Analysis

of

obstetric

complications

and

uterine

connective

tissue

in

tenascin-X-deficient

humans

and

mice

Tenascin-X

-LRB-

TNX

-RRB-

is

a

large

multi-domain

extracellular

matrix

glycoprotein

Complete

deficiency

of

TNX

in

humans

leads

to

a

recessive

form

of

Ehlers-Danlos

syndrome

-LRB-

EDS

-RRB-

and

TNX

haploinsufficiency

is

a

cause

of

hypermobility

type

EDS

EDS

patients

appear

to

have

a

higher

risk

of

several

complications

during

pregnancy

such

as

pelvic

instability

premature

rupture

of

membranes

and

postpartum

hemorrhage

Here

we

present

a

study

of

genitourinary

and

obstetric

complications

in

TNX-deficient

women

of

reproductive

age

We

have

found

complications

such

as

uterus

prolapses

that

are

in

agreement

with

previous

findings

in

other

EDS

types

In

TNX

knockout

-LRB-

KO

-RRB-

mice

we

have

observed

mild

pregnancy-related

abnormalities

Morphological

and

immunohistological

analysis

of

uterine

tissues

has

not

revealed

obvious

quantitative

or

spatial

differences

between

TNX

KO

and

wildtype

mice

with

respect

to

collagen

types

I

III

V

and

XII

or

elastic

fibers

We

conclude

that

TNX-deficient

women

are

at

risk

of

obstetric

complications

but

that

TNX

KO

mice

show

only

a

mild

phenotype

Furthermore

we

show

that

TNX

is

involved

in

the

stability

of

elastic

fibers

rather

than

in

their

initial

deposition

Introduction

Tenascin-X

-LRB-

TNX

-RRB-

is

a

large

multi-domain

extracellular

matrix

-LRB-

ECM

-RRB-

glycoprotein

composed

of

epidermal

growth

factor

-LRB-

EGF

-RRB-

like-repeats

fibronectin

type

III

repeats

and

a

C-terminal

fibrinogen

domain

-LRB-

Bristow

et

al.

Lethias

et

al.

Elefteriou

et

al.

Ikuta

et

al.

Tucker

et

al.

-RRB-

Complete

deficiency

of

TNX

in

humans

leads

to

a

rare

recessive

form

of

Ehlers-Danlos

Syndrome

-LRB-

EDS

-RRB-

and

TNX

haploinsufficiency

is

a

cause

of

hypermobility

type

EDS

Patients

of

both

EDS

types

exhibit

mild

to

severe

joint

hypermobility

The

skin

of

TNX-deficient

patients

is

markedly

lax

with

poor

recoil

properties

and

shows

easy

bruising

The

collagen

density

appears

reduced

in

the

dermis

of

these

patients

and

the

elastic

fibers

are

abnormal

-LRB-

Burch

et

al.

Schalkwijk

et

al.

Zweers

et

al.

Peeters

et

al.

Lindor

and

Bristow

-RRB-

Most

of

the

literature

concerning

pregnancy

in

EDS

uses

the

old

classification

of

nine

subgroups

In

our

study

we

use

the

revised

classification

of

six

subtypes

of

EDS

-LRB-

Beighton

et

al.

-RRB-

Tissue

fragility

is

a

hallmark

of

EDS

and

is

present

in

all

the

different

subtypes

EDS

patients

appear

to

have

a

higher

risk

of

several

complications

during

pregnancy

such

as

pelvic

instability

premature

rupture

of

membranes

and

postpartum

hemorrhage

Pregnancy

in

classical

and

hypermobility

type

EDS

has

a

relative

favorable

maternal

and

neonatal

outcome

Pregnancy

in

vascular

type

EDS

patients

is

associated

with

severe

complications

including

maternal

mortality

-LRB-

Pepin

et

al.

Carley

and

Schaffer

Roop

and

Brost

Wegrowski

et

al.

Ramos-e-Silva

et

al.

Lind

and

Wallenburg

Kuczkowski

Parry

and

Strauss

-RRB-

The

effect

of

TNX

deficiency

on

pregnancy

has

not

been

studied

so

far

and

only

one

case

report

of

pregnancy

in

a

TNX-deficient

woman

has

been

reported

in

the

literature

-LRB-

Lindor

and

Bristow

-RRB-

Here

we

present

a

study

of

all

known

pregnancies

and

genito-urinary

-LRB-

GU

-RRB-

abnormalities

in

TNX-deficient

woman

of

reproductive

age

identified

in

our

clinic

-LRB-

Schalkwijk

et

al.

-RRB-

and

in

the

literature

-LRB-

Lindor

and

Bristow

Schalkwijk

et

al.

-RRB-

Furthermore

we

have

extended

our

studies

to

a

TNX

knockout

-LRB-

KO

-RRB-

mouse

model

in

which

we

have

investigated

uterine

development

during

pregnancy

We

have

previously

established

that

TNX

is

essential

for

the

development

of

tissue

strength

in

the

skin

and

is

able

to

bind

to

elastin

a

major

component

of

elastic

fibers

and

to

collagens

type

I

III

V

and

XII

-LRB-

Egging

et

al.

b

Veit

et

al.

