MATERNAL, UMBILICAL CORD AND NEONATAL

INFLAMMATORY AND HAEMATOLOGICAL MARKERS IN

HISTOLOGIC CHORIOAMNIONITIS

Dr Rebecca A. Howman

(MBBS)

This thesis is presented as part of the requirement for the degree of

Master of Clinical Research

at the University of Western Australia

2009

2

Abstract

Introduction:

Fetal inflammatory response syndrome (FIRS) has only recently been recognised as

an important cause of spontaneous preterm delivery (PTD). In addition, it has been

associated with a number of other short-term and long-term adverse neonatal

outcomes, including early onset neonatal sepsis, necrotising enterocolitis,

periventricular leucomalacia, cerebral palsy, and bronchopulmonary dysplasia,

although the causal mechanisms are unclear. The hallmark of FIRS is histologic

chorioamnionitis (HCA). Mothers with HCA are often asymptomatic and it remains

unclear whether elevated maternal inflammatory markers, such as C-reactive protein

(CRP) and procalcitonin (PCT), are predictive of preterm birth. Furthermore

neonatal inflammatory markers such as CRP, PCT, white cell count (WCC) and

absolute neutrophil count (ANC), are commonly used in clinical practice to diagnose

infection in the neonatal period. Although both intrauterine inflammation and FIRS

may have effects on inflammatory markers for up to 10 days following delivery, the

extent to which intrauterine infection and FIRS confound these diagnostic surrogates

of neonatal infection is unknown.

Hypothesis and Aims:

This work addressed the hypothesis that HCA is associated with inflammatory

changes that may be detected in the: (a) maternal circulation at the time of delivery,

(b) umbilical cord blood at delivery and (c) post-natal circulation within the first 48

hours of life.

The primary aim of this study was to investigate the relationship between the

presence of HCA and maternal inflammatory markers (serum CRP and PCT on the

day of delivery) as well as neonatal inflammatory markers (haematological

parameters, CRP and PCT up to 48 hours following delivery). The secondary aim

was to validate the accuracy of measurement of haematological parameters in

umbilical cord blood samples.

3

Methods:

Study design and measurements:

This was a cross-sectional study of 343 mothers and 421 neonates (including multiple

gestations) recruited at a single tertiary perinatal centre between 11th August 2003

and 19th January 2006. The gold standard for intrauterine inflammation was HCA as

defined by validated histopathological scoring on placental samples. From samples

collected at delivery, maternal ultrasensitive CRP and PCT, and umbilical cord

ultrasensitive CRP, PCT, and haematological parameters were measured.

Ultrasensitive CRP was measured using latex immunonephelometry using the Dade

Behring Nephelometer BN II. Post-natal CRP was measured by enzymatic sandwich

immunoassay using VITROS Chemistry Products CRP Slides. PCT was measured

using the BRAHMS PCT sensitive immunoluminometric assay on a Berthold

Technologies Lumat LB9507 luminometer. Cord and neonatal full blood counts were

measured using a Beckman Coulter HmX analyzer. The quality of all haematological

samples was reviewed and samples with clotting and/or platelet activation were

excluded from analysis. Blood films and manual 100-white cell differentials were

performed on all cord and neonatal haematological samples. Extensive clinical and

laboratory data including demographic data, gestational age (GA), birth weight, mode

of delivery, intra-partum clinical data, antibiotic use, microbiological culture results,

and early onset neonatal sepsis were collected for the first 48 hours of life.

Validation of measurement of haematological parameters in cord blood:

Both accuracy and precision of measurement was assessed. Comparison of paired

umbilical cord values and Day 0 peripheral blood values was used to assess reliability

of haemoglobin (Hb), red cell count (RCC), mean cell volume (MCV), mean cell

haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), and platelet

count. The intra-rater and inter-rater variability for cord white cell count (WCC),

absolute neutrophil count (ANC), nucleated red blood cell count (NRBC) and

immature:total (I:T) ratio assessed measurement precision.

4

Statistical analysis:

Continuous data were summarised using non-parametric summary statistics using

medians, interquartile ranges (IQR) and ranges (R) and Mann-Whitney U tests were

used to compare groups. Nominal data were summarised using frequencies.

Pearson’s chi-square tests (if frequency in each cell >5) or Fisher’s exact test (if

frequency in any cell <5) were used to compare frequencies between groups.

Spearman’s rho was used to correlate non-parametric continuous variables. Linear

regression was used to explore the effect of HCA on maternal, cord and neonatal

outcomes. Logistic regression was performed to identify simultaneous factors

associated with HCA. For all analyses, a p-value of <0.05 was considered statistically

significant. Data were analysed using SPSS statistical software (version 15.0:

Chicago, Illinois). Intraclass Correlation Coefficient (ICC) was used to measure the

absolute agreement between individual blood parameters as scored by different

observers.

Results:

Of 325 deliveries with placental samples available, there were 26 (8%) cases of HCA.

The key findings were that, compared to 299 non-HCA deliveries, HCA is associated

with significantly increased:

(1) maternal usCRP concentrations (median 26 versus 5.6 mg/L; adjusted OR

2.86, 95% CI 1.47-5.57; p=0.002), but not PCT concentrations, on the day of

delivery,

(2) cord usCRP (median 0 mg/L (range 0-45.6) versus median 0 mg/L (range

0-63.9); Mann U-Whitney analysis, p<0.001) and PCT concentrations

(median 0.293 versus 0.064 ug/L; adjusted OR=2.49, 95% CI 1.45-4.28;

p=0.001),

(3) neonatal corrected WCC and ANC within the first 24 hours of delivery

(median 10.3 versus 9.2 x109/L; adjusted OR 1.12, 95% CI 1.03-1.22;

p=0.007, and median 4.5 versus 3.0 x109/L; adjusted OR 2.21, 95% CI 1.20-

4.09; p=0.011, respectively), and

(4) neonatal maximal CRP levels within the first 48 hours of delivery (median

10 versus 6.5 mg/L; adjusted OR 3.26, 95% CI 1.73-6.14; p<0.001): this

effect was most marked in the first 24 hours of life (median 11 versus 6.5

mg/L; adjusted OR 5.39, 95% CI 2.35-12.39, p<0.001).

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The measurement of haematological parameters in the cord blood, MCV, MCH and

MCHC were highly reproducible between paired cord and neonatal samples. Hb and

RCC were affected by differences in the type of sample and fluid shifts in the

newborn. Cord platelet counts were likely affected by platelet activation. For both

intra-rater and inter-rater reproducibility, the corrected WCC, ANC and NRBC were

shown to be reliable with an ICC of >0.90 for all comparisons. However, I:T ratio

was poorly reproducible.

Discussion:

HCA appears to be a minor inflammatory insult for the mother. In the majority of

cases it is asymptomatic and results in minor increases in PCT and CRP levels on the

day of delivery. Conversely HCA results in significant inflammatory changes in the

newborn that can be seen in the cord blood. Sensitive markers of inflammation in the

cord blood are significantly higher in affected infants (CRP and PCT), while less

sensitive markers, such as WCC and ANC are not significantly different. This study

has shown that fetal inflammation has sustained effects on CRP and haematological

parameters in early neonatal life; CRP, WCC and ANC are significantly higher in

newborns exposed to HCA, peaking 24 hours following delivery. These effects may

confound the interpretation of common diagnostic tests for early onset neonatal

sepsis.

Conclusion:

HCA results in mild elevations in CRP and PCT in the cord blood. Over the

subsequent 24 hours CRP, WCC and ANC increase significantly in these neonates.

Intrauterine exposure to HCA may influence surrogate diagnostic markers for early

onset sepsis in newborn infants. Future research to investigate novel diagnostic

markers, such as CD64 and soluble triggering receptor expressed on myeloid cells

(TREM-1), or enhanced microbiological molecular diagnosis, will help distinguish

true invasive infection from HCA-driven inflammation in the newborn infant.

6

Declaration

I declare that this thesis is my own account of my research except where others have

been acknowledged. All work described is original and has not been previously

submitted for a degree at this or any other University.

..........................................

Dr Rebecca A. Howman

Acknowledgements

This work would not have been possible without the assistance and support from

many people.

My supervisors, Professor Catherine Cole and Associate Professor David Burgner,

have been very understanding and supportive of me. I would like to thank them for

their practical help and mentorship through this process. I have learnt much from

both of them about research. I would also like to acknowledge Dr Andrew Barr, my

supervisor in haematology training, who has been very flexible and given me the time

I needed to pursue this project.

This project involved the input of many people who were part of the SPIN (Study of

Postnatal Immunity in the Newborn). I would like to acknowledge the many families

who agreed to be involved in this project, and the investigators and research assistants

who enrolled subjects and collected the data. In particular, I would like to mention

Dr Peter Richmond who was involved in the Ethics approval of this project and

provided the consent documents. In addition, I am grateful for the work of Dr Adrian

Charles, consultant histopathologist, who assessed the placental samples for evidence

of histologic chorioamnionitis, Wanda Randall, who supervised the collection and

measurement of cord blood samples, Paul Chubb and the Fremantle Hospital

Department of Biochemistry, who performed the procalcitonin assays, and Jesper

Jensen and Professor Catherine Cole, who independently scored the cord blood films.

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I would also like to acknowledge BRAHMS who provided the reagent for the

procalcitonin assays without charge.

The assistance of Angela Jacques and Dorota Doherty (Women and Infant Research

Foundation) with the statistical analyses is gratefully acknowledged. Thank you for

your patience and perseverance.

Finally I would like to thank my husband, David Waterhouse and my daughter,

Katelyn. Without your love and support, I would not have managed to do this. Katie,

I hope that one day you will be proud of your mummy.

