Fetal Inflamatory

2013

http://informahealthcare.com/jmfISSN: 1476-7058 (print), 1476-4954 (electronic)

J Matern Fetal Neonatal Med, 2013; 26(8): 795–801! 2013 Informa UK Ltd. DOI: 10.3109/14767058.2013.765404

The fetal inflammatory response in subgroups of women with pretermprelabor rupture of the membranes

Marian Kacerovsky1,2, Teresa Cobo3, Ctirad Andrys4, Ivana Musilova2,5, Marcela Drahosova4, Helena Hornychova6,Petr Janku7, and Bo Jacobsson8,9

1Biomedical Research Center, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic, 2Department of Obstetrics and Gynecology,

Faculty of Medicine in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic, 3Maternal Fetal Medicine Department, Hospital

Clinic, IDIBAPS, Barcelona, Spain, 4Department of Clinical Immunology and Allergy, Faculty of Medicine in Hradec Kralove, University Hospital

Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic, 5Department of Obstetrics and Gynecology, University Hospital

Pardubice, Pardubice, Czech Republic, 6Fingerland’s Department of Pathology, Faculty of Medicine in Hradec Kralove, University Hospital Hradec

Kralove, Charles University in Prague, Hradec Kralove, Czech Republic, 7Department of Obstetrics and Gynecology, University Hospital and Masaryk

University, Brno, Czech Republic, 8Department of Obstetrics and Gynecology, Sahlgrenska University Hospital, Gothenburg, Sweden, and9Department of Public Health, Oslo University, Oslo, Norway

Abstract

Objective: To evaluate the influence of microbial invasion of the amniotic cavity (MIAC) andhistological chorioamnionitis (HCA) on the intensity of the fetal inflammatory response and theoccurrence of fetal inflammatory response syndrome (FIRS) in preterm prelabor rupture ofmembranes (PPROM).Methods: One hundred and forty-nine women with singleton pregnancies complicated byPPROM between the gestational ages 24þ 0 and 36þ 6 weeks were included in the study.Blood samples were obtained by venipuncture from the umbilical cord after the delivery of thenewborn. The umbilical cord blood interleukin (IL)-6 levels were evaluated using ELISA kits. Thefetal inflammatory response was determined by IL-6 levels, and FIRS was defined as anumbilical cord blood IL-6411 pg/mL.Result: IL-6 levels and the occurrence of FIRS were higher in women complicated with bothMIAC and HCA (median IL-6 35.5 pg/mL, FIRS in 68%) than in women with HCA alone (medianIL-6 5.8 pg/mL, FIRS in 36%), MIAC alone (median IL-6 2.8 pg/mL, FIRS in 17%) or womenwithout MIAC or HCA (median IL-6 4.3 pg/mL, FIRS in 29%). There were no differences in IL-6levels or rates of FIRS among women with MIAC alone or HCA alone and women without bothMIAC and HCA.Conclusion: A higher fetal inflammatory response mediated by umbilical cord bloodIL-6 was identified when both MIAC and HCA were detected in pregnancies complicated byPPROM.

Keywords

Infection, inflammation, interleukin-6,preterm delivery

History

Received 3 November 2012Accepted 8 January 2013Published online 1 February 2013

Introduction

Preterm prelabor rupture of membranes (PPROM) is defined

as a leakage of amniotic fluid before 37 gestational weeks that

precedes the onset of regular uterine activity [1]. PPROM is

responsible for 30%–40% of preterm deliveries and belongs to

a group of obstetrical syndromes having multiple etiologies

[1,2]. PPROM is often complicated by microbial invasion of

the amniotic cavity (MIAC) and histological chorioamnionitis

(HCA), observed in 25%–50% and 42%–64% of cases,

respectively [3–8].

Both MIAC and HCA can be responsible for the activation

of fetal innate immunity and lead to the fetal inflammatory

response, which is characterized by elevated levels of

interleukin (IL)-6 and other inflammatory mediators in

umbilical cord blood [9,10]. Although the highly orchestrated

releasing of fetal cytokines, chemokines and other inflamma-

tory mediators is an important part of the fetal defense against

infection, the excessive production of these mediators can

have deleterious consequences for the fetus [10–12]. In 1998,

Gomez et al. [9] defined fetal plasma IL–6 concentrations

higher than 11 pg/mL as the fetal inflammatory response

syndrome (FIRS). This information is clinically relevant

because fetuses affected by FIRS have evidence of multiorgan

involvement and are at increased risk for serious neonatal

morbidity [10,13]. Other inflammatory mediators in fetal

plasma have also been considered in the definition of FIRS

[14–17]. In addition, funisitis along with chorionic plate

vasculitis have been demonstrated as the histopathological

counterpart of FIRS [18].

