containing

and

a

serum-free

mix

-LRB-

SFM

-RRB-

including

transferrin

-LRB-

µg/ml

Sigma

-RRB-

sodium

selenite

-LRB-

ng/ml

Sigma

-RRB-

linoleic

acid

-LRB-

nmol/L

Sigma

-RRB-

bovine

serum

albumin

-LRB-

Sigma

-RRB-

and

insulin

-LRB-

µg/ml

Actrapid

Novo-Nordisk

Pharmaceuticals

Pty

Ltd

Sydney

Australia

-RRB-

for

h

prior

to

treatment

addition

HESC

were

decidualized

by

two

distinct

methods

as

previously

described

-LRB-

Dimitriadis

et

al.

-RRB-

For

the

inhibitor

mRNA

expression

and

protein

secretion

studies

mM

cAMP

-LRB-

Sigma

-RRB-

was

added

to

medium

for

days

Alternatively

when

exogenous

proteins

were

added

cells

were

decidualized

with

-LRB-

M

Sigma

-RRB-

and

MPA

-LRB-

Sigma

-RRB-

for

days

All

media

were

replaced

every

h.RNA

extraction

and

cDNA

synthesisTotal

RNA

was

extracted

from

non-decidualized

and

decidualized

HESC

from

two

separate

biopsies

at

Day

using

the

RNeasy

Minikit

-LRB-

Qiagen

Sciences

Germantown

MD

USA

-RRB-

according

to

the

manufacturer

's

instructions

RNA

from

positive

control

tissues

was

extracted

by

homogenization

in

Trizol

reagent

-LRB-

Qiagen

Sciences

Clifton

Hill

Victoria

Australia

-RRB-

according

to

the

manufacturer

's

instructions

with

the

exception

of

an

additional

chloroform

extraction

step

to

minimize

carryover

of

phenol

into

the

precipitate

The

control

tissues

used

were

term

placenta

-LRB-

activin

βA

βB

-RRB-

pregnant

mouse

endometrium

-LRB-

-RRB-

mature

rat

ovary

-LRB-

-RRB-

cycling

mouse

endometrium

-LRB-

-RRB-

mouse

heart

-LRB-

-RRB-

cells

-LRB-

Nodal

-RRB-

All

samples

were

treated

with

RNase-free

DNase

-LRB-

Ambion

Austin

TX

USA

-RRB-

to

remove

the

possibility

of

genomic

DNA

contamination

RNA

samples

were

then

analysed

by

spectrophotometry

to

determine

RNA

concentration

yield

and

purity

Total

RNA

-LRB-

µg

-RRB-

was

reverse

transcribed

at

°C

for

h

in

µl

reaction

mixture

using

ng

random

hexanucleotide

primers

and

IU

AMV

reverse

transcriptase

-LRB-

Roche

Castle

Hill

Australia

-RRB-

in

the

presence

of

cDNA

synthesis

buffer

-LRB-

Roche

-RRB-

mmol/l

dNTPs

-LRB-

Roche

-RRB-

mmol/l

dithiothreitol

-LRB-

Roche

-RRB-

IU

ribonuclease

inhibitor

-LRB-

RNasin

Promega

Annandale

Australia

-RRB-

The

resultant

cDNA

mixtures

were

heated

at

°C

for

min

before

storage

at

°C

Negative

controls

were

performed

by

omission

of

reverse

transcriptase

Triplicate

RNA

samples

were

reverse

transcribed

in

triplicate

for

each

condition

-LRB-

non-decidualized

and

decidualized

-RRB-

with

the

successful

conversion

to

cDNA

monitored

by

expression

-LRB-

data

not

shown

-RRB-

RT

PCRMessenger

RNA

expression

for

all

named

TGFβ

superfamily

ligand

members

was

determined

using

a

conventional

PCR

block

cycler

-LRB-

Hybaid

Middlesex

UK

-RRB-

All

ligands

-LRB-

excluding

Nodal

-RRB-

used

a

µl

aliquot

of

RT

product

to

be

amplified

in

a

total

volume

of

µl

using

µl

of

RT

single

strength

PCR

buffer

-LRB-

mmol/l

Tris

HCl

mmol/l

mmol/l

KCl

pH

Roche

-RRB-

mmol/l

dNTPs

-LRB-

Gibco

Melbourne

Australia

-RRB-

pmol/µl

sense

and

antisense

primers

-LRB-

Sigma

Genosys

Australia

Pty

Ltd

Castle

Hill

Australia

-RRB-

and

IU

Taq

DNA

polymerase

-LRB-

Roche

-RRB-

For

Nodal

µl

of

RT

was

