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Keywords:

  • Amniotic fluid;
  • biomarkers;
  • calgranulin;
  • defensin;
  • inflammation;
  • interleukin-6;
  • proteomics;
  • umbilical cord blood

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interest
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References

Objective  To determine the relationship between presence of amniotic fluid (AF) biomarkers characteristic of inflammation (defensins 2 and 1 and calgranulins C and A) and fetal inflammatory status at birth.

Design  Prospective observational cohort.

Setting  Tertiary referral University hospital.

Population  One hundred and thirty-two consecutive mothers (gestational age, median [interquartile range]: 29.6 [24.1–33.1] weeks) who had a clinically indicated amniocentesis to rule out infection and their newborns.

Methods  Intra-amniotic inflammation was diagnosed by mass spectrometry surface-enhanced-laser-desorption-ionization time of flight (SELDI-TOF). The AF proteomic fingerprint (mass-restricted [MR] score) ranges from 0–4 (none to all biomarkers present). The intensity of intra-amniotic inflammation was graded based on the number of proteomic biomarkers: MR score 0: ‘no’ inflammation, MR score 1–2: ‘minimal’ inflammation and MR score 3–4: ‘severe’ inflammation. At birth, cord blood was obtained for all women. Severity of histological chorioamnionitis and early-onset neonatal sepsis (EONS) was based on established histological and haematological criteria. Interleukin-6 (IL-6) levels were measured by sensitive immunoassays. The cord blood-to-AF IL-6 ratio was used as an indicator of the differential inflammatory response in the fetal versus the AF compartment.

Main outcome measures  To relate proteomic biomarkers of intra-amniotic infection to cord blood IL-6 and to use the latter as the primary marker of fetal inflammatory response.

Results  Women with intra-amniotic inflammation delivered at an earlier gestational age (analysis of variance, P < 0.001) and had higher AF IL-6 levels (P < 0.001). At birth, neonates of women with severe intra-amniotic inflammation had higher cord blood IL-6 levels (P = 0.002) and a higher frequency of EONS (P = 0.002). EONS was characterised by significantly elevated cord blood IL-6 levels (P < 0.001). Of the 39 neonates delivered by mothers with minimal intra-amniotic inflammation, 15 (39%) neonates had umbilical cord blood IL-6 levels above the mean for the group and 2 neonates had confirmed sepsis. The severity of the neutrophilic infiltrate in the chorionic plate (P < 0.001), choriodecidua (P = 0.002), umbilical cord (P < 0.001) but not in the amnion (P > 0.05) was an independent predictor of the cord blood-to-AF IL-6 ratio. Relationships were maintained following correction for gestational age, birthweight, amniocentesis-to-delivery interval, caesarean delivery, status of the membranes, race, MR score and antibiotics and steroid exposure.

Conclusions  We provide evidence that presence of proteomic biomarkers characteristic of inflammation in the AF is associated with an increased inflammatory status of the fetus at birth. Neonates mount an increased inflammatory status and have positive blood cultures even in the context of minimal intra-amniotic inflammation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interest
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References

Premature birth (PTB) remains a significant public health problem worldwide. In 2006, the prevalence of PTB rose to an unprecedented 12.8%.1 A persistent theme of modern perinatology is that inflammatory intrauterine processes adversely affect the fetus prior to birth.2–5 This notion is based on studies demonstrating that complications of immature organ systems (respiratory, gastrointestinal, immune and central nervous system), which are characteristic of early-onset neonatal sepsis (EONS), are at least partially the result of a fetal inflammatory response in utero and one of the primary causes of the high neonatal morbidity and mortality attendant PTB.5–7

Fetal inflammation represents a highly orchestrated process designed to combat infection and tissue injury.8,9 However, while chemokines and pro-inflammatory cytokines are essential for fetal defence against infection, excessive influx of activated leucocytes coupled with exaggerated production of pro-inflammatory mediators can have deleterious consequences for the host.10 One such pro-inflammatory cytokine, interleukin-6 (IL-6), is traditionally considered an activator of acute-phase responses and of lymphocytes.11 Previous studies have shown that elevated fetal IL-6 levels at birth are a risk factor for sepsis-induced disseminated intravascular coagulation, pneumonia and cerebral palsy.12–15 Therefore, IL-6 is postulated to be an inflammatory marker of EONS, particularly in the preterm neonate.16,17