During the course of MIAC, the fetus is exposed to infected

amniotic fluid. There is evidence that this hostile environment

Address for correspondence: Marian Kacerovsky, MD, PhD, BiomedicalResearch Center, University Hospital Hradec Kralove, Sokolska 581, 50005 Hradec Kralove, Czech Republic. Tel: þ420-777657991, Direct:þ420-495832676. Email: [email protected]

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is associated with the intra-amniotic and fetal inflammatory

response [4,19,20]. Nevertheless, our recent work demon-

strates that the intra-amniotic inflammatory response reflects a

late stage of inflammation, where both the amniotic fluid

(MIAC) and the placenta with fetal membranes (HCA) are

involved [21]. This is an important fact from a clinical point of

view, suggesting that only MIAC leading to the development

of HCA is associated with a strong intra-amniotic inflamma-

tory response. Given that intra-amniotic inflammation is a risk

factor for the development of fetal inflammatory response and

FIRS, we hypothesized that the intensity of the fetal inflam-

matory response and the occurrence of FIRS would be

different in the PPROM subgroup based on the presence or

absence of MIAC and HCA.

Therefore, the purpose of this study was to examine the

intensity of the fetal inflammatory response, characterized by

umbilical cord blood IL-6 levels, and the occurrence of FIRS

(defined as umbilical cord blood IL-6411 pg/mL) according

to the presence of MIAC and HCA in PPROM pregnancies.

Material and methods

Sample collection

Between September 2010 and May 2012, we conducted a

prospective cohort study of pregnant women at gestational

ages of 24þ 0 and 36þ 6 weeks who were admitted to the

Department of Obstetrics and Gynecology, University

Hospital Hradec Kralove, Czech Republic. Women with

singleton pregnancies, PPROM and maternal age 418 years

were eligible for the study. Women with diabetes mellitus,

preeclampsia, signs of fetal growth restriction, the presence of

either congenital or chromosomal fetal abnormalities, signs of

fetal hypoxia or significant vaginal bleeding were excluded

from the study.

Gestational ages were established by first-trimester fetal

biometry. In the Czech Republic, women with PPROM at less

than 34 weeks of gestation are treated with corticosteroids for

the induction of lung maturation (two doses of 14 mg

betamethasone administered intra-muscularly 24 h apart),

tocolytics for 48 h and antibiotics, whereas no treatment

except antibiotics is initiated to delay delivery after 34 weeks.

The management of PPROM in the Czech Republic is not

expectant (except 528 gestational weeks); the induction of

labor or the termination of pregnancy is initiated no later than

72 h after rupture of the membranes, depending on the

gestational age of the pregnancy, the fetal status, the maternal

serum levels of C-reactive protein (CRP) and cervicovaginal

streptococcus � colonization [22].

PPROM was diagnosed by examination with a sterile

speculum to verify the pooling of amniotic fluid in the vagina

after confirming the presence of insulin-like growth factor

binding protein (ACTIM PROM test; MedixBiochemica,

Kauniainen, Finland) in the vaginal fluid.

Ultrasound-guided transabdominal amniocentesis was per-

formed upon admission before the administration of cortico-

steroids, antibiotics or tocolytics; approximately 5 mL of

amniotic fluid was aspirated. The samples were transported to

the laboratory for detection of genital mycoplasmas using

polymerase chain reaction analyses and for aerobic and

anaerobic cultivation.

Umbilical blood samples were obtained by venipuncture

from clamped umbilical cords immediately after the delivery

of the neonates and prior to the delivery of the placenta using

a vacutainer blood collection system. The sample of umbilical

cord blood was centrifuged and aliquoted, and the supernatant

were stored at �70 �C until the samples were assayed.

After delivery, the placentas were fixed in formalin, and

tissue samples from the placenta, umbilical cord and placental

membranes were routinely processed and embedded in

paraffin. Tissue sections were stained with hematoxylin and

eosin for standard histological examination.

To evaluate the fetal inflammatory response and rate of

FIRS, the data were analyzed according to the presence of

both MIAC and HCA, the presence of HCA alone, the

presence of MIAC alone and a group without either MIAC

or HCA.

