ORIGINAL ARTICLE

Changes in Maternal Blood Inflammatory Markers As a

Predictor Of Chorioamnionitis: A Prospective Multicenter Study

de

ric Lirussi2,5,6, Didier Riethmuller7, Nathalie
Isabelle Le Ray1,2,3, Guillaume Mace3,4, Mourad Sediki1, Fre
8
7
9
10
Lentz , Rajeev Ramanah , Toufic Hoyek , Gilles Spagnolo , Nicole Laurent11, Francoise Goirand2,5,6,
Paul Sagot3,4, Marc Bardou1,2,3,6
1

Centre dInvestigations Cliniques 1432 (INSERM CIC 1432), CHU Dijon, Dijon Cedex, France;

de Bourgogne, Dijon, France;
Universite
3

seau National Gyn

Re
ecologie et Obst
etrique des CIC de lINSERM, GO-CIC;
4

cologie et dObste

trique, CHU de Dijon, Dijon, France;
Service de Gyne
5
Laboratoire de Pharmacologie-Toxicologie, CHU de Dijon, Dijon, France;
6

et de la Recherche Me

dicale), Dijon, France;
Centre de Recherche Lipides, Nutrition Cancer U866, INSERM (Institut National de la Sante
7

cologie et dObste

trique, CHU Jean Minjoz, Besancon, France;
Service de Gyne
8

cologie et dObste

trique, CH de Ch^alon-Sur-Sao
^ne;
Service de Gyne
9

cologie et dObste

trique, CH dAuxerre;
Service de Gyne
10

cologie et dObst
etrique, CH de M^acon;
Service de Gyne
11
Service dAnatomo-Pathologie, CHU de Dijon, Dijon, France
2

Keywords
Chorioamnionitis, cohort study, IL-6, IP-10,
MCP-1, PPROM
Correspondence
Prof. Marc Bardou, INSERM CIC 1432, CHU de
Dijon, 14, rue Gaffarel, BP 77908, 21079
DIJON Cedex, France.
E-mail: marc.bardou@u-bourgogne.fr

Problem
To evaluate the inflammatory pattern in maternal circulation from
women with preterm premature rupture of membranes (PPROM) considering the occurrence of histologically confirmed chorioamnionitis
(HCA).

Submission July 22, 2014;

accepted August 26, 2014.

Method of study
A prospective study was conducted in 121 women with PPROM
between 24 and 34 + 0 weeks of gestation. Association between white
blood cells (WBC) count, plasma CRP, IL-6, MCP-1 and IP-10 levels,
and HCA was assessed.

Citation
Le Ray I, Mace G, Sediki M, Lirussi F,
Riethmuller D, Lentz N, Ramanah R, Hoyek T,
Spagnolo G, Laurent N, Goirand F, Sagot P,
Bardou M. Changes in maternal blood
inflammatory markers as a predictor of
chorioamnionitis: a prospective multicenter
study. Am J Reprod Immunol 2015; 73: 7990

Results
The rate of HCA was 44.7% (54/121). During the 5 days preceding
delivery, median CRP, WBC, and IL-6 levels were significantly higher in
the HCA than in no-HCA group (P < 0.001). Variations in IL-6, IP-10
levels, during the 2472 hr before delivery, were predictors of the
occurrence of HCA, but the diagnostic accuracy was low [Receiver Operating Characterictic (ROC) curve, area under the curve (AUC) = 0.56].

doi:10.1111/aji.12323

Conclusion
An increase in IL-6, CRP, IP-10 maternal plasma levels was confirmed
in PPROM women with HCA. Longitudinal follow-up of these markers
did not add valuable information regarding HCA.

