amniotic infection syndrome: nosology and reproducibility...

Amniotic Infection Syndrome: Nosologyand Reproducibility of Placental ReactionPatterns

RAYMOND W. REDLINE,1* ONA FAYE-PETERSEN,2 DEBRA HELLER,3

FAISAL QURESHI,4 VAN SAVELL,5 CAROLE VOGLER,6 AND THE SOCIETY FOR PEDIATRICPATHOLOGY, PERINATAL SECTION, AMNIOTIC FLUID INFECTION NOSOLOGY COMMITTEE1Department of Pathology, University Hospitals of Cleveland and Case Western Reserve University, 11100 EuclidAvenue, Cleveland, OH 44106, USA2Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA3Department of Pathology, University of Medicine and Dentistry of New Jersey—New Jersey Medical School,Newark, NJ, USA4Pathology Department, DMC University Laboratories, Hutzel Site and Wayne State University, Detroit, MI, USA5Department of Pathology, Driscoll Children’s Hospital, Corpus Christie, TX, USA6Department of Pathology, St. Louis University School of Medicine, St. Louis, MO, USA

Received July 1, 2002; accepted May 12, 2003; published online September 11, 2003.

ABSTRACTClinically responsive placental examination seeks to pro-vide useful information regarding the etiology, progno-sis, and recurrence risk of pregnancy disorders. Thepurpose of this study was to assemble and validate acomplete set of the placental reaction patterns seen withamniotic fluid infection in the hope that this might pro-vide a standardized diagnostic framework useful forpracticing pathologists. Study cases (14 with amnioticfluid infection, 6 controls) were reviewed blindly by sixpathologists after agreement on a standard set of diag-nostic criteria. After analysis of initial results, criteriawere refined and a second, overlapping set of cases werereviewed. Majority vote served as the gold standard.Grading and staging of maternal and fetal inflammatoryresponses was found to be more reproducible using atwo- versus three-tiered grading system than a three-versus five-tiered staging system (overall agreement81% vs. 71%). Sensitivity, specificity, and efficiency forindividual observations ranged from 67–100% (24/30� 90%). Reproducibility was measured by unweightedkappa values and interpreted as follows: � 0.2, poor;

0.2–0.6, fair/moderate; � 0.6, substantial. Kappa valuesfor the 12 lesions evaluated in 20 cases by the six pathol-ogists were: acute chorioamnionitis/maternal inflamma-tory response (any, 0.93; severe 0.76; advanced stage,0.49); chronic (subacute) chorioamnionitis (0.25); acutechorioamnionitis/fetal inflammatory response (any,0.90; severe, 0.55; advanced stage, 0.52); chorionic vesselthrombi (0.37); peripheral funisitis (0.84); acute villitis(0.90); acute intervillositis/intervillous abscesses (0.65),and decidual plasma cells (0.30). Adoption of this clearlydefined, clinically relevant, and pathologically reproduc-ible terminology could enhance clinicopathologic corre-lation and provide a framework for future clinical re-search.

Key words: amniotic infection syndrome, chorioamnio-nitis, nomenclature, placenta, reproducibility

INTRODUCTIONThe general sequence of pathologic changes ac-companying amniotic fluid infection has been rec-ognized for some time [1, 2]. Other clinically rele-vant histologic patterns have been added to thisbasic framework [3–9]. Despite a fairly extensive*Corresponding author, e-mail: [email protected]

Pediatric and Developmental Pathology 6, 435–448, 2003

DOI: 10.1007/s10024-003-7070-y

© 2003 Society for Pediatric Pathology

literature on this topic, there has been little changein the prevailing diagnostic terminology which hasbasically been “acute chorioamnionitis with orwithout funisitis.” Recent data has suggested a re-lationship between placental inflammation andimportant clinical outcomes such as neurologicimpairment and chronic lung disease [10–12]. Thishas coincided with a more active clinical approachto the management of chorioamnionitis and pre-term labor [13]. These changes have led to the needfor more detailed description, more specific diag-nostic criteria, and greater standardization of di-agnostic terminology in this area.

As in other organ systems, questions havebeen raised regarding the reliability of placentaldiagnosis, i.e., the ability of expert placental pa-thologists to agree with each other and with theirgeneral pathology colleagues [14–16]. With re-newed interest in the clinical significance of pla-cental pathology, the time seems appropriate tointroduce more precision and uniformity in pla-cental diagnosis. With this in mind, the PerinatalSection of the Society for Pediatric Pathology hasundertaken an initiative to review, define, and val-idate diagnostic criteria for a number of differentplacental reaction patterns. The current report pre-sents the results of the Amniotic Fluid InfectionNosology Committee.