The study was approved by the institutional review board

committee (March 19, 2008; no. 200804 SO1P). All women

provided their written informed consent and were self-

reported as Caucasians.

Diagnosis of MIAC

MIAC was defined as a positive PCR analyses for genital

mycoplasmas (Ureaplasma parvum, Ureaplasma urealyticum

and Mycoplasma hominis) and/or Chlamydia trachomatis and/

or growth of any bacteria in the amniotic fluid except for

coagulase-negative Staphylococcus epidermidis, which was

considered a skin contaminant.

Diagnosis of HCA

The placentas were collected and fixed in 10% neutral

buffered formalin. Tissue samples were obtained from the

placenta (at least two samples), umbilical cord (typically, one

sample) and placental membranes (at least two samples) and

were processed and embedded in paraffin. Sections of tissue

blocks were stained with hematoxylin and eosin. The degree

of neutrophil infiltration was evaluated separately in the free

membranes (amnion and chorion-decidua), in the chorionic

plate, and in the umbilical cord according to the criteria

provided by Salafia [23]. A diagnosis of HCA was made

based on the presence of histological grades of chorion-

decidua 3–4, chorionic plate 3–4, umbilical cord 1–4 and/or

amnion 1–4 [23]. Histological grades of umbilical cord 1–4

were categorized as the presence of funisitis [23].

Histopathological examinations were performed by a single

pathologist, who was blinded to the clinical status of the

patient.

Umbilical cord blood IL-6 levels

IL-6 levels were assessed by enzyme-linked immunosorbent

assays Human IL-6 Quantikine (R&D Systems Inc.,

Minneapolis, MN). The sensitivity of the test was less than

0.70 pg/mL, and the inter-assay and intra-assay coefficients

were less than 10%.

Diagnosis of FIRS

FIRS was diagnosed in the presence of umbilical cord blood

IL-6 concentrations higher than 11.0 pg/mL [9].

796 M. Kacerovsky et al. J Matern Fetal Neonatal Med, 2013; 26(8): 795–801

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Statistical analysis

The demographic and clinical characteristics were compared

using a non-parametric Jonckheere–Terpstra test and pre-

sented as medians (range) for continuous variables.

Categorical variables were compared using the chi-square

test and were presented as numbers (%). The normality of the

data was tested using the D0Agostino and Pearson omnibus

normality test. Because umbilical cord blood concentrations

of IL-6 were not normally distributed, a non-parametric

Kruskal–Wallis and Mann–Whitney U-test was used for the

analyses as appropriate. Differences were considered statis-

tically significant at p50.05. All p values were from two-

sided tests, and all statistical analyses were performed using

SPSS 19.0 for Mac OS X (SPSS Inc., Chicago, IL, USA) and

with GraphPad Prism 5.03 for Mac OS X (GraphPad

Software, La Jolla, CA, USA).

Results

Demographic and clinical characteristics of the studypopulation

In total, 165 women with PPROM at gestational ages between

24þ 0 and 36þ 6 weeks were recruited. Of the 165 women,

amniocentesis failed in 10 (6%) women, and histopathologic

assessment of the placenta was not available for 6 (4%)

women. Thus, 149 women were included in our study. The

maternal and neonatal characteristics of the entire study group

are summarized in Table 1. The overall rate of MIAC was

38% (56/149), and HCA was observed in 56% (83/149) of

women. Both MIAC and HCA were present in 26% (38/149)

of women. HCA alone was present in 30% (45/149) of

women, MIAC alone was present in 12% (18/149) of women,

and 32% (48/149) women exhibited neither MIAC nor HCA.

The differences among the subgroups included the gestational

age at delivery and sampling, the latency between PPROM

and delivery, the antenatal administration of corticosteroids

and maternal serum CRP levels at admission. Women with

both MIAC and HCA were of lower gestational age at

delivery and sampling, had children of lower birth weights,

had a higher rate of corticosteroid administration, higher

maternal serum CRP and the longest latency. In addition,

women with MIAC and HCA had higher rate of funisitis than

those with HCA alone. Nevertheless, the latency from

PPROM to delivery, the gestational age at delivery and the

birth weight cannot be considered relevant clinical findings in

our cohort of women because of the active management

beyond 28 weeks in Czech Republic (128/149) [22].

