The

role

of

metacaspase

in

Plasmodium

berghei

development

and

apoptosis

The

malaria

parasite

encodes

a

wide

range

of

proteases

necessary

to

facilitate

its

many

developmental

transitions

in

vertebrate

and

insect

hosts

Amongst

these

is

a

predicted

cysteine

protease

structurally

related

to

caspases

named

Plasmodium

metacaspase

-LRB-

-RRB-

We

have

generated

Plasmodium

berghei

parasites

in

which

the

sequence

is

removed

and

replaced

with

a

green

fluorescent

reporter

gene

to

investigate

the

expression

of

its

contribution

to

parasite

development

and

its

involvement

in

previously

reported

apoptosis-like

cell

death

of

P.

berghei

ookinetes

Our

results

show

that

the

gene

is

expressed

in

female

gametocytes

and

all

downstream

mosquito

stages

including

sporozoites

but

not

in

asexual

blood

stages

We

failed

to

detect

an

apparent

loss-of-function

phenotype

suggesting

that

constitutes

a

functionally

redundant

gene

We

discuss

these

findings

in

the

context

of

two

other

putative

Plasmodium

metacaspases

that

we

describe

here

Introduction

Malaria

remains

the

most

important

parasitic

human

disease

responsible

for

millions

of

deaths

each

year

The

life

cycle

of

malaria

parasites

is

complex

involving

multiple

life

stages

and

requiring

two

hosts

mosquito

and

human

The

uptake

of

gametocytes

by

the

mosquito

leads

to

rapid

gametogenesis

and

fertilisation

Ookinetes

are

formed

in

the

mosquito

midgut

lumen

which

cross

the

midgut

wall

and

transform

into

oocysts

Each

oocyst

can

produce

thousands

of

sporozoites

which

after

release

traverse

the

mosquito

hemolymph

and

infect

the

salivary

gland

tissues

before

they

can

enter

the

human

host

via

the

mosquito

bite

Following

inoculation

sporozoites

infect

hepatocytes

and

develop

into

liver

schizonts

Liver

schizont

produce

merozoites

which

upon

release

into

the

blood

stream

initiate

the

erythrocytic

cycle

the

prime

cause

of

clinical

symptoms

in

malaria

patients

Existing

malaria

control

strategies

are

hampered

by

increasing

parasite

resistance

in

the

field

to

established

antimalarial

drugs

as

well

as

by

widespread

insecticide

resistance

in

the

mosquito

vectors

Effective

antimalarial

vaccines

are

only

at

the

preliminary

stages

of

development

There

is

thus

a

great

need

to

develop

novel

strategies

for

malaria

control

Proteases

are

attractive

antimalarial

targets

because

of

their

indispensable

roles

in

parasite

development

and

infectivity

A

wide

variety

of

proteases

of

malaria

parasites

have

been

described

-LSB-

-RSB-

Amongst

these

are

cysteine

proteases

belonging

to

the

family

-LRB-

clan

CD

-RRB-

which

include

caspases

Caspases

belong

to

a

distinct

class

of

cysteine

proteases

with

a

so

called

Caspase

Hemoglobinase

Fold

-LRB-

CHF

-RRB-

-LSB-

-RSB-

Recently

two

new

families

of

predicted

CHF

proteases

closely

related

to

the

caspases

were

identified

named

paracaspases

and

metacaspases

-LSB-

-RSB-

In

metazoa

caspases

are

centrally

involved

with

the

molecular

machinery

of

programmed

cell

death

-LRB-

apoptosis

-RRB-

and

are

responsible

for

many

of

the

biochemical

and

morphological

changes

that

accompany

it

They

cleave

a

variety

of

proteins

ultimately

leading

to

the

disintegration

of

the

cell

-LSB-

-RSB-

Metacaspases

have

thus

far

only

been

identified

in

organisms

that

do

not

possess

classic

caspase

genes

plants

fungi

and

the

parasitic

protists

Trypanosoma

Leishmania

and

Plasmodium

and

there

is

some

evidence

linking

metacaspases

to

apoptosis

in

these

organisms

-LSB-

-RSB-

In

Plasmodium

falciparum

a

metacaspase

gene

-LRB-

GenBank

accession

here

named

-RRB-

has

been

described

possessing

consensus

histidine

and

cysteine

residues

that

typically

form

the

catalytic

dyad

in

this

family

of

proteases

-LSB-

-RSB-

suggesting

that

this

parasite

species

may

possess

a

mechanism

of

programmed

cell

death

Indeed

apoptosis-like

DNA

fragmentation

has

been

reported

to

occur

in

blood

stage

parasites

of

P.

falciparum

in

response

to

chloroquine

treatment

-LSB-

-RSB-

In

addition

ookinetes

of

Plasmodium

berghei

were

reported

to

undergo

an

apoptosis-like

mechanism

of

cell

death

resulting

in

very

substantial

parasite

losses

-LSB-

-RSB-

In

this

study

we

investigate

the

role

of

in

the

reported

programmed

cell

death

in

Plasmodium

by

constructing

parasites

We

discuss

our

findings

in

the

context

of

two

additional

Plasmodium

metacaspase-like

proteins

that

we

describe

here

Materials

and

methods

Parasite

maintenance

culture

and

purification

P.

berghei

ANKA

-LRB-

clone

-RRB-

parasites

were

maintained

in

female

TO

mice

by

mechanical

blood

passage

and

regular

transmission

in

Anopheles

stephensi

as

described

-LSB-

-RSB-

Ookinete

cultures

were

set-up

as

described

-LSB-

-RSB-

both

with

and

without

prior

removal

of

white

blood

cells

by

columns

In

general

h

ookinete

cultures

were

centrifuged

at

g

for

min

and

the

cell

pellet

incubated

on

ice

in

volumes

of

M

to

lyse

the

red

blood

cells

In

some

experiments

ookinetes

were

further

purified

on

Nycodenz

cushens

Finally

parasites

were

concentrated

by

centrifugation

at

g

for

min

resuspended

in

RPMI

or

phosphate

buffered

saline

and

immediately

analysed

Generation

of

parasites

A

bp

fragment

was

amplified

by

PCR

from

P.

berghei

genomic

DNA

with

primers

F

-LRB-

TAAACCATTGGTCATACCAAAAAATAATCAAAAAAATAACCAA

-RRB-

and

R

-LRB-

CGGGCCGCTCTAGCATGACCAGGCTCAATAATTGAACA

-RRB-

and

introduced

into

NdeI-digested

via

In-Fusion

PCR

cloning

-LRB-

BD

Biosciences

-RRB-

The

resulting

plasmid

pLP-DHFR/MC

contains

a

loxP-prokaryotic

promotor

cassette

-LRB-

BD

Biosciences

-RRB-

followed

by

a

modified

Toxoplasma

gondii

dihydrofolate

reductase

-LRB-

dhfr

-RRB-

gene

cassette

-LSB-

-RSB-

followed

by

the

UTR

of

A

bp

fragment

was

amplified

by

PCR

from

P.

berghei

genomic

DNA

with

primers

pDNR-ΔMC-F

-LRB-

ACGAAGTTATCAGTCGACGGTACCCCATCATAAAGCAAAAAGC

-RRB-

and

pDNR-ΔMC-R

-LRB-

ATGAGGGCCCCTAAGCTTATTTAACATAAATTTTGTCCATTT

-RRB-

and

introduced

into

SalI/HindIII-digested

pDNR-EGFP

via

In-Fusion

PCR

cloning

The

resulting

plasmid

pDNR-ΔMC/EGFP

contains

two

loxP

sites

flanking

the

UTR

plus

first

codons

of

fused

in-frame

to

the

enhanced

green

fluorescent

protein

-LRB-

egfp

-RRB-

gene

followed

by

the

UTR

of

P.