Introduction
Premature rupture of the membranes (PROM) is
defined as the rupture of the amniotic membranes
American Journal of Reproductive Immunology 73 (2015) 7990
2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

with release of the amniotic fluid (AF) prior to the

onset of labor, and it is deemed preterm (PPROM)
when it occurs prior to 37 weeks of gestation (WG).1
PPROM affects approximately 3% of the pregnancies
79

LE RAY ET AL.

and is associated with one-third of preterm births.13

Several risk factors for PPROM have been described
such as smoking,4,5 previous preterm delivery,6 or
oxidative stress,7 even if antioxidant nutrients have
not been found protective against PROM.8 It remains
associated with higher morbidity and mortality rates
than other etiologies of prematurity.9,10 In the case
of chorioamnionitis (CA), the clinical and biological
signs classically used to diagnose infection have a
poor predictive value, appear late,11 and may even
be absent.3 This is of particular concern as acute histologically confirmed chorioamnionitis (HCA) is a
risk factor for adverse neonatal outcomes, mostly
sepsis,12 and microbial invasion of the amniotic cavity (MIAC) occurs in at least one-third of preterm
deliveries with PPROM despite antibiotic therapy.13,14 In consequence, it is crucial to find effective
tools to predict the presence of HCA before clinical
symptoms appear so as to deliver the fetus in a
timely fashion.
Studies have shown that HCA is associated with
higher levels of several cytokines, including interleukin-6 (IL-6) and MCP-1, in the AF,15,16, fetal cord
blood,17 cervicovaginal fluid,18 and maternal
serum.19 IL-6,20,21 IL-8,22,23 monocyte/macrophage
chemo-attractant protein-1 (MCP-1),15,24 and interferon-gamma-inducible protein-10 (IP-10)25,26 have
thus been assessed in their accuracy to diagnose
MIAC in women with PPROM. Studies, published
after our own work was designed and conducted,
have suggested other markers to be of potential interest, such as IL-8, regulated on activation normal
T-expressed and secreted (RANTES), brain-derived
neurotropic factor (BDNF), IFN-c, GM-CSF, macrophage inflammatory protein-1a (MIP-1a), IL-6, IL-10,
IL-1b, TNF-b, triggering receptor expressed on myeloid cells-1 (TREM-1), neurotropin-3 (NT-3), and soluble TNF receptor-1 (sTNF-R1),27 nevertheless IL-6
and CRP remain among the most validated markers.
However, the different biological markers that
have been evaluated are far from ideal, either
because the diagnostic accuracy is not sufficient to
define a specific threshold28 or because of the invasiveness of AF puncture, which precludes repeated
sampling.26,29 Additionally, in PPROM, a reduced AF
volume often makes AF sampling difficult,30 and few
studies have focused on noninvasive sample, for
example in maternal plasma.27,31 Systematic reviews
have pointed out inconsistencies in studies reporting
on the diagnostic accuracy of biomarkers such as
CRP.32,33
80

Most studies focused on a single measurement of

one, or a combination of several, biological marker,
either at the time of PROM, or just before or at the
time of delivery. Such methods do not take into
account the dynamic process of cytokine synthesis in
response to infectious stimuli.34
Rather than trying to identify new markers of
interest for the diagnosis of HCA, this multicenter
prospective study was designed to determine
whether longitudinal determinations of clinical
parameters (temperature, maternal and fetal heart
rate, uterine tenderness) and changes in maternal
circulating inflammatory markers (white blood
cells (WBC) count, C-reactive protein, IL-6, MCP-1,
IP-10) could accurately predict the presence of histologic CA in patients with PPROM.
Patients and methods
Study Design
This prospective multicentre study included women
hospitalized for preterm premature rupture of the
membranes (PPROM) between 24 and 34 WG,
between 09/2008 and 10/2010. All consecutive
women were to be asked to participate in this study.
Membrane rupture was diagnosed by the presence of
AF at sterile speculum examination or by a positive
Actim PROM TEST (Medix Biochemica, Kauniainen,
Finland) that detects insulin-like growth factor binding protein-1 (IGFBP-1) using vaginal samples.
Clinical CA was defined classically35 as the presence of uterine tenderness and/or purulent or foulsmelling AF with any two of the following criteria:
antepartum temperature of 38.0C or more, maternal tachycardia (more than 120 beats/min), maternal leukocytosis more than 18,000 cells/mm3, or
fetal tachycardia (more than 160 beats/min). CA
was confirmed by histological examination of the
placenta by a single staff pathologist using validated
criteria,36 associated, or not, with positive placental
culture, either monomicrobial or polymicrobial.37
Positive placental cultures with negative histology
were not considered for the diagnosis of HCA as it
is more likely to reflect vaginal contamination at
the time of delivery. Histological examinations were
performed locally as part of the standard management of these women and reviewed centrally by a
single pathologist in the coordinating center.
The pathologist was blinded to the initial results.
Exclusion criteria were delivery within 12 hr after
American Journal of Reproductive Immunology 73 (2015) 7990
2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