The most recent advances in proteomics have generated novel research and diagnostic applications that have only begun to impact on the diagnosis of intra-amniotic inflammation.18–21 However, development of targeted therapies aimed to change neonatal outcome is impossible without a better understanding of the fetal inflammatory response to intra-amniotic infection/inflammation. We have previously shown that proteomic mapping of the amniotic fluid (AF) reveals a proteomic profile (mass-restricted [MR] score) that is highly characteristic of intra-amniotic inflammation.18,19 Previously, we have demonstrated that presence of four inflammatory proteomic biomarkers (neutrophil defensins 2 and 1 and calgranulins C and A) in the AF is highly predictive of histological chorioamnionitis (HCA), funisitis and EONS.22,23 Given our results, we have concluded that proteomic analysis of the AF provides an opportunity for recognition of HCA in utero and that this methodology may in the future identify candidates for antenatal therapeutic interventions.23 The fetal inflammatory response in women displaying a proteomic fingerprint characteristic of inflammation remains unknown. The degree of placental HCA is an important factor in modulating cytokine levels in the fetuses of women whose pregnancies are complicated by intra-amniotic inflammation.10 Therefore, in this study, we sought to determine the relationship between presence of biomarkers characteristic of inflammation in the AF, HCA and cord blood IL-6 and to use the latter as the primary marker of fetal inflammatory status at birth.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interest
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References

Study population and research design

We studied prospectively 132 consecutive preterm singleton neonates born to mothers who had an amniocentesis to rule out infection. Women were recruited following admission to Labour and Birth or to the High-Risk Antepartum Units at Yale New Haven Hospital. Amniocentesis was indicated independent of our study protocol. The Human Investigation Committee of Yale University approved our study.

AF was collected by ultrasound-guided amniocentesis for all the participants, and each woman was followed prospectively to the point of delivery. Inclusion criteria included a singleton fetus, gestational age ≥23.1 weeks, symptoms of preterm labour, preterm prelabour rupture of membranes (PPROM), advanced cervical dilatation (≥3 cm) or uterine contractions intractable to tocolysis. Women with anhydramnios, human immunodeficiency or hepatitis viral infections were not enrolled. Gestational age was determined based on last menstrual period and ultrasound information obtained prior to 20 weeks of gestation.24 Preterm labour was defined as the presence of regular uterine contractions and documented cervical effacement and/or dilatation in women <37 weeks gestational age. The diagnosis of PPROM was confirmed by vaginal AF ‘pooling’, ‘nitrazine’ and ‘ferning’ or by an amniocentesis-dye positive test. In the absence of infection, PPROM was managed expectantly, and no digital exams were permitted. Women received corticosteroids for lung maturity if <32 weeks gestational age and antibiotic therapy as clinically indicated.25 The neonatology resuscitation team was present at the time of delivery for all women.

Laboratory tests performed for the purpose of diagnosing infection/inflammation were glucose, lactate dehydrogenase (LDH), Gram stain and white blood cell (WBC) count. For clinical management, a AF glucose cutoff of ≤15 mg/dl and LDH levels ≥419 units/l were considered suggestive of intra-amniotic infection.26,27 AF not used for clinical purposes was spun at 3000 ×g at 4°C for 20 minute, aliquoted and immediately stored at −80°C until IL-6 levels were measured by a specific and sensitive immunoassay.

Microbiological analysis of AFs by culture

AF was retrieved using sterile techniques and examined immediately for the presence of microorganisms using the traditional culturing method. Briefly, AF was centrifuged and the sediment resuspended in 0.25 ml of supernatant. The concentrated AF was cultured for anaerobic bacteria, Ureaplasma and Mycoplasma spp. using laboratory media such as Chocolate, Martin Lewis, MacConkey, Azidobenzoic acid, Thioglycollate, Bacteroides Bile Esculin/Laked Blood Kanamycin Vancomycin and Columbia CNA agar, Ureaplasma broth and agar. In addition, the AF was cultured in an anaerobic chamber (Forma Anaerobic System; Thermo Electron Co., Waltham, MA, USA) at 37°C. The results of the microbiological tests were available for case management and were reported as final after 5 days of culturing. Presumptive bacterial identification was based on standard microbiological criteria of colonial morphology, medium reaction and Gram stain, and the use of a VITEK 2 automated card system (bioMérieux, Hazelwood, MO, USA; http://www.biomerieux-usa.com). Bacterial identification was based on biochemical tests and antibiotic susceptibility.

MR score

Fresh AF was used to generate the MR score as previously described. The method for generation of the MR score has been previously described.18 The MR score ranges from 0 to 4, depending upon the presence or absence of each of the four protein biomarkers.18 A value of 1 was assigned if a biomarker peak was present and 0 if absent. Based on our previous results, we stratified the study population based on the ‘severity’ of inflammation (MR 0: no inflammation, MR 1–2: minimal inflammation, and MR 3–4: severe inflammation).19 All SELDI assays and scorings of the AF samples were performed by one investigator (I.A.B.) without knowledge of the maternal clinical outcome, the results of the placental histological examination, AF and umbilical cord IL-6 levels and EONS.