amplified

in

a

total

of

µl

using

the

KOD-Taq

PCR

kit

-LRB-

Bioron

Germany

-RRB-

which

included

PCR

KOD

Hot

Start

buffer

mM

dNTPs

pmol/μl

primers

mM

and

IU

Taq

DNA

polymerase

-LRB-

Roche

-RRB-

For

all

ligands

the

PCR

was

performed

in

three

stages

as

follows

the

first

stage

involved

°C

for

min

x

C

for

min

where

x

is

the

annealing

temperature

for

the

individual

primer

pairs

-LRB-

see

Supplementary

data

-RRB-

and

°C

for

min

the

second

stage

involved

cycles

of

°C

for

min

x

C

for

min

and

°C

for

min

and

the

final

stage

was

°C

for

min

PCR

products

including

positive

controls

were

analysed

by

electrophoresis

on

a

agarose

gel

-LRB-

Roche

-RRB-

and

stained

with

ethidium

bromide

Bands

of

interest

were

excised

from

the

gel

purified

-LRB-

DNA

purification

kit

Qiagen

-RRB-

and

directly

sequenced

to

confirm

their

identity.Inhibitor

experimentsFor

examination

of

the

effect

of

inhibitors

on

decidualization

cells

-LRB-

from

n

biopsies

-RRB-

were

plated

at

a

density

of

in

plates

until

confluent

and

then

equilibrated

in

serum-free

medium

containing

SFM

for

h

This

was

designated

Day

and

first

day

of

treatments

HESC

were

exposed

to

varying

doses

of

either

the

inhibitor

-LRB-

-RRB-

-LRB-

and

nM

-RRB-

-LRB-

Harrison

et

al.

-RRB-

or

the

inhibitor

-LRB-

and

µM

-RRB-

-LRB-

TOCRIS

Bioscience

Northpoint

UK

-RRB-

Following

h

incubation

with

either

inhibitor

mM

cAMP

-LRB-

Sigma

-RRB-

was

added

as

decidualizing

stimulus

Each

treatment

was

performed

in

triplicate

wells

of

a

plate

and

the

experiments

ran

for

days

with

media

collection

and

replenishment

-LRB-

including

inhibitors

-RRB-

every

h

In

each

case

the

final

medium

collection

represented

the

final

h.

Each

experiment

included

a

medium-only

and

a

cAMP-only

control

Cells

were

photographed

for

morphological

analysis

and

cells

from

triplicate

wells

were

pooled

for

trypan

blue

cell

exclusion

to

check

cell

viability

at

the

end

of

each

experiment

Five

separate

cultures

were

conducted

for

each

inhibitor.TGFβ

superfamily

assaysCulture

medium

from

triplicate

wells

from

three

tissue

biopsies

-LRB-

n

separate

decidualization

experiments

-RRB-

were

collected

pooled

and

concentrated

to

measure

-LRB-

ELISA

R&D

Systems

-RRB-

and

-LRB-

ELISA

Ray

BioTech.

Norcross

GA

USA

-RRB-

according

to

the

manufacturer

's

instructions

Mean

sensitivities

of

the

assays

were

pg/ml

pg/ml

pg/ml

pg/ml

with

intra-assay

CV

and

inter-assay

CV

for

each

assay

Mean

sensitivity

for

was

pg/ml

and

intra

and

inter-assay

variabilities

were

and

respectively

and

ELISAs

measured

only

the

activated

protein

form

and

required

activation

steps

prior

to

assay

Dimeric

activin

A

secretion

from

stromal

cells

was

measured

by

Activin

A

Immunofluorometric

Assay

-LRB-

IFMA

-RRB-

-LRB-

Harrison

et

al.

-RRB-

The

working

range

of

the

assay

is

ng/well

with

a

sensitivity

of

ng/well

All

assays

were

read

at

nm.Addition

of

exogenous

and

during

decidualizationHESC

from

three

separate

cultures

were

decidualized

using

and

MPA

as

described

above

in

the

presence

of

varying

doses

of

either

recombinant

human

-LRB-

rh

-RRB-

-LRB-

ng/ml

R&D

Systems

-RRB-

or

ng/ml

PeproTech

Inc.