8

Table of Contents

Abstract.....................................................................................................................2

Declaration................................................................................................................6

Acknowledgements ...................................................................................................6

Hypothesis and Aims...........................................................................................12

CHAPTER 1: Literature Review ............................................................................13

1.1 The importance of preterm delivery (PTD) ....................................................14

1.2 What causes spontaneous preterm delivery?...................................................14

1.3 How does intrauterine infection/inflammation cause spontaneous PTD?........15

1.3.1 Pathways of infection..............................................................................15

1.3.2 Fetal inflammatory response syndrome (FIRS)........................................16

1.3.3 Consequences of the fetal inflammatory response syndrome (FIRS)........17

1.4 The relationship of placental histology with the fetal inflammatory response

syndrome.............................................................................................................18

1.4.1 Definition of histologic chorioamnionitis (HCA).....................................18

1.4.2 Correlation of histologic chorioamnionitis with the fetal inflammatory

response syndrome ..........................................................................................20

1.5 Does the fetal inflammatory response syndrome affect surrogate markers for

infection in the early neonatal period? .................................................................20

1.5.1 Recognition of sepsis in neonate .............................................................20

1.5.2 I:T ratio, total white cell count, and absolute neutrophil count.................21

1.5.3 C-Reactive Protein (CRP) .......................................................................25

1.5.4 Procalcitonin...........................................................................................28

1.5.5 Summary of neonatal inflammatory markers in HCA..............................31

1.6 Can we detect mothers with intra-amniotic infection and predict the risk of pre-

term delivery? .....................................................................................................32

1.6.1 Why do we want to predict preterm birth ................................................32

1.6.2 Chorioamnionitis is a chronic infection ...................................................32

1.6.3 Clinical signs ..........................................................................................32

1.6.4 Amniotic fluid cultures ...........................................................................33

1.6.5 Use of other biomarkers to detect risk of preterm birth............................33

1.6.6 Is maternal serum C-reactive protein a marker for intrauterine

inflammation? .................................................................................................33

1.6.7 Is maternal serum PCT a marker for intrauterine inflammation?..............34

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1.6.8 Summary of maternal inflammatory markers in HCA .............................35

CHAPTER 2: Methods............................................................................................36

2.1 Study Design and Inclusion Criteria...............................................................37

2.2 Research Governance and Ethics ...................................................................37

2.2.1 Confidentiality ........................................................................................38

2.2.2 Seeking consent from the mother alone...................................................38

2.2.3 Timing of consent ...................................................................................38

2.2.4 Risks associated with the study ...............................................................39

2.2.5 Research involving fetal/placental tissue .................................................39

2.3 Recruitment and consent................................................................................39

2.4 Sample collection and preparation .................................................................41

2.5 Measurement .................................................................................................42

2.5.1 Clinical data............................................................................................42

2.5.2 Placental histology and definition of histologic chorioamnionitis ............43

2.5.3 Ultrasensitive CRP..................................................................................43

2.5.4 Procalcitonin...........................................................................................44

2.5.5 Cord full blood counts and blood films ...................................................44

2.5.6 Neonatal CRP .........................................................................................47

2.6 Statistical Analysis ........................................................................................48

CHAPTER 3: Results correlation of HCA with maternal, cord and neonatal outcome

variables..................................................................................................................51

3.1 Study Cohort .................................................................................................52

3.2 Placental Histology........................................................................................53

3.3 Comparison of maternal characteristics in cases with and without histologic

chorioamnionitis..................................................................................................54

3.4 Comparison of neonatal characteristics in cases with and without histologic

chorioamnionitis..................................................................................................57

3.5 Histologic chorioamnionitis and maternal usCRP and PCT............................59

3.6 Histologic chorioamnionitis and cord/neonatal outcome variables..................60

3.6.1 Cord usCRP and PCT .............................................................................60

3.6.2 Cord and neonatal haematologic parameters............................................62

3.6.3 Neonatal CRP .........................................................................................65

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CHAPTER 4: The reliability of the umbilical cord full blood picture as a measure of

haematological parameters in the immediate post-natal period.................................69

4.1 Introduction...................................................................................................70

4.1.1 Red cells .................................................................................................70

4.1.2 Leukocytes..............................................................................................71

4.1.3 Platelets ..................................................................................................72

4.1.4 Summary ................................................................................................72

4.2 Methods.........................................................................................................72

4.2.1 Accuracy of red blood cell parameters and platelet counts.......................72

4.2.2 Precision of corrected WCC, ANC, I:T ratio and NRBC .........................73

4.3 Results...........................................................................................................74

4.3.1 Accuracy of red blood cell parameters and platelet counts.......................74

4.3.2 Precision of corrected WCC, ANC, I:T ratio and NRBC .........................77

4.4 Discussion .....................................................................................................78

4.4.1 Accuracy of red cell parameters and platelet counts ................................78

4.4.2 Precision of corrected WCC, ANC, I:T ratio and NRBC .........................82

4.4.3 Limitations of analysis ............................................................................83

4.4.4 Conclusion..............................................................................................84

CHAPTER 5: General discussion ...........................................................................85

5.1 Summary and discussion of findings..............................................................86

5.1.1 Maternal inflammatory markers ..............................................................87

5.1.2 Cord inflammatory markers ....................................................................89

5.1.3 Neonatal inflammatory markers ..............................................................93

5.2 Clinical and diagnostic implications...............................................................98

5.3 Limitations of the study ............................................................................... 100

5.3.1 Study cohort, reduced statistical power ................................................. 100

5.3.2 Neonatal CRP data................................................................................ 101

5.3.3. Limitations of the study design ............................................................ 102

5.4 Future directions.......................................................................................... 103

5.5 Concluding comments ................................................................................. 103

Appendix 1 – Preliminary information sheet for parents .................................... 104

Appendix 2 – Full information sheet for parents and consent form..................... 105

References......................................................................................................... 108

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Table of Figures

Figure 1.3.1 Stages of ascending infection of intrauterine cavity............................ 16

Figure 1.5.2 Reference ranges for neutrophil counts in first 60 hours after birth.... 23

Figure 1.5.4a PCT levels in term (>36 weeks) infants.............................................. 29

Figure 1.5.4b PCT levels in preterm (<36 weeks) infants......................................... 30

Figure 2.3.1 Procedure for enrolment into study, collection and handling of samples

and formal consent..................................................................................................... 40

Figure 4.3.1 Bland Altman plots comparing cord and neonatal results..................... 76

Figure 4.3.2 Intra-observer correlation for observer 1............................................... 78

Table of Tables

Table 1.4.1 Diagnostic criteria for histologic chorioamnionitis............................ 19

Table 2.5.5 CV(%) for parameters measured on the Beckman Coulter HmX...... 45

Table 2.6 Maternal, cord and neonatal outcome variables................................. 49

Table 3.1.1 SPIN study cohort: gestational age groups......................................... 52

Table 3.1.2 SPIN study cohort: mode of delivery................................................. 53

Table 3.1.3 SPIN study cohort: singleton or multiple gestation............................ 53

Table 3.3 Demographic and clinical features of mothers.................................... 56

Table 3.4 Demographic and clinical features of infants...................................... 58

Table 3.5.1 Maternal PCT and usCRP concentrations in subjects with HCA..... 59

Table 3.5.2 Linear regression for the effect of HCA on maternal PCT & usCRP 60

Table 3.6.1a Cord PCT and usCRP level in subjects with and without HCA......... 61

Table 3.6.1b Linear regression for the effect of HCA on cord usCRP and PCT..... 62

Table 3.6.2a Cord and neonatal haematologic parameters in subjects with HCA.. 63

Table 3.6.2b Linear regression for the effect of HCA on neonatal WCC and ANC..64

Table 3.6.3a Availability of neonatal CRP measures within the first 48 hours of

delivery...................................................................................................................... 65

Table 3.6.3b Neonatal CRP levels in cases with HCA............................................. 67

Table 3.6.3c Linear regression for maximal neonatal CRP levels............................. 68

Table 4.3.1 Mean Hb, RCC, MCV, MCH, MCHC and platelet counts in paired cord

and neonatal samples................................................................................................. 75

Table 4.3.2 Intra- and inter-observer reliability for corrected WCC, ANC, I:T ratio

and NRBC.................................................................................................................. 77

12

Hypothesis and Aims

The hallmark of chronic intrauterine inflammation is histologic chorioamnionitis.

This study addresses the hypothesis that histologic chorioamnionitis is associated

with inflammatory changes that may be detected in (a) the mother at the time of

delivery, and (b) the umbilical cord blood and neonatal blood within the first 48 hours

of life.

To test this hypothesis, the objectives were as follows:

1. To establish whether haematologic parameters can be accurately and precisely

measured in the umbilical cord blood using a routine haematology cell counter.

2. To investigate whether there is a correlation between the presence of histologic

chorioamnionitis and:

a. maternal serum usCRP at delivery

b. maternal PCT at delivery

3. To investigate whether there is a correlation between the presence of histologic

chorioamnionitis and:

a. umbilical cord haematologic parameters

b. umbilical cord ultrasensitive C-reactive protein (usCRP) levels

b. umbilical cord procalcitonin (PCT) levels

d. neonatal haematologic parameters in the first 24 hours of life

e. neonatal CRP levels in the first 48 hours of life

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CHAPTER 1:

Literature Review

14

1.1 The importance of preterm delivery (PTD)

Preterm birth (before 37 completed weeks of gestation) is the leading cause of

perinatal morbidity and mortality in developed countries [1]. In Australia,

approximately 8.1% of births are preterm, yet 81.4% of perinatal mortality and 78.0%

of admissions to level III neonatal intensive care units occur in this group [2].

Improvements in neonatal care, such as the increased use of antenatal steroids and

artificial pulmonary surfactant, have led to a decline in the neonatal mortality rate

amongst preterm and very preterm infants [3, 4]. However, survival amongst infants

born at low gestational ages is associated with long-term disability and impairment,

particularly in the areas of neuromotor function, mental development, and language

and speech development [5]. Major costs to the health system [6] and the wider

community are incurred as a result of the morbidity associated with preterm birth.

1.2 What causes spontaneous preterm delivery?

PTD may be iatrogenic or spontaneous. Approximately 30% of preterm deliveries

are induced or delivered by caesarean section for maternal and/or fetal indications

[1]. Of the remainder, approximately two thirds of PTD result from the spontaneous

onset of labour with intact membranes, and one third of PTD are preceded by preterm

premature rupture of membranes (PPROM) [1].

Although the exact mechanism of spontaneous PTD is not apparent in most cases,

intrauterine infection and/or inflammation is now recognised as an important

pathophysiological factor [1]. The risk factors for PTD include PPROM, cervical

insufficiency, uterine over-distension, uterine anomalies, intrauterine

infection/inflammation, and other maternal factors such as stress and smoking [7].

Intrauterine infection or inflammation is implicated in approximately 25% of cases of

spontaneous preterm birth [1, 8, 9]. This proportion is even higher in neonates born

at very early gestational age (GA), with approximately 80% of births at less than 28

to 30 weeks GA associated with either histologic chorioamnionitis or organisms in

the placental membranes [10].

15

1.3 How does intrauterine infection/inflammation cause spontaneous preterm

delivery?

1.3.1 Pathways of infection

The amniotic cavity is normally sterile, and sterility is maintained by the action of

antimicrobial proteins and peptides in the cervical mucous plug, innate immune

responses in the uterine epithelium and placental tissues, and the physical barrier of

fetal membranes [11]. Nonetheless, micro-organisms may access the uterine cavity

by various mechanisms, which include: (1) ascending infection from the vagina and

the cervix; (2) contamination during invasive procedures such as chorionic villus

sampling or amniocentesis; or (3) retrograde spread from the abdominal cavity

through the fallopian tubes [1, 9]. Recent reports indicate that haematogenous spread

from distant sites, such as the oral cavity, might also be possible [12]. The usual

pathway is ascending vaginal infection, as evidenced by microbiological culture

studies of amniotic fluid. The most common isolates are vaginal commensal

organisms, such as Ureaplasma urealyticum, Mycoplasma hominis, Gardnerella

vaginalis, peptostreptococci, and bacteroides species [8, 9].

The current model for the mechanism of ascending vaginal infection was developed

by Romero and Mazor (Figure 1.3.1) [13]. According to this model, pathogenic

organisms become commensal in the vagina (Stage I), pass through the cervix to

colonise the decidua (Stage II), and then invade the amniotic cavity to cause intra-

amniotic infection (Stage III). Organisms may then directly infect the fetus (Stage

IV). It is the fetal response to these pathogens that is implicated in spontaneous

preterm birth. This fetal response is known as the fetal inflammatory response

syndrome and is discussed in the next section.