Umbilical cord blood

Umbilical cord blood was obtained by aseptic puncture of the clamped umbilical vein at the time of delivery. Immediately following collection, the cord blood was centrifuged at 1000 ×g for 15 minutes. Serum was aliquoted in sterile polypropylene tubes and stored at −80°C until IL-6 levels were examined.

Immunoassays for IL-6

Enzyme-linked immunosorbent assay (ELISA) for human IL-6 (Pierce Endogen, Rockford, IL, USA) was performed in duplicate according to manufacturers’ instructions by investigators unaware of the AF and umbilical cord blood sample origin. The minimal detectable concentration was 1 pg/ml, and the inter- and intra-assay coefficients of variation were <10%.

Histological evaluation of the placenta and diagnosis of inflammation

In all 132 women, haematoxylin- and eosin-stained sections of extraplacental membranes (amnion and choriodecidua), chorionic plate, choriodecidua and umbilical cord were examined systematically for inflammation. Three histological stages of chorioamnionitis28 (stage I: intervillositis, stage II: chorionic inflammation and stage III: full-thickness inflammation of both chorion and amnion) were complemented by a previously described histological grading system that includes four grades of inflammation of the amnion, choriodecidua and umbilical cord.29

Evaluation of EONS

Neonatal haematological indices and sepsis categorisation were assessed from blood specimens and cultures obtained within 2 hours from the time of birth by an investigator (V.B.) unaware of the results of the umbilical cord IL-6 levels, proteomic profiling of the AF or histological evaluation of the placenta.19,22 All 132 neonates were admitted to the Yale Newborn Special Care Unit. Neonatal sepsis was defined as the presence of confirmed or suspected sepsis at ≤72 hours after birth. Confirmed sepsis represented the presence of a positive blood culture. A diagnosis of EONS was based on clinical symptoms corroborated with haematological laboratory results.30,31 The following haematological criteria were used as indicators of EONS: (1) absolute neutrophil count of <7500 or >14 500 cells/mm3, (2) absolute band count >1500 cells/mm3, (3) immature/total (I:T) neutrophil ratio >0.16, (4) platelet count <150 000 cells/mm3.32 Sepsis was suspected in the presence of two or more haematological criteria in the absence of a positive blood culture. EONS was dichotomised into present (when sepsis was either confirmed or suspected) or absent. All neonates with confirmed or suspected sepsis received antibiotic therapy per institutional protocol.

The results of the AF IL-6 levels, histological examination of the placenta and presence or absence of EONS for 79 women were previously used in studies aimed to explore the maternal and fetal response to intrauterine infection/inflammation.19,22,23,33 The relationship between the MR score and cord blood IL-6 levels, the results of the AF and cord blood IL-6 levels, histological examination of the placenta and specific haematological indices suggestive of EONS for 53 subjects included in the current submission have not been reported before. The relationship between cord blood IL-6 levels, EONS and absolute neutrophil count as well as the results of the correlations between cord blood-to-AF IL-6 ratio and histological examination of the placenta for all 132 women included in the present analysis are novel.

Statistical analysis

Statistical analyses were performed with Sigma Stat, version 2.03 (SPSS Inc., Chicago, IL, USA) and MedCalc (Mariakerke, Broekstraat, Belgium) statistical softwares. Normality, testing was performed using the Kolmogorov–Smirnov test. Data were compared with one-way analysis of variance (ANOVA), followed by Dunnett’s tests (parametric) or Kruskal–Wallis on ranks, followed by Dunn’s tests (nonparametric) to adjust for multiple comparisons as appropriate. The IL-6 concentrations were presented as arithmetic means with interquartile range. Statistical analysis was completed before (Kruskal–Wallis ANOVA) or after (one-way ANOVA) logarithmic transformation of data. Pearson correlations were used to measure colinearity between the selected independent variables and other relevant relationships between dependent and independent variables. Comparisons between proportions were performed with chi-square test. Stepwise multivariable regression analysis was used to determine concurrent relationships between variables and to correct for possible influences of gestational age and birthweight. P < 0.05 was considered significant throughout the analysis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interest
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References

In Table 1, we present the clinical characteristics of the study subjects at the time of enrolment and their pregnancy outcomes. In this cohort, women of African-American descent were more frequently diagnosed with severe (MR score 3–4) intra-amniotic inflammation. Women with MR scores 3–4 were of lower gestational age and more often had symptoms of clinical chorioamnionitis compared with the group of women with no (MR score 0) or minimal inflammation (MR score 1–2). Similarly, women with severe inflammation had a shorter amniocentesis-to-delivery interval and, consequently, gave birth at an earlier gestational age compared with the other two groups. At birth, neonates of mothers with severe inflammation had lower birthweight and Apgar scores. Women with minimal and severe inflammation of the AF more often underwent Caesarean section delivery.