METHODSA study set of 20 cases was assembled from the filesof University Hospitals of Cleveland. Three slidesfrom each case (umbilical cord, placental mem-brances, and one full-thickness section of placentalparenchyma) were selected. Gestational age andplacental weight were provided for each case.Fourteen placentas were originally diagnosed byone of the pathologists (R.W.R.) with one or morefindings consistent with amniotic fluid infection.The remaining six placentas had other pathologicdiagnoses, but lacked signs of amniotic fluid infec-tion. Placental reaction patterns relevant to amni-otic fluid infection were chosen for evaluation.These patterns are described below, summarizedin Table 1, and illustrated in Figures 1–3. All caseswere examined in a blinded fashion by the sixmembers of the study group using a previouslyagreed upon set of diagnostic criteria (round 1).Terminology was chosen to correspond as closely

as possible to that proposed by the College ofAmerican Pathologists consensus group on placen-tal diagnosis [17]. Tabulated results from the pre-liminary round (round 1) were circulated and dis-cussed. Representative photomicrographs of eachlesion were reviewed at a meeting of the Society forPediatric Pathology—Perinatal Section. After theinitial review a second revised set of diagnosticcriteria were proposed and agreed upon. Twelve ofthe original cases were retained, eight new caseswere added (five new cases and three new con-trols), and the numerical order was changed. Thissecond overlapping set of 20 cases was then circu-lated for reexamination followed by an analysis ofindividual performance and reproducibility (round2).

The revised diagnostic criteria as agreed uponby the study participants before the final evalua-tion (round 2) were as follows:

Maternal inflammatory responsesStage/progression of disease

Stage 1 (acute subchorionitis/early acute chorion-itis) � patchy-diffuse accumulations of neutro-phils in the subchorionic plate fibrin (Fig. 1a)and/or membranous chorionic trophoblast layer (afew scattered neutrophils in the lower half of cho-rionic plate and/or the membranous chorionicconnective tissue are allowed) (Fig. 1b). Stage 2(acute chorioamnionitis) � more than a few scat-tered neutrophils in the chorionic plate or mem-branous chorionic connective tissue and/or theamnion (Fig. 1c). Stage 3 (necrotizing chorioam-nionitis) � degenerating neutrophils (karyorrhe-xis), thickened eosinophilic amniotic basementmembrane, and at least focal amnionic epithelialdegeneration/sloughage (Fig. 1d).

Grade/intensity

Grade 1 (mild–moderate) � individual or smallclusters of maternal neutrophils diffusely infiltrat-ing amnion, chorionic plate, chorion laevae, and/orsubchorionic fibrin (Fig. 1c). Grade 2 (severe) �

three or more chorionic microabscesses (microab-scess � confluent neutrophils measuring at least10 � 20 cells in extent) between chorion anddecidua in the membranes and/or under the cho-rionic plate (Fig. 1e) or a continuous band ofconfluent chorionic polymorphonuclear leuko-

436 R.W. REDLINE ET AL.

Tab

le1.

Pla

cen

tare

acti

on

pat

tern

sre

late

dto

amn

ioti

cfl

uid

infe

ctio

n:

no

men

clat

ure

and

defi

nit

ion

s

Dia

gno

stic

cate

gori

esS

ugg

este

dd

iagn

ost

icte

rmin

olo

gyD

efin

itio

ns

Mat

ern

alin

flam

mat

ory

resp

onse

Sta

ge 1—E

arly

Acu

tesu

bch

orio

nit

isor

chor

ion

itis

PM

Nin

sub

chor

ion

icfi

bri

nan

d/o

rm

emb

ran

etr

oph

obla

st(F

ig.

1a,b

)

2—In

term

edia

teA

cute

chor

ioam

nio

nit

isD

iffu

se-p

atch

yP

MN

infi

bro

us

chor

ion

and

/or

amn

ion

(Fig

.1c

)

3—A

dva

nce

dN

ecro

tizi

ng

chor

ioam

nio

nit

isP

MN

kary

orrh

exis

,am

nio

cyte

nec

rosi

s,an

d/o

ram

nio

nb

asem

ent

mem

bra

ne

thic

ken

ing/

hyp

ereo

sin

oph

ilia

(Fig

.1d

)

Gra

de 1—

Mil

d–m

oder

ate

No

spec

ial

term

inol

ogy

requ

ired

Not

seve

reas

defi

ned

bel

ow

2—S

ever

eS

ever

eac

ute

chor

ioam

nio

nit

isor

wit

hsu

bch

orio

nic

mic

roab

sces

ses

Con

flu

ent

PM

N(�

10�

20ce

lls

inex

ten

t)b

etw

een

chor

ion

and

dec

idu

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olat

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cior

con

tin

uou

sb

and

(Fig

.1e

)

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(or

sub

acu

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chor

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nio

nit

isS

ub

amn

ion

icm

onon

ucl

ear

cell

infi

ltra

tew

ith

occa

sion

alP

MN

(mec

oniu

man

dh

emos

ider

in-l

aden

mac

rop

hag

esex

clu

ded

)(F

ig.