-RRB-

We

have

investigated

the

expression

of

these

molecules

at

the

protein

level

Materials

and

methods

TNX-deficient

patients

We

investigated

pregnancies

and

GU

abnormalities

in

all

currently

identified

female

tenascin-X

deficient

patients

of

reproductive

age

identified

in

our

clinic

and

in

the

literature

The

study

protocol

was

approved

by

the

local

medical

ethics

committee

and

written

informed

consent

was

obtained

from

the

patients

Experimental

animals

TNX

knockout

-LRB-

KO

-RRB-

mice

were

obtained

as

described

previously

-LRB-

Mao

et

al.

-RRB-

For

all

studies

we

used

TNX

KO

mice

that

were

crossed

back

with

six

generations

of

mice

Wildtype

-LRB-

WT

-RRB-

mice

were

used

as

a

control

All

mice

investigated

for

pregnancy

abnormalities

and

morphological

changes

in

uterus

tissue

were

between

and

months

of

age

reflecting

a

normal

age

distribution

of

a

breeding

mice

population

Skin

samples

were

taken

from

and

mice

and

aortic

tissue

was

obtained

from

mice

The

experimental

design

was

approved

by

the

Animal

Use

Committee

of

Radboud

University

Nijmegen

Breeding

Breeding

pairs

were

kept

in

a

female

male

ratio

Females

were

inspected

daily

for

vaginal

plugs

and

those

with

plugs

were

isolated

and

inspected

each

day

for

abnormalities

and

progression

of

pregnancy

Litter

size

was

determined

after

birth

Neonatal

survival

is

defined

as

the

difference

in

litter

size

after

partus

and

weeks

postpartum

Processing

of

samples

for

-LRB-

immuno

-RRB-

histochemistry

Mice

were

killed

in

a

sealed

compartment

by

exposure

to

a

mixture

of

carbogen

gas

and

increasing

concentrations

of

Skin

samples

for

frozen

sections

were

embedded

in

Tissue-Tek

O.C.T.

compound

-LRB-

Sakura

Finetek

Europe

The

Netherlands

-RRB-

and

snap

frozen

in

liquid

nitrogen

Skin

samples

for

paraffin

sections

were

fixed

in

buffered

formalin

for

h.

Affinity-purified

TNX

antibody

production

TNX

FNIII

repeats

were

amplified

by

polymerase

chain

reaction

-LRB-

PCR

forward

primer

reverse

primer

-RRB-

by

using

a

previously

described

human

TNX

cDNA

as

a

template

-LRB-

Morel

et

al.

-RRB-

The

PCR

product

was

ligated

into

the

TOPO

vector

-LRB-

Invitrogen

Breda

The

Netherlands

-RRB-

according

to

the

manufacturer

's

instructions

for

easy

digestion

with

restriction

enzymes

The

TOPO

vector

with

insert

was

digested

with

EcoRI

and

SalI

The

region

coding

for

was

inserted

into

the

EcoRI/SalI

site

of

the

-LRB-

a

-RRB-

plasmid

-LRB-

Brunschwig

Chemie

Amsterdam

The

Netherlands

-RRB-

The

sequence

of

the

TNX

domains

was

verified

by

dideoxy

sequencing

with

a

DNA

analyzer

-LRB-

Applied

Biosystems

Nieuwekerk

a/d

IJssel

The

Netherlands

-RRB-

TNX

protein

was

expressed

and

purified

according

to

the

manufacturer

's

instructions

-LRB-

Brunschwig

Chemie

-RRB-

Purified

TNX

protein

was

used

for

immunization

of

a

rabbit

Aliquots

of

μg

TNX

in

μl

phosphate-buffered

saline

were

mixed

with

an

equal

volume

of

Freund

's

complete

adjuvant

for

the

first

injection

and

Freund

's

incomplete

adjuvant

for

boosters

Three

injections

were

administered

subcutaneously

at

intervals

Polyclonal

antibodies

against

TNX

were

purified

by

affinity

chromatography

on

a

column

with

the

antigen

coupled

to

CNBr-activated

sepharose

-LRB-

GE

Healthcare

Life

Sciences

Diegem

Belgium

-RRB-

Histopathology

histochemistry

and

immunohistochemistry

Staining

of

elastic

fibers

was

performed

by

modified

Hart

's

stain

on

paraffin

sections

-LRB-

μm

Starcher

et

al.

-RRB-

Paraffin

sections

were

also

stained

with

hematoxylin

and

eosin

-LRB-

H&E

-RRB-

stain

to

assess

uterus

morphology

and

pathology

in

collaboration

with

a

professional

pathologist

Frozen

sections

-LRB-

μm

-RRB-

were

stained

with

antibodies

against

collagen

types

I

III

and

V

-LRB-

SouthernBiotech

USA

-RRB-

and

elastin

-LRB-

Elastin

Products

USA

-RRB-

as

previously

described

by

Egging

et

al.