MATERNAL INFLAMMATORY MARKERS IN PPROM

admission, hemorrhagic placenta previa, and HIV

infection.
Maternal temperature, heart rate and arterial pressure, and fetal heart tones were measured twice
daily for 2 days and daily thereafter. The amniotic
fluid index (AFI), assessed by ultrasonography, and
CRP levels were measured at a frequency defined by
the local protocols. Tocolysis was used in women
who did not exhibit clinical and biological signs of
CA. Labor was induced, or C-section was performed,
in patients with clinical signs of CA. In case of subacute CA, that is, women who did not exhibit the
full set of diagnosis criteria, induction was chosen
for women with favorable cervix, otherwise it was
C-section. Early neonatal sepsis was diagnosed by
the pediatrician as neonatal infection within 72 hr
after delivery38 and was considered confirmed if
there were positive cultures of serum or cerebrospinal fluid associated with clinical signs of infection.
Probable neonatal infection was defined by clinical
signs of sepsis and CRP 10 mg/L, or CRP >10 mg/L
and bacteria found on the skin or in orifices (nose,
ears).
To determine serum cytokine concentrations,
fresh blood samples were collected longitudinally
by venipuncture according to the schedule presented in Table I. Maternal venous blood samples
were drawn into sterile anticoagulant-containing
tubes and stored at 4C. Specimens were centrifuged at 2800 g for 10 min at 4C, and the aliquots
of plasma were frozen at 80C until the analyses.
Blood samples were taken twice daily during the
first 2 days after admission, once daily from day 3
(D3) to day 7 (D7), every other day from D8 to
D14, twice weekly from D15 to D21 and then
weekly up to delivery. This timing was decided to
have a close monitoring after admission followed
by a more acceptable frequency of venipuncture as
days passed on.
Progesterone was not a part of the standard of
care for women with history of preterm labor; antibiotics were prescribed in all women with positive
vaginal or urinary analysis. Apart from clinical and
biological longitudinal follow-up according to study
protocol, management of women was left upon local
standard of care.
The study protocol was approved by the French
Medicines Agency (registration number 2006-A0065942 and NCT01903759) and the local ethics committee
(CPP-Est 1, Dijon, France). Written informed consent
was obtained from all patients.
American Journal of Reproductive Immunology 73 (2015) 7990
2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Biochemical Analyses
Standard laboratory parameters (CRP, WBCs count),
urine and vaginal bacteriological examinations were
performed locally in each of the five participating
centers.
Maternal serum levels of IL-6, MCP-1, and IP-10
were determined with enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis,
MN, USA). IL-6 concentrations were determined
using an ultrasensitive ELISA assay with a lower
limit of detection of 0.156 pg/mL and inter- and
intra-assay variations of <10%. Lower limits of
detection and inter- and intra-assay variations for
MCP-1 and IP-10 were 31.2 pg/mL and 7.8 pg/mL;
and <8 and 9%, respectively. Analysis were preformed centrally, and results were not a part of the
decision-making process to manage the patients.
Statistical Analysis
Sample size calculation was based on previous report
suggesting a 4050% rate of histologically proven
CA in women with PROM,39,40 to have about 60
women with HCA. Descriptive statistics (means with
standard deviations and numbers with percentages
for categorical variables) were generated for each of
the variables. Observations were stratified on the
basis of the presence or absence of HCA. Tests were
performed using the t-test for continuous variables
and the chi-square test for categorical variables. Differences between groups were considered significant
if P < 0.05.
The evolution of cytokine, CRP and WBC levels
measured during the 5 days preceding delivery was
described and compared between the HCA and noHCA groups by multivariate analysis of variance
(MANOVA) for time and group. When the MANOVA
gave significant results (P < 0.05) for the group, a
Wilcoxon test was used to compare the values at
each time point between the HCA and no-HCA
group.
As variations in a biological marker might be more
informative than a specific threshold to improve the
diagnosis of HCA, we thus created a variable called
delta. For each biological parameter, cytokines of
interest, CRP or WBC, delta was the difference
between two values obtained for the same patient
measured at a time interval of between 24 and
72 hr. As the objective was to determine whether or
not a patient would develop CA within 72 hr, deltas
81

LE RAY ET AL.