1f)

Fet

alin

flam

mat

ory

resp

onse

Sta

ge 1—E

arly

Wit

hch

orio

nic

vasc

uli

tis

oru

mb

ilic

alp

hle

bit

isIn

tram

ura

lP

MN

-ch

orio

nic

vess

els

and

/or

um

bil

ical

vein

(Fig

.2a

,b)

2—In

term

edia

teW

ith

um

bil

ical

vasc

uli

tis

(on

eor

two

arte

ries

�ve

in)

oru

mb

ilic

alp

anva

scu

liti

s(a

llve

ssel

s)In

tram

ura

lP

MN

-um

bil

ical

arte

ryor

arte

ries

(�u

mb

ilic

alve

in)

(Fig

.2c

)

3—A

dva

nce

dW

ith

(su

bac

ute

)n

ecro

tizi

ng

fun

isit

isor

wit

hco

nce

ntr

icu

mb

ilic

alp

eriv

ascu

liti

sP

MN

�as

soci

ated

deb

ris

inco

nce

ntr

icb

and

s-ri

ngs

-hal

osar

oun

don

eor

mor

eu

mb

ilic

alve

ssel

s(F

ig.

2d)

Gra

de 1—

Mil

d–m

oder

ate

No

spec

ial

term

inol

ogy

requ

ired

Not

seve

reas

defi

ned

bel

ow

2—S

ever

eW

ith

ase

vere

feta

lin

flam

mat

ory

resp

onse

orw

ith

inte

nse

chor

ion

ic(u

mb

ilic

al)

vasc

uli

tis

Nea

rco

nfl

uen

tin

tram

ura

lP

MN

-ch

orio

nic

and

/or

um

bil

ical

vess

els

wit

hat

ten

uat

ion

/deg

ener

atio

nof

VS

MC

(Fig

.2e

,f)

Oth

erW

ith

asso

ciat

edfe

tal

vess

elth

rom

bi

Rec

ent

thro

mb

osis

asso

ciat

edw

ith

intr

amu

ral

PM

N(F

ig.

2f)

Oth

ersp

ecifi

cfe

atu

res

Per

iph

eral

fun

isit

isF

ocal

aggr

egat

esof

PM

Nat

the

um

bil

ical

cord

surf

ace

(Fig

.3a

)

Acu

tevi

llit

isP

MN

invi

llou

sst

rom

a(o

rb

etw

een

trop

hob

last

and

stro

ma)

(Fig

.3b

)

Acu

tein

terv

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sw

ith

inte

rvil

lou

sab

sces

ses

Pat

chy-

dif

fuse

PM

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inte

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ssp

ace

(Fig

.3c

)

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ace

lls

Un

equ

ivoc

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lasm

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lls

ind

ecid

ua

bas

alis

orca

psu

lari

s(F

ig.

3d)

PM

N,

pol

ymor

ph

onu

clea

rle

uko

cyte

;V

SM

C,

vasc

ula

rsm

ooth

mu

scle

cell

.

AMNIOTIC INFECTION SYNDROME 437

cyte (PMN) more than 10 cells in width occupy-ing more than half of the subchorionic fibrin orone revolution of the membrane role. Chronic(or subacute) chorioamnionitis � more than oc-

casional maternally derived mononuclear cells(usually macrophages) in the chorionic plate(most frequently below the amnion) in caseswithout exogenous pigment deposition (i.e.,

Figure 1. Histologic features of reaction patterns relatedto the maternal inflammatory response to amniotic fluidinfection. a. Acute subchorionitis (maternal stage 1):neutrophils emanating from the intervillous space arediffusely scattered in the subchorionic fibrin. b. Acutechorionitis of chorion laevae (maternal stage 1): a bandof neutrophils emanating from decidual small venulesare gathered at the chorion laevae trophoblast layerwithout spread to the overlying amnion. c. Acute chorio-amnionitis, not otherwise specified (maternal stage 2,grade 1): neutrophils are seen in both chorionic and am-nionic connective tissue in the placental membranes. d.

Necrotizing (maternal stage 3) chorioamnionitis: degen-erating subamnionic neutrophils, thickened amnionicbasement membrane, and desquamation of amnionic epi-thelial cells are seen in placental membranes. e. Severe (ma-ternal grade 2) acute chorioamnionitis: (sub)chorionicmicroabscesses are depicted comprised of confluent ag-gregates of maternal neutrophils, at least 10 � 20 cells inextent. f. Chronic (subacute) chorioamnionitis: patchymaternally derived histiocytic infiltrate of the chorionicplate concentrated immediately below the amnionic sur-face (left). Occasional neutrophils are also present.


meconium, hemosiderin). Neutrophils may berare or abundant, but a coexistent acute chorio-amnionitis should be present in at least one sec-tion (Fig. 1f).