16

Figure 1.3.1 Stages of ascending infection of intrauterine cavity

Most intrauterine infection arises from ascending vaginal infection. In stage I micro-organisms colonise the birth canal, stage II micro-organisms pass through the cervix and colonise the decidua, stage III micro-organisms invade the amniotic cavity and infect the amniotic fluid, and in stage IV there is direct infection of the fetus. This figure was reproduced from Romero and Mazor, Clin Obstet Gynecol, 1988 [13].

1.3.2 Fetal inflammatory response syndrome (FIRS)

The fetal response to microbial products such as endotoxin or micro-organisms, is a

systemic inflammatory response, similar to the systemic inflammatory response seen

in adults. The fetal inflammatory response syndrome is characterised by (i) changes

in the amniotic fluid, such as increased levels of interleukin-6 (IL-6) [14-16], tumour

necrosis factor (TNFα) [17], interleukin-1β (IL-1β) [17], and white blood cell count

[17]; (ii) changes in the cord blood such as increased levels of IL-6 [17, 18]; and/or

(iii) by histologic evidence of inflammation in the chorionic plate vessels of the

placenta and/or umbilical cord (fetal vasculitis). Previous studies show a clear

relationship between intra-amniotic infection, a fetal inflammatory response (as

defined by increased cord levels of IL-6), the development of early onset neonatal

17

sepsis or bacteraemia [19], and severe neonatal morbidity [18]. According to the

Romero model of ascending infection (Figure 1.3.1), the inflammatory process in

stage II is maternal in origin. In stage III, the intra-amniotic inflammatory process is

of fetal in origin, rather than maternal origin [13]. Support for this model has been

provided by studies in which neutrophils in the amniotic cavity of mothers carrying

male fetuses stained for the Y chromosome, indicating a fetal inflammatory response

[20]. Further, infants with histologic fetal inflammatory response (stage III) show a

higher morbidity and mortality than those exhibiting a maternal inflammatory

response only (stage II) [21].

1.3.3 Consequences of the fetal inflammatory response syndrome (FIRS)

FIRS is associated with short and long term adverse neonatal outcomes. Firstly, there

are now substantial data that demonstrate that intrauterine inflammation plays an

important role in spontaneous preterm labour [9, 22]. Occult bacterial infection

results in activation of the innate immune response and release of inflammatory

mediators from the decidua and chorioamnionic membranes [22]. These mediators

include IL-1β, IL-6 and TNF-α, prostaglandins and metalloproteases, which together

stimulate myometrial contractility, ripen the cervix and weaken chorioamnionic

membranes [23]. Inflammation is also implicated in spontaneous labour at term and

it is postulated that normal labour is initiated by a similar, although less intense,

inflammatory process [22].

In addition to the relationship with spontaneous PTD, the fetal inflammatory response

syndrome is independently associated with other adverse neonatal outcomes, such as

periventricular leucomalacia and cerebral palsy [18, 24-28], bronchopulmonary

dysplasia [18, 29-31], necrotising enterocolitis [18], and early onset neonatal sepsis

[18, 32]. These associations underscore the importance of identifying neonates

exposed to intrauterine inflammation who are at increased risk of long-term

morbidity and may benefit from therapeutic intervention in utero or early in life.

18

1.4 The relationship of placental histology with the fetal inflammatory response

syndrome

1.4.1 Definition of histologic chorioamnionitis (HCA)

Acute inflammatory changes within the placenta and chorioamnionic membranes

arise from intrauterine infection. For many years the diagnostic terminology used by

histopathologists for these changes was “acute chorioamnionitis with or without

funisitis (inflammation of the umbilical cord)” [33]. This terminology was re-

evaluated following research into PTD and recognition of FIRS. In 2003 Redline et

al. devised new diagnostic criteria for histologic chorioamnionitis [33] (Table 1.4.1).

In this schema, placental reactions for the maternal and fetal compartment are

considered separately, which fits more comfortably with the model proposed by

Romero et al. [13]. The maternal inflammatory response is assessed by stage of

progression of inflammation from venules near the decidua capsularis through the

chorion to the amnion. The fetal inflammatory response is determined by degree of

involvement of the chorionic and umbilical vessels. The intensity of the maternal and

fetal inflammatory responses is graded according to the density of infiltrating mature

neutrophils.

These criteria were shown to be reproducible, with substantial interobserver

agreement (kappa >0.60) for the presence of any maternal inflammatory response,

any fetal inflammatory response, severe (grade 2) maternal inflammatory response,

peripheral funisitis, acute villitis, and acute intervillositis/intervillous abscesses [33].

19

Table 1.4.1 Diagnostic criteria for histologic chorioamnionitis

This table shows the diagnostic criteria for histologic chorioamnionitis as published by Redline et al.[33].

20

1.4.2 Correlation of histologic chorioamnionitis with the fetal inflammatory

response syndrome

The strength of the evidence that links HCA with FIRS and PTD, is dependent on the

definition of HCA and the patient population studied (preterm, term, excluded

subgroups etc.) [34]. When HCA is defined by the presence of chorionic vasculitis,

funisitis or the fetal neutrophilic response (as in the Redline criteria) [33], there is a

strong correlation between HCA and FIRS [19, 35-38]. Furthermore there is a

relationship between HCA and PTD [39, 40] and the long-term sequelae of FIRS [37,

41-43]. Data from a rabbit model has shown the introduction of infection into

amniotic fluid can induce HCA in the fetal and maternal compartments as well as

spontaneous preterm birth [44]. As such, HCA is considered the histologic hallmark

of FIRS.

1.5 Does the fetal inflammatory response syndrome affect surrogate markers for

infection in the early neonatal period?

1.5.1 Recognition of sepsis in neonate

Recognition of the infection in the early neonatal period is difficult due to non-

specific and poorly localised clinical signs [45, 46]. Yet, with the emergence of

multi-drug resistant bacteria, the ability of clinicians to accurately diagnose or rule-

out infection is critical in limiting inappropriate use of antibiotics [47]. Blood culture

remains a key investigation for the correct diagnosis of neonatal sepsis [48].

Unfortunately, blood culture lacks sensitivity due to low volume samples [49, 50],

low bacterial-density [50] and antenatal administration of antibiotics [45]. C-reactive

protein (CRP), total white cell count (WCC), absolute neutrophil count (ANC) and

immature:total granulocyte ratio (I:T) are the most widely used surrogate markers of

infection in this population and often employed to guide the use and duration of

intravenous antibiotics.

However, the diagnostic specificity of these investigations may be confounded by

concurrent causes of inflammation or antenatal/perinatal factors that do not

necessarily herald true bacteraemia. FIRS is an example of a potential source of

antenatal inflammation that may interfere with the results of these investigations.

21

Recent evidence shows that inflammation associated with PTD can have sustained

effects on inflammatory markers for up to 10 days following delivery [51, 52]. One

of the principal aims of this thesis was to determine the extent to which surrogates of

neonatal infection may be confounded by intrauterine inflammation.

1.5.2 I:T ratio, total white cell count, and absolute neutrophil count

I:T ratio

The immature:total granulocyte ratio (I:T) is a commonly employed surrogate of

bacterial infection. The ratio is based on the presence of increased numbers of

immature or non-segmented neutrophils in the circulation (‘left-shift’) in the setting of

bacterial sepsis [53], particularly bacterial infection in newborn infants [54-56]. After

manual assessment of the blood film, the I:T ratio is calculated by adding the number

band forms plus any immature granulocytes then dividing by the total granulocyte

count (mature neutrophils, bands and immature granulocytes).

The I:T ratio is used exclusively in the setting of neonatal infection [57]. Manroe et

al. [58] found the normal I:T ratio in healthy newborns was <0.16 in the first 72 hours

after birth. Schelonka et al. have since shown healthy neonates may have an I:T ratio

up to 0.27 [59]. The commonly cited thresholds for the diagnosis of neonatal sepsis

are 0.16 [60, 61] and 0.2 [62]. This cut-off has a sensitivity of 13-86% and specificity

of 51-96% [62] for the diagnosis of neonatal sepsis.

The accuracy of the I:T ratio is dependent on the ability of technicians to reliably

differentiate band forms and immature granulocytes from segmented neutrophils. A

2006 survey of haematology laboratories in the Netherlands found wide intra- and

inter-observer variation in discriminating band cells from segmented neutrophils [63].

Schelonka et al. [64] demonstrated correlation coefficient (r2) values of 0.10-0.28

among highly experienced operators. There is evidence that there may be even

greater variation when examining neonatal blood films [57, 65, 66]. Despite this, I:T

ratio is still considered a validated measure by many researchers and has appeared in a

number of publications since 2006 [67-70].

22

There is little information in the literature on I:T ratio in HCA. In a study of infants

exposed to clinically-diagnosed chorioamnionitis, Jackson et al. [71] found a

significantly higher proportion of symptomatic neonates had abnormal I:T ratios when

compared to neonates with no sign of bacterial infection (p=0.002), although 58% of

asymptomatic neonates also had abnormal I:T ratios. No study has compared the I:T

ratio in newborns exposed to HCA versus newborns not exposed to HCA.

Total White Cell Count (WCC) and Absolute Neutrophil Count (ANC)

Neutrophils are central to neonatal innate immune function. Deficiencies in number

and function contribute to immunodeficiency, particularly in preterm infants [11]. In

routine clinical practice, neonatal neutrophil and total white cell count are utilised as

an adjunct in the diagnosis of neonatal sepsis [62, 72]. The total white cell count

(WCC) is derived from the measured total nucleated cell count after adjustment for

the number of circulating nucleated red blood cells [58, 73]. The absolute neutrophil

count (ANC) is calculated from the proportion of neutrophils (segmented plus band

forms) per 100 white cells multiplied by the total white cell count. Normal values in

the neonatal period are highly dependent on GA, birth weight and postnatal age [58,

74-76] (see Figure 1.5.2a and 1.5.2b). In healthy newborns, term and preterm,

neutrophil counts increase in the early neonatal period and peak at approximately 24

hours following delivery.

In neonatal sepsis, the WCC and ANC may increase or decrease. Leucopaenia and

neutropaenia at diagnosis may signify overwhelming sepsis and carry a poor

prognosis [77, 78]. The fetal response to intrauterine infection appears to be an

increase in the WCC and ANC [79]. However, due to a reduced storage pool, mature

neutrophils are quickly exhausted and a decrease in WCC and ANC follows. In

neonatal sepsis, a decrease in the WCC and ANC is frequently observed, particularly

in gram-negative bacterial infection [11, 54, 72, 80, 81].

23

Figure 1.5.2 Reference ranges for neutrophil counts in first 60 hours after birth

Reference ranges for total neutrophil values in the first 60 hours after birth [74]. The solid lines depict the boundaries of the revised reference range of Mouzinho et al. [75] in infants <1500 g. The dotted lines depict the reference range of Manroe et al. [58]. Total neutrophil counts increase in normal infants in the first 24 hours following delivery.

In a systematic review on the accuracy of haematological variables in the diagnosis of

bacterial infection from birth to 90 days, Fowlie et al. [62] showed that various

studies have used many different thresholds of WCC and ANC as indicators of sepsis.