-LRB-

-RRB-

Collagen

XII

was

stained

as

previously

described

by

Veit

et

al.

-LRB-

-RRB-

TNX

was

stained

with

affinity-purified

antibodies

directed

against

of

TNX

Detection

was

performed

with

antibodies

labeled

with

fluorescein

isothiocyanate

-LRB-

FITC

green

DakoCytomation

Denmark

-RRB-

and

Alexa

Fluor

-LRB-

red

Molecular

Probes

The

Netherlands

-RRB-

Cell

nuclei

were

made

visible

with

-LRB-

DAPI

blue

Molecular

Probes

-RRB-

counterstaining

Assesment

of

immunostaining

was

performed

as

previously

described

-LRB-

Egging

et

al.

-RRB-

Images

were

captured

and

processed

with

Axiovision

software

-LRB-

Carl

Zeiss

The

Netherlands

-RRB-

Electron

microscopy

For

electron

microscopy

WT

and

TNX

KO

samples

from

uterine

wall

at

weeks

post-partum

skin

from

and

mice

and

aortas

from

mice

were

fixed

in

glutaraldehyde

in

M

sodium

cacodylate

overnight

and

washed

in

M

sodium

cacodylate

buffer

The

tissues

were

then

sequentially

treated

with

osmium

tetroxide

tannic

acid

and

uranyl

acetate

and

then

dehydrated

and

embedded

in

Epon

as

previously

described

-LRB-

Davis

-RRB-

Thin

sections

-LRB-

nm

-RRB-

were

counterstained

with

uranyl

acetate

in

absolute

methanol

and

lead

citrate

and

examined

in

a

Tecnai

transmission

electron

microscope

at

kV

Statistics

For

statistical

analyses

Fisher

's

exact

or

Chi-square

tests

were

used

where

appropriate

for

proportional

data

Student

's

t-test

was

used

to

compare

mean

litter

size

after

partus

and

neonatal

survival

-LRB-

difference

in

litter

size

after

partus

and

weeks

postpartum

-RRB-

A

P-value

of

was

considered

statistically

significant

Results

Pregnancy

in

TNX-deficient

patients

We

investigated

pregnancy

and

GU

abnormalities

in

TNX-deficient

woman

of

reproductive

age

General

reproduction

characteristics

and

GU

abnormalities

of

female

TNX-deficient

patients

are

presented

in

Table

Maternal

and

neonatal

outcome

was

generally

normal

one

out

of

pregnancies

resulted

in

an

intrauterine

death

of

the

fetus

Tissue

laxity

of

TNX-deficient

patients

was

demonstrated

by

the

occurrence

of

vaginal

uterine

and

rectal

prolapses

even

at

a

relative

young

age

One

case

of

postpartum

hemorrhage

was

observed

in

childbirths

of

our

patients

had

urinary

incontinence

symptoms

TNX-deficient

EDS

is

an

autosomal

recessive

disorder

therefore

all

offspring

of

a

TNX-deficient

patient

and

an

unaffected

individual

are

obligatory

heterozygotes

We

have

previously

found

that

approximately

of

adult

females

that

are

haploinsufficient

for

TNX

suffer

from

hypermobility

type

EDS

or

benign

joint

hypermobility

syndrome

-LRB-

Zweers

et

al.

-RRB-

We

found

no

obvious

abnormalities

-LRB-

e.g.

floppy

infant

syndrome

premature

birth

-RRB-

in

the

neonates

-LRB-

obligatory

heterozygotes

-RRB-

of

our

TNX-deficient

population

however

some

TNX-deficient

patients

themselves

were

born

prematurely

-LRB-

Table

Lindor

and

Bristow

-RRB-

Four

out

of

six

of

the

TNX-deficient

women

had

postpartum

complications

-LRB-

one

of

the

patients

had

no

pregnancies

-RRB-

This

was

in

agreement

with

the

findings

in

the

literature

of

an

increase

in

maternal

GU

complications

in

EDS-deficient

women

compared

with

the

control

population

-LRB-

Ainsworth

and

Aulicino

Carley

and

Schaffer

Kuczkowski

Lind

and

Wallenburg

Ramos-e-Silva

et

al.

-RRB-

Table

in

TNX-deficient

woman

-LRB-

VUE

vaginal

uterine

extirpation

IUD

intra-uterine

death

CAH

congenital

adrenal

hyperplasia

-RRB-

Age

represents

the

age

of

the

patient

at

the

anamnesisPatient

IDAge

-LRB-

years

-RRB-

Gravida

number/Para

numberGenito-urinary

-LRB-

sister

of

patient

-RRB-

VUE

after

uterine

prolapse

-LRB-

at

age

-RRB-

Vaginal

prolapse

weeks

after

born

prematurely

-LRB-

week

-RRB-

with

shoulder

luxation

rectal

prolapse

at

age

-LRB-

sister

of

patient

-RRB-

One

incident

of

hemorrhage

postpartum

-LRB-

ml

blood

loss

-RRB-

IUD

-LRB-

weeks

-RRB-

with

relatively

large

blood

lossDeceased

at

age

severe

co-morbidity

including

cardiomyopathy

arrhythmia

arteriosclerosis

and

amputation

of

left

leg

-LRB-

Schalkwijk

et

al.