Fetal inflammatory responsesStage/progression of diseaseStage 1 (chorionic vasculitis/umbilical phlebitis) �

neutrophils in the wall of any chorionic plate vessel

Figure 2. Histologic features of reaction patterns relatedto the fetal maternal inflammatory response to amnioticfluid infection. a. Acute chorionic vasculitis (fetal stage 1,grade 1): neutrophils are scattered throughout the up-per amnionic aspect of a major chorionic plate vessel. b.Umbilical phlebitis (fetal stage 1): neutrophils are iden-tified between the widely separated smooth muscle fas-cicles of the umbilical vein. c. Umbilical arteritis (fetalstage 2): neutrophils infiltrate the subendothelial cush-ion and tightly packed smooth muscle cells of the umbil-

ical artery. d. Subacute necrotizing funisitis (fetal stage3): an arc of degenerating neutrophils and mineralizedcell debris in Wharton’s jelly surrounds an umbilical ves-sel. e. Intense chorionic vasculitis (fetal grade 2): nearconfluent fetal neutrophils infiltrate the outer (am-nionic) wall of a chorionic vessel. f. Chorionic vasculitiswith adherent fetal vessel thrombus: a recent nonocclu-sive thrombus adheres to the outer (amnionic) wall of anacutely inflamed chorionic vessel.


(Fig. 2a) or the umbilical vein (Fig. 2b). Extensionof neutrophils into Wharton’s jelly is allowed if notaggregated in a concentric band, ring, or haloaround the umbilical vein. Stage 2 (umbilical vas-culitis) � neutrophils in one or both umbilicalarteries � vein (Fig. 2c). Extension of neutrophilsinto Wharton’s jelly is allowed if not aggregated ina concentric band, ring, or halo around the umbil-ical vessel(s). Stage 3 (necrotizing funisitis or con-centric umbilical perivasculitis) � neutrophils, cel-lular debris, eosinophilic precipitate, and/ormineralization arranged in a concentric band,ring, or halo around one or more umbilical vessels(generally most severe on the side nearest the pe-riphery of the cord) (Fig. 2d).

Grade/intensity

Grade 1 (mild–moderate) � scattered neutrophilicinfiltrate in the subendothelial or intramural por-tions of any chorionic (or umbilical) vessel (Fig.

2a). Grade 2 (severe) � chorionic plate (or umbil-ical) vessels with near confluent neutrophils plusattenuation and/or degeneration of vascularsmooth muscle cells on the side facing the amni-otic cavity (Fig. 2e). Associated fetal vessel thrombi� recent thrombosis of chorionic plate (or umbil-ical) vessel(s) in the context of a fetal inflammatoryresponse (Fig. 2f).

Other specific featuresPeripheral funisitis � small punctate microab-scesses with neutrophils on the outer surface ofcord (Fig. 3a). Acute villitis � neutrophils in thevillous stroma and/or the adjacent subtrophoblas-tic space (Fig. 3b). Acute intervillositis/intervillousabscesses � neutrophils within perivillous fibrin orthe intervillous space (Fig. 3c). Decidual plasmacells � unequivocal plasma cells in either the de-cidua basalis or capsularis (Fig. 3d).

Figure 3. Histologic features of other specific features ofamniotic fluid infection. a. Peripheral funisitis: a triangu-lar neutrophilic microabscess is seen just below the um-bilical surface epithelium. b. Acute villitis: fetal neutro-phils are present in the villous stroma andsubtrophoblastic space of terminal villi. Occasional neu-

trophils are also present in the intervillous fibrin. c. Acuteintervillositis/intervillous abscesses: near confluent neu-trophils are noted in the intervillous space with only fo-cal involvement of adjacent villous stroma. d. Decidualplasma cells: small aggregates of plasma cells are presentin the decidua basalis.


Data analysisThe gold standard for the presence or absence of adiagnosis in each case was the group consensus.The diagnosis from the original institution was tobe used as the “tie breaker” in split cases, but wasnot required for any diagnosis in any case. Individ-ual variations from consensus grades and stageswere tabulated for rounds 1 and 2 and the percentagreement was determined. The final analysis(round 2) evaluated the following 12 reaction pat-terns: any maternal of fetal inflammatory response(grade and stage not � 0), severe maternal or fetalinflammatory response (grade 2), advanced stagematernal or fetal inflammatory response (stage 3),and the six other features listed above. Collectiveperformance of the group was assessed by combin-ing the individual diagnoses to derive specificity(true negatives/true negatives plus false positives),sensitivity (true positives/true positives plus falsenegatives), and efficiency (true positives plus truenegatives/total observations) relative to the goldstandard. Interobserver reliability was assessed byunweighted kappa analysis [18]. Kappa analysis isa measurement of agreement between observerson a case to case basis. Kappa values can varyfrom �1 to �1, with �1 indicating perfect in-verse correlation; 0, no correlation; and �1, per-fect positive correlation. Interpretation of kappavalues can vary. We used the following guide-lines: � 0.2, poor agreement; 0.2–0.6, fair/moder-ate agreement; � 0.6 substantial agreement [18].