Table I Baseline Characteristics at the Time of Admission

n
Age (years)
BMI
<25
2529.9
30
Tobacco use
Remote
During pregnancy
Parity
0
12
3
Pregnancy characteristics
Gestational age at admission (weeks of gestation)
<26
2629
3032
Preterm labor history
Preterm premature rupture of membrane history
First trimester vaginal bleeding
Cystitis during pregnancy
Vaginosis during pregnancy
Uterine contractions
Delay to care >12 hr
Clinical characteristics
Meconium in amniotic fluid
Amniotic fluid index
<5 cm
58 cm
>8 cm
Fetal heart tone abnormality
Vaginal bacterial infection
Positive CBEU
Clinical CA

P-value

Total

No-HCA

HCA

121
30.43 (5.76)

67
29.73 (6.08)

54
31.29 (5.27)

83 (69)
16 (13)
22 (18)

47 (39)
7 (6)
13 (11)

36 (30)
9 (7)
9 (7)

15 (12)
32 (26)

10 (8)
16 (13)

5 (4)
16 (13)

41 (34)
63 (52)
17 (14)

27 (22)
39 (32)
1 (1)

14 (12)
24 (20)
16 (13)

22
57
42
19
12
19
5
9
57
16

7
29
31
8
4
9
5
8
34
9

15
28
11
11
8
10
0
1
23
7

0.134
0.63

0.563

<0.0001

0.004
(18)
(47)
(35)
(16)
(10)
(16)
(4)
(7)
(47)
(13)

4 (3)
20
25
75
5
51
9
6

(6)
(24)
(26)
(12)
(6)
(13)
(7)
(12)
(51)
(13)

1 (1)

(17)
(21)
(62)
(96)
(42)
(8)
(5)

7
10
50
2
28
5
1

(6)
(8)
(42)
(3)
(42)
(7)
(1)

(12)
(23)
(9)
(20)
(15)
(19)
(0)
(2)
(43)
(13)

3 (6)
13
15
25
3
23
4
5

(11)
(12)
(21)
(6)
(43)
(7)
(9)

0.310
0.189
0.608
0.064
0.056
0.464
0.871
0.323
0.008

0.655
1
1
0.089

HCA, histologically confirmed horioamnionitis; BMI, Body Mass Index; CBEU, cytobacteriological examination of urine.
Data are expressed as numbers and percentages (for categorical variables), or as means and standard deviations for continuous variables.
HCA and no-HCA are groups of women with and without a histological/bacteriological diagnosis of CA.

for which the second sample was taken during the

2472 hr before delivery in a patient with HCA were
called imminent CA (iHCA). All other deltas,
obtained in women with or without CA, were called
no imminent CA (no-iHCA) deltas. As patients had
multiple blood samples, there were numerous deltas
per patient, reflecting the longitudinal follow-up,
which is the main strength of our paper. The relationship between the delta value and inclusion in
the iHCA or no-iHCA group was analyzed by
patient-stratified logistic regression. Variables with
82

P < 0.20 were retained in the multivariate model,

which included clinical and biological data.
All analyses were performed using R software version 2.14.1 (2011-12-22).
Results
Baseline Clinical Characteristics
The study included 121 consecutive patients with
PPROM, of whom 54 (45%) had a final diagnosis of
American Journal of Reproductive Immunology 73 (2015) 7990
2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