For WCC, a low count threshold was <5-7 x109/L and a high count was >21-30

x109/L. For ANC, a low count was <1-5 x109/L and a high count was >5.4-11.4

x109/L. Not surprisingly these studies found dramatically difference sensitivities and

specificities for the diagnosis of neonatal sepsis. For WCC the sensitivity varied

between 18-63% and specificity was between 80-98%. For ANC, sensitivity varied

between 1-89% and specificity 44-93%. The wide variation in results is due to the

use of different cut-off values to define sepsis, the variable incidence of sepsis in the

study population, comparison to different control groups (e.g. perfectly well infants or

neonates with suspected sepsis), and a lack of consideration of appropriate reference

24

ranges for GA and postnatal age. Other factors affecting ANC in newborn infants

include sampling site (arterial, venous, or capillary), stress (pain and crying),

dexamethasone therapy and mode of delivery [76, 82]. In order to improve the

diagnostic utility of haematologic variables, some authors have proposed the use of

scoring systems that take into account total WCC, ANC, I:T ratio, platelet count and

toxic changes [46, 61], but generally these have not performed well in clinical settings

[62].

Neutrophils are an essential component of the innate immune response and a major

effector cell of the inflammatory response. Fetal neutrophils are implicated in fetal

compartment HCA [33]. Hence, it is reasonable to expect a relationship between cord

ANC and HCA. Only two studies have investigated the strength of relationship

between neonatal neutrophil counts and HCA. De Dooy et al. [52] assessed the effect

of HCA (n=34) on the neonatal WCC and ANC on Day 0, 1 and 2. Umbilical cord

parameters were not measured in this study. The results show that HCA is associated

with a significantly higher WCC on Day 0, and higher WCC and ANC on the first and

the second postnatal days. Jackson et al. [71], examined neonatal neutrophil counts in

asymptomatic term and near-term neonates exposed to suspected clinical

chorioamnionitis (n=856). They found that 99% of asymptomatic neonates exposed

to clinical chorioamnionitis had at least one abnormal neutrophil count on the first day

of life. Umbilical cord neutrophil counts were not measured in this study. Further,

although histologic chorioamnionitis was not assessed in this study, a previous report

from the same institution found that HCA was prevalent in women with clinical

chorioamnionitis (27-80% of women with PPROM) [71, 83]. A possible explanation

for the abnormal neutrophil results observed in this study is the influence of HCA on

early neutrophil counts. Gotsch et. al. suggest that these parameters are significantly

higher in neonates with FIRS (as defined by elevated fetal plasma IL-6

concentrations), although they do not specify whether this observation is seen in cord

blood or samples from the early neonatal period [84]. No previous study has

investigated the direct relationship between umbilical cord ANC and WCC and the

presence of HCA.

25

1.5.3 C-Reactive Protein (CRP)

What is CRP?

Serum CRP is an established and widely available diagnostic test. It has been used in

clinical practice for decades as an adjunctive test in the diagnosis of inflammation,

sepsis and infection. CRP itself is an acute phase reactant produced by the liver,

predominantly in response to IL-6 [85]. Its synthesis is synergistically enhanced by

the presence of IL-1ß. CRP binds to polysaccharides in pathogens and activates the

classical complement pathway. Levels are elevated as early as 12-24 hours from the

onset of inflammation, when clinical signs may still be unclear, and remain elevated

until the stimulus resolves. The serum CRP concentration may increase up to 1000

fold in the presence of infection, surgery, trauma and acute inflammatory events [86]

and it is well-described as a marker for bacterial infection in neonates and children

[62, 87, 88].

Sensitivity and specificity of CRP in neonatal sepsis

The roles of serum CRP measurement in the neonatal intensive care unit include (1)

diagnosis of neonatal sepsis, (2) exclusion of neonatal sepsis, and (3) determining

response to therapy and duration of antibiotic treatment [89]. Given that the mortality

rate associated with neonatal sepsis is 5 to 50% [89], a diagnostic test in this setting

must have a high sensitivity to ensure true cases recognised and appropriately treated

with antibiotics. The sensitivity of CRP as an early marker for infection is poor (40-

65%), as levels may not be elevated within the first 24 hours of onset of infection [90,

91]. Its performance improves in the later phase of infection. At 24-48 hrs from the

initial evaluation, individual CRP measurements have a sensitivity of 80-90% [91].

Three serial CRP measurements over the first 72 hours have a sensitivity of 90-98%

[91]. Furthermore, two CRP levels of <10 mg/L obtained 24 hours apart, 8 to 48

hours after the initial evaluation, indicate that bacterial infection is unlikely and that

antibiotic therapy can be stopped [89, 91].

Serum CRP is not recommended as a sole diagnostic test for neonatal sepsis as it is

only moderately specific for bacterial infection in this setting. The specificity of a

single CRP measurement is 69-90% at the initial presentation and 75-84% at 24-48

26

hours [91]. This is because there are other factors that may elevate CRP levels in

neonates. These include perinatal asphyxia, fetal distress during delivery,

periventricular and intraventricular haemorrhage, pneumothoraces, and meconium

aspiration pneumonitis [89, 92, 93]. Interestingly maternal fever during labour and

PROM are also associated with elevated neonatal CRP levels [89, 92, 93]. It is

unclear whether this is due to inflammation as a result of the fetal inflammatory

response syndrome, or transportation of CRP from the mother to the fetus across the

placenta.

As described in section 1.3.2, IL-6 is one of the key cytokine mediators of the fetal

inflammatory response syndrome. Further, it is known that IL-6 does not cross the

placental barrier [94]. Several previous studies have shown that IL-6 is elevated in

the umbilical cord blood of neonates with HCA or funisitis [19, 36, 95-98]. As IL-6

is also a direct stimulator of CRP, it is expected that CRP will be elevated in the cord

blood of neonates with histologic chorioamnionitis, although data are sparse.

Ultrasensitive serum CRP measurement

With standard methods for CRP measurement the lower limit of detection is 5-10

mg/L. More recently, highly sensitive assays for CRP have been developed that allow

precise and accurate measurement of CRP in the range between 0 and 10 mg/L [86].

It is now recognised that chronic subclinical inflammation can cause low level

increases in CRP concentrations (in the range of 0-10 mg/L) and can be associated

with adverse outcomes, for example, the metabolic syndrome, the development of

atherosclerosis and coronary artery disease [99].

Umbilical cord CRP as an indicator for the presence of histologic chorioamnionitis

There are only five studies that have examined whether the presence of HCA is

associated with elevated umbilical cord serum CRP levels. All of these studies have

shown that HCA/funisitis is associated with elevated umbilical cord CRP levels when

measured by high-sensitivity methods (lower limit of detection 0.2-1.0 mg/L) [100-

104]. Furthermore, the intensity of the cord CRP response correlates with whether or

not amniotic fluid infection is demonstrated [101].

27

Relationship of cord CRP and neonatal CRP in neonates exposed to histologic

chorioamnionitis

The relationship between cord and neonatal CRP in neonates exposed to HCA

remains largely unexplored. There are two studies that provide indirect evidence that

there may be a relationship. De Dooy et al. [52], found that neonatal CRP levels on

Day 0 and Day 1 were significantly higher in neonates with HCA. Interestingly, a

CRP of >14mg/L on Day 1 of life was independently associated with HCA on

multivariate logistic regression analyses, whereas the Day 0 CRP level was not. The

sensitivity of the CRP assay used in this study is not specified and CRP was not

measured in the umbilical cord sera. In contrast, this thesis investigated whether there

is a direct relationship between HCA and usCRP levels, measured in cord blood, or

conventional CRP measured in peripheral blood on Day 0, 1 or 2.

Skogstrand et al. [51], showed that neonatal cytokine levels measured from dried

blood spots collected at mean 5 days following delivery were significantly higher in

preterm and very preterm infants. This study did not correlate cytokine levels with

the presence of HCA, but provides evidence that the elevated cytokine levels

associated with preterm birth are sustained into early neonatal life. CRP levels were

also measured in this study and the results are intriguing. CRP levels were

significantly lower in very preterm and preterm neonates when compared to term

neonates (p<0.0001, Mantel-Haenzel trend test). The authors contend that, as only 2

of 160 neonates in the study had sepsis, this result may reflect constitutively lower

CRP levels in preterm and very preterm infants when compared to term infants

without infection. It is unclear whether this is due to differences in liver maturity,

CRP gene polymorphisms, or other yet to be defined mechanisms.

No previous studies have directly examined the association of HCA with cord and

neonatal CRP levels. As CRP is one of the major diagnostic tests used for bacterial

infection in early neonatal period [62, 88], it is important for clinical practice to

establish the influence of HCA on neonatal CRP results.

28

1.5.4 Procalcitonin

What is procalcitonin?

Procalcitonin (PCT) is an acute phase reactant that is generated by similar

inflammatory pathways as CRP [105]. It is the peptide prohormone of calcitonin and

is normally produced by C-cells in the thyroid gland [106]. During sepsis, a number

of non-thyroidal tissues including circulating monocytes [107], renal cells, pancreatic

cell, adipose tissue, and hepatocytes [108] produce procalcitonin in direct response to

endotoxins [109] and inflammatory cytokines, such as TNFα and IL-6 [105].

Comparison of the properties of PCT versus CRP as a marker for inflammation

and/or sepsis

Procalcitonin may have advantages over CRP as a marker for inflammation or sepsis.

Levels increase more rapidly than CRP (2-4 hours) in response to systemic

inflammation or infection [110]. PCT circulates at very low levels in healthy subjects

as it is rapidly metabolised in the thyroid gland to calcitonin, an N-terminal residue,

and calcitonin carboxyterminus peptide I (CCP-I) (also known as katacalcin) [106]. In

the setting of sepsis, there is a marked increase in production of PCT with levels

increasing from the picogram range up to concentrations of up to 1000 ng/mL [111].

PCT levels remain elevated during the inflammatory process and rapidly return to

baseline on resolution of the stimulus [109, 110]. Thus PCT levels more accurately

reflect the real-time extent of inflammation and may be more useful than CRP in the

early diagnosis of infection and monitoring of disease.

PCT and CRP were compared as diagnostic tests for bacterial infection in two

systematic reviews and meta-analyses, one in adults and one that combined neonatal,

paediatric and adult studies. Uzzan et al. [112] found that PCT was a better

diagnostic marker for sepsis, severe sepsis or septic shock than CRP (OR 15.7 [95%

CI 9.1-27.1] versus OR 5.4 [95% CI 3.2-9.2]) in critically ill adult patients (33

studies, 3943 intensive care patients). Similarly, Simon et al. [113] found that PCT

was more sensitive (88% [95% CI 80-93%] versus 75% [62-84%]) and more specific

(81% [95% CI 67-90%] versus 67% [95% CI 56-77%]) than CRP for differentiating

bacterial infection from other causes of inflammation in hospitalised patients (12

29

studies; 46 neonates, 638 children, and 702 adults in different hospital settings).

These data indicate that PCT is better than CRP at differentiating bacterial infection

from other causes of inflammation.

PCT increases physiologically in the neonate during the first few days of life

The use of PCT for the diagnosis of sepsis in the perinatal period is not

straightforward; a physiological increase in PCT concentrations occurs in healthy

neonates the first few days of life. In both term and preterm neonates, PCT increases

during the first 48 hours with a peak between 18 and 30 hours of life and returns to

normal levels between 42 and 48 hours of life [114-116]. Interpretation of normal

values relies on use of nomograms that take into account time since birth and GA

[114, 115] (Figures 1.5.4a and 1.5.4b). The adult reference range is generally used 3

days after birth.