RESULTSPreliminary analysisThe initial study set of 20 cases, 14 with featuresassociated with amniotic fluid infection and 6 con-trols, was circulated to group members for blindedreview and coding on standardized forms (round1). Analysis of the first round results revealed un-acceptable interobserver variation. A revised andsimplified set of definitions was circulated andagreed upon and a second overlapping set of studycases was circulated for review (round 2). The le-sions, terminology, and definitions used in the finalround are described in the Methods section, sum-marized in Table 1, and illustrated in Figures 1, 2,3.

Grading and stagingAll possible histologic gradations of intensity(grade) and progression of disease (stage) for ma-ternal and fetal inflammatory responses are poten-tially relevant to clinical outcome. However, exces-sive subclassification in the absence ofdocumented clinical significance is one cause ofunacceptable interobserver variability amongst pa-thologists. Table 2 compares the group perfor-mance using two different classification schemes,and shows the results from round 1 using a slightmodification of a previously described classifica-tion system used in research studies correlatingplacental diagnosis with clinical outcome [19].This scheme utilized three-tiered grading and five-tiered staging of both maternal and fetal inflam-matory responses. Overall agreement using thissystem was modest (71%) and particular problemsexisted in separating intermediate and low gradesof inflammation (Table 2). These distinctions areof no documented clinical significance. Most ofdisagreement was the result of minor disagree-ments (� 1) suggesting that a simplified schememight yield better reproducibility without sacrific-ing the clinical significance associated with highgrade and stage. For round 2, the number of gradeswas reduced to two, retaining the high grade cate-gories because of their established clinical signifi-cance (see Discussion). The number of stages wasreduced to three, distinguishing only those caseswith findings suggestive of early and advancedstage infection from those without these character-istics. The results shown in Table 2 showed a sub-stantial improvement in overall agreement (81%).

Final group survey results (round 2)A complete analysis of the group’s performance isshown in Table 3. Twelve reaction patterns wereevaluated: presence of any maternal or fetal in-flammation, presence of severe (grade 2) maternalor fetal inflammation, presence of advanced (stage3) maternal or fetal inflammation, chronic (sub-acute) chorioamnionitis, fetal vessel thrombi, pe-ripheral funisitis, acute villitis, acute intervillositis/intervillous abscesses, and decidual plasma cells.The gold standard for the presence or absence ofeach condition (prevalence) in the 20 study caseswas group consensus. Group consensus was unan-imous for 43/60 (72%) diagnoses and near unani-


Table 2. Stage and grade of infection, preliminary and final rounds, comparing expanded versuscontracted scales

Stage/grade No. of casesconsensuspositive

Individual deviations from consensusa

(% of observers)

Initial evaluation (round 1): three-tieredgrading/five-tiered staging

-2 -1 0 �1 �2

Maternal inflammatory response

Grade

1 2 — 8 67 8 17

2 2 0 8 50 42 —

3 10 3 17 80 — —

Combined 14 2 14 74 8 2

Stage

1 1 — 17 83 0 0

2 4 0 25 67 8 0

3 5 0 7 70 17 6

4 3 0 17 67 16 —

5 1 16 17 67 — —

Combined 14 1 16 68 13 2

Fetal inflammatory response

Grade

1 4 — 8 54 17 21

2 4 0 13 54 33 —

3 5 0 3 97 — —

Combined 14 0 8 68 17 7

Stage

1 4 — 8 88 4 0

2 1 0 50 50 0 0

3 2 0 0 83 17 0

4 4 3 11 83 3 —

5 1 0 0 100 — —

Combined 14 1 10 83 6 0

Overall performance (total observations) (324) 1 14 71 11 3

Maternal inflammatory response

Grade

1 5 — 3 84 13 —

2 9 0 11 89 — —

Combined 14 0 8 87 5 —

Stage

1 2 — 8 59 25 8

2 8 0 6 86 8 —

3 4 0 17 83 — —

Combined 14 0 10 81 8 1

Fetal inflammatory response

Grade

1 8 — 7 67 26 —

(continued)