MATERNAL INFLAMMATORY MARKERS IN PPROM

CA and 67 (55%) did not (Table I). Therefore, the

majority of women with PPROM did not experience
CA (Table I).
Two intramuscular injections of betamethasone
(12 mg) were given at an interval of 24 hr for fetal
lung maturation. All of the patients except four
received intravenous antibiotics for a mean duration
of 102 hr (range: 9528). A combination of third
generation cephalosporins and metronidazole was
the most commonly used antibiotics regimen. All of
the patients except ten received tocolysis (nicardipine, atosiban, either alone or sequentially).
There were no differences between the study
groups for age and BMI. However, it was a greater
proportion of women with parity 3 in the CA
group, as among these 17 patients, 16 had histological evidence of CA. Gestational age at admission was
significantly associated with CA (P < 0.01), as CA
was confirmed in 15 of the 22 women (68%) with a
gestational age of <26 WG, but only in 11 of the 42
women (26%) above 32 WG, with an unadjusted
relative risk of 2.60 (95% confidence interval: 1.45
4.66, P = 0.0027) (Table I).
Clinical Characteristics at the Time of Delivery
A positive placental culture was found in 26
patients, 23 with only one pathogen and three with
two pathogens. Escherichia coli was the most prevalent pathogen followed by Bacteroides fragilis. Histology was negative in eight women with positive
placental culture (Table II).
The placenta was deemed positive for CA after histological assessment in 53 cases, among which 18
had a positive placental culture. Finally, 8 of the 26
women with positive placental culture had a negative histological assessment.
There was no significant difference between the
HCA and no-HCA group for the latency period (time
between hospitalization and delivery). However, gestational age at delivery was significantly lower in the
HCA group than in the no-HCA group (P < 0.01).
Early neonatal infection, either confirmed or probable, correlated strongly with a diagnosis of CA
(P < 0.001) (Table II).
Biological Data at Admission
Among the biological characteristics at admission,
only WBC count was higher in the HCA than in
no-HCA group (P = 0.013). The mean IL-6 and CRP
American Journal of Reproductive Immunology 73 (2015) 7990
2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Table II Characteristics at the Time of Delivery and Newborns

Outcome
No-HCA
n
Caesarean section (n)
Delay between preterm
premature rupture of
membrane and
delivery (days)
Gestational age at
delivery (weeks)
<26
2629
3034
35
Birthweight (g)
Apgar 5 min <7
Apgar 10 min <7
Arterial pH
Venous pH
Oxygenotherapy at
day 28
Oxygenotherapy at
day 36
Neurological
impairment
Early neonatal infection
Death

67
22 (33)
13.76 (20.07)

P-value

HCA
54
20 (37)
8.98 (10.84)

0.702
0.098

0.004
3
19
39
6
1970.7
2
1
7.33
7.37
7

(2)
(16)
(32)
(5)
(619.3)
(3)
(1)
(0.06)
(0.06)
(10)

9
23
22
0
1593.9
7
2
7.28
7.35
12

(7)
(19)
(18)
(0)
(558.7)
(13)
(4)
(0.09)
(0.08)
(22)

<0.001
0.076
0.579
0.005
0.108
0.075

2 (3)

4 (7)

0.401

5 (7)

10 (19)

0.056

2 (3)
1 (1)

21 (39)
5 (9)

<0.0001
0.088

HCA, histologically confirmed chorioamnionitis.

Data are expressed as numbers and percentages (for categorical
variables), or as means and standard deviations for continuous
variables.
HCA and no-HCA are groups of women with and without a histological/bacteriological diagnosis of CA.

levels were numerically higher in the HCA than the

in the no-HCA group, but the difference did reach
statistical significance (P = 0.051 and P = 0.071,
respectively). IP-10 and MCP-1 levels in women
with and without CA were not different (Table III).
Biological Parameters During the 5 Days Before
Delivery
Fig. 1 presents the median levels of CRP, WBC, and
the cytokines (IL-6, IP-10, MCP-1) on the 5 days
preceding delivery. A progressive and significant
increase was observed for CRP and IL-6 in the HCA
group, and the difference with the no-HCA group
became statistically significant 2 days before delivery
(MANOVA, P < 0.001). The trend was less marked
for the median WBC count. Indeed, Although the
83

LE RAY ET AL.

Table III Biological Characteristics of the Population Upon

Admission

n
White blood cells
(n 9 103/mm3)
CRP (mg/L)
IL-6 (pg/mL)
IP-10 (pg/mL)
MCP-1 (pg/mL)

P-value

No-HCA

HCA

67
11.66 (3.08)

54
13.3 (3.89)

0.013

7.72
1.41
89.51
108.97

12.69
3.09
71.72
92.18

0.071
0.051
0.265
0.520

(8.42)
(1.74)
(92.8)
(140.47)

(18.46)
(5.17)
(53.38)
(105.69)

HCA, histologically confirmed chorioamnionitis.