Figure 1.5.4a PCT levels in term (>36 weeks) infants

This figure shows procalcitonin (PCT) levels in term infants versus time since delivery. Note the Y axis is a logarithmic scale. PCT concentrations increase following delivery, peaking at approximately 24 hours [114].

30

Figure 1.5.4b PCT levels in preterm (<36 weeks) infants

This figure shows procalcitonin (PCT) levels in preterm infants versus time since delivery. In contrast to figure 1.5.4b, the Y axis is not a logarithmic scale. PCT levels are shown to increase following delivery, peaking at approximately 24 hours [115].

The post-natal surge in PCT levels arises from endogenous production of PCT, rather

than transplancental transport. Evidence for this was provided by Assumma et al.

who found PCT values in paired maternal and cord sera at birth were positively

correlated, however the post-natal increase is unaffected by maternal PCT [117]. This

observation was later confirmed by Kordek et al. [118] who found that maternal PCT

concentrations do not correlate with the umbilical cord or neonatal PCT

concentrations.

The results of studies on the use of PCT as an early marker for neonatal sepsis are

contradictory. In a recent review, Van Rossum et al. [111] summarised the evidence

for PCT as a marker for neonatal sepsis. Of 17 studies identified, the majority have

found that serum PCT concentrations are an early and specific marker for neonatal

Cover up

31

sepsis. However, six studies concluded that PCT is not a better marker than CRP in

neonatal sepsis[119-124]. Possible explanations for these conclusions include a lack

of correction for neonatal reference values [120-123], and a lower specificity of PCT

for bacterial infection in this setting. Other non-infective sources of inflammation

that increase PCT concentrations in neonates include perinatal asphyxia, intracranial

haemorrhage, and maternal pre-eclampsia [122, 124-127].

Umbilical cord serum PCT concentrations in HCA

It seems plausible that the presence of HCA is a further variable that affects early

neonatal PCT concentrations, but there are few studies that examined this

relationship. In a small study of only 8 neonates with HCA, Janota et al. found that

HCA did not significantly influence cord PCT levels, but was associated with a more

pronounced post-natal increase in PCT at 72 hours and 7 days [121]. Three studies

[88, 118, 128] have found that PCT levels are higher (>0.5-1.2 ug/L) in newborn

infants that subsequently develop early onset neonatal sepsis compared with

uninfected neonates, however these studies did not correlate PCT levels with the

results of placental histology.

1.5.5 Summary of neonatal inflammatory markers in HCA

At birth, the best measures of antenatal events are found in the cord blood.

Inflammatory mediators associated with intrauterine inflammation are maximal at the

time of delivery. To date there is little information concerning the effect of

intrauterine inflammation on cord WCC, ANC, I:T ratio, and levels of usCRP and

PCT. All of these markers are used in clinical practice to assist in the diagnosis of

neonatal sepsis, hence it is important to establish the extent to which these surrogates

are influenced by the presence of HCA.

32

1.6 Can we detect mothers with intra-amniotic infection and predict the risk of

pre-term delivery?

1.6.1 Why do we want to predict preterm birth

The ability to identify pregnancies at risk of preterm birth due to intrauterine

inflammation is important for several reasons. First, it may enable use of specific

treatments to prevent PTD. Second, it may define a population of neonates at risk of

long-term sequelae as a result of exposure to intra-uterine inflammation. Finally, if a

population at high risk of preterm birth can be detected, it may allow further insights

and understanding of the mechanisms that lead to preterm birth and the development

of preventative interventions.

1.6.2 Chorioamnionitis is a chronic infection

Current evidence suggests that the intrauterine infection/inflammation is often chronic

and asymptomatic [9, 22]. Supportive data includes results from second trimester

studies of amniotic fluid cytokine levels and bacterial culture results. Gray et al.

[129] found that women without clinical symptoms undergoing amniocentesis for

genetic diagnosis during the second trimester had positive amniotic fluid culture

results, yet did not deliver or miscarry until up to 4 weeks later. Furthermore,

concentrations of inflammatory cytokines, such as MMP-8 [130], IL-6 [131] and

TNFα [8], measured in the amniotic fluid obtained during second trimester

amniocentesis are elevated in those who deliver preterm, but the delivery may not

occur for many weeks.

1.6.3 Clinical signs

A minority of women with intrauterine inflammation/HCA have clinical signs of

infection. Clinical signs of chorioamnionitis include intrapartum fever, uterine

tenderness, maternal tachycardia, fetal tachycardia and/or foul-smelling amniotic

fluid. These signs have a poor sensitivity for the presence of HCA. In a study of 52

women with HCA, 9.6% had fever, 5.8% had uterine tenderness, and 11.5% had post-

partum endometritis [132]. Moreover, while signs of clinical chorioamnionitis may

be diagnosed in 0.9-10% of pregnancies [133, 134], these signs are non-specific, as

33

38.1% of women with symptoms of clinical chorioamnionitis have no evidence of

histologic placental inflammation [133].

1.6.4 Amniotic fluid cultures

Positive amniotic fluid cultures are associated with preterm birth [14, 15]. However,

intra-amniotic inflammation can be present with or without microbiologically-proven

amniotic fluid infection [14, 15]. Less than half of women with intra-amniotic

inflammation have positive amniotic fluid cultures [135, 136]. Furthermore, the

maternal and neonatal outcome for women with intra-amniotic inflammation is

similar, regardless of whether or not amniotic fluid infection is demonstrated [101,

135, 136]. Therefore, the detection of intrauterine inflammation, rather than infection

per se, may be more important in predicting risk of PTD.

1.6.5 Use of other biomarkers to detect risk of preterm birth

The lack of sensitivity of amniotic fluid culture or clinical signs for identifying

pregnancies complicated by intra-amniotic inflammation, has led to an explosion of

research into other maternal biomarkers as predictors of preterm birth. These include

cytokines (IL-6, IL-10), complement levels, white cell growth factors and products

(lactoferrin, defensins, granulocyte colony stimulating hormone), matrix

metalloproteinases, acute phase reactants (C-reactive protein, ferritin), as well as

molecular markers [10, 22, 137]. C-reactive protein and procalcitonin are examples

of acute phase reactants, already widely available to clinicians, that, if predictive of

preterm birth, could be readily incorporated into clinical practice. In the following

section, the evidence for maternal CRP and PCT levels as markers for intrauterine

inflammation is discussed.

1.6.6 Is maternal serum C-reactive protein a marker for intrauterine inflammation?

Previous studies have shown consistently that elevated maternal CRP levels are

correlated with HCA in a variety of different study populations (women with

PPROM, suspected PROM, intact membranes, PTD, term delivery), using differing

methodologies for measuring CRP and differing definitions of HCA [138-143].

34

Trochez-Martinez et al. [144] recently published a systematic review of the use of

CRP as a predictor of chorioamnionitis in PPROM. All 8 eligible studies found a

positive association between CRP and the presence of HCA, despite varying

methodologies for CRP measurement and CRP cut-off values. However, the

conclusions from these studies were contradictory with regard to whether CRP was a

useful predictor of chorioamnionitis; three studies concluded that CRP measurement

is useful in predicting the presence of chorioamnionitis and the remaining 5 did not.

Evidence from the literature shows a consistent association between CRP and HCA.

However due to the lack of specificity, an elevated maternal CRP concentration is

may not be useful as a predictor for the presence of HCA.

1.6.7 Is maternal serum PCT a marker for intrauterine inflammation?

There is very little evidence in the literature on whether maternal PCT is associated

with intrauterine inflammation or HCA. In healthy women who deliver at term, PCT

concentrations are similar to the normal healthy adult population [117]. Assumma et

al. found that in women without evidence of clinical chorioamnionitis, or non-

infectious complications of pregnancy and who deliver at term, PCT concentrations

did not appear to be affected by mode of delivery, use and type of anesthesia, duration

of active labor, rupture of membranes >18 hours, maternal group B streptococcus

colonisation or intrapartum antibiotics administration [117]. Torbé´ et al. observed

that in apparently healthy women with preterm labour and no clinical sign of

infection, PCT levels were significantly higher than in healthy pregnant women who

delivered at term (p<0.05) [145]. Furthermore, women who delivered prematurely

(n=20) had a non-significant trend towards higher PCT concentrations in preterm

labour than women who responded to tocolytic treatment and delivered at term

(n=33). However, this study was underpowered to detect a difference in PCT levels

between women in preterm labour who delivered prematurely or at term. Clinically

silent intrauterine inflammation, causing preterm labour and raised PCT

concentrations, could possibly account for these results.

A further study by the same group showed that a PCT >1.9 ng/mL predicted HCA

with a sensitivity of 75%, specificity of 45%, PPV 35% and NPV 82% [146].

35

Although they conclude that PCT was unsatisfactory in predicting the PPROM-to-

delivery [146] or admission-to-delivery intervals in women with intact membranes

[145], the role of PCT in the pathophysiology of preterm labour warrants further

study.

1.6.8 Summary of maternal inflammatory markers in HCA

Histologic choriomanionitis is clinically silent and may be present many weeks before

PTD occurs. The detection of intrauterine inflammation, rather than infection, may be

more important in predicting risk of PTD. Maternal biomarkers that predict the risk

of PTD are an area of active research. Current evidence shows that maternal serum

usCRP and PCT are associated with the presence of HCA, however there is

uncertainty with regard to the ability to predict subsequent PTD.

36

CHAPTER 2:

Methods

37

The primary aim of this study was to investigate markers of inflammation in mothers

and infants exposed to HCA. This chapter outlines the study design, research

governance, recruitment procedures, sample collection and preparation, measurement

and statistical methods.

2.1 Study Design and Inclusion Criteria

This was a cross-sectional study of newborns and their mothers, who delivered at

King Edward Memorial Hospital for Women (KEMH), Western Australia, between

August 2003 and January 2006. KEMH is the only tertiary obstetric and neonatal

hospital in Western Australia. As such, the study population was a heterogenous mix

of high-risk infants, born both at term and preterm. This project was part of a larger

study, known as the Study of Postnatal Immunity in the Newborn (SPIN), which

aimed to investigate innate immune function in newborns.

All live infants who were delivered at KEMH and their mothers were eligible for

inclusion in the study. The only mothers excluded were those whose understanding of

English was insufficient for full informed consent.

2.2 Research Governance and Ethics

This research was conducted with regard for the principles of respect for the truth,

scientific integrity, scientific rigour, and respect for research participants, in

accordance with the National Health and Medical Research Council’s “Australian

Code for the Responsible Conduct in Research (2007)” [147] and “National Statement

on Ethical Conduct in Human Research” [148]. The KEMH ethics committee

approved the project. Specific ethical issues that were considered were confidentiality,

seeking consent from the mother alone, timing of consent, risks associated with the

study, and research involving fetal/placental tissue. These issues are discussed in

sections 2.2.1 – 2.2.5.