Table 1.  Demographic and clinical characteristics of women and their newborns
VariableMR scoreP value
MR 0 (n = 26)MR 1–2 (n = 39)MR 3–4 (n = 67)
  • The MR score ranges from 0 to 4. MR score 0 indicates no inflammation, MR score 1–2 indicates minimal inflammation and MR score 3–4 indicates severe inflammation.

  • *

    Data presented as median (interquartile range) and analysed by Kruskal–Wallis one-way ANOVA on Ranks test.

  • **

    Data presented as n (%) and analysed by chi-square test.

Maternal characteristics at enrolment and hospital course
Age (years)*28 (24–32)29 (22–32)28 (22–36)0.737
Gravidity*2 (2–3)2 (1–4)2 (2–5)0.411
Parity*1 (0–1)0 (0–1)1 (0–2)0.269
Race** 
 Caucasian14 (55)26 (66)15 (23)<0.001
 African-American4 (15)8 (21)34 (51)<0.001
 Hispanic4 (15)5 (13)13 (19)0.060
 Others4 (15)0 (0)5 (7)0.052
Gestational age (weeks)*30.8 (29.5–32.4)29.6 (25.7–32.1)27.4 (24.7–30.1)<0.001
Ruptured membranes**21 (81)23 (59)38 (57)0.089
Uterine contractions**10 (63)16 (70)33 (49)0.553
Clinical chorioamnionitis**0 (0)0 (0)11 (16)0.003
History of preterm birth**10 (63)7 (18)20 (30)0.176
Cervical dilatation*0 (0–2)1 (0–3)2 (0–4)0.101
Steroid exposure during pregnancy**22 (85)34 (87)61 (91)0.250
Prenatal antibiotic treatment**23 (88)32 (82)57 (85)0.777
Outcome characteristics
Amniocentesis to delivery (hours)*53 (16–144)46 (11–135)8 (4–19)<0.001
Amniocentesis to delivery—% delivery within 72 hours**16 (62)21 (54)62 (92)<0.001
Gestational age at delivery (weeks)*32.1 (30.1–33.2)30.6 (27.5–32.5)27.5 (25.1–30.1)<0.001
Birthweight (g)*1802 (1400–2145)1630 (1135–2815)1030 (772–1492)<0.001
Caesarean delivery**5 (19)19 (49)30 (45)0.040
Apgar score at 1 minute*8 (6–9)7 (5–8)6 (3–8)0.011
Apgar score at 5 minutes*9 (8–9)9 (8–9)8 (6–9)0.019

We present the results of the AF analysis in Table 2. Women with severe intra-amniotic inflammation had lower AF glucose, higher LDH and higher WBC counts compared with the women with no or minimal inflammation. This finding was consistent with a higher prevalence of a positive Gram stain and a positive microbial culture of the AF in the group of women with severe inflammation. There was a significant inverse correlation between gestational age at enrolment and intra-amniotic infection as determined by a positive microbial culture (R =−0.407, P < 0.001). Pathological examination of the placenta demonstrated that severe inflammation of the AF was associated with a higher incidence and grades/stages of amniochorion, chorionic plate and umbilical cord histological inflammation (Table 3).

Table 2.  AF analysis
VariableMR scoreP value
MR 0 (n = 26)MR 1–2 (n = 39)MR 3–4 (n = 67)
  • The MR score ranges from 0 to 4. MR score 0 indicates no inflammation, MR score 1–2 indicates minimal inflammation and MR score 3–4 indicates severe inflammation.

  • *

    Data presented as median (interquartile range) and analysed by Kruskal–Wallis one-way ANOVA on Ranks test.

  • **

    Data presented as n (%) and analysed by chi-square test.

AF
Glucose (mg/dl)*27 (19–40)25 (15–36)4 (2–15)<0.001
LDH (units/l)*122 (104–198)198 (150–266)718 (441–1145)<0.001
WBC (cells/mm3)*4 (2–10)8 (2–25)756 (123–1570)<0.001
Positive Gram stain**1 (4)4 (11)34 (51)<0.001
Positive cultures**1 (4)5 (13)45 (67)<0.001
Table 3.  Placental pathology
VariableMR scoreP value
MR 0 (n = 26)MR 1–2 (n = 39)MR 3–4 (n = 67)
  • The MR score ranges from 0 to 4. MR score 0 indicates no inflammation, MR score 1–2 indicates minimal inflammation and MR score 3–4 indicates severe inflammation.