Data are expressed as means and standard deviations.
HCA and no-HCA are groups of women with and without a histological/bacteriological diagnosis of CA.

median WBC count was always numerically higher

in the HCA than in the no-HCA group on the 5 days
preceding delivery, the difference was not always
statistically significant (MANOVA, P < 0.0001), but the
difference was statistically significant for day 3 and
day 1 before delivery (P < 0.01).
In contrast, there were no significant differences,
or even trends toward differences, between the two
groups for median IP-10 and MCP-1 levels.
Assessment of Predictors of Delivery Associated
With CA During the 2472 hr Before Delivery
We then calculated variations, so called deltas (see
methods section) for each of our five biological
markers, CRP, WBC, IL-6, IP-10, and MCP-1, and
compared those in which the second sample was
obtained during the 2472 hr before delivery in
women with HCA (iHCA deltas) with all other deltas
obtained in women with or without HCA (no-iHCA
deltas). Delta values for the biological parameters
tested in the iHCA and no-iHCA groups are presented in Table IV. Table IV shows that delta values
are numerically very close and that a decrease in the
plasma level of the markers of interest (evidenced by
negative deltas in the first quartile) can be observed
in both groups.
Table V shows that despite the variations in IL-6,
CRP and IP-10 levels are statistically associated with
CA after multivariate analysis, the low-level variation in each parameter tested during the 2472 hr
before delivery made it impossible to discriminate
between PPROM with HCA and PPROM without
84

HCA. This is shown in Fig. 2, the ROC curve, which

shows an area under the curve (AUC) of 0.56.
In summary, our results showed that the median
CRP, WBC and IL-6 levels were higher in the HCA
than in the no-HCA group during the days before
delivery. However, longitudinal follow-up of the
variations in the tested parameters did not make it
possible to identify patients who were about to
develop HCA, even when samples were taken
between 24 and 72 hr before delivery.
Discussion
In this prospective longitudinal follow-up of 121
women admitted with PPROM between 24 and
34 weeks of gestation we described that almost half
of the women had a confirmed CA (45%). Gestational age and parity were strongly associated with
the diagnosis of CA. This is consistent with previous
studies showing that gestational age at both sampling and delivery were statistically lower in women
with CA or MIAC than without,12,18 even if other
authors did not report such differences in gestational
age.41,42 Our finding that more multiparous women
were in the HCA group might be, although not
formally, explained by some kind of cervical insufficiency that is a recognized risk factor for intra-amniotic inflammation.43 This is, nevertheless, not an
unexpected finding as multiparous women are consistently found more numerous in the groups with
CA than without, even if the difference is generally
not statistically significant.12,18,41 We describe a progressive and significant increase in CRP and IL-6 in
the HCA group, with a difference with the no-HCA
group becoming statistically significant 2 days
before delivery. No such findings were observed for
WBC, IP-10 and MCP-1 levels. Univariate analysis
regression showed variations in IL-6, IP-10, and CRP
levels, during the 2472 hr before delivery to be
predictors of the occurrence of iHCA. After adjustment only IL-6 (1.062; 1.02151.1041, P = 0.003)
and IP-10 (1.0021; 1.00071.0035, P = 0.0044)
remained significant predictors, but the diagnostic
accuracy was low (ROC curve, AUC = 0.56). The
recently published study by Cobo et al.27 strengthens the interest of our longitudinal follow-up.
Indeed, they found no difference in the maternal
plasma level of several inflammatory markers,
including IL-6 and IP-10 between women with and
without MIAC,27 likely because they collected only
one sample of maternal blood. There are, to our
American Journal of Reproductive Immunology 73 (2015) 7990
2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

MATERNAL INFLAMMATORY MARKERS IN PPROM

(a)

(b)

(c)

(d)

(e)

Fig. 1 (ae) Mean values for the measurement of the five biological parameters of interest on the 5 days preceding delivery. Black boxes
represent values for women without chorioamnionitis (no-HCA) and gray boxes for those with HCA. All comparisons are for HCA versus no-HCA,
**P < 0.01, ***P < 0.001.

knowledge, very few reports of longitudinal monitoring of maternal inflammatory markers in women
with PPROM, and in our own study, statistical
American Journal of Reproductive Immunology 73 (2015) 7990
2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

significance between HCA and no-HCA was not

reached at the time of admission, but only close to
delivery.
85

LE RAY ET AL.