mous (5/6) for another 12%. Overall agreementbetween group prevalence and a priori prevalence(the original diagnosis of the submitting patholo-gist, R.W.R.) for the 10 lesions in the 20 cases was87%. The range of individual prevalences for eachlesion among the 20 cases was generally narrow.Group performance was evaluated in two ways.First, each individual response was classified astrue or false positive, or true or false negative,relative to the group consensus and overall sensi-tivity, specificity, and diagnostic efficiency. Overalldiagnostic efficiency was 84% or greater for alllesions studied. Sensitivity and specificity werealso high for the majority of diagnoses. Problemareas included relatively low sensitivity (� 85%)for advanced (stage 3) fetal inflammatory re-sponse, fetal vessel thrombi, decidual plasma cells,and chronic chorioamnionitis, and relatively lowspecificity (� 85%) for decidual plasma cells. Thesecond measure of individual performance was toevaluate overall agreement using the unweightedkappa (k) statistic. For this analysis, �-values wereinterpreted as follows: 0.00–0.20, poor; 0.21–0.60,fair/moderate; 0.61–1.00, substantial/near perfect[18]. By these criteria, none of the diagnoses fellinto the poor category. Reproducibility for pres-ence of any maternal inflammatory response, pres-ence of any fetal inflammatory response, severe(grade 2) maternal inflammatory response (Fig.1e), peripheral funisitis (Fig. 3a), acute villitis (Fig.3b), and acute intervillositis/intervillous abscesses(Fig. 3c) was substantial to near perfect. Reproduc-ibility for the remaining diagnoses—advanced

(stage 3) maternal inflammatory response (Fig.1d), severe (grade 2) fetal inflammatory response(Fig. 2e) advanced (stage 3) fetal inflammatory re-sponse (Fig. 2d), chronic (subacute) chorioamnio-nitis (Fig. 1f), fetal vessel thrombi (Fig. 2f), anddecidual plasma cells (Fig. 3d)—was fair to mod-erate.

DISCUSSIONPremature delivery is the major cause of perinatalmorbidity and mortality in the United States ac-counting for 70% of deaths, nearly half of cerebralpalsy, and a substantial proportion of other disor-ders including chronic lung disease, mental retar-dation, and sensorineural impairment [20]. Infec-tion of the amniotic fluid is either the initiatingevent or the final common pathway leading to de-livery in more than a third of these patients [21,22]. The term amniotic infection syndrome wasoriginally coined to encompass the circumstancesin which microorganisms enter the normally ster-ile amniotic sac, the sequence of maternal and fetalinflammatory reactions these organisms elicit, andthe adverse consequences of infection and inflam-mation for the mother and fetus [1]. Pathologistscan provide important information related to allthree components. In terms of predisposing condi-tions, pathologists contribute by recognizing bac-terial vaginosis on pap smear, detecting cervico-vaginal group B streptococci by culture, and by thediagnosis of subacute or chronic deciduitis (endo-metritis) in the delivered placenta [20, 23, 24].With respect to the sequence of maternal and fetal

Table 2. (Continued)

Stage/grade No. of casesconsensuspositive

Individual deviations from consensusa

(% of observers)

2 6 0 14 86 — —

Combined 14 0 10 76 14 —

Stage

1 4 — 17 83 0 0

2 7 0 11 79 10 —

3 3 0 28 72 — —

Combined 14 0 16 79 5 0

Overall performance (total observations) (336) 0 11 81 8 � 1

aPositive and negative numbers specify the number of stages or grades over or under the consensus score for each individual observer.


Tab

le3.

Fin

algr

ou

psu

rvey

resu

lts,

rou

nd

2

Les

ion

pre

vale

nce

for

the

20st

ud

yca

ses

Gro

up

con

sen

sus

Co

llec

tive

ind

ivid

ual

per

form

ance

(20

case

ssc

ore

dfo

r12

lesi

on

s)In

tero

bse

rver

rep

rod

uci

bil

ity

Pla

cen

tal

lesi

on

reac

tio

np

atte

rnA

pri

ori

Gro

up

Ind

ivid

ual

ran

ge6/

65/

64/

65S

ensi

tivi

tyS

pec

ifici

tyE

ffici

ency

Kap

pa

Mat

ern

alin

flam

mat

ory

resp

onse

An

y0.

700.

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inflammation, histologic chorioamnionitis has re-peatedly been shown to be both sensitive and spe-cific for infection and is considered the gold stan-dard against which other clinical predictors ofinfection should be measured [25–28]. Finally, asdescribed below, certain reaction patterns such asseverity of inflammation, associated fetal vascularthrombosis, patterns suggestive of a specific caus-ative organism, and estimation of the risk for neo-natal sepsis may identify infants at increased riskfor specific perinatal complications.

Most attempts to subclassify the inflamma-tory responses to amniotic fluid infection havebuilt upon the observations of Blanc who de-scribed what he believed to be the anatomic se-quence of events [29]. In this scheme, stage 1 wasdefined by neutrophils in the subchorionic fibrin,stage 2 by neutrophils in the chorionic plate, andstage 3 by neutrophils in the amnion. Van Hoevenand colleagues later found that amnionic necrosis(necrotizing chorioamnionitis), a late complica-tion of inflammation in the amnion, was associ-ated with an increased risk of perinatal death andpreterm delivery [9]. For this reason, they arguedthat this feature should be specified in the finalpathologic diagnosis. Mueller-Heubach and co-workers also described associations of necrotizingchorioamnionitis with preterm labor, prematurerupture of membranes, and decreased gestata-tional age in very low birthweight infants [7].