  • *

    Data presented as median (interquartile range) and analysed by Kruskal–Wallis one-way ANOVA on Ranks test.

  • **

    Data presented as n (%) and analysed by chi-square test.

Presence and degree of placental inflammation
Chorionic plate inflammation (stage)*0 (0–0)1 (0–2)3 (2–3)<0.001
Chorionic plate inflammation (stage II–III)**6 (23)12 (31)54 (81)<0.001
Amnionitis (grade)*0 (0–0)0 (0–1)3 (1–3)<0.001
Amnionitis (grade 2–4)**2 (8)9 (23)49 (73)<0.001
Choriodeciduitis (grade)*0 (0–2)1.5 (0–3)3 (3)<0.001
Choriodeciduitis (grade 2–4)**9 (35)19 (49)61 (91)<0.001
Funisitis (grade)*0 (0–0)0 (0–1)2 (0–4)<0.001
Funisitis (grade 1–4)**3 (12)11 (28)39 (58)<0.001

We found that neonates delivered by mothers with MR scores 3–4 were more often anaemic and had leucocytosis (Table 4). These neonates were lymphopenic and had bandaemia and a higher I:T neutrophil ratio. Overall, six neonates had confirmed sepsis documented by a positive blood culture. Two of the neonates were delivered by mothers with negative AF cultures but had MR scores of 2. All mothers with MR scores 3–4 who delivered neonates with proven sepsis (n = 4) had positive AF cultures (Escherichia coli [n = 3]; mixed flora: Staphylococcus aureus and Corynebacterium [n = 1]). Neonates delivered by mothers with AF MR score 3–4 more often had a haematological work up suggestive of EONS compared with neonates delivered by mothers with MR score 0 or 1–2. This relationship persisted after adjusting for gestational age at birth. The overall prevalence of EONS in our cohort was 26% (34/132), of which 56% of instances occurred in the context of PPROM. Interestingly, five of the six women with proven sepsis documented by positive blood cultures occurred in the context of intact membranes.

Table 4.  Results of neonatal haematological indices
VariableMR scoreP value
MR 0 (n = 26)MR 1–2 (n = 39)MR 3–4 (n = 67)
  • ABC, absolute neutrophil count; ANC, absolute neutrophil count.The MR score ranges from 0 to 4. MR score 0 indicates no inflammation, MR score 1–2 indicates minimal inflammation and MR score 3–4 indicates severe inflammation.

  • *

    Data presented as median (interquartile range) and analysed by Kruskal–Wallis one-way ANOVA on Ranks test.

  • **

    Data presented as n (%) and analysed by chi-square test.

Haematological indices 
Haematocrit (%)*48 (44–54)48 (44–53)44 (40–49)0.030
Haemoglobin (g/dl)*16 (14–17)15 (14–17)14 (13–16)0.005
WBC (cells × 1000/mm3)*9 (7–12)10 (7–12)13 (2–38)0.061
Platelets (cells × 1000/mm3)*246 (209–300)250 (212–293)261 (222–332)0.698
Segmented (%)*32 (26–45)31 (22–38)33 (24–42)0.455
Lymphocytes (%)*45 (32–59)49 (44–59)33 (24–47)<0.001
ANC (cells/mm3)*3054 (2185–4818)3317 (2023–4113)4030 (2016–7040)0.234
ABC (cells/mm3)*186 (0–592)316 (80–677)668 (266–2592)<0.001
I:T ratio (%)*5 (2–9)5 (2–7)9 (4–19)0.017
Positive blood cultures**0 (0)2 (5)4 (6)0.453
EONS**4 (18)4 (11)26 (39)0.002

The AF IL-6 levels of women with MR scores 3–4 were significantly higher compared with that of women with no or minimal inflammation (median [interquartile range], MR score 0: 0.3 [0.1–0.7] versus MR score 1–2: 1.3 [0.5–3.9] versus MR score 3–4: 22.1 [8.5–57.5] ng/ml, Kruskal–Wallis ANOVA, P < 0.001) (Figure 1A). Neonates delivered by mothers with MR scores 3–4 had higher umbilical cord IL-6 compared with neonates delivered by mothers with no or minimal inflammation (MR score 0: 5.9 [5.5–8.5] versus MR score 1–2: 6.9 [5.4–97.6] versus MR score 3–4: 28.9 [8.5–161.3] pg/ml, P < 0.001) (Figure 1B).

image

Figure 1. Relationship between presence of biomarkers characteristic of intra-amniotic inflammation in the AF and AF and umbilical cord blood (CB) IL-6 levels. (A) AF IL-6 levels in women with and without intra-amniotic inflammation. (B) Umbilical cord blood IL-6 levels in neonates delivered by women with and without intra-amniotic inflammation. The MR score ranges from 0 to 4. MR score 0 indicates no inflammation, MR score 1–2 indicates minimal inflammation and MR score 3–4 indicates severe inflammation. Data presented in logarithmic format.