NJ

USA

-RRB-

added

every

h

with

media

and

decidualizing

stimulus

replenishment

At

Day

cells

were

morphologically

assessed

and

conditioned

media

were

collected

for

PRL

measurement.PRL

and

protein

assaysPRL

production

by

HESC

was

assayed

in

duplicate

by

ELISA

-LRB-

Bioclone

Australia

Pty

Ltd

Sydney

Australia

-RRB-

to

determine

the

extent

of

decidualization

-LRB-

Dimitriadis

et

al.

-RRB-

Media

collected

at

the

end

of

the

experiment

were

concentrated

for

PRL

measurement

A

quality

control

sample

-LRB-

culture

medium

from

a

single

endometrial

cell

culture

-RRB-

was

included

in

every

assay

The

lower

detection

limit

of

the

assay

was

mIU/l

The

inter

and

intra-assay

variablilities

were

and

respectively

PRL

concentrations

-LRB-

mIU/l

-RRB-

were

corrected

for

the

amount

of

protein

-LRB-

μg/μl

-RRB-

determined

using

the

Bradford

reagent.Statistical

analysisData

were

expressed

as

mean

SEM

Statistical

significance

for

inhibitor

studies

was

determined

following

confirmation

of

normal

distribution

by

one-way

ANOVA

followed

by

Dunnett

's

multiple

comparisons

test

Data

from

ELISA

and

recombinant

protein

studies

were

analysed

by

Student

's

t-test

A

P-value

of

was

considered

statistically

significant

ResultsLocalization

of

TGFβ

superfamily

ligands

in

human

endometrium

and

first

trimester

deciduaAll

TGFβ

superfamily

members

studied

except

Nodal

were

detected

in

mid-late

secretory

endometrium

and

first

trimester

decidua

As

previously

described

-LRB-

Jones

et

al.

-RRB-

activin

A

and

activin

B

were

localized

to

the

morphologically

distinct

decidual

cells

-LRB-

Fig.

and

B

-RRB-

glandular

and

luminal

epithelial

cells

and

some

leukocytes

-LRB-

data

not

shown

-RRB-

Some

glands

had

punctate

staining

for

-LRB-

Fig.

-RRB-

but

was

most

strongly

stained

in

decidual

cells

in

mid-late

secretory

endometrium

-LRB-

Fig.

-RRB-

In

first

trimester

decidua

protein

was

detected

in

both

the

decidual

cells

-LRB-

Fig.

inset

-RRB-

and

glandular

epithelium

while

the

vessels

were

devoid

of

stain

-LRB-

Fig.

-RRB-

protein

was

observed

in

the

decidualized

stromal

cells

-LRB-

Fig.

and

G

-RRB-

and

was

very

low

in

the

glands

-LRB-

Fig.

-RRB-

and

luminal

epithelium

-LRB-

data

not

shown

-RRB-

Similarly

was

very

strongly

expressed

by

the

decidua

of

first

trimester

placenta

as

well

as

the

glandular

epithelium

-LRB-

Fig.

inset

-RRB-

was

present

in

decidualized

stromal

cells

-LRB-

Fig.

and

J

-RRB-

with

minimal

staining

evident

in

the

glandular

epithelium

-LRB-

Fig.

-RRB-

As

previously

described

-LRB-

Graham

et

al.

-RRB-

was

detected

in

decidual

cells

and

extravillous

trophoblasts

of

first

trimester

placenta

-LRB-

Fig.

-RRB-

was

not

detected

in

the

glandular

or

luminal

epithelium

but

was

present

in

the

cytoplasm

of

both

non-decidualized

and

decidualized

stromal

cells

-LRB-

Fig.

and

M

-RRB-

However

in

first

trimester

placenta

was

reduced

in

the

decidual

cells

but

strongly

produced

by

the

glandular

epithelium

-LRB-

Fig.

inset

-RRB-

was

detected

in

the

stroma

and

not

in

the

glands

in

mid-late

secretory

endometrium

-LRB-

Fig.

-RRB-

Stromal

staining

for

did

not

appear

to

be

decidual

cell

specific

however

in

highly

decidualized

tissue

stronger

vesicle

staining

was

evident

-LRB-

Fig.