berghei

dhfr

The

insert

contained

within

pDNR-ΔMC/EGFP

was

introduced

into

pLP-DHFR/MC

via

Cre-loxP

site-specific

recombination

-LRB-

BD

Biosciences

-RRB-

to

give

the

transfection

vector

pLP-ΔMC/EGFP

Prior

to

performing

transfections

this

plasmid

was

digested

with

KpnI

and

NotI

to

remove

the

vector

backbone

Parasite

transfection

pyrimethamine

selection

and

dilution

cloning

were

performed

as

previously

described

-LSB-

-RSB-

Genomic

DNA

extraction

and

Southern

blot

were

performed

as

previously

described

-LSB-

-RSB-

Apoptosis

assays

Phosphatidylserine

-LRB-

PS

-RRB-

translocation

was

studied

with

fluorescein

isothiocyanate

-LRB-

FITC

-RRB-

conjugated

Annexin

V

in

conjunction

with

propidium

iodide

by

using

the

Annexin-FITC

Apoptosis

Detection

Kit

-LRB-

Sigma

Aldrich

-RRB-

according

to

manufacturer

's

instructions

DNA

condensation

in

the

nucleus

was

assessed

using

acridine

orange

staining

as

described

-LSB-

-RSB-

DNA

fragmentation

was

assessed

by

in

situ

terminal

deoxynucleotidyl

transferase

mediated

dUTP-biotin

nick

end

labelling

-LRB-

TUNEL

-RRB-

by

using

the

ApopTag

Fluorescein

In

Situ

Apoptosis

Detection

Kit

-LRB-

Chemicon

International

-RRB-

according

to

manufacturer

's

instructions

Caspase

activation

was

assessed

with

sulphorhodamine-VAD-FMK

inhibitor

of

caspases

by

using

the

CaspaTag

Pan-Caspase

In

Situ

Assay

Kit

-LRB-

Chemicon

International

-RRB-

according

to

manufacturer

's

instructions

Results

Description

of

Plasmodium

metacaspases

Using

BLAST

searches

of

available

Plasmodium

genome

sequences

with

the

amino

acid

sequence

of

we

identified

orthologues

in

P.

berghei

and

other

Plasmodium

species

including

P.

chabaudi

P.

yoelii

P.

knowlesi

P.

vivax

and

P.

gallinaceum

Multiple

alignment

of

the

predicted

proteins

revealed

conserved

amino

and

carboxy-terminal

domains

separated

by

a

region

of

variable

length

and

sequence

These

amino

and

carboxy-terminal

domains

had

in

Pfam

and

SMART

searches

significant

homologies

with

a

domain

-LRB-

a

dependent

membrane

targeting

module

Pfam

identifier

-RRB-

and

with

the

Caspase

domain

-LRB-

Pfam

identifier

-RRB-

respectively

-LRB-

Fig.

-RRB-

We

also

identified

two

paralogues

of

-LRB-

named

and

-RRB-

in

all

Plasmodium

species

each

containing

a

region

of

significant

homology

to

the

Caspase

domain

in

Pfam

and

SMART

searches

In

contrast

to

the

proteins

however

sequence

conservation

in

the

much

larger

and

proteins

appeared

to

be

limited

to

their

C-terminal

regions

containing

the

putative

caspase

domains

-LRB-

Fig.

-RRB-

Multiple

alignment

of

the

predicted

caspase

domains

of

and

from

P.

berghei

P.

falciparum

and

P.

vivax

along

with

a

metacaspase

from

yeast

-LRB-

-RRB-

-LSB-

-RSB-

shed

further

light

on

their

structures

-LRB-

Fig.

-RRB-

Like

all

orthologues

possess

histidine

and

cysteine

residues

predicted

to

form

the

catalytic

dyad

in

this

family

of

proteases

It

is

interesting

to

note

that

has

the

predicted

catalytic

cysteine

located

one

residue

upstream

of

the

consensus

position

for

this

amino

acid

with

a

proline

in

the

consensus

position

-LRB-

Fig.

-RRB-

The

same

is

true

for

and

-LRB-

data

not

shown

-RRB-

In

contrast

has

two

adjacent

cysteines

at

the

consensus

position

and

the

preceding

residue

-LRB-

Fig.

-RRB-

The

same

is

found

in

and

-LRB-

data

not

shown

-RRB-

Catalytic

dyad

histidine

and

cysteine

residues

are

absent

in

the

consensus

positions

in

the

and

proteins

-LRB-

Fig.

-RRB-

However

and

have

a

cysteine

at

the

preceding

amino

acid

position

similar

to

the

situation

in

of

rodent

malaria

species

Generation

and

molecular

analyses

of

knockout

parasites

Because

proteins

display

the

greatest

sequence

similarity

with

the

consensus

domain

and

of

the

three

Plasmodium

metacaspases

described

here

are

therefore

most

likely

to

be

active

enzymes

we

decided

to

investigate

the

expression

of

and

its

contribution

to

P.

berghei

parasite

development

To

this

purpose

we

generated

genetically

modified

parasites

in

which

the

coding

sequence

was

removed

and

replaced

with

a

reporter

gene

enhanced

green

fluorescent

protein

-LRB-

EGFP

-RRB-

In

the

strategy

used

the

UTR

and

UTR

of

the

gene

allow

for

double

homologous

recombination

-LSB-

-RSB-

introducing

the

EGFP

coding

sequence

downstream

of

the

first

codons

of

-LRB-

coding

for

MSLQMDKIYVK

-RRB-

as

well

as

inserting

a

modified

T.

gondii

dihydrofolate

reductase/thymidylate

synthase

-LRB-

tgdhfr/ts

-RRB-

gene

cassette

conferring

resistance

to

the

antimalarial

drug

pyrimethamine

into

the

locus

-LRB-

Fig.

-RRB-

After

transfection

of

purified

P.

berghei

schizonts

-LSB-

-RSB-

we

readily

obtained

pyrimethamine-resistant

parasites

Diagnostic

PCR

amplification

across

the

predicted

integration

sites

showed

that

correct

integration

of

the

DHFR/TS

cassette

into

the

locus

had

occurred

-LRB-

data

not

shown

-RRB-

This

was

confirmed

by

assessing

the

integrity

of

a

clonal

population

of

the

genetically

modified

parasite

-LRB-

named

-RRB-

by

Southern

blot

analysis

of

HincII-digested

genomic

DNA

Accordingly

a

probe

corresponding

to

the

UTR

plus

coding

region

gave

rise

to

expected

bands

of

and

kb

in

the

parental

wild-type

-LRB-

WT

-RRB-

parasites

-LRB-

Fig.

-RRB-

In

contrast

in

the

parasites

expected

bands

of

and

kb

were

observed

The

kb

fragment

corresponds

to

bp

of

the

UTR

of

its

first

codons

plus

bp

of

the

egfp

gene

-LRB-

Fig.

-RRB-

The

and

kb

bands

are

more

weakly

labelled

than

the

kb

band

because

a

smaller

portion

of

the

probe

anneals

to

these

fragments

-LRB-

Fig.

-RRB-

A

probe

corresponding

to

the

tgdhfr/ts

gene

gave

rise

to

bands

of

and

kb

in

the

parasites

but

no

signal

in

the

WT

parasite

sample

as

expected

-LRB-

Fig.

-RRB-

These

combined

results

are

fully

consistent

with

the

structural

removal

of

the

coding

sequence

and

the

integration

of

the

reporter

and

selectable

marker

genes

into

the

locus

expression

and

loss-of-function

phenotype

parasites

developed

normally

in

mice

and

were

morphologically

indistinguishable

from

WT

parasites

in

Giemsa-stained

blood

smears

Using

UV

microscopy

EGFP

fluorescence

was

readily

observed

in

a

subpopulation

of

parasitized

erythrocytes

Upon

gametocyte

activation

-LRB-

by

placing

the

infected

blood

in

ookinete

medium

-RRB-

the

large

majority

-LRB-

-RRB-

of

fluorescent

parasites

emerged

from

the

host

cell

-LRB-

clearly

visible

by

the

rounding

up

of

the

parasite

-RRB-

by

min

typical

of

gametogenesis

The

number

of

remaining

intracellular

fluorescent

parasites

was

considerably

lower

than

the

percentage

of

trophozoites

present

in

the

sample

as

assessed

by

Giemsa

staining

These

are

therefore

likely

to

constitute

immature

gametocytes

or

gametocytes

that

had

failed

to

emerge

We

were

further

able

to

distinguish

between

male

and

female

gametocytes

by

observing

exflagellation

-LRB-

i.e.

the

release

of

male

gametes

-RRB-

this

involved

gametocytes

that

were

not

green

From

these

combined

observations

we

conclude

that

the

gene

is

expressed

only

in

female

gametocytes

-LRB-

Fig.

-RRB-

As

far

as

we

could

ascertain

all

females

were

positive

and

all

males

negative

Normal

differentiation

of

gametocytes

into

ookinetes

both

in

vitro

and

in

vivo

was

observed

These

ookinetes

again

displayed

bright

EGFP-based

fluorescence

under

UV

light

-LRB-

Fig.

-RRB-

WT

and

parasite-infected

A.

stephensi

mosquitoes

developed

comparable

numbers

of

oocysts

indicating

that

the

ookinetes

are

capable

of

normal

midgut

invasion

and

subsequent

ookinete-to-oocyst

transition

on

the

hemocoel

side

of

the

midgut

wall

Examination

of

oocysts

on

the

midgut

wall

by

UV

microscopy

displayed

EGFP

fluorescence

in

oocysts

and

the

sporozoites

contained

within

-LRB-

Fig.