-RRB-

Undefined

prolapse

-LRB-

at

age

-RRB-

Precipitious

second

stage

at

term

for

all

birthsLindor

and

Bristow

Uterine

prolapse

-LRB-

at

age

-RRB-

also

recurrent

rectal

prolapses

Pregnancy

in

TNX

KO

mice

We

investigated

GU

and

pregnancy-related

abnormalities

in

TNX

KO

and

WT

mice

ranging

between

months

of

age

Litter

size

-LRB-

TNX

KO

pups

WT

pups

-RRB-

and

neonatal

survival

-LRB-

TNX

KO

WT

-RRB-

did

not

differ

significantly

-LRB-

Student

's

t-test

-RRB-

GU

abnormalities

of

the

TNX

KO

and

WT

mice

are

presented

in

Table

Overall

we

noted

relatively

few

pregnancy-related

abnormalities

Obstruction

of

the

uterine

or

vaginal

canals

during

partus

resulting

in

a

failure

to

deliver

pups

was

noted

more

often

in

WT

mice

compared

with

TNX

KO

mice

but

the

difference

was

not

significant

-LRB-

Chi-square

test

-RRB-

The

length

of

term

did

not

differ

significantly

for

the

TNX

KO

mice

-LRB-

mean

days

-RRB-

compared

with

WT

mice

-LRB-

mean

days

Fisher

's

exact

test

-RRB-

A

striking

difference

however

between

TNX

KO

and

WT

was

found

for

the

location

of

the

vaginal

plug

that

was

present

after

mating

Generally

the

vaginal

plug

in

TNX

KO

mice

was

located

much

deeper

in

the

vaginal

canal

than

in

WT

mice

This

made

identification

of

vaginal

plugs

in

TNX

KO

mice

markedly

more

difficult

resulting

in

a

significant

increase

in

pregnancies

with

unknown

length

of

pregnancy

-LRB-

Fig.

Chi-square

test

-RRB-

No

uterine

or

rectal

prolapses

were

observed

in

this

particular

study

-LRB-

pregnancies

of

KO

and

WT

mice

-RRB-

We

did

however

observe

rectal

prolapses

in

the

breeding

colony

of

our

TNX

KO

mice

population

-LRB-

shown

in

Fig.

-RRB-

albeit

at

a

low

frequency

-LRB-

-RRB-

We

did

not

observe

rectal

prolapses

in

our

WT

mice

population

Table

in

TNX-deficient

miceGenito-urinary

complicationsTNX

KO

miceWT

miceNumber

of

miceNumber

of

miceMaternal

death

during

during

bleeding

of

vagina

-LRB-

as

seen

at

vaginal

plug

check

-RRB-

after

number

of

evaluated

KO

mice

abnormalities

Vaginal

plugs

which

are

present

after

mating

are

much

more

difficult

to

asses

in

TNX

KO

mice

than

in

WT

mice

This

resulted

in

a

significant

increase

of

pregnancies

in

which

the

date

of

impregnation

was

unknown

-LRB-

a

P

Chi-square

test

-RRB-

Total

evaluations

TNX

KO

mice

n

WT

mice

n

In

this

study

no

uterine

prolapses

were

observed

although

some

TNX

KO

mice

suffered

from

a

rectal

prolapse

-LRB-

b

-RRB-

Rectal

prolapses

were

also

observed

in

TNX-deficient

patients

although

the

incidence

in

TNX

KO

mouse

was

lower

-LRB-

in

mice

-RRB-

Structure

of

mouse

uterus

The

mouse

uterus

consists

of

three

layers

an

outer

layer

which

includes

the

single-cell

layered

perimetrium

and

a

thin

layer

of

connective

tissue

a

muscular

layer

the

myometrium

composed

of

two

oppositely

orientated

layers

of

muscle

and

an

endometrium

which

undergoes

remodeling

during

the

menstrual

cycle

and

pregnancy

The

endometrium

contains

luminal

and

glandular

epithelial

cells

stromal

cells

resident

immune

cells

of

various

types

and

endothelial

cells

No

differences

in

the

overall

morphology

of

the

uterine

tissues

were

noticeable

between

TNX

KO

and

WT

mice

-LRB-

Fig.

uteri

on

day

postpartum

-RRB-

as

assessed

by

light

microscopy

of

H&E

stained

sections

As

we

mainly

focused

on

the

ECM

compartment

of

the

uterus

we

did

not

attempt

to

make

quantitative

analyses

of

the

cell

type

distribution

Fig.

of

TNX

KO

and

WT

uterus

H&E

stained

sections

of

the

uterus

of

WT

mice

-LRB-

a

-RRB-

and

of

TNX

KO

mice

-LRB-

b

-RRB-

No

differences

in

the

structure

of

the

uteri

were

noticeable

between

TNX

KO

and

WT

mice

The

mouse

uterus

consists

of

a

thin

outer

layer

the

perimetrium

-LRB-

P

-RRB-

A

muscle

layer

the

myometrium

-LRB-

M

-RRB-

consists

of

two

oppositely

orientated

layers

of

muscle

although

this

is

difficult

to

distinguish

in

H&E

stained

slides

-LRB-

E

endometrium

L

lumen

-RRB-

Bars

mm

TNX

distribution

in

mouse

uterus

TNX

was

present

throughout

the

uterus

of

virgin

mice

and

in

the

uterus

during

and

after

pregnancy

-LRB-

Fig.