-RRB-

Similar

but

faint

punctate

staining

for

was

detected

around

spiral

arterioles

of

first

trimester

placenta

but

not

specifically

in

decidual

cells

-LRB-

Fig.

inset

-RRB-

was

also

strongly

localized

to

glandular

and

luminal

epithelium

-LRB-

Fig.

-RRB-

immunostaining

in

mid-late

secretory

endometrium

showed

minimal

glandular

and

luminal

epithelial

staining

and

intense

stromal

cell

cytoplasmic

staining

in

both

non-decidualized

and

decidualized

cells

-LRB-

Fig.

-RRB-

immunolocalized

to

glandular

epithelium

and

endothelial

cells

but

was

very

low

in

the

stroma

-LRB-

Fig.

-RRB-

and

in

first

trimester

placenta

showed

similar

staining

patterns

to

-LRB-

data

not

shown

-RRB-

No

immunoreactive

Nodal

was

detected

in

late

secretory

endometrium

-LRB-

Fig.

-RRB-

although

the

immunostaining

protocol

was

verified

by

positive

staining

in

term

placenta

-LRB-

not

shown

-RRB-

Figure

Localization

of

TGFβ

superfamily

members

during

decidualization

in

mid-late

secretory

endometrium

and

first

trimester

decidua.Photomicrographs

are

representatives

of

immunostaining

for

activin

A

-LRB-

A

-RRB-

activin

B

-LRB-

B

-RRB-

-LRB-

C

E

-RRB-

-LRB-

F

H

-RRB-

-LRB-

I

K

-RRB-

-LRB-

L

N

-RRB-

-LRB-

O

Q

-RRB-

-LRB-

R

-RRB-

-LRB-

S

-RRB-

Nodal

-LRB-

T

-RRB-

In

mid-late

secretory

endometrium

scale

bar

µm

-LRB-

C

F

I

L

O

R

S

-RRB-

scale

bar

µm

-LRB-

D

G

J

M

P

T

-RRB-

In

first

trimester

decidua

scale

bar

µm

-LRB-

E

H

K

N

Q

-RRB-

inserts

show

higher

power

images

of

same

tissue

-LRB-

E

H

K

N

Q

-RRB-

Negative

controls

for

each

antibody

are

shown

in

inserts

-LRB-

C

F

I

L

O

R

S

T

-RRB-

Arrows

highlight

decidualized

-LRB-

D

-RRB-

non-decidualized

-LRB-

ND

-RRB-

stromal

cells

and

glands

-LRB-

G

-RRB-

TGFβ

superfamily

mRNA

expression

in

non-decidualized

and

decidualized

HESCBy

RT

PCR

mRNA

expression

was

evident

for

activin

βA

activin

βB

and

in

both

non-decidualized

and

decidualized

HESC

-LRB-

Fig.

-RRB-

Nodal

mRNA

was

undetectable

in

both

As

conventional

RT

PCR

was

used

only

the

presence/absence

of

each

gene

was

assessed

Positive

control

PCR

products

-LRB-

Fig.

see

supplementary

data

-RRB-

confirmed

sequence

identity

and

no

signals

were

detected

when

reverse

transcriptase

was

omitted

-LRB-

Fig.

-RRB-

Figure

mRNA

expression

for

TGFβ

superfamily

members

by

decidualized

and

non-decidualized

HESC.HESC

were

either

non-decidualized

or

decidualized

with

cAMP

for

days

Total

RNA

was

extracted

from

cells

for

RT

PCR

Representative

products

demonstrating

Activin

βA

Activin

βB

and

Nodal

mRNA

are

shown

in

non-decidualized

-LRB-

ND

-RRB-

and

cAMP-decidualized

-LRB-

D

-RRB-

HESC

ve

positive

control

RT

negative

control

Data

shown

is

from

a

single

experiment

which

is

representative

of

independent

culture

and

significantly

decreases

PRL

secretion

during

HESC

decidualizationTo

establish

whether

HESC

decidualization

could

be

blocked

or

reduced

the

newly

developed

activin

type

II

receptor

antagonist

or

the

type

I

receptor

kinase

inhibitor

were

used

in

an

in

vitro

decidualization

model

with

PRL

secretion

a

marker

of

decidualization

as

the

end-point

These

inhibitors

affect

activin

and

various

other

TGFβ

family

members

-LRB-

see

Table

I

-RRB-

Cells

maintained

in

medium

alone

-LRB-

non-decidualized

-RRB-

showed

non-detectable

PRL

levels

over

the

final

h

of

culture

-LRB-

Fig.