38

2.2.1 Confidentiality

Maintaining confidentiality was of utmost importance. Samples and clinical data

were coded with a unique alphanumeric identifier, allowing identification of

maternal/neonatal pairs. All data were potentially identifiable (i.e. coded but re-

identifiable) so that further data could be obtained as needed. As multiple studies

were undertaken on the same biobank, this aspect enabled new data to be linked to

existing data. All biological samples were analysed only with reference to the

alphanumeric identifier and not to the participant’s name. Linking information was

stored securely as a hard copy (not electronically), with only the principal investigator

having access to the linking information. No individual data was released to study

participants or their families except where this information was of direct clinical

relevance.

2.2.2 Seeking consent from the mother alone

This project required samples from the infant and mother. Collection of blood

samples from fathers was not required to address the study hypothesis. Thus, the

mother alone gave consent for infant samples. In some cases, the father was not

present at the time of delivery or was no longer in a relationship with the mother at

the time the child was born. Fathers, if present, were encouraged to discuss the study

both with the mother and, if necessary, with the investigators, but ultimately the

decision to give consent for enrolment rested with the mother. Making enrolment

contingent on consent from both parents may have created additional difficulties for

parents if their relationship was sub-optimal and would lead to the exclusion of many

mother-infant pairs simply on the grounds that the father was absent.

2.2.3 Timing of consent

In the majority of cases, women gave full consent prior to delivery. However, in

women who presented with threatened or actual preterm labour it was not always

possible, or appropriate, to obtain consent prior to delivery. Consequently, these

women were given a brief explanatory leaflet about the study and the need to collect

cord and maternal samples at delivery (see Appendix 1). A tick box allowed women

to indicate provisional willingness to have samples taken. In these circumstances, if

39

women had indicated willingness to enrol, samples were collected and processed.

Formal consent was then sought post-partum. If consent was not given, the research

samples were discarded.

2.2.4 Risks associated with the study

The risks associated with this study were minimal. Minor discomfort was associated

with maternal blood sampling. Where possible, these samples were collected from

intravenous lines, or during routine sampling at the time of delivery. The cord blood

and placental samples were taken from material that would normally be discarded.

Neonatal blood samples were predominantly collected from indwelling venous lines.

Minor discomfort was associated with neonatal samples that were collected by heel-

prick or venepuncture, however these were only collected where clinically indicated.

Placental histology is part of routine clinical practice in infants born at less than 32

weeks gestation.

2.2.5 Research involving fetal/placental tissue

The use of placental tissue and cord blood was essential to this project. These

samples were prepared for histological examination and were not cultured or used for

stem cell research. Participants in the study gave specific consent for research on

placental tissue and cord blood.

2.3 Recruitment and consent

343 mothers and 421 neonates (including multiple gestations) were recruited between

11th August 2003 and 19th January 2006, according to the procedure shown in Figure

2.3. Research assistants provided preliminary information to all women attending the

antenatal clinics at KEMH, as well as women who presented with threatened or actual

preterm labour (see Appendix 1). A tick box allowed women to indicate provisional

willingness to be involved in the study. Full information sheets were then provided to

those women (see Appendix 2). In the majority of cases, neonatal fellows, or

neonatal research nurses who were familiar with the study protocol, obtained formal

40

consent prior to delivery (see Appendix 3). In some cases of spontaneous preterm

birth, women gave preliminary consent prior to delivery and full-consent was sought

after delivery. Women who had twins or triplets were asked to sign one consent form

for each infant.

Participating mothers were free to withdraw from the study at any time without

affecting their normal care or that of their infants. Importantly, recruitment was

dependent on the availability of research assistants, research nurses and neonatal

fellows.

Figure 2.3 Procedure for enrolment into study, collection and handling of samples, and formal consent

41

2.4 Sample collection and preparation

A number of samples were collected from mothers and infants for the SPIN study.

The samples that are relevant to this particular project were as follows:

(a) Placental samples

Full, partial or sampled placental specimens were collected at delivery and submitted

for histological examination. These specimens were first examined macroscopically.

Specimens were then sampled and fixed in 10% buffered formalin. The fixed tissue

blocks were embedded into paraffin wax. Five-micrometer thick sections were

mounted onto glass slides. After dewaxing, the samples were stained with

haemotoxylin and eosin. As part of routine care, tissue samples of cases of HCA

were also cultured using standard media (chocolate agar and CLED (Cysteine,

Lactose and Electrolyte Deficient) plates) for aerobic bacteria, selective agar for

mycoplasma and ureaplasma urealyticum, and selective agar and enrichment broth for

listeria.

(b) Cord blood samples

To minimise the risk of sample contamination with maternal blood, neonatal registrars

or fellows collected cord blood samples by direct sampling from the umbilical vessels

at delivery. Cord blood was collected into tubes containing ethylene diamine

tetraacetic acid (EDTA) (5 mL) and lithium-heparin gel (5mL). EDTA samples were

used for cord full blood counts, and blood smears. Blood smears were made and

stained with May Grünwald-Giemsa. Lithium-heparin gel tubes were used for

procalcitonin and usCRP measurement. Serum was prepared from these tubes by

centrifugation for 10 mins at 3000 rpm. Arterial and venous cord blood samples were

collected, as per routine clinical practice at KEMH, for measurement of blood gases

and cord pH.

42

(c) Maternal samples

At delivery, 5 mL of maternal peripheral blood was collected by venepuncture into

tubes containing lithium-heparin gel. Serum samples were prepared by centrifugation

for 10 mins at 3000 rpm and were used for procalcitonin, and usCRP measurements.

(d) Neonatal samples

Where clinically indicated, neonatal samples (0.2-0.5 mL) were collected for

measurement of full blood counts (0-24 hrs post-delivery) and standard CRP

concentrations (Day 0 (0-1 hr post-delivery), Day 1 (>1-24hrs), and Day 2 (24-48

hrs)). Clinical indications included investigation for suspected early-onset neonatal

sepsis and routine monitoring in neonates at high risk of early-onset neonatal sepsis

(e.g. preterm delivery). These samples were collected in EDTA and lithium-heparin

gel, respectively. Serum samples were prepared from lithium-heparin gel samples by

centrifugation for 3 mins at 1300 rpm.

2.5 Measurement

2.5.1 Clinical data

Demographic and clinical data on mothers and infants were collected at the time of

delivery. For mothers, these data included maternal age, maternal ethnic origin,

smoking status, number of previous deliveries, pregnancy complications, premature

rupture of membranes, clinical features of delivery (including length of labour,

intrapartum fever, use of antibiotics), antenatal steroids, and mode of delivery. For

neonates, the data that was collected included gestation age (GA) (as estimated by

date of the last menstrual period, in utero ultrasound, or if no antenatal information

available, the Ballard score at delivery [149], sex, birth weight, head circumference,

length, 1, 5 and 10 minute Apgar scores, the need for, nature and duration of

ventilatory support, neonatal sepsis, time to discharge, and neonatal deaths.

43

2.5.2 Placental histology and definition of histologic chorioamnionitis

An experienced consultant histopathologist examined the placental sections, blinded

to haematological and biochemical data. In approximately two-thirds of cases, three

sections of cord and membrane, and two sections of placenta with chorionic plate

were assessed. Where smaller samples were submitted to histopathology, a single

section of cord, placenta and membrane was assessed. The sections were scored

according to the Redline criteria (see Table 1.4.1). These criteria are validated and

widely accepted [33, 41].

HCA was considered a binary variable; that is, it was either present or absent.

According to the Redline criteria, placental fetal or maternal responses are graded

(range 0 - 4) and staged (range 0 - 4). An abnormal maternal response was

grade/stage >1 and an abnormal fetal response was grade/stage >0. Thus, the

presence of HCA was defined as an abnormal maternal and/or fetal response.

2.5.3 Ultrasensitive CRP

Ultrasensitive CRP was measured by latex immunonephelometry using the Dade

Behring Nephelometer BN II (Dade Behring, Marburg, Germany). The methods used

were consistent with the manufacturer’s instructions and used the manufacturers

specific reagents, calibrators and controls for ultrasensitve CRP measurement. The

precision of this method was assessed by the intra-assay and inter-assay coefficients

of variation (CV) at two different CRP concentrations. At a CRP level of 0.5 mg/L,

the intraassay CV was 3.1% (n=20) and the interassay CV was 2.5% (n=10). At a

CRP level of 2.1 mg/L, the intra-assay CV was 3.4% (n=20) and the inter-assay CV

was 2.1% (n=10).

The lower limit of detection of the usCRP assay was 0.15 mg/L. For the purpose of

statistical analysis, values <0.15 mg/mL were set to zero.

44

2.5.4 Procalcitonin

Procalcitonin was measured by the BRAHMS PCT sensitive immunoluminometric

assay on a Berthold Technologies Lumat LB9507 luminometer (Berthold

Technologies, Bad Wildbad, Germany). The manufacturers reagents, calibrators and

controls were used and the methods were in keeping with the manufacturers

recommendations. The precision of this method was assessed by the intra-assay and

inter-assay CVs different PCT concentrations. The intraassay CV was 5.6% at 0.10

ug/L (n=25) and 4.8% at 2.8 ug/L (n=25), while the interassay CV was 2.8% at 0.27

ug/L (n=5).

The lower limit of detection of the PCT assay was 0.01 ug/L. There were no samples

with measured values less than 0.01ug/L.

2.5.5 Cord full blood counts and blood films

Full blood counts

Cord and neonatal full blood counts were measured using a Beckman Coulter HmX

analyzer (Fullerton, California, USA). The instrument was calibrated 6 monthly using

an instrument specific calibration product. The daily controls were the Coulter 5C

cell control and a within-run drift control.

The coefficient of variation (CV) (as calculated from the standard deviation divided

by the mean) is a normalised measure of dispersion and was used to assess precision

of measured haematologic parameters. The coefficients of variation for these

parameters are described in Table 2.5.5. The results show that these parameters were

measured precisely with coefficients of variation of <5% over a range of absolute

values for white cell count, red cell count, haemoglobin, mean cell volume and

platelet count. This table also shows that for all parameters (except MCV) the range

of absolute values assessed was inclusive of values observed in neonatal samples. For

MCV, neonatal values were slightly higher than the values used for precision testing.

However, as the Beckman Coulter HmX has a linear performance for MCV over the

range of 50-150 fL [150], further precision testing was not required.

45

Table 2.5.5 Coefficient of Variation (CV) (%) for parameters measured on the Beckman Coulter HmX This table shows the coefficients of variation for the various haematologic parameters at typical measured values. The median and interquartile range (IQR) for the neonatal data are shown. *At least 31 replicate determinations were performed on the same sample.

Haematologic parameter Measured value

CV (%)*

Median (IQR)

for neonatal data in this study

White cell count (WCC x109/L)

4.0 – 15. 0 ≤2.5

7.3 (5.6-9.7)

Red cell count (RCC x1012/L)

3.0 – 6.0 ≤2.0

3.9 (3.7-4.7)

Haemoglobin

(Hb g/L) 120 – 180 ≤1.5

150 (141-167)

Mean cell volume

(MCV fL) 80.0 – 100.0 ≤2.0

113 (109-117)

Platelet count (Plt x109/L)

200 – 500 ≤5.0

243 (205-299)

46

Haematocrit (Hct), Mean Corpuscular Haemoglobin (MCH), and Mean Corpuscular

Haemoglobin Concentration (MCHC) are derived from measured parameters (Table

2.5.5), and are described as follows:

Hct is the relative volume of packed erythrocytes to whole blood, and is

calculated as:

MCH is the quantity of haemoglobin within red cells, and is calculated as:

MCHC is the average concentration of haemoglobin per erythrocyte, and is

calculated as:

Blood films

The quality of each cord blood sample was assessed on the blood film. Samples that

showed platelet clumping, fibrin clots, or poor staining were excluded from further

analysis.