Umbilical cord blood IL-6

We observed differences in umbilical cord blood IL-6 levels

among the subgroups based on the presence or absence of

MIAC and HCA (p¼ 0.001). The women with both MIAC

and HCA exhibited the highest median level of IL-6 levels

among the subgroups (both MIAC and HCA 35.5 pg/mL,

HCA alone 5.8 pg/mL, MIAC alone 2.8 pg/mL, and without

both MIAC and HCA 4.3 pg/mL), and differences in the

Table 1. Maternal and neonatal characteristics in the different subgroup of PPROM pregnancies.

The presence ofMIAC and HCA

(n¼ 38)

The presence ofHCA alone

(n¼ 45)

The presence ofMIAC alone

(n¼ 18)

The absence ofMIAC and HCA

(n¼ 48) p Value

Maternal age 29 (18–40) 34 (22–44) 32 (24–44) 30 (19–40) 0.50Prepregnancy BMI 22.0 (17.0–35.7) 24.0 (17.5–40.6) 21.3 (16.3–29.4) 21.4 (16.4–38.6) 0.56Smoking 10 (26%) 6 (13%) 5 (28%) 8 (17%) 0.36Gestational age at admis-

sion (weekþ days)31þ 1 (24þ 5–35þ 4) 33þ 5 (25þ 0–36þ 4) 34þ 3 (26þ 2–36þ 3) 34þ 1 (24þ 0–36þ 6) 50.0001

Gestational age at delivery(weekþ days)

31þ 3 (25þ 0–35þ 5) 34þ 0 (25þ 4–36þ 6) 34þ 4 (26þ 2–36þ 4) 34þ 2 (24þ 0–36þ 6) 50.0001

Latency from PPROM toamniocentesis (h)

7 (2–23) 6 (1–18) 4 (1–23) 7 (1–24) 0.22

Latency from PPROM todelivery (h)

54 (8–244) 45 (6–128) 17 (5–114) 22 (7–242) 0.005

CRP levels at admission(mg/L)

8.8 (0–82.0) 7.9 (1.0–72.0) 4.0 (1.0–13.0) 5.0 (0–71.3) 0.01

WBC count ad admission(�109 L)

13.4 (7.0–27.0) 12.0 (6.0–20.0) 11.0 (7.0–19.0) 12.0 (7.0–24.0) 0.12

Administration ofcorticosteroids

27 (71%) 22 (49%) 7 (39%) 18 (38%) 0.01

Administration ofantibiotics

38 (100%) 43 (96%) 18 (100%) 46 (96%) 0.48

Induction of labor 19 (50%) 14 (31%) 9 (50%) 16 (33%) 0.20Spontaneous vaginal

delivery26 (68%) 31 (69%) 14 (78%) 35 (73%) 0.87

Cesarean section 11 (29%) 14 (31%) 4 (22%) 13 (27%) 0.83Forceps delivery 1 (3%) 0 (0%) 0 (0%) 0 (0%) 0.40Birth weight (g) 1570 (710–2570) 2160 (640–3150) 2225 (1040–1870) 2248 (750–3120) 50.0001Funisitis 16 (42%) 6 (13%) – – 0.0035 min Apgar score57 2 (5%) 3 (7%) 0 (0%) 1 (2%) 0.54

PPROM: Preterm prelabor rupture of membranes; MIAC: microbial invasion of amniotic cavity; HCA: histological chorioamnionitis; CRP: C-reactiveprotein; WBC: white blood cells.

Continuous variables were compared using a non-parametric Jonckheere–Terpstra test and presented as medians (range). Categorical variables werecompared using Chi-square test and presented as numbers (%). Statistically significant differences are marked in bold.

DOI: 10.3109/14767058.2013.765404 Fetal inflammatory response and PPROM 797

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umbilical cord blood IL-6 levels were observed between

women with both MIAC and HCA and other women (MIAC

and HCA versus HCA alone: p¼ 0.01; MIAC and HCA

versus MIAC alone: p¼ 0.0006; MIAC and HCA versus

without MIAC and HCA: p¼ 0.0004); see Figure 1. No

differences in umbilical cord blood IL-6 levels were observed

among the women with HCA alone, MIAC alone or without

MIAC and HCA (HCA alone versus MIAC alone: p¼ 0.15;

HCA alone versus without MIAC and HCA: p¼ 0.45; MIAC

alone versus without MIAC and HCA: p¼ 0.35); see Figure 2.