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Of the 39 neonates delivered by mothers with MR score 1–2, 15 (39%) had umbilical cord blood IL-6 levels above the mean for the group. Two neonates had confirmed sepsis. The two neonates with positive blood cultures delivered by mothers with MR score 1–2 had umbilical cord blood IL-6 levels considerably above the mean for their group (IL-6 absolute values: 49 834 and 1533 pg/ml, respectively). This occurred in the context of low AF IL-6 levels (IL-6 absolute values: 0.134 and 3.9 ng/ml, respectively). Both neonates had an amniocentesis-to-delivery interval less than 4 hours. Evaluation of white matter disorders showed that both neonates developed periventricular leucomalacia. In addition, the neurodevelopmental outcome assessment at 18 months of age showed that both children had cerebral palsy. Two of the four neonates delivered by mothers with MR score 3–4 who had documented positive blood cultures expired soon after delivery (umbilical cord IL-6 absolute levels: 19 817 and 77.2 pg/ml, respectively). The other two neonates did not develop periventricular leucomalacia (umbilical cord IL-6 absolute levels: 90.2 and 460.6 pg/ml, respectively). The results of the neurodevelopmental outcome at 18 months of age for the first infant showed mild gross motor delay for which the infant is currently getting therapy. The results of the neurodevelopmental outcome for the second infant were not available at the time of this report. The amniocentesis-to-delivery interval was less than 10 hours for all the four neonates.

Of all the haematological indices that comprise EONS criteria, cord blood IL-6 correlated best with the I:T neutrophil ratio (R = 0.501, P < 0.001) and the absolute band count (R = 0.330, P < 0.001). Cord blood IL-6 correlated inversely with the degree of anaemia as measured by neonatal haemoglobin concentration (R=−337, P < 0.001) and haematocrit (R =−0.300, P < 0.001). Neonates diagnosed with EONS had significantly higher cord blood IL-6 levels (No EONS: 7.9 [5.8–36.8] versus Yes EONS: 83.7 [17.3–361.6] pg/ml, P < 0.001) (Figure 2A). At birth, there was a significant inverse correlation between the absolute neutrophil count and cord blood IL-6 levels, which was maintained following correction for gestational age and birthweight (R=−0.439, P < 0.001) (Figure 2B).

image

Figure 2. Relationship between the umbilical cord blood (CB) IL-6 levels, EONS and absolute neutrophil count. (A) Umbilical cord blood IL-6 levels in neonates diagnosed with or without EONS. (B) Distribution of cord blood IL-6 levels on the y-axis in relationship to the absolute neutrophil count on the x-axis. Data presented in logarithmic format.

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Overall, there was a significant correlation between the AF and cord blood IL-6 levels (R = 0.415, P < 0.001) as shown in Figure 3. However, in 94% of the women (124/132), the absolute value of AF IL-6 concentration was higher than that measured in the umbilical cord blood. When separated by severity of intra-amniotic inflammation as depicted by the MR score, a disproportionate increase in AF IL-6 level compared with that of the fetal compartment was seen in women complicated by severe AF inflammation. This leads to an apparent decrease in the proportion of cord blood IL-6 relative to that in AF (cord blood-to-AF IL-6 ratio: MR score 0: 2.8% [1–5] versus MR score 1–2: 1.4% [0.4–6.4] versus MR score 3–4: 0.2% [0.1–1.2], P < 0.001).

image

Figure 3. Relationship between the AF and the umbilical cord (CB) blood IL-6 levels. Distribution of the AF (y-axis) in relationship to the cord blood IL-6 levels on the x-axis. Data presented in logarithmic format.

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Because of the variability in the fetal response to the severity of intra-amniotic inflammation, we searched for factors that may impact on the degree to which AF inflammation leads to an increase in cord blood IL-6 levels. Multivariate stepwise analysis was conducted, with the cord blood-to-AF IL-6 ratio as the dependent variable and parameters such as HCA and funisitis, gestational age at delivery, birthweight, amniocentesis-to-delivery interval, caesarean delivery, status of the membranes, race, MR score and antibiotics and steroid exposure as independent variables. We found that the intensity of intra-amniotic inflammation as determined by the MR score in conjunction with the degree of both HCA and funisitis predicted the cord blood-to-AF IL-6 ratio (F ratio: 16.6, R = 0.540, P < 0.001). This suggests that the degree of placental inflammation is an important factor in modulating cytokine levels in the fetuses of women whose pregnancies are complicated by intra-amniotic inflammation. To further explore this possibility, the relationship between the extent of the inflammatory infiltrate in the amnion, choriodecidua, chorionic plate blood vessels (stages of HCA) and umbilical cord (grades of funisitis) was examined, yielding a significant increase in cord blood-to-AF IL-6 ratio in the context of stages II and III neutrophil infiltration of the chorionic plate (Figure 4A, ANOVA, P < 0.001) and grades 3–4 choriodeciduitis (Figure 4B, P = 0.002) and grades 2–4 funisitis (Figure 4C, P<0.001). Conversely, the cord blood-to-AF IL-6 ratio was not influenced by the degree of amnionitis (P > 0.05).