-RRB-

Fluorescent

sporozoites

were

observed

in

the

mosquito

salivary

glands

-LRB-

Fig.

-RRB-

which

were

infectious

to

mice

upon

infected

mosquito

bite

These

combined

results

show

that

the

gene

is

expressed

in

female

gametocytes

and

all

downstream

mosquito

stages

including

sporozoites

but

not

in

the

asexual

blood

stages

The

apparently

normal

development

of

the

parasite

both

in

the

vertebrate

and

insect

hosts

shows

that

is

not

an

essential

gene

and

may

be

functionally

redundant

Apoptosis

in

ookinetes

It

has

been

reported

that

in

P.

berghei

a

large

proportion

of

ookinetes

undergo

cell

death

displaying

typical

apoptosis-like

features

such

as

PS

translocation

nuclear

condensation

and

DNA

fragmentation

-LSB-

-RSB-

As

caspases

are

well

known

to

play

a

central

role

in

apoptosis

we

wanted

to

assess

the

effect

of

knockout

on

this

process

Initially

we

assessed

the

level

of

apoptosis-like

cell

death

in

WT

ookinetes

Annexin

V

conjugated

to

FITC

-LRB-

staining

green

-RRB-

which

specifically

binds

to

PS

was

used

to

assess

the

proportion

of

ookinetes

displaying

PS

translocation

to

the

outer

leaflet

of

the

cell

membrane

Propidium

iodide

-LRB-

staining

red

-RRB-

was

used

simultaneously

to

assess

plasma

membrane

integrity

-LRB-

cell

viability

-RRB-

Across

experiments

conducted

we

observed

an

average

viable

ookinetes

-LRB-

no

staining

-RRB-

while

an

average

appeared

dead

-LRB-

red

only

staining

-RRB-

-LRB-

Fig.

-RRB-

The

average

proportion

of

ookinetes

displaying

PS

translocation

-LRB-

green

staining

-RRB-

was

less

than

Only

of

ookinetes

examined

showed

PS

translocation

and

were

propidium

iodide

negative

-LRB-

green

only

staining

-RRB-

-LRB-

Fig.

-RRB-

In

addition

to

this

assay

we

used

acridine

orange

staining

to

look

for

nuclear

condensation

and

TUNEL

to

assess

DNA

fragmentation

in

P.

berghei

ookinetes

as

described

-LSB-

-RSB-

We

found

no

evidence

for

either

of

these

processes

occurring

Given

the

low

numbers

of

positive

cells

in

the

above

assays

we

decided

to

look

for

the

activation

of

caspases

a

key

event

in

caspase-dependent

apoptosis

by

using

a

fluorochrome-labelled

inhibitor

of

caspases

-LRB-

CapaTag

-RRB-

In

mammalian

cells

these

labelled

inhibitors

are

permeant

and

covalently

bind

to

the

active

centres

of

caspases

with

a

stoichiometry

In

this

assay

WT

ookinetes

displayed

an

average

of

positive

cells

-LRB-

two

experiments

-RRB-

after

h

of

culture

which

at

h

had

gone

up

to

an

average

-LRB-

four

experiments

-RRB-

Under

the

same

conditions

ookinetes

showed

an

average

positive

cells

-LRB-

two

experiments

-RRB-

at

h

and

-LRB-

five

experiments

-RRB-

at

h

of

culture

We

also

used

a

green

fluorescent

P.

berghei

parasite

with

an

intact

gene

-LRB-

PbGFPCON

-LSB-

-RSB-

-RRB-

that

displayed

an

average

and

of

positive

ookinetes

at

and

h

respectively

Thus

knockout

of

does

not

appear

to

significantly

affect

the

percentage

of

ookinetes

that

bind

CaspaTag

Discussion

In

this

paper

we

describe

the

generation

of

P.

berghei

parasites

in

which

the

caspase-like

protein

is

replaced

by

the

fluorescent

reporter

EGFP

allowing

us

to

study

both

its

expression

profile

and

its

loss-of-function

phenotype

using

a

single

genetically

modified

parasite

line

Our

EGFP

reporter

data

show

the

gene

to

be

transcribed

and

probably

translated

in

female

gametocytes

and

all

downstream

mosquito

stages

including

sporozoites

but

not

in

asexual

blood

stages

However

we

can

not

rule

out

that

the

gene

may

be

subject

to

translational

repression

There

are

many

examples

of

genes

that

are

transcribed

in

the

female

gametocyte

but

which

have

a

translational

block

and

are

not

translated

until

later

in

development

as

was

recently

published

-LSB-

-RSB-

Interestingly

is

not

one

of

the

genes

identified

by

these

authors

supporting

a

scenario

of

direct

translation

Knockout

of

expression

does

not

seem

to

adversely

affect

any

stage

of

parasite

development

at

least

in

vivo

in

the

mouse

and

mosquito

or

in

the

culture

systems

used

in

this

study

This

was

surprising

because

is

a

highly

conserved

single

copy

Plasmodium

gene

and

because

is

predicted

to

possess

consensus

catalytic

dyad

residues

and

thus

is

likely

to

possess

caspase

activity

Although

the

two

paralogues

and

do

not

share

the

typical

consensus

catalytic

dyad

residues

of

the

Caspase

family

it

is

possible

that

they

constitute

active

zymogens

by

using

alternate

functionally

equivalent

catalytic

residues

Indeed

functional

replacement

between

cysteine

serine

or

threonine

residues

has

been

reported

for

other

cysteine

and

serine

proteases

-LSB-

-RSB-

If

this

is

the

case

it

is

conceivable

that

one

or

both

of

the

paralogues

could

carry

out

the

role

of

hence

leading

to

functional

redundancy

and

resulting

in

an

apparently

normal

mutant

phenotype

as

observed

Interestingly

five

metacaspase

genes

have

been

identified

in

Trypanosoma

brucei

and

two

of

these

also

lack

a

conserved

histidine

residue

-LRB-

replaced

by

a

tyrosine

-RRB-

and/or

cysteine

residue

-LRB-

replaced

by

a

serine

and

-RRB-

-LRB-

reviewed

in

-LSB-

-RSB-

-RRB-

Whether

any

of

these

molecules

are

active

enzymes

remains

to

be

determined

It

has

been

previously

shown

in

plants

and

fungi

-LSB-

-RSB-

that

metacaspases

can

be

involved

in

the

regulation

or

the

execution

of

programmed

cell

death

Because

an

apoptosis-like

mechanism

of

cell

death

was

reported

to

take

place

in

ookinetes

of

P.

berghei

-LSB-

-RSB-

we

hypothesized

that

could

be

involved

in

this

phenomenon

Of

three

typical

apoptotic

markers

PS

translocation

nuclear

condensation

and

DNA

fragmentation

only

PS

translocation

gave

positive

results

in

our

hands

PS

translocation

does

also

occur

when

cells

die

from

accidental

cell

death

-LRB-

oncosis

-RRB-

in

which

case

membrane

integrity

is

lost

concomitantly

Accordingly

we

can

not

truly

determine

whether

ookinetes

that

in

our

assay

stained

doubly

positive

for

both

Annexin

V

and

propidium

iodide

were

in

the

later

stages

of

apoptosis

or

whether

they

died

from

an

unrelated

death

mechanism

The

fact

that

a

subset

of

ookinetes

stained

positive

for

Annexin

V

suggests

that

the

low

numbers

of

positive

cells

observed

are

not

a

result

of

a

reduced

PS

content

in

the

parasite

plasma

membrane

Overall

the

results

based

on

these

three

apoptotic

markers

indicate

that

apoptosis-like

death

in

ookinetes

in

our

hands

is

low

When

we

monitored

for

potential

apoptotic

ookinetes

using

CaspaTag

we

obtained

up

to

positive

cells

Surprisingly

no

reduction

of

CaspaTag

labelling

was

observed

in

parasites

Although

is

the

most

likely

target

of

the

CaspaTag

inhibitor

it

is

possible

that

other

targets

of

CaspaTag

are

present

in

ookinetes

which

could

mask

any

reduction

in

CaspaTag

binding

in

the

parasite

Clearly

the

other

two

putative

metacaspases

are

prime

candidates

Indeed

P.

falciparum

transcriptome

and

proteome

analyses

indicate

that

is

expressed

in

gametocytes

and

sporozoites

-LSB-

-RSB-

Hence

its

expression

in

other

mosquito

stages

including

ookinetes

is

likely

This

scenario

could

also

explain

the

normal

development

of

the

mutant

parasites

by

assuming

a

level

of

functional

redundancy

between

the

three

Plasmodium

metacaspases

Expression

and

knock

out

experiments

with

the

other

metacaspases

are

underway

to

test

this

hypothesis

Caspase

activation

is

one

of

the

earliest

events

in

apoptotic

mammalian

cells

Hence

early

apoptotic

cells

have

activated

caspases

but

display

no

changes

in

plasma

membrane

integrity

-LSB-

-RSB-

It

was

therefore

notable

that

no

EGFP-based

fluorescence

was

observed

in

CaspaTag

positive

ookinetes

neither

in

the

nor

PbGFPCON

parasite

lines

indicating

that

these

cells

had

lost

plasma

membrane

integrity

before

CaspaTag

labelling

occurred

This

is

consistent

with

results

from

Al-Olayan

et

al.