-RRB-

In

contrast

cells

treated

with

cAMP

secreted

PRL

-LRB-

mean

mIU/l

-RRB-

and

morphologically

changed

from

elongated

spindle-shaped

cells

to

typical

enlarged

polygonal

cells

demonstrating

successful

decidualization

as

previously

described

-LRB-

Dimitriadis

et

al.

-RRB-

Stromal

cells

cultured

with

cAMP

and

showed

a

significant

dose-dependent

decrease

in

PRL

secretion

at

and

nM

with

no

further

decrease

at

nM

-LRB-

Fig.

-RRB-

At

the

nM

dose

a

reduction

in

PRL

secretion

was

seen

compared

with

cAMP-treated

cells

without

inhibitor

which

was

similar

to

decidualization

reduction

seen

with

follistatin

Similarly

cells

co-cultured

with

cAMP

and

showed

a

dose-dependent

decrease

in

PRL

secretion

Addition

of

at

a

concentration

of

µM

significantly

reduced

PRL

secretion

compared

with

cAMP-only

treated

cells

-LRB-

Fig.

-RRB-

Further

significant

decreases

were

observed

with

doses

ranging

from

µM

-LRB-

Fig.

-RRB-

At

µM

reduced

PRL

secretion

by

compared

with

cAMP

alone

-LRB-

Fig.

-RRB-

Higher

doses

were

not

tested

When

either

inhibitor

-LRB-

or

-RRB-

was

administered

to

cells

in

the

absence

of

cAMP

PRL

was

not

detected

in

culture

medium

In

addition

cell

viability

was

tested

using

the

trypan

blue

exclusion

method

at

the

end

of

the

experiment

and

was

found

to

not

differ

between

treatments

with

medium-only

cAMP

and

either

inhibitor

Microscopic

visualization

indicated

cell

morphology

was

not

altered

by

inhibitor

use

suggesting

this

effect

was

not

due

to

toxicity

to

cells.Figure

and

inhibitors

significantly

decrease

HESC

decidualization.Confluent

stromal

cells

were

cultured

for

days

with

cAMP

with

medium

changes

every

days

-LRB-

A

-RRB-

was

added

at

doses

and

nM

to

cAMP-treated

cultures

-LRB-

B

-RRB-

was

added

at

doses

and

µM

Controls

-LRB-

not

decidualized

ND

-RRB-

and

cAMP

-LRB-

decidualized

D

-RRB-

PRL

secretion

from

cells

was

used

as

a

marker

of

decidualization

and

values

were

corrected

for

total

protein

All

data

-LRB-

mean

SEM

-RRB-

is

expressed

as

change

from

control

-LRB-

-RRB-

Results

are

combined

data

from

triplicate

wells

of

five

independent

experiments

for

each

inhibitor

P

and

P

compared

with

cAMP

alone.Secretion

of

activin

A

and

but

not

or

from

non-decidualized

and

decidualized

HESC

in

vitroTGFβ

ligands

are

active

as

secreted

proteins

Therefore

we

measured

from

three

different

cultures

by

ELISA

and

activin

A

by

IFMA

-LRB-

n

-RRB-

in

serum-free

culture

medium

There

was

a

increase

in

dimeric

activin

A

secreted

from

decidualized

-LRB-

-RRB-

compared

with

non-decidualized

cells

-LRB-

-RRB-

supporting

previous

published

data

from

our

laboratory

-LRB-

Jones

et

al.

-RRB-

Non-decidualized

and

decidualized

HESC

secreted

and

each

in

the

range

of

cells

-LRB-

Fig.

-RRB-

and

levels

were

below

the

sensitivity

of

the

assays

-LRB-

data

not

shown

-RRB-

Decidualized

stromal

cells

secreted

significantly

higher

amounts

of

and

compared

with

non-decidualized

cells

-LRB-

versus

versus

-RRB-

-LRB-

Fig.

-RRB-

secretion

was

increased

slightly

with

decidualization

-LRB-

versus

-RRB-

but

this

was

not

significant

-LRB-

Fig.