Severity of the maternal inflammatory re-sponse has been estimated in a couple of ways.keenan and colleagues in 1977 showed that sever-ity, as determined by the formation of subchori-onic microabscesses, was associated with an in-creased risk of presumed or documented fetalsepsis [4]. Muller-Heubach and colleagues pro-posed an alternative numerical system in which �

30 neutrophils in the upper half of the chorionicplate was considered severe [7]. However, severitydefined in the latter fashion has not been predictiveof clinically significant complications [10, 11, 30].This numerical definition was also poorly repro-ducible in our study (round 1). Better reproducibil-ity and the clinical significance described byKeenan et al. [4] lead us to recommend this as amore useful criterion for severity.

In Blanc’s original formulation, a fetal in-flammatory response was indicated by the pres-

ence of neutrophils in the walls of large fetal ves-sels in the chorionic plate and umbilical cord [1,29]. The presence of umbilical cord inflammationhas been shown to be a risk factor for clinicalmanifestations of the fetal inflammatory responsesyndrome including intraventricular hemorrhageand CNS echolucencies in preterm infants [6, 31].A greater association with complications in pre-term infants may relate in part to an increasedthreshold for mounting fetal inflammatory re-sponses in the less mature fetus [32]. It has gener-ally been observed that chorionic plate vesselsand/or the umbilical vein are involved prior to theumbilical arteries. Support for this sequence hasrecently been published by two recent studies dem-onstrating increased fetal cytokine levels and in-creased morbidity in patients with inflammation inone or both umbilical arteries compared to thosewith involvement of the vein alone [33, 34].Keenan et al. also demonstrated the additionalclinical significance of arterial inflammation byshowing that umbilical panvasculitis (veins plusarteries) was an independent risk factor for neona-tal sepsis [4]. In view of this data, it seems impor-tant to differentiate between involvement of theumbilical vein alone (phlebitis) and umbilical vas-culitis with arterial involvement. Use of the genericterm “funisitis” is not sufficiently specific andshould be discouraged.

Nararro and Blanc were also the first to de-scribe a later stage of fetal inflammation, subacutenecrotizing funisitis [3]. In this lesion, fetal neu-trophils migrate into the umbilical stroma (Whar-ton’s jelly), arranging themselves in a circular arearound the vessel. This pattern has been attributedto the precipitation of immune complexes formedby microbial antigens diffusing in from the amni-otic fluid and maternal antibodies diffusing outfrom the umbilical vessels [5]. Such immune com-plexes are chemotactic for fetal neutrophils. Mat-suda and coworkers later showed that infants withthis lesion are at significantly increased risk forchronic lung disease [35]. These observations de-fine three temporal stages of fetal involvement:stage 1, neutrophils in chorionic and/or umbilicalvein, stage 2, neutrophils in an umbilical artery,and stage 3, concentric arcs of degenerating neu-trophils in Wharton’s jelly.


Severity of fetal inflammation was found bySpong and coworkers to be a risk factor for severevariable decelerations and increased circulatingNRBC in very low birth weight infants [36]. Otherstudies by Redline et al. have shown that severefetal inflammation is a risk factor for neurologicimpairment in both very low birth weight and terminfants [11, 19]. While severity is more easily as-sessed and of better documented significance inchorionic plate vessels, umbilical vessels can alsobe assessed in cases where the chorionic plate hasnot been sampled. Much of the effect of severe fetalinflammation in the second group of studies citedabove was attributable to coexisting chorionic ves-sel thrombosis. Reproducibility for the diagnosisof fetal vessel thrombi was somewhat disappoint-ing in the present study, largely owing to ambigu-ity in cases with small foci of endothelial fibrindeposition. For diagnostic purposes, we recom-mend close adherence to the classic criteria for theidentification of true thrombi (glassy red-bluestaining, lamination, and adhesion to the vesselwall) when making this diagnosis (Fig. 2f).

Peripheral funisitis, the formation of superfi-cial umbilical cord abscesses, was a highly repro-ducible finding (kappa � 0.84). This pattern, whendiffusely distributed over the cord, is virtually pa-thognomonic for candida infection [5, 37]. Whenthe peripheral inflammatory response is focal andlocated near the placental cord insertion, the asso-ciation with candida is less specific. Our study setcontained one case of each type and both wererecognized by 100% of observers. Because of thisoverlap, a final diagnosis of candidal chorioamnio-nitis requires demonstration of fungal hyphae intissue sections.