-LSB-

-RSB-

who

reported

that

their

caspase

inhibitor-positive

ookinetes

were

propidium

iodide

permeable

Moreover

we

found

that

pre-treatment

with

the

unlabelled

inhibitor

failed

to

reduce

subsequent

binding

of

CaspaTag

-LRB-

data

not

shown

-RRB-

In

mammalian

cells

this

has

been

reported

to

reduce

CaspaTag

binding

by

over

-LSB-

-RSB-

We

should

thus

consider

the

possibility

that

these

caspase

inhibitors

may

have

different

specificities

in

Plasmodium

than

they

do

in

mammalian

cells

It

is

difficult

to

explain

why

in

our

hands

the

number

of

potentially

apoptotic

ookinetes

detected

was

far

below

the

numbers

previously

reported

-LSB-

-RSB-

Despite

numerous

attempts

we

were

unable

to

find

conditions

that

led

to

increased

numbers

of

ookinetes

displaying

apoptotic

markers

We

can

not

rule

out

that

small

differences

in

the

experimental

set-ups

might

explain

the

conflicting

findings

Alternately

the

level

of

apoptosis

detected

in

P.

berghei

ookinetes

may

depend

on

multiple

factors

that

are

still

to

be

defined

for

instance

the

exact

conditions

under

which

the

gametocytes

are

generated

in

the

mouse

host

A

not

dissimilar

discrepancy

also

exists

for

the

reported

apoptosis-like

DNA

fragmentation

that

occurs

in

blood

stage

P.

falciparum

parasites

in

response

to

chloroquine

treatment

-LSB-

-RSB-

an

observation

that

was

not

corroborated

in

a

more

recent

study

-LSB-

-RSB-

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

plasmodium berghei
metacaspase
apoptosis


Preconception and the Young Cancer Survivor Introduction While the diagnosis of cancer is not common in young men and women , cancer survivors younger than 40 are likely to be very concerned about the impact of their cancer on future fertility , their risk of cancer recurrence after treatment and risks to future offspring . It is estimated that 1 in 900 persons between 16 and 44 are cancer survivors -LSB- 1 -RSB- , and that there are at least 250,000 women age 40 and younger who are breast cancer survivors in the United States . As cancer survival increases , the focus on quality of life issues increases . When questioned , at least 73 % of young breast cancer survivors reported concerns about infertility due to cancer treatment , and 29 % reported that this concern impacted their choice of cancer treatment -LSB- 2 -RSB- . Premature ovarian failure In women , the options for preserving fertility and risk of ovarian failure depends on the age at the time the cancer is treated , the type and location of cancer , and the specific treatment chosen . Some treatments , such as bone marrow transplantation -LRB- BMT -RRB- are accompanied by very high rates -LRB- > 99 % -RRB- of ovarian failure -LSB- 3 -RSB- . Even for those women who resume normal menstruation after chemotherapy , premature ovarian failure -LRB- POF -RRB- is not uncommon -LSB- 4 -RSB- , and the risk of premature ovarian failure is 8-fold higher in cancer survivors -LSB- 5 -RSB- . The risk of POF increases with the woman 's age , with less than 20 % of women under 30 years experiencing POF , whereas most women older than 40 will become menopausal after chemotherapy -LSB- 6 -RSB- . POF and menopause are dependent on the type of therapy used . Rates of ovarian failure range from 15 % for acute myelocytic leukemia -LRB- AML -RRB- to 50 % for breast cancer , with the overall rate of ovarian failure of 34 % -LSB- 4 -RSB- . The chemotherapy agents with the highest risk for ovarian failure were the alkylating agents . The higher the dose of these drugs , the higher the likelihood of ovarian failure . Ovarian tissue damage is well documented with the use of radiation therapy . Radiotherapy is used for treatment of several cancer types affecting pre-menopausal women . The degree of damage to ovarian tissue is related to a woman 's age , the total dose of radiation used , and the number of exposures . In one study , when the same total dose of radiation was delivered , exposure to multiple fractionated doses versus a single dose resulted in less damage to the ovaries -LSB- 4 -RSB- . When used in conjunction with chemotherapy , the risk of POF increases . Byrne et al. assessed the risk of premature ovarian failure in childhood cancer survivors and found the relative risk of early menopause was 9.2 for those treated with chemotherapy alone , and 27 for those treated with radiation plus chemotherapy -LSB- 5 -RSB- . Fertility preservation techniques Although an increasing number of techniques have become available for the preservation of fertility , many are experimental and have not been tested in randomized controlled trials . Table 1 reviews the types of options available and their benefits and limitations . Table 1Fertility preservation options for women with malignanciesMethodBenefitsLimitationsIn vitro Fertilization -LRB- IVF -RRB- Most successful , widespreadDelay of therapy , use of fertility medicationsFrozen oocytesNo partner is neededDifficult to preserveOvarian tissue preservationLess tissue damage from freezing , can be used in pre-menarchal and young womenExperimentalTransposition of ovarian tissueLess radiation exposureLimited to women having radiation therapyPharmacological protectionLimit damage to ovarian tissueAdequate prospective studies needed In vitro fertilization -LRB- IVF -RRB- The best-studied and most successful option for preserving fertility in female cancer patients is ovarian stimulation followed by IVF and embryo cryopreservation . Ovarian stimulation needs to occur prior to cancer therapy because treatment can adversely affect ovarian reserve and response . The pregnancy rate with frozen embryos is lower compared to fresh IVF embryo transfer . A meta-analysis by the American Society for Reproductive Medicine reviewed over 88000 cycles of assisted reproductive technology -LRB- ART -RRB- treatment . When fresh oocytes were used , a delivery rate of 41.8 % was found in more than 6500 cycles analyzed , compared to a delivery rate of 18.6 % for frozen embryos -LSB- 7 -RSB- . One concern about ovarian stimulation with IVF is that it requires a delay of therapy , which could impact growth rate of tumors and survival rates . In addition , concerns have been voiced that the medications used to stimulate ovarian production could impact on estrogen-receptor positive tumors such as breast carcinoma . Anti-estrogens , such as tamoxifen , or aromatase inhibitors , -LRB- e.g. letrozole -RRB- , have both been used in ovarian stimulation in breast cancer patients to maintain low estradiol levels during IVF cycles . Frozen oocytes Frozen oocytes are more difficult to preserve , resulting in a lower survival , fertilization and pregnancy rates . Damage is in part due to ice formation during the freezing process . Unfertilized frozen oocytes have a low success rate for live birth -LRB- 2 % per oocyte -RRB- -LSB- 8 -RSB- , although the rates are now increasing . Rapid freezing of eggs by vitrification limits damage , which improves oocyte survival . Using intracytoplasmic sperm injection -LRB- ICSI -RRB- increases the fertilization rate even further . Ovarian tissue cryopreservation For pre-menarchal girls , young women with POF , women that can not delay therapy or those in whom high doses of fertility medications are contraindicated , ovarian tissue cryopreservation is offered in some centers . Unlike frozen oocytes , primordial follicles in ovarian tissue are smaller and have less follicular fluid , and therefore may be at less risk from damage from ice crystals that form during freezing . It is hoped that transplanting thawed ovarian tissue will result in the recovery of fertility . Transplantation should be used with caution in women with ovarian or metastatic cancer , to prevent transferring malignant cells back after treatment . This treatment is still considered experimental . Although success in animals has been demonstrated , transplantation of cryopreserved ovarian tissue has resulted in only two livebirths -LSB- 9 , 10 -RSB- . Ovarian transposition For women having pelvic radiation , transposition of ovarian tissue outside of the field of radiation successfully preserves ovarian function . The radiation dose to the transposed ovary is approximately 5 -- 10 % the level of the non-transposed ovary -LSB- 11 -RSB- . Pharmacological protection To limit or prevent chemotherapy-induced POF , some centers use gonadatropin releasing hormone -LRB- GnRH -RRB- agonists to reduce gonadatropins . This results in mimicking a pre-pubertal state , because the ovary appears to be less sensitive to gonadotoxic drugs prior to menarche . Because dividing cells are more susceptible to damage from cytotoxic agents , ovarian follicular cells are vulnerable . The mechanism of protection by GnRH agonists is unknown . One prospective clinical case series found that 70 of 75 women -LRB- 93 % -RRB- co-treated with GnRH agonists and chemotherapy resumed cyclic ovarian function verses 38 of 82 women -LRB- 46 % -RRB- treated with chemotherapy alone -LSB- 12 -RSB- . Adequately controlled prospective studies still are required , although data from animal studies are promising . Anti-apoptotic agents , such as sphingosine-1-phosphate -LRB- SIP -RRB- , have prevented radiation-induced oocyte loss in mice -LSB- 13 -RSB- . However , since apoptosis is needed for the treatment of cancer , this method faces some obstacles for implementation . Male fertility In men , sperm banking is routine and should be offered to all men prior to undergoing cancer treatment . When combined with intracytoplamic sperm injection -LRB- ICSI -RRB- , sperm banking should preserve fertility for almost all males . There is no increased risk for birth defects when sperm is banked prior to therapy -LSB- 14 , 15 -RSB- . Even in azoospermic males , testicular sperm extraction -LRB- TESE -RRB- with ICSI has resulted in successful pregnancies . By 2005 , 11 of 29 men with azoospermia that had used TESE with ICSI had achieved a pregnancy which resulted in a livebirth -LSB- 16 -RSB- . For pre-pubertal males , sperm banking is not available because there is no spermatogenesis . The possibility of cryobanking of testicular tissue is being explored -LSB- 16 , 17 -RSB- . An increase in sperm aneuploidy is seen up to 18 months post-chemotherapy , so it has been suggested that a delay of conception for 12 -- 18 months after therapy is completed is reasonable -LSB- 18 -RSB- . Pregnancy after cancer Once a woman has recovered from cancer treatment , no clear guidelines exist about the length of time to wait before attempting to become pregnant . Effect of cancer treatment on pregnancy Once a cancer survivor achieves pregnancy , her health and the fetal health may be adversely affected by her prior treatment for cancer . Clinically significant heart disease occurs in 0.5 -- 1 % of women treated with anthracycline-based chemotherapy drugs , and may not develop until months or years after therapy is completed -LSB- 19 -RSB- . Also , women who have radiation to the left side of the abdomen or chest are at risk for heart damage , which could worsen during pregnancy or labor . Women with radiation to the abdomen or pelvic area or total body radiation are at increased risk for miscarriage , preterm birth and delivery of low birthweight infants -LSB- 20 -RSB- . A report from the Childhood Cancer Survivor Study -LRB- CCSS -RRB- did not show any increase in adverse pregnancy outcomes in women treated with most chemotherapy agents . However , an increased risk of stillbirth in women treated with abdominal radiation for Wilm 's tumor has been noted -LSB- 21 -RSB- . Women who have completed treatment for breast cancer are often placed on selective estrogen receptor modulators -LRB- SERMs -RRB- such as tamoxifen to reduce the incidence of a second cancer . However , tamoxifen use during pregnancy is to be avoided because it is potentially teratogenic . Effect of pregnancy on cancer Some women are concerned that the hormonal changes from pregnancy may increase the risk of cancer recurrence , but pregnancy after breast cancer management does not increase the risk of cancer recurrence or cancer-related death -LSB- 22 , 23 -RSB- . However , women who are carriers of BRCA1 or BRCA2 germline mutations and who have had a full-term pregnancy are at significantly higher risk of developing breast cancer by age 40 than mutation carriers who do not have children -LSB- 24 -RSB- . The risk for breast cancer is increased by 70 % in the first 2 years after pregnancy for BRCA2 carriers , but not BRCA1 carriers -LSB- 25 -RSB- . Risk to offspring Since cancer treatment by radiation and many chemotherapy agents are known to cause DNA mutations , cancer survivors are often concerned that the treatment may increase the risk for birth defects , genetic conditions or cancer in their offspring . Several studies have found no increased risk of congenital abnormalities in the offspring of cancer survivors . Epidemiologic data from Japanese survivors of the atomic bomb and their children did not show any evidence for new mutations . The CCSS also did not find any link between radiation therapy or chemotherapy for childhood cancer and genetic diseases or birth defects -LSB- 26 , 27 -RSB- . Risk of cancer in offspring No increased risk for childhood cancer was identified in the offspring of childhood cancer survivors , but additional follow-up is needed -LSB- 26 -RSB- . In rare instances , the childhood cancer may be hereditary , with a risk of passing on the mutated gene to the offspring . Some cancers , such as breast , colon or ovarian cancer , are more likely to be hereditary when the cancer occurs at an earlier age . For women with breast cancer diagnosed prior to age 40 and no other family history of early breast cancer , the risk of a mutation in one of the hereditary breast cancer genes , BRCA1 or BRCA2 is estimated to be at least 13 % -LSB- 28 -RSB- . Summary All women should be counseled about the impact of cancer treatment on fertility prior to undergoing chemotherapy , ovarian surgery or BMT , and offered fertility preservation options , of which ovarian stimulation with IVF is the most successful . Once women achieve pregnancy , the risk for birth defects or genetic conditions is not increased because of either radiation or chemotherapy . Because treatment options may increase the risk for adverse pregnancy outcome , review of therapy records is essential for determining potential risk to the pregnancy . For most young cancer survivors , fertility can be preserved and a successful pregnancy achieved , but discussing these issues prior to treatment is essential .