-RRB-

Western

blot

analysis

was

performed

for

and

using

µg

of

cell

lysates

from

non-decidualized

and

decidualized

HESC

however

these

proteins

were

below

the

level

of

detection

Positive

controls

-LRB-

rat

heart

and

human

term

placenta

human

term

placenta

and

recombinant

human

and

recombinant

human

respectively

data

not

shown

-RRB-

validated

the

technique.Figure

and

protein

secretion

by

HESC

during

and

protein

were

measured

in

culture

medium

from

non-decidualized

-LRB-

dark

-RRB-

and

decidualized

-LRB-

grey

-RRB-

cells

at

Day

of

culture

Results

are

combined

data

from

n

independent

culture

experiments

and

data

-LRB-

mean

SEM

-RRB-

is

represented

as

difference

from

control

-LRB-

non-decidualized

cells

defined

as

-RRB-

P

and

P

compared

to

control.Exogenous

and

increases

HESC

decidualization

in

vitroTo

assess

whether

addition

of

and/or

to

HESC

enhances

decidualization

in

vitro

cells

were

decidualized

with

and

MPA

under

serum-free

conditions

for

days

and

PRL

secretion

measured

At

Day

PRL

secretion

from

non-decidualized

cells

was

minimal

but

was

increased

when

cells

were

decidualized

-LRB-

Fig.

-RRB-

When

-LRB-

or

ng/ml

-RRB-

was

added

to

decidualizing

HESC

PRL

secretion

was

significantly

increased

at

the

ng/ml

dose

compared

with

the

decidualized

control

-LRB-

P

-RRB-

The

addition

of

at

or

ng/ml

to

decidualizing

HESC

however

this

was

only

significant

at

the

ng/ml

dose

-LRB-

P

-RRB-

compared

with

decidualized

cells

alone.Due

to

the

variability

of

endometrial

biopsies

only

one

dose

for

each

and

has

resulted

in

a

significant

promotion

of

decidualisation

Also

as

the

is

known

to

be

quite

potent

this

result

could

be

explained

by

the

fact

that

at

the

higher

doses

it

might

be

negatively

affecting

the

response

for

example

receptor

and

downstream

signalling

disturbances.Figure

Effect

of

exogenous

and

on

PRL

secretion

from

HESC.Cells

were

untreated

or

treated

with

-LRB-

M

-RRB-

MPA

-LRB-

M

-RRB-

alone

or

combined

with

recombinant

human

-LRB-

rh

-RRB-

-LRB-

ng/ml

-RRB-

-LRB-

A

-RRB-

-LRB-

ng/ml

-RRB-

-LRB-

B

-RRB-

for

day

PRL

was

measured

in

cultured

medium

from

the

last

h

of

treatment

Data

are

represented

as

percent

fold

change

from

control

-LRB-

decidualized

D

-RRB-

defined

as

ND

non-decidualized

HESC

Results

are

combined

data

from

triplicate

wells

of

three

independent

cultures

P

and

P

compared

with

D.

DiscussionThis

is

the

first

study

to

identify

and

protein

in

secretory

phase

human

endometrium

and

show

the

mRNA

expression

of

these

ligands

in

cultured

HESC

In

addition

the

current

study

has

immunolocalized

and

protein

to

decidualized

stromal

cells

and

provided

further

evidence

for

a

role

for

secreted

activin

and

in

decidualization

through

the

use

of

novel

inhibitors

and

a

well-established

ex

vivo

decidualization

model.Decidualization

of

endometrial

stromal

cells

is

a

pivotal

event

in

the

preparation

for

blastocyst

implantation

Although

this

process

is

governed

by

hormonal

regulation

it

is

becomingly

increasingly

evident

that

a

multitude

of

factors

including

cytokines

and

growth

factors

are

essential

in

ensuring

this

process

occurs

successfully

such

that

endometrial

stromal

cells

are

differentiated

into

the

implantation-favourable

decidual

cells

The

identification

of

and

protein

in

mid-secretory

human

endometrial

tissue

together

with

the

increased

secretion

of

both

and

from

stromal

cells

following

decidualization

suggests

these

TGFβ

family

members

are

hormonally

regulated

and

important

for

decidualization.The

finding

that

secretion

from

HESC

increases

as

they

decidualize

in

vitro

and

that

addition

of

further

drives

decidualization

as

determined

by

secretion

of

PRL

extends

the

recent

study

-LRB-

Li

et

al.