image

Figure 4. Relationship between the umbilical cord blood (CB)-to-AF IL-6 ratio and histological inflammation of the placenta. (A) CB-to-AF IL-6 ratio in women with stages I–III histological inflammation of the chorionic plate. (B) CB-to-AF IL-6 ratio in women with grades 0–4 choriodeciduitis. (C) CB-to-AF IL-6 ratio in women with grades 0–4 funisitis. Data for the CB and AF IL-6 levels are presented in logarithmic format.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interest
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References

In this study, we sought to determine the relationship between the presence of four AF proteomic biomarkers (human neutrophil defensins 2 and 1 and calgranulins C and A) characteristic of intra-amniotic inflammation and the likelihood and severity of the fetal inflammatory response. We determined that although there is a direct correlation between the AF and cord blood IL-6 levels, the intensity of the fetal inflammatory response can not always be predicted by the severity of intra-amniotic inflammation. This conclusion is based on our analysis showing that only neonates delivered in the context of severe intra-amniotic inflammation (MR score 3–4) have significantly elevated cord blood IL-6 levels but not those delivered by women with no (MR score 0) or minimal (MR score 1–2) inflammation. Similar to other studies, we have shown that neonates with haematological indices suggestive of EONS have higher umbilical cord IL-6 concentrations, which correlates best with the I:T ratio.14,30,34–38 We further provide evidence that a significant inverse correlation between the absolute neutrophil count and umbilical cord IL-6 levels exists. Lastly, we demonstrated that the cord blood-to-AF IL-6 ratio (an indicator of the differential inflammatory response in the fetal versus the AF compartment) is highly correlated with the MR score and highly dependent on the severity of histological inflammation of the chorionic plate, choriodecidua and funisitis but not that of the amnion.

In prior studies, we described that the components of a given MR score have an order in appearance (first defensins, followed by calgranulins).19 Our approach allowed us to objectively stratify the intensity of intra-amniotic inflammation and simultaneously assess the degree of fetal inflammation. In the present study, we reveal that the fetal inflammatory status in women with MR score 1–2 (defensin stage) is overall negligible. Because the strength of the fetal inflammatory response is highly associated with the degree of placental HCA, we can conclude that at the stage of minimal AF inflammation (MR score 1–2) the fetus is most commonly protected. However, detection of a positive blood culture and umbilical cord IL-6 levels well above the mean in several neonates delivered by women with MR scores 1–2 shows that this protection is not always complete. Several explanations may be possible.

Experimental data indicate that the inflammatory process of the placenta and its membranes is generally regarded as a continuum.39 It is believed that organisms first colonise and spread along the chorioamniotic structure. Thus, the initial inflammatory response is thought to be maternal in its origin.40,41 The next step involves infiltration of the membranous chorioamnion, followed by widespread inflammation within the AF cavity and development of necrotising chorioamnionitis. This view holds true in light of studies that genotyped the amniochorion neutrophils and demonstrated that these cells matched the maternal genotype in all women.42 Lastly, placental lesions related to alterations in the uteroplacental vascular pathology of the chorionic plate (chorionitis, vasculitis and moderate-to-severe villous oedema) are regarded as an advanced stage of placental inflammation with mixed maternal and fetal origin.42–45 That the above is consistent with experimental data in animals, it stands to reason that the haematogenic infection of the human fetus and mounting of a fetal inflammatory response, in the absence of concurrent AF colonisation and inflammation, are possible.46–49

Another possible explanation for the differential maternal and fetal inflammatory response may rest with our genetic make up.50 Deciphering the human genome has provided partial explanation for the extent to which the genetic make up differs from person to person. The working model for inflammation-induced PTB suggests that mothers and fetuses have a genetic predisposition towards either a hyperreactive or a hyporeactive immune system.51 The implications of this dissimilarity make a strong case for the variant maternal and fetal inflammatory response observed in this study and may provide clarification for our finding that at birth, several fetuses had positive blood cultures and an overwhelming inflammatory response in the context of minimal AF inflammation.