For each remaining cord blood film, a manual 100-cell white cell differential was

performed. This count was used to estimate the percentage of mature neutrophils,

Hct (%) = RCC x MCV

10

MCH (pg) = Hb x10

RCC

MCHC (g/dL) = Hb x100

Hct

47

band cells, immature granulocytes, monocytes, eosinophils, basophils and

lymphocytes, as well as the number of nucleated red blood cells per 100 white cells.

A band cell was defined as a neutrophil in which the width of the narrowest segment

of the nucleus was not less than one-third the broadest segment [54].

The corrected white blood count (WBC) was calculated as follows:

The absolute neutrophil count was calculated as follows:

The immature:total ratio (I:T) was calculated as follows:

The I:T ratio was calculated as follows:

2.5.6 Neonatal CRP

In the postnatal samples, CRP was measured by enzymatic sandwich immunoassay

using VITROS Chemistry Products CRP Slides (Ortho-Clinical Diagnostics,

Rochester, New York, USA). The within-run imprecision was 1.3mg/L at 8 mg/L and

2.68 mg/L at 21.5 mg/L, while the within-lab precision is 25.1% at 4 mg/L and 7.4%

at 14 mg/L (CV; N=20 samples/day determined on 80 days).

total nucleated cell count x100

corrected WBC result =

100 + number of NRBC/100 WBC

cell % x corrected WBC

absolute neutrophil count =

100

number of bands cells + immature granulocytes

I:T ratio =

number of mature neutrophils + band cells + immature granulocytes

48

From the beginning of the study until 16th May 2005, the lower limit of detection for

the assay was 7 mg/L. After this time, a more sensitive slide technique was employed

and the lower limit of detection was 3 mg/L. For the purpose of analysis, values that

were less than the reference range were analysed in two ways. In the first approach

all values that were less than the reference range were set to 0 mg/L. In the second

approach values were set to 6.5 mg/L and 2.5 mg/L, respectively. The first approach

inflates the difference between detectable and undetectable values and is therefore

more likely to detect a difference. Results of these two approaches were presented to

highlight that the definition of values less than the reference range did not impact on

overall results.

For analysis, the maximum CRP value was the maximal CRP value recorded within

the first 48 hours post-delivery even if this was the only available CRP value.

2.6 Statistical Analysis

The assistance of Angela Jacques and Dorota Doherty (Women and Infant Research

Foundation) with the statistical analyses is gratefully acknowledged. Continuous data

were summarised using non-parametric summary statistics via medians, interquartile

ranges (IQR) and ranges (R), and Mann-Whitney U tests were used to compare

groups. Nominal data were summarised using frequencies. Pearson’s chi-square tests

(if frequency in each cell >5) or Fisher’s exact test (if frequency in any cell <5) were

used to compare frequencies between groups. Spearman’s rho was used to correlate

non-parametric continuous variables.

Linear regression was used to explore the effect of HCA on maternal, cord and

neonatal outcomes. Logistic regression was performed to identify simultaneous

factors associated with HCA. Regression models for maternal outcomes were

adjusted for co-existing conditions that are associated with a maternal inflammatory

response and/or increased levels of maternal cytokines (Table 2.6). These variables

were the presence of premature rupture of membranes (PROM), intrapartum fever, or

the duration of labour before delivery [151-153]. Cord and neonatal outcomes were

adjusted for factors that are associated with differences in cytokine levels in newborn

49

infants (Table 2.6). These were GA, whether small for GA according to Australian

normative data [154, 155], and duration of labour. In addition to these variables, cord

and neonatal NRBC count were also adjusted for hypoxia, as fetal hypoxia is

associated with elevated NRBC counts in newborns [156]. Cord and neonatal WCC

and ANC were adjusted for use of antenatal steroids as this is associated with higher

newborn WCC and ANC [157]. The only case with early onset sepsis was excluded

from the neonatal analysis. Analysis of residuals was used to evaluate the assumption

of normality of data. Non-normal continuous outcome variables were log-

transformed (natural logarithim) to achieve data normality (see Table 2.6). Extreme

outliers (>3 standard deviations) were excluded as necessary. Coefficient of

determination (R2) was used to measure what proportion of variance was explained by

the variables in the model. Odds ratios (OR) were used to summarise effect sizes.

For all analyses, a p-value of <0.05 was considered statistically significant. Data was

analysed using SPSS statistical software (version 15.0: Chicago, Illinois).

50

Outcome Potential confounders Log (ln) transformation required

usCRP PROM hours, length of labour, intrapartum fever Yes

Maternal

PCT PROM hours, length of labour, intrapartum fever Yes

usCRP GA, small for GA, length of labour Yes

PCT GA, small for GA, length of labour Yes

Neutrophil count GA, small for GA, length of labour Antenatal steroids Yes

Cord

NRBC count GA, small for GA, length of labour Hypoxia No

First CRP within 48 hours of delivery GA, small for GA, length of labour Yes

Maximal CRP within 48 hours of delivery GA, small for GA, length of labour No

Neutrophil count within 24 hours of

delivery

GA, small for GA, length of labour Antenatal steroids Yes

Neonatal

NRBC count within 24 hours of delivery

GA, small for GA, length of labour Hypoxia Yes

Table 2.6 Maternal, cord and neonatal outcome variables

This table shows the maternal, cord and neonatal outcome variables. The potential confounders that were considered in multivariate analysis are specified in the second column. Data that required logarithmic transformation are detailed in the third column. PROM hours = duration of premature rupture of membranes, GA=gestational age, usCRP = ultrasensitive C-reactive protein concentration, PCT=procalcitonin concentration, NRBC count = no. of nucleated red blood cells per 100 white cells

51

CHAPTER 3:

Results correlation of HCA with maternal, cord and neonatal

outcome variables

52

3.1 Study Cohort

Between 11th August 2003 and 19th January 2006, 343 mothers and 421 neonates were

recruited to the SPIN study. Neonates in whom no placental sample was submitted

were excluded from further analysis (n=96), leaving 325 neonates. Statistics from the

KEMH perinatal database show that 11560 infants were born at KEMH during this

time period; the SPIN study therefore recruited 2.8% of the total number of infants

born during the study period (Table 3.1.1). A greater proportion of preterm infants

from the total preterm population were recruited (222 recruited of 2630 total, 8.3%),

particularly in the 28 to 32 week GA group (90 recruited of 459 total, 19.6%),

reflecting the broader immunological aims of the SPIN study. Of the very premature

infants (< 28 weeks), only 19 infants were recruited (19 recruited of 372 total, 5.1%).

Table 3.1.1 SPIN study cohort: gestational age groups This table shows the SPIN study cohort in gestational age groups by number (% total) (<28 weeks (wks), 28-32 wks, 32-37 wks and >37 wks). It also shows the total population of births at King Edward Memorial Hospital during the study period number (% total) of the total population of births at the study period (11th August 2003 to 19th January 2006). The SPIN study as a proportion of the total population at KEMH is shown in the far right column.

Gestational Age Group SPIN study Total population Proportion recruited to

SPIN study of total population (%)

< 28 wks 19 (6%) 372 (3%) 5.1%

28 – 32 wks 90 (28%) 459 (4%) 19.6%

32 – 37 wks 113 (35%) 1849 (16%) 6.1%

Term > 37 wks 98 (30%) 8880 (77%) 1.1%

Preterm < 37 wks 222 (68%) 2680 (23%) 8.3%

Data not available 5 (2%) - -

Total 325 (100%) 11560 (100%) 2.8%

53

Tables 3.1.2 and 3.1.3 show the mode of delivery and number of multiple births.

Approximately two thirds of the study population were delivered by elective

caesarean section, with high proportion (39%) of multiple gestation pregnancies.

Table 3.1.2 SPIN study cohort: Mode of delivery This table shows the mode of delivery of neonates in the SPIN study cohort (number (% total)).

Table 3.1.3 SPIN study cohort: Singleton or multiple gestation

This table shows the number (% total) in the SPIN study cohort who were singletons or multiple gestations

3.2 Placental Histology

Of the 325 placental samples available, there were 26 cases of HCA. In the majority

of cases, both a maternal response and fetal response was seen (n=18). There were 2

cases where there was a maternal response without a fetal response, and 6 cases where

there was a fetal response without a maternal response. For analysis, HCA was

considered present in any case with a maternal and/or fetal response (n=26).

Table 3.2 (page 55) describes each of these cases in detail: specifically, the strength of

fetal and/or maternal response, GA at delivery, whether the gestation was single or

multiple, type of placental sample, and results of bacterial culture. The median GA

Mode of delivery No. of neonates (%)

Spontaneous vaginal delivery 87 (28%)

Assisted vaginal delivery 22 (7%)

Emergency caesarean section 3 (1%)

Elective caesarean section 208 (64%)

Data not available 5 (2%)

Total 325 (100.0%)

Single or multiple No. of neonates (%)

Singleton 192 (61%)

Multiple births 128 (39%)

Data not available 5 (2%)

Total 325 (100.0%)

54

was 30 weeks (interquartile range 27.5 to 32 weeks). The majority of cases with

HCA were single gestations (n=23). Placental culture results were available in 17

cases. Of these, 64.7% (11/17) were positive, with the most common isolates being

Mycoplasma hominis (n=5), Ureaplasma urealyticum (n=2), or mixed bacterial

species (n=4).

3.3 Comparison of maternal characteristics in cases with and without histologic

chorioamnionitis

Table 3.3 (page 55) compares maternal characteristics in subjects with (n=26) and

without HCA (n=299). Maternal ethnicity had an affect on chorioamnionitis

(p=0.01), with mothers of Aboriginal or Torres Strait Island descent being

disproportionately represented (6/26 (23%) versus 20/299 (7%); p=0.004). Smoking

mothers also had a significantly higher rate of chorioamnionitis (10/26 (39%) versus

42/299 (14%); p=0.001). Mothers with HCA were more likely to have premature

rupture of membranes (PROM) (19/26 (73%) versus 100/299 (34%); p<0.001), with a

significantly longer duration of PROM prior to delivery (median 0.50 hours versus

0.15 hours; p=0.014). Seven of 26 (27%) of mothers with HCA had symptoms of

clinical chorioamnionitis, including intrapartum fever, whereas 4 of 299 (1%) of

mothers without HCA had symptoms of clinical chorioamnionitis (p<0.001). Mothers

with HCA were more likely to be treated with antibiotics for PROM (11/26 (42%)

versus 31/299 (11%); p<0.001); either prior to the onset of labour (11/26 (42%)

versus 26/299 (9%); (p<0.001) or during labour (13/26 (50%) versus 50/299 (17%);

p<0.001). The mode of delivery differed significantly between cases with and without

HCA. Fifteen of 26 (58%) of cases with HCA were delivered by spontaneous vaginal

delivery, compared to 72/299 (25%) of cases without HCA (p=0.001). Furthermore,

23/26 (89%) of mothers with HCA experienced labour prior to delivery (vaginal

delivery or caesarean section), compared to 133/299 (45%) of mothers without HCA

(p<0.001). For women who experienced labour there was no difference in the

duration of labour before delivery (5.90 hours for women with HCA versus 5.73

hours for women without HCA; p=0.586).