Table IV Description of Deltas of the Biological Markers During the 2472 hr Before Delivery
iHCA deltas
Median
IL-6 (pg/mL)
IP-10 (pg/mL)
CRP (mg/L)
WBC (n 9 103/mm3)
MCP-1 (pg/mL)

No-iHCA deltas
1st quartile

0.337
8.463
0
0.2
10.168

0.1967
7.803
1.6
1.6
3.998

3rd quartile
1.2694
32.803
2.45
1.8
41.601

Median
0.1273
6.234
0
0.4
8.882

1st quartile
0.4765
10.046
1.5
1.7
10.171

3rd quartile
0.7327
27.612
1.2
0.8
44.838

WBC, white blood cells.

Data are expressed as medians (1st3rd quartiles).
iHCA are deltas where the second sample was obtained during the 2472 hr prior to delivery with confirmed HCA, and no-iHCA are all other
deltas obtained in women without or with a histological/bacteriological diagnosis of HCA.

Table V Evaluation of Predictors of Delivery During the 2472 hr Before Delivery and Associated with Chorioamnionitis
Univariate analysis
OR, 95% CI
IL-6
IP-10
MCP-1
Clinical chorioamnionitis
WBC
CRP

1.0113
1.0006
1.0001
1.0309
1.0108
1.0045

(1.0021.0208)
(1.00011.0010)
(0.99971.0006)
(0.80441.3213)
(0.9981.023)
(1.00051.0085)

Multivariate analysis
P-value
0.021
0.019
0.542
0.810
0.085
0.031

OR, 95% CI

P-value

1.0098 (1.00091.0189)
1.0005 (1.00001.0010)

0.034
0.042

1.0029 (0.99891.0069)

0.154

WBC, white blood cells; OR, odds ratio; CI, confidence interval.

Fig. 2 Receiveroperator characteristics curve for the multivariate

model (ROC).

In this later paper, Cobo et al. slightly contradicted

their study published 1 year before where they
described IL-6 as the only biomarker that increased
significantly in preterm labor with MIAC.41
86

Our study assessed the pertinence of determining

variations in a combination of clinical and biological
markers to predict HCA among women with
PPROM. Such predictors are needed to assist obstetricians to manage PPROM and to induce delivery in
a timely fashion, that is, to say when the balance
between the risks of preterm delivery and maternofetal sepsis is optimal.
The wide variations in most of the biological
markers of potential clinical interest make it difficult
to define relevant thresholds, and the invasiveness
of AF sampling makes it inappropriate for repeated
measurements. Cobo et al.27 have suggested cervical
mucus to be an appropriate matrix to test for MIAC,
but once again not with repeated measurement during pregnancy. As a result, no single marker, or
combination of markers of CA, has been validated. It
is of interest to note that a clinical diagnosis of CA at
the time of delivery, based on clinical exam and routine blood tests had, a sensitivity and specificity of
0.57 and 0.81, respectively, for the presence of HCA.
This poor diagnostic accuracy might be explained, at
American Journal of Reproductive Immunology 73 (2015) 7990
2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

MATERNAL INFLAMMATORY MARKERS IN PPROM

least partly, as even a proven CA can be associated

with a decrease in CRP and WBC during predelivery
follow-up (cf. Table IV, first quartile values).
In our study, we found that gestational age and parity were strongly associated with the diagnosis of CA,
as reported by Park et al.44, who described a model
based on maternal blood CRP, WBC, parity, and gestational age with good discrimination for the diagnosis
of intra-amniotic infection (IAI) in women with
PPROM. In the Park et al.44 study, the diagnosis of IAI
was based on either a positive AF culture and/or an
AF IL-6 level >2.6 ng/mL, and thus did not overcome
the limitation of the invasive and single assessment of
CA. The same criticism can be made about another
recently reported study by the same team, suggesting
that a combination of cervicovaginal IL-6 and gestational age was accurate (AUC of 0.807) in predicting
IAI.18 From a clinical perspective, a single calculation
of the risk of IAI with a sensitivity of 60% and a specificity of 70% does not appear to be really useful, especially for very early PPROM, and given that 29% of
women presenting with PPROM were, for different
reasons, not included in the study.18 Therefore, using
the model proposed by Ryu et al.18 might increase the
rate of preterm delivery, at least in countries where
antibiotic prophylaxis is the standard treatment for
women with PPROM,45 which is not always the case
as two recently published randomized clinical trials
showed that antibiotics were given to only 4050% of
women with PPROM.46,47
Increasing prematurity might be of bigger concern
than CA, as a recently published study suggested
that in preterm infants (<37 weeks of gestation and
2500 g at birth), a documented diagnosis of clinical
CA was not associated with adverse neurodevelopmental outcomes.48 This study, despite several limitations, principally the lack of power, suggested that
preterm delivery rather than CA aggravated the risk
of long-term adverse outcomes.48 This finding is in
support of our approach, which involves assessing
variations in biological makers close to delivery, to
delay delivery as long as possible and to deliver only
when CA likely to be diagnosed.
Interestingly, in the study by Cobo et al., they
suggested that increases in serum markers (IL-18) in
MIAC can only be observed before 32 weeks of
gestation.41 This may explain why our biological follow-up did not improve the diagnostic yield of
serum markers.
Cytokine levels in general, and IL-6 in particular,
are very low, in the picogram range, with wide
American Journal of Reproductive Immunology 73 (2015) 7990
2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