-RRB-

which

showed

an

increase

of

mRNA

with

decidualization

in

a

similar

model

and

of

PRL

mRNA

following

treatment

of

HESC

with

This

is

important

given

that

translation

and

secretion

do

not

necessarily

follow

increases

in

mRNA

The

inhibitor

which

acts

primarily

on

those

ligands

acting

through

the

activin

type

receptor

-LRB-

Table

I

-RRB-

is

not

known

to

block

the

action

of

which

acts

through

the

BMP

type

II

receptor

-LRB-

BMPRII

-RRB-

Therefore

the

inability

of

to

fully

block

the

decidualization

process

suggests

that

factors

other

than

activins

and

GDFs

are

likely

to

be

important

to

this

process

is

a

likely

candidate.Interestingly

in

mice

uterine-specific

conditional

ablation

of

resulted

in

uterine

stromal

disruption

and

an

inability

to

undergo

normal

decidual

reactions

and

pregnancy

progression

-LRB-

Lee

et

al.

-RRB-

Our

demonstration

of

in

human

endometrium

in

vivo

with

the

strongest

immunostaining

in

decidualized

cells

in

the

mid-late

secretory

phase

and

in

the

decidua

of

early

pregnancy

confirms

the

biological

relevance

in

women

Given

this

data

in

conjunction

with

our

functional

studies

of

human

decidualization

there

appears

to

be

little

doubt

that

is

an

important

mediator

of

both

mouse

and

human

decidualization.This

study

also

demonstrates

for

the

first

time

the

presence

of

in

human

endometrium

and

first

trimester

placenta

where

it

is

intensely

and

specifically

stained

in

the

decidual

cells

is

best

known

for

its

role

in

early

chondrogenesis

and

joint

formation

being

involved

in

inducing

cartilage

differentiation

growth

and

maturation

-LRB-

Mikic

-RRB-

as

well

as

bone

formation

and

angiogenesis

-LRB-

Yamashita

et

al.

-RRB-

Differentiation

and

angiogenesis

are

the

main

events

of

decidualization

Unfortunately

assays

for

were

not

available

to

measure

the

secretion

from

decidualized

cells

The

decrease

in

decidualization

seen

following

administration

of

was

not

likely

to

be

due

to

blockade

as

this

ligand

has

a

relatively

low

if

any

affinity

for

the

activin

type

II

receptor

Future

studies

should

address

whether

has

a

functional

role

in

decidualization

whether

it

be

in

promoting

differentiation

or

in

the

formation

of

associated

blood

vessels

to

support

the

blastocyst

in

early

pregnancy

and

throughout

gestation.Although

the

TGFβs

have

been

previously

detected

in

the

human

endometrium

the

information

regarding

their

role

during

decidualization

is

contentious

as

studies

report

contrasting

effects

on

PRL

production

by

endometrial

stroma

and

during

decidualization

in

vitro

-LRB-

Kubota

et

al.

Kim

et

al.

-RRB-

We

showed

here

that

under

serum-free

conditions

secretion

increases

during

cAMP-induced

decidualization

in

vitro

Microarray

studies

have

likewise

shown

increased

and

mRNA

when

cAMP

or

progesterone

is

used

as

decidual

stimuli

-LRB-

Popovici

et

al.

Tierney

et

al.

-RRB-

The

protein

for

both

isoforms

localizes

to

stromal

cells

in

human

endometrium

throughout

the

menstrual

cycle

-LRB-

Gold

et

al.

Godkin

and

Dore

-RRB-

although

decidualized

stromal

cells

were

not

shown

In

contrast

the

present

study

identified

protein

in

decidualized

stromal

cells

in

human

endometrium

and

its

secretion

increased

in

cAMP-decidualized

HESC

Administration

of

which

effectively

blocks

the

TGFβs

as

well

as

activins

by

blocking

their

structurally

similar

and

receptors

-LRB-

Inman

et

al.