The presence of neutrophils in the villousstroma may be found in two distinct patterns. Thefirst pattern, acute villitis with a minimal intervil-lous component, is believed to reflect overwhelm-ing fetal sepsis, often caused by gram negativebacilli such as E. coli [17]. The histologic findingsare acute capillaritis with emigration of fetal neu-trophils to villous stroma. Clusters of neutrophilsin acute villitis often accumulate between villousstroma and the villous trophoblast basement mem-brane (Fig. 3b). Bacteria are usually evident byroutine hematoxylin and eosin (H&E) stain. Thesecond pattern is acute villitis accompanied by in-

tervillositis and intervillous abscess formation(Fig. 3c). Acute villitis in these cases is less exten-sive than the intervillositis and is presumed to rep-resent secondary spread to villi. This pattern ismost commonly seen in infections caused by Lis-teria monocytogenes [38]. Campylobacter fetus anda variety of rare infections, such as coccidiomyco-sis, psittacosis, tularemia, and brucellosis, mayalso manifest some or all of these findings [39, 40].In the present study, the shared feature of acutevillitis was easily recognized and agreed upon byvirtually all observers (kappa � 0.90). The addi-tional finding of acute intervillositis/intervillousabscesses was recognized by all observers, but wasoccasionally overdiagnosed in cases of acute villitiswith occasional perivillous neutrophils leading to alower kappa value (0.65).

The final two patterns, decidual plasma cellsand chronic (subacute) chorioamnionitis, wereless reproducible and often missed in consensus-positive cases. Plasma cells in decidua, like those innonpregnant endometrium, are notoriously diffi-cult to detect. Overdiagnosis of cells lacking one ormore of the classic morphologic features can alsobe a problem. Chronic (subacute) chorioamnioni-tis requires the observer to recognize an underly-ing histiocytic infiltrate in membranes already suf-fused with neutrophils. It is possible that specialstains such as syndecan-1 for plasma cells andCD68 for histiocytes could improve reproducibil-ity. Plasma cells in particular, and B-lymphocytesin general, are always abnormal in the endometrialcavity [41]. Their presence is an indicator of inap-propriate antigenic exposure. One source of anti-gen is bacterial subacute endometritis, which maylead to recurrent acute chorioamnionitis in subse-quent pregnancies [20]. Uterine plasma cells in theabsence of acute inflammation are also associatedwith infertility, spontaneous abortion, and chronicvillitis [42–44]. We believe that the presence ofplasma cells should always be noted in the finaldiagnosis [45]. Chronic chorioamnionitis, on theother hand, is a heterogeneous entity that can ac-company either acute chorioamnionitis or chronicvillitis [46, 47]. This lesion overlaps with a veryrecently characterized lesion known as subacutechorioamnionitis [48]. In the latter report, pub-lished after completion of our study, subacute cho-rioamnionitis when combined with subacute ne-


crotizing funisitis (our fetal Stage 3chorioamnionitis) was found to be a significantrisk factor for chronic lung disease. In view of itsevolving status and the lack of reproducibility inthe present study, we recommend that the terms“chronic” or “subacute” chorioamnionitis be usedsparingly and as descriptive adjuncts rather thanprimary diagnoses [49].

Reliability of placental diagnosis has not beenextensively examined. In one study, it was foundthat interobserver agreement between subspecialtyperinatal pathologists was better than that be-tween general surgical pathologists and perinatalsubspecialists [14]. Discrepancy rates in the lattersituation ranged from 32–59%. Some reliabilitystudies have been performed with other primarygoals, such as demonstrating that gestational agecannot be specified based on placental histologyalone and developing a working definition ofchronic deciduitis [15, 50]. Statistical measures ofreliability were not performed in these studies. Toour knowledge, only two studies have directly mea-sured reproducibility. Both were performed by ep-idemiologists for the purpose of validating the useof archived placental material in retrospectivestudies [16, 51]. Both concluded that reliability isexcellent for many placental lesions including thevarious stages of the maternal response in acutechorioamnionitis and the presence or absence of afetal response. While other diagnoses were lessreproducible, it was shown that reliability could beimproved through the use of a standardized studyprotocol. Our study is the first designed by pathol-ogists for the purpose of developing and standard-izing diagnostic criteria that included statisticalevaluation of reproducibility. Furthermore, it isthe first to comprehensively address all pathologicfindings related to a single perinatal process, inthis case amniotic fluid infection. Additional stud-ies are in progress to apply the same procedure totwo other processes—maternal vascular underper-fusion and fetal thromboocclusive disease.

In conclusion, we have proposed and vali-dated a system for describing the maternal andfetal inflammatory responses to amniotic fluid in-fection plus a number of other reaction patternsuseful for defining the etiology and clinical conse-quences of infection. We have attempted to utilizeterminology and criteria that conform as closely as

possible to previously reported schema. Westrongly advocate the use of this, or a similarlyconstructed, system to help standardize diagnosesbetween hospitals, provide useful information toclinicians, and serve as a valuable tool for conduct-ing future studies relating placental risk factors toadverse perinatal outcome.

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