confidence intervals. In the paper by Gulati et al.,20

even though the authors described statistically significant differences, IL-6 levels were 3.98  3.99
and 20.09  16.83 pg/mL in women with PPROM
without and with CA, respectively. In this study,
the authors found, as we did, an increase in IL-6
levels from admission to the 7th day after admissiononset of labor, which was observed irrespective
of the presence of CA. Surprisingly, this increase
was more pronounced in women without than in
those with CA (3.5 versus 2.8-fold increase, respectively). Besides variability between measurements,
there are also discrepancies in the level of IL-6
expression in maternal blood that can be explained
either by the sensitivity of the assays used or by
the different cytokine expressor phenotype.49 Furthermore, the exclusion/inclusion of elective labor
induction, which is likely to affect cytokine levels
at delivery,50 might contribute to this variability.51
We have recently shown that HCA was not associated with significant changes in maternal plasma
level of MCP-1-expressing monocytes or MCP-1,52
suggesting that the immune response is preferentially located on the fetal side, as previously
reported.53
In agreement with two meta-analyses, which concluded that CRP was not a valid predictor of CA in
PPROM, we did not find longitudinal monitoring of
CRP to be predictor of HCA.32,33 Nevertheless, a
recent paper from the same group suggest CRP level
to be associated with an increased risk for neonatal
sepsis (OR: 1.01 per mmol/L) in women with late
RPM.54
The strength of our study lies in the fact that we
prospectively assessed the interest of close monitoring, rather than taking a single measurement, of several maternal plasma markers.48 As sampling AF
carries a significant risk of complications,55 it is unlikely to become a more accurate diagnostic tool to
monitor women with PPROM. By very close prospective monitoring of a significant number of
women with PPROM, we generated a significant
body of data and confirmed that close to delivery,
IL-6, CRP or WBC counts vary whether or not the
women have confirmed HCA.
The negative conclusion of our study can be considered as a weakness; nevertheless, it is an important finding for the obstetricians.
Analyses could have been stratified according specific risk factors, such as BMI, history of previous
PPROM, but as none of this characteristics was
87

LE RAY ET AL.

statistically different between HCA and no-HCA

women, this might not be a major limitation of our
study. Furthermore, our study was not powered
enough for such stratification.
Conclusion
Despite IL-6 and IP-10 independent positive association with HCA, following the comprehensive clinical
and biological monitoring of 121 women, we suggest
that none of these markers alone is likely to be of
clinical value to help obstetricians to decide when to
induce labor. Other clinically helpful predictors still
need to be found, as recent studies assessing new
markers such as Toll-like receptor 2,56 or RANTES,27
were negative. It might also be of interest to assess
such a prospective follow-up restricted to PPROM
before 28 WG.
Acknowledgments
This work was supported by a grant from the French
Ministry for Health (PHRC interregional 2006). The
authors would like to thank the women who agreed
to take part of the study, the midwives and obstetricians who included and followed these women. Special thanks for the staff of the Clinical Research Unit
(Centre dInvestigations Cliniques plurith

ematique
1432, formerly 803) for the logistics of this trial.
Final thanks go to Philip Bastable for English editing.
Disclosure of interests
Authors have no conflict of interest relevant to this
paper.
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