FIRS and subgroup of PPROM

FIRS was identified in 68% (26/38) of women with both MIAC

and HCA, 36% (16/45) of women with HCA alone, 17% (3/18)

of women with MIAC alone, and 29% (14/48) of women

without MIAC and HCA. We observed statistically significant

difference in the rates of FIRS based on the presence or

absence of MIAC and HCA (p50.0001). Women with both

MIAC and HCA exhibited higher FIRS rates than did other

women (HCA alone: p¼ 0.004; MIAC alone: p¼ 0.0004;

without MIAC and HCA: p¼ 0.0004); see Figure 3. No

differences in the rates of FIRS were found among women

with HCA alone, MIAC alone and women without MIAC and

HCA (HCA alone versus MIAC alone: p¼ 0.22; MIAC alone

versus without MIAC or HCA: p¼ 0.36; HCA alone versus

without MIAC and HCA: p¼ 0.66); see Figure 4.

Discussion

MIAC and HCA are two of the most common pathological

conditions that determine the outcome of PPROM pregnan-

cies. Understanding the influence of these conditions on the

development of the fetal inflammatory response is essential

for better management of this pregnancy complication. The

following are the key findings from this study: (i) there was a

difference in the fetal inflammatory response and the rate of

FIRS based on the presence of MIAC and/or HCA, (ii) a

higher fetal inflammatory response, as determined by umbil-

ical cord blood IL-6 levels, was detected in the presence of

both MIAC and HCA in PPROM pregnancies, (iii) the highest

rate of FIRS was observed in PPROM pregnancies compli-

cated by both MIAC and HCA and (iv) no differences in the

fetal inflammatory response and rate of FIRS were noted

among groups with HCA alone, MIAC alone, and those

without MIAC and HCA.

Microorganisms that are inside the amniotic cavity can

access the fetus by various entry points, including the skin,

Figure 1. The umbilical cord blood IL-6levels with respect to the presence or absenceof MIAC and/or HCA. Pregnancies with bothMIAC and HCA exhibited a higher medianIL-6 value than did those with HCA alone,MIAC alone, and those without MIAC andHCA.


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respiratory and gastrointestinal tract, and the ear [24,25].

Microorganisms, their parts or their products can be

recognized by specific tools of fetal innate immune system

and trigger a systemic inflammatory response with the main

aim of eliminating this threat. Moreover, the fetal inflamma-

tory response can occur as a consequence of non-infectious

insults, such fetal anemia due to Rh alloimunization [26]. Lee

et al. [27] described the link between intra-amniotic and fetal

inflammatory response in PPROM pregnancies. Our group

recently reported that the presence of MIAC leading to the

development of HCA in PPROM pregnancies is associated

with stronger intra-amniotic inflammatory responses compare

to other PPROM pregnancies [21]. With this knowledge, we

hypothesized that MIAC leading to HCA is related to the fetal

inflammatory response. Moreover, to date, there was a

paucity of information regarding the fetal inflammatory

response in PPROM pregnancies complicated by both

MIAC and HCA, HCA alone, MIAC alone and pregnancies

not complicated by either condition.

Based on our results regarding intra-amniotic inflamma-

tory responses, we determined that the presence of both

MIAC and HCA in pregnancies complicated by PPROM was

associated with a higher fetal inflammatory response

mediated by umbilical cord IL-6. On the other hand, we did

not observe any difference in umbilical cord blood IL-6 levels

among the subgroups with MIAC alone, HCA alone, and

without MIAC and HCA. This finding is clinically relevant,

suggesting high umbilical cord blood IL-6 levels as marker of

the presence of both MIAC and HCA in PPROM pregnancies.

From a clinical point of view, FIRS is considered an

extensive fetal inflammatory response to both inflammatory

and non-inflammatory stimuli. The presence of FIRS has

Figure 2. The umbilical cord blood IL-6 levels with respect to thepresence or absence of MIAC and/or HCA. No differences in umbilicalcord blood IL-6 levels were observed among women with HCA alone,MIAC alone, and those without MIAC and HCA.

Figure 3. Women with both MIAC and HCA exhibited higher rates ofFIRS that did those with HCA alone, MIAC alone, and those withoutMIAC and HCA.

Figure 4. No differences in rates of FIRS were observed among womenwith HCA alone, MIAC alone, and those without MIAC and HCA.