-RRB-

Immunostaining

for

TNX

was

discontinuous

in

the

perimetrium

-LRB-

Fig.

f

-RRB-

TNX

was

detected

in

the

endometrium

although

the

epithelium

lining

the

endometrium

was

negative

for

TNX

-LRB-

Fig.

-RRB-

Furthermore

TNX

was

present

in

the

layers

of

connective

tissue

ensheathing

the

muscle

bundles

of

the

myometrium

-LRB-

Fig.

-RRB-

Overall

no

change

in

the

localization

of

TNX

immunostaining

was

observed

during

and

after

pregnancy

The

specificity

of

our

TNX

antibody

was

demonstrated

by

the

complete

absence

of

positive

staining

in

the

tissues

of

the

TNX

KO

mice

-LRB-

Fig.

g

-RRB-

Fig.

of

TNX

in

the

uterus

-LRB-

P

perimetrium

M

myometrium

showing

longitudinal

muscle

bundles

M

myometrium

showing

transverse

muscle

bundles

E

endometrium

Epi

epithelium

of

the

lumen

L

lumen

-RRB-

TNX

-LRB-

green

-RRB-

is

present

throughout

the

uterus

of

virgin

mice

-LRB-

a

-RRB-

and

in

the

uterus

during

and

after

pregnancy

shown

for

uteri

at

days

pregnant

-LRB-

b

-RRB-

and

weeks

postpartum

-LRB-

c

-RRB-

Cell

nuclei

are

stained

with

DAPI

-LRB-

blue

-RRB-

TNX

is

present

in

the

endometrium

-LRB-

d

-RRB-

and

the

layers

of

connective

tissue

ensheathing

muscle

bundles

of

the

myometrium

-LRB-

f

-RRB-

TNX

immunostaining

of

the

perimetrium

is

relatively

weak

-LRB-

a-c

f

-RRB-

The

epithelium

of

the

lumen

is

negative

for

TNX

-LRB-

d

-RRB-

The

specificity

of

the

TNX

antibody

is

demonstrated

in

e

g.

d

g

Uteri

at

weeks

postpartum

Bars

μm

Collagen

distribution

in

mouse

uterus

We

have

previously

demonstrated

the

binding

and

the

colocalization

of

TNX

with

collagen

types

I

II

and

V

in

the

skin

-LRB-

Egging

et

al.

b

-RRB-

In

the

uterus

TNX

also

colocalizes

with

major

fibrillar

collagens

types

I

III

and

V

as

is

shown

for

uteri

weeks

postpartum

-LRB-

Fig.

-RRB-

The

collagens

are

present

in

the

perimetrium

the

endometrium

and

the

layers

of

the

connective

tissue

ensheathing

the

muscle

bundles

of

the

myometrium

Similar

results

have

been

obtained

for

uteri

from

virgin

pregnant

and

postpartum

mice

No

differences

in

collagen

type

I

III

and

V

immunostaining

are

found

between

WT

and

TNX

KO

mice

-LRB-

data

not

shown

-RRB-

which

is

in

agreement

with

our

observations

in

skin

-LRB-

Egging

et

al.

-RRB-

TNX

does

not

exclusively

colocalize

with

collagens

however

because

TNX

also

cololalizes

with

other

ECM

components

such

as

-LRB-

tropo

-RRB-

elastin

Collagen

type

XII

an

interaction

partner

of

TNX

-LRB-

Veit

et

al.

-RRB-

is

present

throughout

the

uterus

-LRB-

Fig.

-RRB-

Collagen

type

XII

occurs

in

the

transverse

muscle

bundles

of

the

myometrium

around

blood

vessels

and

around

the

lumen

although

the

signal

intensity

is

not

completely

continuous

around

the

lumen

and

blood

vessels

TNX

and

collagen

type

XII

are

both

present

throughout

the

uterus

No

differences

in

the

collagen

type

XII

localization

in

the

uterus

between

WT

-LRB-

Fig.

c

e

g

-RRB-

and

TNX

KO

-LRB-

Fig.

d

f

h

-RRB-

mice

have

been

observed

Fig.