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

preconception
fertility preservation
ovarian failure
pregnancy after cancer


Health

Care

Provider

Knowledge

and

Practices

Regarding

Folic

Acid

United

States

Objective

To

assess

health

care

providers

-LRB-

HCP

-RRB-

knowledge

and

practices

regarding

folic

acid

-LRB-

FA

-RRB-

use

for

neural

tube

defect

-LRB-

NTD

-RRB-

prevention

Methods

Two

identical

surveys

were

conducted

among

obstetricians/gynecologists

-LRB-

OB/GYNs

-RRB-

and

family/general

physicians

-LRB-

FAM/GENs

-RRB-

-LRB-

-RRB-

and

physician

assistants

-LRB-

PAs

-RRB-

nurse

practitioners

-LRB-

NPs

-RRB-

certified

nurse

midwives

-LRB-

CNMs

-RRB-

and

registered

nurses

-LRB-

-RRB-

to

ascertain

knowledge

and

practices

regarding

FA

For

analysis

T-tests

univariate

and

multivariate

logistic

regression

modeling

were

used

Results

Universally

providers

knew

that

FA

prevents

birth

defects

Over

knew

when

a

woman

should

start

taking

folic

acid

for

the

prevention

of

NTDs

and

over

knew

FA

supplementation

beyond

what

is

available

in

the

diet

is

necessary

However

only

half

knew

that

of

all

pregnancies

in

the

United

States

are

unplanned

Women

heard

information

about

multivitamins

or

FA

most

often

during

well

woman

visits

in

obstetrical/gynecology

-LRB-

ob/gyn

-RRB-

practice

settings

-LRB-

-RRB-

and

about

of

the

time

during

well

woman

visits

in

family/general

-LRB-

fam/gen

-RRB-

practice

settings

and

of

the

time

at

gynecology

visits

-LRB-

both

settings

-RRB-

Among

all

providers

did

not

know

the

correct

FA

dosage

-LRB-

μg

daily

-RRB-

HCPs

taking

multivitamins

were

more

than

twice

as

likely

to

recommend

multivitamins

to

their

patients

-LRB-

Odds

Ratio

-LSB-

OR

-RSB-

Confidence

Interval

-LSB-

CI

-RSB-

-RRB-

HCPs

with

lower

income

clients

-LRB-

OR

CI

-RRB-

and

HCPs

with

practices

having

more

than

minorities

-LRB-

OR

CI

-RRB-

were

more

likely

to

recommend

supplements

NPs

in

ob/gyn

settings

were

most

likely

and

FAM/GENs

were

least

likely

to

recommend

supplements

-LRB-

OR

CL

and

OR

CL

respectively

-RRB-

Conclusions

Knowledge

about

birth

defects

and

the

necessity

of

supplemental

FA

was

high

Increasing

knowledge

about

unintended

pregnancy

rates

and

correct

dosages

of

FA

is

needed

The

strongest

predictor

for

recommending

the

use

of

FA

supplements

was

whether

the

provider

took

a

multivitamin

Introduction

Birth

defects

continue

to

be

the

leading

cause

of

infant

morta-lity

in

the

United

States

-LSB-

-RSB-

and

those

of

the

nervous

system

including

neural

tube

defects

-LRB-

NTDs

-RRB-

are

the

third

leading

type

of

birth

defect

-LSB-

-RSB-

NTDs

are

serious

birth

defects

of

the

brain

-LRB-

anencephaly

-RRB-

and

spine

-LRB-

spina

bifida

-RRB-

affecting

appro-ximately

pregnancies

each

year

in

the

United

States

-LSB-

-RSB-

Studies

show

that

of

these

birth

defects

can

be

prevented

if

a

woman

consumes

adequate

folic

acid

-LRB-

FA

-RRB-

daily

before

conception

and

throughout

the

first

trimester

of

pregnancy

-LSB-

-RSB-

The

U.S.