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largely been observed in pregnancies complicated with

MIAC, but non-infectious conditions are able to mount

FIRS as well [9,26]. Our findings are consistent with this

evidence; FIRS was observed in all subgroups of PPROM

independent of the presence of either MIAC or HCA. The

highest rates of FIRS, as we expected, were found in the

subgroup with both MIAC and HCA. On the other hand, no

differences in the rate of FIRS were found among the other

subgroups. Interestingly, the presence of FIRS in the

subgroups without MIAC (HCA alone and without both

MIAC and HCA) was associated with a lower gestational age

(p50.0001 and p¼ 0.02, respectively; data not shown). We

can only hypothesize which non-infectious factor in low

gestational age is responsible for the development of FIRS.

The intensity of the fetal inflammatory response can be

assessed by measuring the levels of different inflammatory

mediators in umbilical cord blood. Unfortunately, there is

little information regarding the dynamics of the levels of these

mediators in umbilical cord blood during advanced preg-

nancy. Therefore, we decided to evaluate IL-6 because its

umbilical cord blood levels do not change during the

advanced pregnancy [28]. Moreover, Mestan et al. [15]

reported that IL-6 is elevated with fetal inflammatory

response regardless of gestational age. The stable umbilical

cord IL-6 levels during pregnancy appear to be an advantage

for the description of the intensity of the fetal inflammatory

response because there is no need for any adjustment for

gestational age.

The majority of studies focusing on the evaluation of

umbilical cord blood mediators used samples obtained from

the umbilical cord after the delivery of the newborn because

of the non-invasive nature of this approach. Unfortunately,

sampling at the time of delivery does not allow researchers to

obtain umbilical cord blood samples from women who were

not treated prenatally with corticosteroids when the gesta-

tional age was less than 34 weeks. Therefore, the next issue

that should be mentioned is the potential influence of

antenatal corticosteroids on fetal IL-6 production. This

effect has been little studied. Kramer et al. [29] previous

reported a transient reduction in IL-6 production after the

administration of betamethasone in a lamb model. In addition,

Caldas et al. [30] reported that a complete antenatal course of

betamethasone can affect umbilical serum IL-6 levels [30].

On the other hand, Kavelaars et al. [31] did not observe any

effect of antenatal glucocorticoids on the levels of fetal IL-6

production. However, IL-6 levels in umbilical cord blood

were highest in the subgroup (both MIAC and HCA positive)

with the highest rate of antenatal administration of cortico-

steroids. Therefore, it is unlikely that the potential transient

suppression of IL-6 levels affects the difference among

subgroups. Moreover, the results remain significant after

controlling for antenatal administration of corticosteroids

(p¼ 0.004).

The principal strength of this study resides in the active

management of PPROM (except pregnancies under 28 weeks)

in the majority of women [91% (135/149)] exhibiting latency

between amniocentesis and delivery less than 96 h. Therefore,

we hypothesize that the inflammatory status of the placenta

and fetal membrane at delivery are very similar to their status

at the time of amniocentesis. This hypothesis is supported by

the work of Ghidini et al. [32], who provide evidence that

there is a lack of association between HCA and the latency

period between rupture of the membranes and delivery. The

other strength of this study is the relatively large cohort of

women with a specific phenotype of spontaneous preterm

delivery (PPROM) from a single institution.

Our study also exhibited some limitations. We did not

use the non-cultivation technologies for the detection of

microorganisms others than Ureaplasma spp., M. hominis and

C. trachomatis. Therefore, we cannot exclude the presence of

non-cultivated and/or difficult to cultivate bacteria in the

amniotic fluid. Accordingly, the size of the subgroups with

HCA alone and without both MIAC and HCA might have

been overestimated, while the subgroups with MIAC alone

and with both MIAC and HCA might have been under-

estimated. On the other hand, modern non-cultivation tech-

niques are rarely available or used in clinics, and clinicians

largely base their decisions on classical cultivation

techniques.

In summary, the presence MIAC and HCA is associated

with a higher fetal inflammatory response and FIRS in

pregnancies complicated by PPROM. Therefore, this specific

subgroup of PPROM represents the worst scenario for

newborns. These results support the urgent need for the

identification of robust, clinically applicable biomarkers for

the early detection of MIAC leading to HCA.

Declaration of interest

This work was supported by a grant from the Ministry of

Health of the Czech Republic (NS 13461-4/2012), Faculty

Hospital in Hradec Kralove (long-term organization develop-

ment plan) and by Charles University in Prague, Faculty of

Medicine in Hradec Kralove, Czech Republic, project

‘‘PRVOUK’’ P37/10.

The authors report no conflicts of interest. The authors

alone are responsible for the content and writing of the paper.

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Fetal Inflamatory

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