colocalizes

with

major

fibrillar

collagens

types

I

III

and

V

-LRB-

P

perimetrium

M

myometrium

with

longitudinal

muscle

bundles

M

myometrium

with

transverse

muscle

bundles

E

endometrium

Epi

epithelium

of

the

lumen

L

lumen

-RRB-

TNX

-LRB-

b

e

h

green

-RRB-

and

collagen

types

I

-LRB-

a

-RRB-

III

-LRB-

d

-RRB-

and

V

-LRB-

g

red

-RRB-

colocalize

-LRB-

c

f

i

yellow

orange

magenta

-RRB-

in

uteri

weeks

postpartum

Similar

results

have

been

obtained

for

uteri

from

virgin

pregnant

and

postpartum

mice

No

differences

in

collagen

type

I

III

and

V

immunostaining

are

apparent

between

WT

and

TNX

KO

mice

-LRB-

data

not

shown

-RRB-

In

k

l

m

higher

magnification

images

of

the

myometrium

are

shown

to

demonstrate

the

colocalization

of

the

fibrillar

collagens

with

TNX

-LRB-

collagen

type

III

is

used

as

an

example

-RRB-

Bars

μmFig

of

collagen

type

XII

in

uterus

-LRB-

P

perimetrium

M

myometrium

with

longitudinal

muscle

bundles

E

endometrium

-RRB-

Collagen

XII

-LRB-

red

-RRB-

is

present

throughout

the

uterus

Immunostaining

appears

to

be

the

strongest

in

the

transverse

muscle

bundles

of

the

myometrium

-LRB-

M

-RRB-

around

blood

vessels

-LRB-

BV

-RRB-

and

around

the

lumen

-LRB-

L

-RRB-

although

the

signal

intensity

is

not

always

completely

continuous

No

differences

in

the

collagen

type

XII

localization

in

the

uterus

between

WT

-LRB-

a

c

e

g

-RRB-

and

TNX

KO

-LRB-

b

d

f

h

-RRB-

mice

are

observed

Bars

μm

Elastic

fibers

in

mouse

tissues

Elastic

fibers

in

the

skin

of

TNX-deficient

patients

are

known

to

be

abnormally

shaped

-LRB-

Zweers

et

al.

-RRB-

and

thus

we

have

investigated

their

structure

in

TNX

KO

mouse

tissues

Elastic

fibers

in

the

uterus

are

mostly

located

in

the

myometrium

and

perimetrium

-LRB-

Fig.

b

-RRB-

The

endometrium

contains

few

elastic

fibers

as

shown

for

WT

-LRB-

Fig.

-RRB-

and

TNX

KO

-LRB-

Fig.

-RRB-

mice

In

the

myometrium

and

perimetrium

positive

staining

for

elastin

is

seen

predominantly

in

the

layers

of

connective

tissue

ensheathing

the

transverse

muscle

bundles

-LRB-

Fig.

d

-RRB-

Despite

the

relative

low

abundance

of

mature

elastic

fibers

in

the

endometrium

the

presence

of

elastin

can

be

detected

by

immunohistochemistry

possibly

a

consequence

of

the

sensitivity

of

the

detection

methods

-LRB-

Fig.

-RRB-

Elastin

localization

is

similar

for

WT

-LRB-

Fig.

-RRB-

and

TNX

KO

-LRB-

Fig.

-RRB-

mice

We

have

previously

demonstrated

binding

of

TNX

with

elastin

and

the

colocalization

of

these

two

proteins

in

skin

-LRB-

Egging

et

al.

b

-RRB-

Consistent

with

these

findings

elastin

also

-LRB-

partially

-RRB-

colocalizes

with

TNX

in

the

uterus

as

shown

in

the

myometrium

-LRB-

Fig.

-RRB-

Colocalization

of

TNX

with

elastin

is

not

complete

since

TNX

also

colocalizes

with

fibrillar

collagens

however

all

elastin-reactive

material

colocalizes

with

TNX

In

previous

work

we

have

showed

an

increase

in

elastin-positive

material

in

the

skin

of

aging

TNX

KO

mice

-LRB-

Egging

et

al.

b

-RRB-

Although

by

light

microscopy

the

elastic

fibers

in

the

TNX

KO

mouse

skin

do

not

appear

to

be

abnormally

shaped

an

increase

in

staining

might

have

resulted

from

an

increase

in

less

mature

or

less

organized

elastin

fibers

an

increase

in

soluble

tropoelastin

within

the

matrix

and/or

the

consequence

of

more

immunoreactive

epitopes

being

available

because

of

an

increased

susceptibility

for

degradation

These

results

are

consistent

with

our

past

observations

of

elastic

fibers

in

the

skin

of

TNX-deficient

patients

in

whom

irregular

and

immature

elastic

fibers

and

fibers

devoid

of

microfibrils

can

be

seen

at

the

ultrastructural

level

-LRB-

Egging

et

al.

Zweers

et

al.

-RRB-

Thus

to

expand

these

findings

further

in

skin

and

to

investigate

the

elastic

fibers

in

the

uterus

we

have

performed

an

ultrastructural

analysis

of

the

elastic

fibers

in

several

tissues

Elastic

fibers

in

WT

-LRB-

Fig.

-RRB-

and

TNX

KO

mice

-LRB-

Fig.

-RRB-

uteri

do

not

appear

to

differ

in

shape

or

size

-LRB-

shown

for

uterus

weeks

postpartum

-RRB-

Similarly

in

aorta

the

elastic

laminae

of

WT

-LRB-

Fig.