-RRB-

resulted

in

a

reduction

in

decidualization

a

greater

decrease

than

that

seen

with

-LRB-

this

study

-RRB-

and

follistatin

-LRB-

Tierney

and

Giudice

-RRB-

This

along

with

our

demonstration

that

addition

of

during

progesterone-induced

decidualization

enhances

decidualization

provides

convincing

evidence

to

suggest

is

important

during

decidualization

The

level

of

activated

was

undetectable

by

ELISA

in

cAMP-decidualized

endometrial

stromal

cells

and

therefore

this

ligand

is

unlikely

to

contribute

to

the

decidual

response.The

identification

of

and

during

the

mid-secretory

phase

is

also

an

important

finding

In

addition

to

decidualization

there

are

many

remodelling

processes

occurring

in

the

endometrium

during

this

time

Both

and

protein

showed

intense

immunolocalization

to

the

cytoplasm

of

the

stroma

irrespective

of

whether

or

not

the

cells

were

decidualized

This

was

verified

for

by

the

minimal

difference

in

secretion

levels

between

non-decidualized

and

decidualized

cells

in

vitro

mRNA

expression

has

been

localized

to

the

vascular

endothelial

cells

in

pregnant

mice

-LRB-

Ying

and

Zhao

-RRB-

and

in

the

non-pregnant

rat

uterus

where

it

is

constitutively

expressed

throughout

the

cycle

-LRB-

Erickson

et

al.

-RRB-

This

differs

to

the

localization

of

protein

shown

here

in

human

endometrium

It

may

be

that

the

mRNA

localized

to

blood

vessels

in

the

rodent

studies

is

not

translated

into

functional

-LRB-

more

commonly

referred

to

as

Myostatin

-RRB-

is

a

negative

regulator

of

skeletal

muscle

but

more

recently

has

been

shown

to

be

involved

in

glucose

metabolism

-LRB-

McPherron

and

Lee

-RRB-

particularly

in

placenta

-LRB-

Mitchell

et

al.

-RRB-

This

study

verified

the

presence

of

protein

in

first

trimester

placenta

and

showed

for

the

first

time

its

presence

in

secretory

phase

endometrium

and

in

HESC

Given

the

lack

of

specific

decidual

staining

it

is

unlikely

that

the

response

from

inhibitor

was

due

to

blocking

in

HESC

even

though

the

predominant

receptor

for

is

the

activin

type

II

receptor

The

presence

of

in

non-muscular

tissue

and

organs

is

rare

and

future

experiments

should

determine

whether

it

has

a

role

in

endometrial

biology

as

it

does

throughout

gestation

in

the

placenta.Vesicular

staining

for

-LRB-

or

Osteogenic

-RRB-

was

evident

in

highly

decidualized

cells

during

the

secretory

phase

of

human

endometrium

with

minimal

staining

in

selected

glandular

and

luminal

epithelial

cells

has

been

identified

in

the

uterine

epithelium

of

non-pregnant

mice

and

rat

uterus

-LRB-

Ozkaynak

et

al.

Erickson

et

al.

-RRB-

with

localization

remaining

unchanged

across

the

cycle

In

pregnant

mice

however

mRNA

was

detected

in

the

decidualizing

stromal

cells

surrounding

the

blastocyst

with

no

staining

in

the

epithelial

cells

-LRB-

Ying

and

Zhao

-RRB-

In

the

present

study

secretion

from

HESC

was

undetectable

by

ELISA

making

it

difficult

to

assess

whether

it

may

have

a

functional

role

during

decidualization.In

conclusion

this

study

has

identified

that

a

wide

range

of

TGFβ

ligands

are

present

in

mid-late

secretory

human

endometrium

but

of

these

in

addition

to

activins

only

and

increase

in

stromal

cells

as

they

undergo

decidualization

and

along

with

activins

are

important

in

driving

the

decidualization

process

Functions

for

the

remaining

ligands

remain

to

be

determined

but

they

are

likely

to

participate

in

the

extensive

tissue

remodelling

that

occurs

in

this

highly

dynamic

tissue

as

it

prepares

for

blastocyst

implantation

and

pregnancy

Supplementary

materialSupplementary

material

is

available

at

HUMREP

Journal

online

FundingThis

work

was

funded

by

the

National

Health

and

Medical

Research

Council

of

Australia

-LRB-

-RRB-

and

a

Prince

Henry

's

Institute

Postgraduate

Scholarship

Supplementary

Material

-LSB-

Supplementary

Data

-RSB-

O O O O O O O O O O B I O B O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

human endometrium
implantation
decidualization