Public

Health

Service

-LRB-

USPHS

-RRB-

-LRB-

-RRB-

and

Institute

of

Medicine

-LRB-

IOM

-RRB-

-LRB-

-RRB-

issued

separate

recommendations

that

all

women

of

childbearing

age

consume

μg

of

FA

daily

for

the

prevention

of

NTDs

-LSB-

-RSB-

Women

can

obtain

adequate

amounts

of

FA

by

taking

a

folic

acid

supplement

taking

a

multivitamin

containing

folic

acid

or

by

eating

cereal

grain

products

fortified

with

of

the

RDA

of

folic

acid

In

the

U.S.

Food

and

Drug

Administration

required

mandatory

fortification

of

enriched

cereal

grains

with

μg

of

FA

per

g

of

cereal

grain

product

in

an

effort

to

reduce

the

occurrence

of

NTDs

-LSB-

-RSB-

It

was

estimated

that

FA

fortification

at

this

level

would

provide

about

mg

of

additional

folic

acid

in

the

average

diet

Recent

evidence

from

the

literature

suggests

that

fortification

has

in

fact

doubled

this

initial

projection

-LSB-

-RSB-

Fortification

of

cereal

grain

products

has

resulted

in

a

reduction

in

NTDs

in

the

United

States

-LSB-

-RSB-

which

is

relatively

consistent

with

the

observed

population

's

increase

of

folic

acid

by

mg/d

-LSB-

-RSB-

While

fortification

has

made

a

significant

contribution

in

preventing

NTDs

most

women

in

the

U.S.

still

do

not

get

enough

FA

from

fortification

alone

In

a

recent

study

examining

serum

folate

levels

of

women

of

childbearing

age

post

fortification

less

than

of

women

achieved

a

red

blood

cell

folate

level

that

is

associated

with

a

significant

decrease

of

NTDs

through

diet

alone

-LSB-

-RSB-

Therefore

it

is

still

necessary

for

women

of

childbearing

age

to

consume

vitamins

containing

FA

to

get

the

daily

recommended

amount

of

FA

-LRB-

μg

-RRB-

Since

the

March

of

Dimes

-LRB-

MoD

-RRB-

and

the

Gallup

Organization

have

conducted

an

annual

national

survey

among

women

of

childbearing

age

regarding

their

awareness

knowledge

and

consumption

of

FA

Early

results

indicated

a

lack

of

awareness

about

FA

among

women

of

childbearing

age

In

in

response

to

these

results

the

MoD

the

Centers

for

Disease

Control

and

Prevention

-LRB-

CDC

-RRB-

and

the

National

Council

on

Folic

Acid

-LRB-

NCFA

-RRB-

began

a

concerted

effort

to

increase

FA

awareness

through

education

campaigns

targeting

women

and

health

care

providers

-LRB-

HCPs

-RRB-

Based

on

the

MoD

survey

FA

awareness

in

women

of

childbearing

age

has

risen

from

in

to

in

-LSB-

-RSB-

Lagging

behind

the

increase

in

awareness

daily

consumption

of

a

multivitamin

containing

FA

increased

only

slightly

from

in

to

in

-LSB-

-RSB-

When

asked

what

types

of

things

would

encourage

them

to

take

a

multivitamin

daily

unprompted

of

women

who

were

aware

of

but

not

consuming

FA

said

they

would

take

a

multivitamin

if

advised

to

do

so

by

an

HCP

-LSB-

-RSB-

Additionally

in

the

HealthStyles

Survey

of

women

through

years

of

age

agreed

or

strongly

agreed

that

they

would

take

a

multivitamin

daily

to

prevent

birth

defects

during

future

pregnancies

if

their

doctor

encouraged

them

to

do

so

-LRB-

unpublished

data

-RRB-

-LSB-

-RSB-

Two

recent

studies

examined

HCP

practices

regarding

FA

in

the

United

States

-LSB-

-RSB-

While

providers

were

generally

aware

of

folic

acid

's

role

in

NTD

prevention

only

half

of

providers

surveyed

in

both

studies

discussed

folic

acid

on

a

regular

basis

with

their

patients

of

childbearing

age

In

the

MoD

in

partnership

with

the

CDC

initiated

this

project

to

examine

HCP

practices

regarding

FA

The

objectives

of

this

research

are

to

Assess

HCP

knowledge

of

FA

recommendations

including

dosage

and

timing

for

prevention

of

NTDs

among

different

provider

types

obstetricians/gynecologists

-LRB-

OB/GYNs

-RRB-

family/general

physicians

-LRB-

FAM/GENs

-RRB-

nurse

practitioners

-LRB-

NPs

-RRB-

certified

nurse

midwives

-LRB-

CNMs

-RRB-

physician

assistants

-LRB-

PAs

-RRB-

and

registered

nurses

-LRB-

RNs

-RRB-

Assess

current

practices

among

HCPs

with

respect

to

recommending

FA

to

women

of

childbearing

age.Identify

the

best

means

of

supporting

HCP

efforts

to

promote

FA

consumption

in

female

patients

of

childbearing

age

Methods

Random

sample

telephone

surveys

lasting

an

average

of

minutes

in

length

were

conducted

in

and

among

HCPs

working

in

either

obstetrical/gynecology

-LRB-

ob/gyn

-RRB-

practice

settings

or

family/general

-LRB-

fam/gen

-RRB-

practice

settings

These

practice

settings

were

selected

because

they

represent

the

settings

where

the

highest

proportion

of

women

of

childbearing

age

receive

care

on

a

regular

basis

Geographic

quotas

were

established

to

ensure

a

representation

of

respondents

proportional

to

the

actual

distribution

of

office-based

providers

nationwide

Providers

were

identified

through

random

sampling

lists

purchased

from

American

Medical

Information

Inc.

a

division

of

InfoUSA

To

identify

sufficient

representation

of

providers

based

in

federally

qualified

health

centers

a

list

of

centers

from

the

Health

Resources

and

Services

Administration

's

Bureau

of

Primary

Health

Care

website

-LRB-

-RRB-

was

added

to

the

sampling

frame

The

identities

of

respondents

were

not

revealed

This

research

met

the

requirements

of

Title

CFR

part

for

the

protection

of

human

subjects

To

encourage

participation

respondents

were

offered

a

honorarium

The

identity

of

the

sponsors

-LRB-

CDC

and

MoD

-RRB-

was

not

divulged

to

respondents

A

cover

letter

from

the

research

firm

was

faxed

to

participants

who

requested

additional

background

information

about

the

survey

Health

care

providers

were

eligible

to

participate

if

they

personally

saw

adult

female

patients

for

office

or

clinic

visits

and

worked

in

ob/gyn

or

fam/gen

medicine

practices

The

surveys

were

presented

as

a

series

of

true/false

multiple

choice

and

open-ended

questions

The

survey

sought

to

determine

the

level

of

HCP

knowledge

regarding

the

role

of

FA

in

the

prevention

of

NTDs

when

HCPs

recommended

FA

and

how

often

HCPs

recommended

FA

to

women

of

childbearing

age

Data

were

analyzed

using

SPSS

software

-LRB-

SPSS

SPSS

Inc.