-RRB-

and

TNX

KO

mice

-LRB-

Fig.

-RRB-

are

similar

in

shape

and

number

-LRB-

aorta

of

mice

-RRB-

In

skin

of

older

TNX

KO

mice

-LRB-

as

shown

for

mice

-RRB-

however

a

difference

in

elastic

fiber

ultrastructure

is

apparent

compared

with

WT

skin

In

addition

to

normal-appearing

elastic

fibers

irregular

elastin

aggregates

as

seen

in

the

skin

of

TNX-deficient

patients

-LRB-

Zweers

et

al.

-RRB-

can

be

observed

-LRB-

Fig.

f

-RRB-

These

elastin

aggregates

are

not

found

in

the

skin

of

TNX

KO

mice

or

in

WT

mice

Interestingly

elastic

fibers

in

the

skin

of

TNX

KO

mice

often

appear

larger

than

those

of

WT

mice

of

the

same

age

-LRB-

compare

Fig.

h

-RRB-

Fig.

and

elastic

fibers

in

the

uterus

-LRB-

L

lumen

-RRB-

Elastic

fibers

-LRB-

purple

modified

Hart

's

staining

-RRB-

are

mostly

located

in

the

myometrium

-LRB-

M

M

-RRB-

and

perimetrium

-LRB-

P

-RRB-

whereas

the

endometrium

-LRB-

E

-RRB-

appears

to

contain

fewer

elastic

fibers

in

WT

-LRB-

a

-RRB-

and

TNX

KO

-LRB-

b

-RRB-

mice

No

elastic

fiber

abnormalities

are

found

in

the

TNX

KO

mice

Elastin

immunostaining

-LRB-

red

-RRB-

is

observed

in

the

myometrium

predominantly

in

the

transverse

bundles

-LRB-

M

in

c

d

-RRB-

The

layers

of

connective

tissue

ensheathing

the

muscle

bundles

of

the

myometrium

-LRB-

M

-RRB-

and

perimetrium

-LRB-

P

-RRB-

are

stained

positively

for

elastin

-LRB-

red

-RRB-

Strong

elastin

staining

is

also

seen

in

the

endometrium

-LRB-

E

-RRB-

Elastin

immunoreactivity

is

similar

for

WT

-LRB-

c

-RRB-

and

TNX

KO

-LRB-

d

-RRB-

mice

Elastin

-LRB-

red

-RRB-

colocalizes

-LRB-

orange

-RRB-

with

TNX

-LRB-

green

-RRB-

in

the

myometrium

-LRB-

M

in

e

-RRB-

Not

all

TNX

colocalizes

with

elastin

as

TNX

also

colocalizes

with

different

collagen

types

-LRB-

Fig.

-RRB-

Bars

μm

Fig.

evaluation

of

elastic

fibers

Elastic

fibers

in

WT

-LRB-

a

-RRB-

and

TNX

KO

-LRB-

b

-RRB-

mouse

uterus

do

not

appear

to

differ

in

shape

or

size

The

dark

structures

-LRB-

arrows

-RRB-

are

elastic

fibers

-LRB-

shown

for

uterus

at

weeks

postpartum

-RRB-

The

elastic

laminae

-LRB-

arrows

-RRB-

in

the

aorta

of

WT

-LRB-

c

-RRB-

and

TNX

KO

-LRB-

d

-RRB-

mice

are

similar

in

shape

and

number

-LRB-

aorta

of

mice

-RRB-

Skin

of

older

TNX

KO

mice

-LRB-

months

old

-RRB-

shows

differences

in

elastic

fibers

from

those

of

WT

skin

Irregular

elastin

aggregates

can

be

observed

in

the

TNX

KO

mouse

skin

-LRB-

e

near

a

sebaceous

gland

-RRB-

A

higher

magnification

of

an

elastin

aggregate

-LRB-

arrow

-RRB-

is

shown

in

f

These

aggregates

were

not

found

in

skin

of

TNX

KO

mice

or

in

WT

mice

No

irregularities

in

the

shape

of

elastic

fibers

are

observed

in

TNX

KO

mice

skin

however

larger

elastic

fibers

than

in

WT

mice

-LRB-

g

-RRB-

are

often

observed

in

the

TNX

KO

mice

-LRB-

h

arrows

elastic

fibers

-RRB-

Bars

μm

-LRB-

a

b

-RRB-

μm

-LRB-

c

d

-RRB-

μm

-LRB-

e

g

h

-RRB-

μm

-LRB-

f

-RRB-

Discussion

GU

complications

occur

frequently

in

patients

with

various

types

of

EDS

-LRB-

Carley

and

Schaffer

Ramos-e-Silva

et

al.

Lind

and

Wallenburg

Kuczkowski

Parry

and

Strauss

Pepin

et

al.

-RRB-

In

classical

and

hypermobility

types

of

EDS

which

are

the

most

common

the

outcome

of

pregnancy

is

generally

favorable

Maternal

complications

however

such

as

postpartum

hemorrhage

and

pelvic

instability

are

more

common

than

in

the

general

population

-LRB-

Ramos-e-Silva

et

al.