The results of the microbiological studies pointed out that in approximately 30% of the women, the bacterial cultures were negative, although proteomic analysis of the AF implied severe intra-amniotic inflammation. This should not come as a surprise since factors other than infection (i.e. bleeding) may be responsible for triggering intra-amniotic inflammation.52 Lastly, although microbial cultures of the AF are highly regarded as the ‘gold standard’ for detection of intra-amniotic infection, ‘difficult-to-cultivate’ or ‘uncultivated’ bacteria may not be found if the clinicians are relying on culture conditions alone.53 The recent development in DNA-based culture-independent technology (16S rRNA) enables detection of previously unidentified uncultivated or difficult-to-cultivate bacterial species and may replace in the future the microbiological cultures to allow for a highly accurate diagnosis of intra-amniotic infection.53

The presence of molecular pattern recognition receptors, notably Toll-like receptors (TLR) 2 and 4 on human trophoblast, and the existence of the S100A12/ENRAGE (RAGE system) in the placental vascular tissue prove that the placenta is prepared to respond to a bacterial attack through an outpouring of inflammatory cytokines.33,54 Our results imply that in the initial phase of the inflammatory response, the placenta plays an important protective role for the fetus. Yet, in a more advanced stage of inflammation, the placenta becomes an important regulator of the differential immune response observed in the AF versus the fetal compartment. Although the activity of TLR seems to exhibit genetic control, the literature continues to be conflicted in regard to the existence of placental-fetal or placental-AF cytokine trafficking.55–57 Although it has been demonstrated that radiolabeled IL-6 crosses the rat fetus in mid gestation, studies showed that in vitro, there is either minimal or no transfer of proinflammatory cytokines across the human placenta.57,58 If this hypothesis is true, our data seem to suggest that this transfer is overwhelmingly directed towards the AF and not the fetal compartment.

Compelling evidence suggests that intra-amniotic inflammation and HCA are risk factors for perinatal injury that includes white matter injury and development of periventricular leucomalacia.2,59 Because a significant number of fetuses exposed to intra-amniotic inflammation and elevated AF cytokine levels do not develop cerebral palsy, it appears reasonable to assume that together with genetic predisposition, several other factors may be involved in fetal defence. Still, the decision to actively intervene and deliver a fetus subjected to a hostile intrauterine inflammatory process should take into account the risks and benefits for both the mother and her very preterm neonate. A major clinical concern remains early and accurate recognition of the fetuses with positive blood cultures who have already mounted a robust inflammatory response in the context of minimal intra-amniotic inflammation. Unfortunately, neither the biophysical profile nor the evaluation of the fetal heart rate reliably provides early identification and therefore needs for immediate delivery of a sick fetus.60–62 Yet, ultrasound evaluation of the fetal adrenal gland volume has just been proposed as a useful clinical marker for early recognition of an increased fetal inflammatory status in utero.63 A rapid intervention based on this marker (i.e. delivery versus anti-inflammatory treatment) may prove beneficial in the future for prevention of cerebral palsy. Until its clinical usefulness is proven in large randomised studies or other clinically relevant genetic markers identified for women with severe intra-amniotic inflammation, a reasonable approach may involve removing the fetus from a septic environment if birth in the very short term appears most likely.25

Disclosure of interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interest
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References

I.A.B. is named coinventor on a pending patent application related to the use of proteomics analysis of AF. Neither I.A.B. nor any of the authors served as consultants or received research funds from a third party interested in biomarker discovery including Ciphergen Biosystems.

Contribution to authorship

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interest
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References

C.S.B. and I.A.B. designed the study, collected, analysed and interpreted the data and drafted the manuscript. V.B. collected, analysed and interpreted the neonatal data. A.T.D. and G.Z. conducted ELISA assays and collected part of the proteomics data. C.S.B., A.T.D., S.A-R., S.L. and E.J.H. recruited women, collected biological specimens prospectively and reviewed the data analysis. All the coauthors participated with aspects of study design, critical interpretation of the data, contributed in writing the paper and have reviewed and approved the final version.

Funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interest
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References

The funding source had no involvement in study design, interpretation of data, writing of the report or decision to submit the paper for publication.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interest
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References

C.S.B. is supported by National Institutes of Health Eunice Kennedy Shriver/National Institute of Child Health and Human Development (NIH/NICHD) grant RO3 HD 50249 and the Yale WRHR Career Development Center (K12 HD 1027766). This work was also supported from NIH/NICHD grant RO1 HD 047321 (I.A.B.).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Disclosure of interest
  8. Contribution to authorship
  9. Details of ethics approval
  10. Funding
  11. Acknowledgements
  12. References
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