Chicago

IL

-RRB-

T-tests

univariate

comparisons

and

multivariate

logistic

regression

modeling

of

the

data

were

conducted

All

analyses

were

tested

for

significance

at

the

confidence

level

-LRB-

p-value

-RRB-

Results

Investigators

surveyed

physician

HCPs

in

and

nonphysician

HCPs

in

-LRB-

Table

-RRB-

Table

type

and

practice

settingPractice

settingObstetrics/Family/GeneralProvider

missing

provider

variables

not

categorized.aProvider

type

abbreviations

OB/GYN

obstetrician/gynecologist

FAM/GEN

family

or

general

practice

physician

PA

physician

assistant

NP

nurse

practitioner

CNM

certified

nurse

midwife

RN

registered

nurse

In

physicians

were

screened

as

potentially

qualified

respondents

Of

these

physicians

-LRB-

-RRB-

did

not

participate

in

surveys

-LRB-

-RRB-

requested

additional

information

but

did

not

schedule

an

interview

-LRB-

-RRB-

did

not

meet

practice

setting

requirements

-LRB-

-RRB-

had

fax

numbers

as

the

primary

contact

-LRB-

-RRB-

were

qualified

refusals

and

-LRB-

-RRB-

had

appointments

set

but

not

kept

The

response

rate

for

eligible

physicians

was

In

nonphysician

HCPs

were

screened

as

potentially

qualified

respondents

Of

these

nonphysician

HCPs

-LRB-

-RRB-

did

not

participate

in

surveys

and

-LRB-

-RRB-

were

qualified

refusals

The

response

rate

for

eligible

nonphysician

HCPs

was

The

majority

of

physicians

were

male

-LRB-

-RRB-

younger

than

years

of

age

-LRB-

-RRB-

and

in

practice

over

years

-LRB-

-RRB-

Nonphysician

HCPs

were

typically

female

-LRB-

-RRB-

and

younger

than

years

of

age

-LRB-

-RRB-

had

been

in

practice

over

years

Nonphysician

HCPs

were

two

times

more

likely

than

physician

HCPs

to

work

with

underserved

populations

-LRB-

Odds

Ratio

-LSB-

OR

-RSB-

Confidence

Interval

-LSB-

CI

-RSB-

-RRB-

Nonphysician

HCPs

saw

more

women

of

childbearing

age

in

their

practices

-LRB-

-RRB-

than

physicans

-LRB-

-RRB-

-LRB-

p-value

-RRB-

The

following

results

were

obtained

from

a

series

of

true/false

questions

In

general

knowledge

that

women

need

a

supplemental

source

of

FA

was

high

-LRB-

-RRB-

Universally

HCPs

in

both

practice

settings

-LRB-

-RRB-

knew

correctly

that

FA

can

prevent

some

birth

defects

However

only

of

all

providers

knew

that

most

grain

products

in

the

United

States

are

fortified

with

FA

In

addition

there

were

significant

differences

in

response

to

this

question

among

provider

types

according

to

practice

setting

ranging

from

of

RNs

in

ob/gyn

settings

to

of

PAs

in

fam/gen

settings

-LRB-

p-value

-RRB-

The

following

data

were

obtained

from

multiple

choice

questions

Providers

in

ob/gyn

settings

were

not

more

knowledgeable

than

providers

in

fam/gen

settings

about

when

a

woman

should

start

taking

FA

for

the

prevention

of

NTDs

-LRB-

at

least

one

month

prior

to

conception

through

the

first

trimester

of

pregnancy

-RRB-

-LRB-

vs.

respectively

p-value

-RRB-

Because

the

majority

of

respondents

knew

the

correct

timing

for

a

woman

to

start

taking

FA

providers

were

asked

to

identify

what

percentage

of

pregnancies

they

thought

were

unintended

in

the

United

States

Only

about

half

of

HCPs

in

both

settings

knew

that

approximately

of

all

pregnancies

in

the

United

States

are

unplanned

-LSB-

-RSB-

The

most

knowledgeable

group

was

NPs

in

ob/gyn

settings

-LRB-

-RRB-

and

the

least

knowledgeable

group

was

RNs

in

ob/gyn

settings

-LRB-

-RRB-

-LRB-

p-value

-RRB-

Providers

answering

incorrectly

were

more

likely

to

underestimate

rather

than

overestimate

the

unintended

pregnancy

rate

Although

a

woman

's

receptiveness

to

the

FA

message

might

increase

during

a

preconception

care

visit

HCPs

providing

prenatal

care

saw

only

a

quarter

of

their

pregnant

patients

for

a

preconception

care

visit

Over

half

-LRB-

-RRB-

of

all

providers

were

knowledgeable

about

the

amount

of

FA

-LRB-

μg

-RRB-

a

woman

needs

daily

for

prevention

of

NTDs

Providers

in

ob/gyn

settings

were

more

knowledgeable

than

providers

in

fam/gen

settings

The

most

knowledgeable

group

was

CNMs

with

knowing

the

correct

daily

dosage

of

FA

the

least

knowledgeable

group

was

FAM/GEN

physicians

-LRB-

-RRB-

-LRB-

p-value

-RRB-

When

asked

about

the

amount

of

FA

a

woman

with

a

prior

NTD

affected

pregnancy

should

take

to

prevent

the

recurrence

of

a

NTD

-LRB-

mg

-RRB-

overall

knowledge

dropped

dramatically

with

only

about

of

all

providers

knowing

the

correct

dose

Providers

in

ob/gyn

settings

were

more

knowledgeable

than

providers

in

fam/gen

settings

The

most

knowledgeable

group

was

OB/GYNs

-LRB-

-RRB-

and

the

least

knowledgeable

were

RNs

in

fam/gen

settings

-LRB-

-RRB-

-LRB-

p-value

-RRB-

Of

critical

importance

to

increasing

consumption

of

FA

among

women

of

childbearing

age

is

whether

FA

is

incorporated

into

preventive

health

care

messages

during

provider/patient

encounters

Providers

were

asked

if

they

always

usually

sometimes

or

never

addressed

multivitamins

or

FA

during

well-woman

examinations

More

providers

in

ob/gyn

settings

than

in

fam/gen

settings

stated

they

regularly

-LRB-

always/usually

-RRB-

addressed

multivitamins

or

FA

during

well-woman

visits

-LRB-

vs.