Kuczkowski

Parry

and

Strauss

-RRB-

Furthermore

EDS

patients

appear

to

have

an

elevated

risk

of

uterine

prolapse

-LRB-

Carley

and

Schaffer

Lind

and

Wallenburg

-RRB-

Pregnancy

in

patients

with

vascular

type

EDS

may

even

lead

to

maternal

death

through

uterus

or

vessel

rupture

-LRB-

Pepin

et

al.

-RRB-

We

have

investigated

pregnancies

and

GU

abnormalities

in

TNX-deficient

woman

of

reproductive

age

Generally

pregnancy

is

without

major

complications

in

TNX-deficient

patients

apart

from

one

noted

incident

of

postpartum

hemorrhage

However

uterine

and

vaginal

prolapse

regularly

occur

in

TNX-deficient

women

even

at

a

young

age

suggesting

laxity

of

GU

tissues

Premature

rupture

of

fetal

membranes

is

a

risk

in

pregnancy

with

EDS

affected

fetuses

-LRB-

Lind

and

Wallenburg

Parry

and

Strauss

-RRB-

No

premature

births

have

been

observed

in

the

offspring

of

the

TNX-deficient

patients

however

some

TNX-deficient

patients

had

been

born

prematurely

Obviously

some

caution

must

be

taken

in

making

conclusions

and

extrapolating

data

from

such

a

small

group

of

TNX-deficient

woman

Complete

TNX

deficiency

in

humans

is

a

rare

condition

and

so

far

only

a

few

patients

have

been

identified

We

have

therefore

investigated

pregnancy

and

uterine

tissue

structure

in

our

TNX

KO

mouse

model

No

gross

significant

differences

have

been

found

with

regard

to

abnormalities

during

pregnancy

or

reproduction

between

the

TNX

KO

and

WT

mice

A

trend

toward

a

reduction

in

the

length

of

pregnancy

in

TNX

KO

mice

The

only

significant

difference

has

been

observed

in

the

location

of

the

vaginal

plugs

which

suggests

laxity

in

the

vaginal

wall

In

a

previous

study

we

have

not

found

any

differences

in

cutaneous

collagen

deposition

between

TNX

KO

and

WT

mice

-LRB-

Egging

et

al.

-RRB-

as

has

been

observed

in

the

skin

of

TNX-deficient

patients

-LRB-

Schalkwijk

et

al.

Zweers

et

al.

-RRB-

Alterations

in

collagen

deposition

in

the

skin

of

TNX

KO

mice

is

however

a

matter

of

debate

-LRB-

Egging

et

al.

Mao

et

al.

Minamitani

et

al.

-RRB-

In

the

present

study

we

have

found

elastin

aggregates

and

enlarged

elastic

fibers

in

skin

of

TNX

KO

mice

at

the

ultrastructural

level

this

is

in

accordance

with

our

previously

published

data

concerning

increased

elastin

staining

in

aging

TNX

KO

mouse

skin

-LRB-

Egging

et

al.

-RRB-

We

have

detected

no

differences

in

elastic

fibers

in

the

uterus

or

elastic

laminae

of

the

abdominal

aorta

between

TNX

KO

and

WT

mice

suggesting

a

specific

role

for

TNX

in

the

maturation

or

maintenance

of

elastic

fibers

in

skin

However

abnormal

elastic

fibers

might

not

be

present

in

the

uterus

since

the

ECM

is

constantly

being

remodeled

during

the

menstrual

cycle

and

pregnancy

Although

the

TNX

KO

mice

appear

to

have

a

much

milder

phenotype

compared

with

TNX-deficient

humans

the

skin

of

TNX

KO

mice

is

significantly

weaker

and

more

lax

compared

with

WT

mice

-LRB-

Bristow

et

al.

Burch

et

al.

Egging

et

al.

Lindor

and

Bristow

Mao

et

al.

Matsumoto

et

al.

Minamitani

et

al.

Schalkwijk

et

al.

Zweers

et

al.

-RRB-

Our

data

suggest

a

more

lax

vaginal

canal

in

TNX-deficient

patients

and

to

a

certain

degree

in

TNX

KO

mice

An

attractive

speculation

considering

the

observations

from

our

studies

in

mice

is

that

TNX

is

essential

for

tissue

integrity

independent

of

the

quantity

of

collagen

deposition

or

elastic

fiber

structure

In

conclusion

we

would

advise

obstetricians

and

gynecologists

to

monitor

TNX-deficient

patients

closely

as

they

are

likely

to

have

weaker

GU

connective

tissue

and

could

therefore

be

at

risk

of

GU

complications

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 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 B 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 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 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 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 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 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 B 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 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 B 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 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 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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B 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 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 B 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 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 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 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 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 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 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 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 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 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 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 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 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 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 B 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 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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 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 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 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 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

ehlers-danlos syndrome
tenascin-x
mouse (tnx knockout; c57bl/6)