respectively

-RRB-

Women

were

times

more

likely

to

hear

about

multivitamins

or

FA

during

encounters

in

ob/gyn

settings

than

in

fam/gen

settings

-LRB-

CI

-RRB-

The

group

most

likely

to

talk

to

patients

about

multivitamins

and

FA

was

NPs

in

ob/gyn

settings

-LRB-

-RRB-

the

least

likely

were

FAM/GEN

physicians

-LRB-

-RRB-

-LRB-

p-value

-RRB-

Fewer

counseling

opportunities

were

taken

at

other

types

of

examinations

Women

heard

the

FA

message

about

half

the

time

from

providers

in

both

settings

at

routine

gynecology

visits

As

expected

the

fewest

counseling

opportunities

taken

were

at

episodic

visits

in

both

settings

Predictors

for

recommending

a

multivitamin

at

a

well-woman

visit

were

determined

The

type

of

provider

and

clinic

setting

variables

were

combined

as

the

base

variable

with

OB/GYNs

as

the

referent

group

The

dependant

variable

was

whether

the

HCP

always

or

usually

recommended

a

multivitamin

during

a

well-woman

visit

Variables

examined

were

number

of

weekly

visits

provider

age

sex

personal

vitamin

use

provider

's

years

in

practice

ethnicity

of

patients

practice

setting

socioeconomic

status

of

patient

base

patient

age

clinic

setting

-LRB-

urban

suburban

or

rural

-RRB-

and

region

of

practice

Clinic

setting

-LRB-

urban

suburban

or

rural

-RRB-

provider

age

region

of

practice

provider

's

years

in

practice

number

of

weekly

visits

and

patient

age

variables

were

not

statistically

significant

in

univariate

analysis

Table

represents

the

variables

included

in

the

logistic

regression

model

In

comparison

with

OB/GYNs

FAM/GEN

physicians

were

statistically

less

likely

-LRB-

OR

CI

-RRB-

to

recommend

multivitamins

while

NPs

in

ob/gyn

settings

were

three

times

more

likely

-LRB-

OR

CI

-RRB-

than

their

OB/GYN

physician

counterparts

to

provide

counsel

on

multivitamins

While

not

quite

reaching

significance

CNMs

were

times

more

likely

-LRB-

OR

CI

-RRB-

than

OB/GYNs

to

provide

counsel

on

multivitamins

All

other

groups

were

not

significantly

different

from

OB/GYNs

Interestingly

whether

a

provider

personally

took

a

multivitamin

was

one

of

the

strongest

predictors

of

recommending

multivitamin

use

to

patients

Providers

who

regularly

took

a

multivitamin

were

times

more

likely

-LRB-

OR

CI

-RRB-

to

recommend

multivitamin

use

to

their

patients

Additional

predictors

of

recommending

multivitamin

use

were

whether

a

provider

stated

that

his

or

her

practice

's

patient

base

was

classified

as

poverty

or

low

income

or

if

the

practice

consisted

of

at

least

minorities

Being

a

female

provider

was

also

correlated

with

recommending

multivitamin

use

to

women

of

child-bearing

age

Table

associated

with

recommending

multivitamins

at

well-woman

visits

included

in

multivariate

logistic

regression

modelVariablesOdds

Confidence

intervalProvider

Type

OB/GYNReferentReferent

NP

in

ob/gyn

PA

in

ob/gyn

RN

in

ob/gyn

NP

in

fam

prac

PA

in

fam

prac

RN

in

fam

prac

Gender

MaleReferentReferent

Provider

takes

a

multivitamin

NoReferentReferent

SES

of

patient

base

Middle

or

high

incomeReferentReferent

Poverty

or

low

Minorities

minoritiesReferentReferent

Denotes

statistical

significance

Approximately

three-quarters

of

all

respondents

had

seen

information

about

FA

in

the

last

year

Unprompted

both

physician

and

nonphysician

HCPs

reported

that

the

organization

most

likely

to

have

provided

that

information

was

the

MoD

-LRB-

and

respectively

-RRB-

Medical

journals

were

identified

most

frequently

as

a

way

to

effectively

reach

providers

with

information

about

FA

-LRB-

physicians

nonphysicians

-RRB-

Nonphysician

providers

reported

the

Internet

as

the

second

most

effective

way

-LRB-

-RRB-

and

physicians

reported

either

medical

associations

or

conferences

as

the

second

most

effective

way

to

reach

them

with

information

about

FA

-LRB-

and

respectively

-RRB-

Providers

identified

up

to

two

types

of

resources

they

would

like

to

have

to

promote

FA

intake

among

patients

Printed

materials

were

the

most

popular

type

of

resource

requested

by

HCPs

Sixty

percent

-LRB-

-RRB-

of

physician

and

of

nonphysician

providers

requested

brochures

to

distribute

to

their

patients

Additional

print

resource

requests

included

charts

and

articles

Free

vitamins

for

patients

were

also

requested

-LRB-

data

not

shown

-RRB-

Discussion

These

findings

suggest

that

physician

and

nonphysician

providers

in

both

ob/gyn

and

fam/gen

settings

know

that

FA

can

prevent

NTDs

and

that

FA

must

be

used

from

at

least

one

month

before

pregnancy

and

continued

through

the

first

trimester

Consistent

with

other

studies

provider

knowledge

of

FA

did

not

necessarily

translate

into

counseling

patients

about

its

benefits

-LSB-

-RSB-

Providers

in

ob/gyn

settings

were

almost

twice

as

likely

as

providers

in

fam/gen

settings

to

regularly

address

multivitamin

or

FA

use

during

well-woman

visits

Although

NPs

and

CNMs

in

ob/gyn

settings

were

most

likely

to

regularly

address

multivitamin

or

FA

use

with

their

patients

these

providers

might

not

have

as

many

opportunities

to

counsel

women

who

are

not

contemplating

pregnancy

Because

providers

in

fam/gen

settings

provide

routine

care

to

large

numbers

of

women

of

childbearing

age

they

play

an

important

role

in

counseling

women

about

FA

consumption

Healthy

People

calls

for

an

increase

in

the

number

of

pregnancies

begun

with

an

optimum

FA

level

Providers

in

fam/gen

settings

are

critical

participants

in

the

process

of

making

the

goals

of

Healthy

People

a

reality

-LSB-

-RSB-

Greater

opportunities

for

promoting

FA

exist

through

changing

provider

practice

behavior

in

fam/gen

settings

It

also

appears

that

well-woman

examinations

in

fam/gen

settings

and

routine

gynecology

visits

in

both

settings

are

important

yet

underutilized

opportunities

for

HCPs

to

counsel

women

about

multivitamin

and

FA

use

The

episodic

visit

was

the

least

utilized

but

with

all

the

competing

priorities

at

these

types

of

encounters

counseling

of

multivitamins

might

not

take

precedence

over

other

topics

to

be

covered

within

limited

timeframes

Providers

in

ob/gyn

settings

were

more

likely

than

providers

in

fam/gen

settings

to

know

the

correct

amount

of

FA

needed

for

NTD

prevention

-LRB-

μg

daily

-RRB-

Less

than

a

third

of

all

providers

knew

the

correct

amount

of

FA

needed

for

the

prevention

of

recurrence

-LRB-

mg

-RRB-

Because

women

with

a

previous

NTD-affected

pregnancy

are

at

an

increased

risk

for

another

NTD-affected

pregnancy

it

is

important

for

all

providers

to

know

the

correct

amount

of

FA

needed

for

recurrence

prevention

Only

about

half

of

providers

in

both

practice

settings

knew

that

approximately

of

pregnancies

in

the

United

States

are

unplanned

Without

screening

their

female

patients

about

pregnancy

intention

perhaps

providers

should

be

recommending

FA

to

all

women

of

childbearing

age

One

of

the

most

interesting

findings

was

that

the

providers

surveyed

practice

what

they

preach

Providers

who

regularly

took

multivitamins

were

more

likely

to

recommend

multivitamins

to

their

patients

Studies

looking

at

other

health

behaviors

have

shown

this

as

well

-LSB-

-RSB-

Thus

including

personal

health

behavior

change

messages

in

HCP

outreach

about

multivitamins

and

FA

might

be

advantageous

In

this

dataset

a

practice

that

served

predominantly

poverty

or

low

income

-LRB-

low

socioeconomic

status

-RRB-

clientele

or

a

practice

consisting

of

at

least

minorities

predicted

recommending

a

multivitamin

Among

minorities

and

women

with

low

socio-economic

status

critical

gaps

in

knowledge

and

practice

on

the

benefits

of

periconceptional

folic

acid

have

been

documented

especially

among

Hispanics

who

are

most

vulnerable

for

NTDs

-LSB-

-RSB-

The

findings

in

this

survey

that

minorities

and

women

of

lower

socio-economic

status

do

appear

to

be

targeted

is

encouraging

and

underscores

the

importance

of

preconception

care

education

programs

for

healthcare

providers

serving

high-risk

populations

This

study

has

certain

limitations

Physician

HCPs

had

a

low

response

rate

compared

to

nonphysician

HCPs

We

can

not

comment

on

the

providers

who

did

not

respond

to

the

survey

Providers

answering

the

survey

might

differ

in

knowledge

and

practice

than

non-responding

providers

Additionally

one

provider

group

surveyed

-LRB-

PAs

-RRB-

had

small

numbers

Many

of

the

variables

examined

were

associated

with

one

another

In

the

logistic

regression

modeling

female

sex

of

the

HCP

was

a

significant

factor

in

predicting

recommending

multivitamins

to

female

patients

of

childbearing

age

This

is

most

likely

because

the

majority

of

the

providers

surveyed

in

were

women

Conclusions

While

it

would

be

most

beneficial

for

women

of

childbearing

age

to

get

adequate

folic

acid

from

fortified

foods

alone

alternatives

include

efforts

to

educate

all

childbearing

aged

women

Several

approaches

to

educating

and

promoting

folic

acid

consumption

are

endorsed

by

the

U.S.

Public

Health

Service

These

approaches

include

direct

to

consumer

marketing

public

health

awareness

activities

health

care

provider

outreach

and

policy

strategies

This

study

examined

just

one

aspect

-LRB-

HCPs

-RRB-

of

the

multifaceted

approach

needed

to

increase

FA

intake

among

women

of

childbearing

age

Collaboration

between

public

health

and

medical

care

services

is

critical

in

educating

women

about

FA

and

multivitamin

use

as

health

care

providers

are

trusted

professionals

Mobilizing

folic

acid

awareness

efforts

to

target

HCPs

might

assist

providers

with

patient

preconception

care

education

Training

providers

while

in

school

or

residency

programs

or

by

offering

continuing

education

courses

that

cover

methods

of

applied

behavior

change

and

health

education

might

be

useful

for

increasing

the

numbers

of

women

hearing

about

folic

acid

from

their

health

care

provider

Additionally

further

research

on

what

barriers

HCPs

experience

when

providing

preventive

care

such

as

provider

time

constraints

self-efficacy

and

what

their

perceptions

are

on

the

benefits

of

preventive

care

is

needed

As

many

preventive

behavior

and

wellness

messages

exist

providers

are

forced

to

prioritize

according

to

the

length

of

and

reason

for

patient

visits

Finding

a

creative

way

to

make

FA

counseling

a

priority

is

critical

to

making

the

FA

message

a

consistent

part

of

the

patient/health

care

provider

dialogue

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

health care provider
folic acid
knowledge
behavior