55

56

Histologic Chorioamnionitis

Maternal characteristics No Yes p-value

n=299 (92%) n=26 (8%)

Maternal age* (yrs) 29 (26-34) [14-45]

29 (22-34) [16-43]

0.779

Maternal ethnic origin 0.010

Aboriginal/TSI 20 (7%) 6 (23%) 0.004

Caucasian 241 (82%) 19 (73%)

Other 33 (11%) 1 (4%)

Smoker 42 (14%) 10 (39%) 0.001

Primigravid 92 (31%) 7 (27%) 0.393

Antenatal steroids 95 (32%) 15 (58%) 0.009

PROM† 100 (34%) 19 (73%) <0.001

Period of PROM (h) †# 0.15 (0.05-0.24) [0-13]

0.50 (0.09-3.45) [0-9]

0.014

Antibiotics for PROM 31 (11%) 11 (42%) <0.001

Antibiotics prior labour onset 26 (9%) 11 (42%) <0.001

Antibiotics during labour 50 (17%) 13 (50%) <0.001

Clinical chorioamnionitis 4 (1%) 7 (27%) <0.001

Intrapartum fever >38oC 4 (1%) 6 (23%) <0.001

Mode of delivery 0.001

Spontaneous VD 72 (25%) 15 (58%)

Assisted VD 19 (7%) 3 (12%)

Emergency CS 3 (1%) -

Elective CS 200 (68%) 8 (31%)

Any labour (1st + 2nd stage) 133 (45%) 23 (89%) <0.001

Length of labour (h)*## 5.73 5.90 0.586

(3.36-9.70) (3.37-11.80)

[0.02-25.58] [1.93-30.17]

Multiple births

Multiples 125 (43%) 3 (11%) 0.002

Table 3.3 Maternal characteristics of cases of histologic chorioamnionitis

Frequencies expressed as n(%) unless specified *Median (Interquartile Range) [Range] †PROM = Premature rupture of membranes #PROM cases only ## Labour cases only

57

3.4 Comparison of neonatal characteristics in cases with and without histologic

chorioamnionitis

Table 3.4 compares neonatal characteristics in subjects with (n=26) and without HCA

(n=299). Cases of HCA were more likely to be born preterm (23/26 (89%) versus

199/299 (68%); p=0.028). Overall, cases of HCA had a median GA of 30 weeks

compared with a median GA of 34 weeks in those without HCA (p<0.001). There

was a higher representation of cases with HCA in the 28-32 week gestation group

(7/26 (27%) versus 12/299 (4%); p<0.001) and <28 week gestation group (11/26

(42%) versus 79/299 (27%); p<0.001). Cases with HCA had a significantly lower

birth weight (median 1575g versus median 2158g; p=0.002), expected birth weight

(median 1403g versus median 2282g; p<0.001), head circumference (median 29cm

versus median 31cm; p=0.001), length (median 40cm versus median 45cm; p=0.001).

The proportion considered small for GA were not significantly different, but the low

numbers of cases in each group may have meant that a true difference was not

detected. Neonates exposed to HCA were more likely to have Apgar scores of <6 at 1

min (12/26 (46%) versus 53/295 (18%); p=0.001) and 5 mins (3/26 (12%) versus

(10/295 (3%); p = 0.029). These neonates were significantly more likely to develop

neonatal sepsis (positive cultures from a sterile site), particularly late onset neonatal

sepsis (5/26 (19%) versus 17/299 (6%); p=0.009). Neonates exposed to HCA spent a

longer time in hospital (median 32 days versus median 19 days; p=0.014) and had a

higher proportion of neonatal deaths (2/26 (8%) versus 1/299 (<1%); p<0.001).

58

Histologic Chorioamnionitis

Neonatal characteristics No Yes p-value

n=299 (92%) n=26 (8%)

Gestational age * 34 (31-38) [26-42]

30 (27-32) [23-40]

<0.001

<28w 12 (4%) 7 (27%) <0.001

28-32w 79 (27%) 11 (42%)

32-37w 108 (37%) 5 (19%)

37+w 95 (32%) 3 (12%)

Preterm 199 (68%) 23 (89%) 0.028

Male gender 132 (45%) 10 (62%) 0.527

Birth weight*

2158 (1510-3081) [495-4945]

1575 (1048-2151) [585-3565]

0.002

Expected birthweight* 2282 (1581-3187) [865-3739]

1403 (999-1432) [591-3463]

<0.001

Head circumference*

31 (28-34) [21-39]

29 (25-30) [21-37]

0.001

Length *

45 (40-49) [30-59]

40 (36-45) [30-51]

0.001

SGA (<10%ile)‡ 41 (14%) 2 (8%) 0.551

Apgar Scores

Apgar 1 minute <6 53 (18%) 12 (46%) 0.001

Apgar 5 minute <6 10 (3%) 3 (12%) 0.029

Any ventilation 133 (45%) 19 (73%) 0.005

Neonatal sepsis (early & late onset)

17 (6%)

5 (19%) 0.009

Neonatal early onset sepsis (<72h)

1 (<1%)

- 0.768

Time to discharge (d)* 19 (5-39) [0-123]

32 (14-62) [0-97]

0.014

Neonatal death 1 (<1%) 2 (8%)

<0.001

Table 3.4 Demographic and clinical characteristics of infants with and without

histologic chorioamnionitis Frequencies expressed as n(%) unless specified *Median (Interquartile Range) [Range] ‡SGA = Small for gestational age (<10th percentile)

59

3.5 Histologic chorioamnionitis and maternal usCRP and PCT

Table 3.5.1 shows maternal usCRP and PCT results in cases with and without HCA.

The median maternal usCRP level was significantly higher in mothers with HCA (26

mg/L versus 5.6 mg/L; p<0.001), but no difference was seen in median PCT levels

(median PCT in HCA 0.046 ug/L versus median non-HCA 0.037 ug/L, p=0.572).

Histologic Chorioamnionitis

Maternal outcome variable No Yes p-value

n=299 n=26

PCT* (ug/L)

0.037 (0.025-0.060) [0.011-1.565]

0.046 (0.022-0.060) [0.014-0.342]

0.572

usCRP*# (mg/L)

5.64 (2.45-13.58)

[0-108]

26 (12.58-61.28)

[1.99-107]

<0.001

*Median (Interquartile Range) [Range] #including undetectable values (set to zero) Table 3.5.1 Maternal PCT and usCRP concentrations in subjects with and without HCA

Linear regression analysis was used to investigate the association between HCA and

maternal outcome variables (i.e. usCRP and PCT). Overall results are shown in table

3.5.2. Maternal linear models were adjusted for potential confounders that may

increase maternal cytokine levels and consequently affect maternal usCRP and PCT.

These were duration of PROM (hours), length of labour and intrapartum fever >38C.

There was a statistically significant linear relationship between HCA and maternal

usCRP (correlation r=0.47, p<0.001), but no statistically significant relationship

between HCA and maternal PCT (correlation r=0.16, p=0.697). Duration of PROM,

length of labour, and intrapartum fever explained 17.3% of the variance in the model.

The addition of HCA explained a further 4.3% of the model variation and HCA was a

significant variable in the model (p=0.023). PCT was not a significant variable in the

model and explained a further 2.7% of variation in the model. Correlation analysis

between maternal CRP and PCT indicated a weak but significant correlation

(Spearman’s rho=0.146, p=0.031). Logistic regression analysis with HCA as the

60

outcome variable showed maternal usCRP was significantly higher in mothers with

HCA compared to those without HCA (OR 2.86; 95% CI 1.47-5.57; p=0.002).

Outcome N:n Model R2 R2 change p-value

PROM, length of labour, intrapartum fever* 0.173 <0.001

* + HCA (p=0.023) 0.216 0.043 <0.001

usCRP

(mg/L)

256:24

* + HCA (p=0.034) + lnPCT (p=0.099) 0.243 0.027 <0.001

PROM, length of labour, intrapartum fever* 0.026 0.534

* + HCA (p=1.0) 0.027 0.001 0.697 PCT

(ug/L) 201:18

* + HCA + usCRP 0.060 0.033 0.787

N = Number of cases without HCA, n = number of cases with HCA PROM = duration of premature rupture of membranes, HCA = histologic chorioamnionitis, R2 = Coefficient of Determination, R2 change = additional variability explained by the adding a further variable into the model

Table 3.5.2 Linear regression analysis for the effect of HCA on maternal usCRP and PCT

3.6 Histologic chorioamnionitis and cord/neonatal outcome variables

3.6.1 Cord usCRP and PCT

Table 3.6.1a shows cord usCRP and PCT levels in cases with and without HCA. The

median cord PCT level was significantly higher in subjects with HCA (0.293 ug/L in

those with HCA versus 0.064 ug/L in those without HCA; p<0.001). There was no

difference in the median usCRP level in those with and without HCA as the median

result for both groups was 0 i.e. undetectable. However, Mann-U Whitney analysis

shows that usCRP are significantly higher in cases of HCA when compared to those

without HCA (p<0.001).

61

Histologic Chorioamnionitis

Cord outcome No Yes p-value

n=299 n=26

PCT (ug/L)

0.064

(0.047-0.102) [0.014-5.242]

0.293

(0.084-0.442) [0.050-27.372]

<0.001

usCRP# (mg/L)

0 (0-0)

[0-45.6]

0 (0-2.9) [0-63.9]

<0.001

# undetectable values (set to zero)

Table 3.6.1a Cord PCT and usCRP levels in subjects with and without HCA

Linear regression analysis was used to investigate the association between HCA and

cord outcome variables (i.e. usCRP and PCT). Results of regression modelling are

shown in Table 3.6.1b. Linear regression models were adjusted for factors that are

known to cause differences in cytokines levels in newborns. These variables were

GA, being small for GA (< 10th percentile) and length of labour. For usCRP, GA,

small for GA and length of labour accounted for 3.7% of variation in the model. The

addition of HCA explained a further 9% of the variation and HCA was a significant

variable in the model (p<0.001). The addition of PCT to the model explained a

further 41.6% of variation in the model, indicating a strong relationship between cord

PCT and cord usCRP. Logistic regression analysis with HCA as the outcome variable

and cord usCRP, GA, small for GA and length of labour as the predictors, showed

cord usCRP was significantly higher in infants exposed to HCA (OR 2.54; 95% CI

1.32-4.89; p=0.005).

For cord PCT, GA, small for GA and length of labour explained 30.7% of variation in

the model. The addition of HCA, explained a further 10.5% of the model variance

and HCA was a significant variable in the model (p<0.001). The addition of ln

usCRP to the model explained a further 28% of variation in the model. This finding

further confirmed the strong relationship between cord usCRP and PCT. Logistic

regression analysis with histological chorioamnionitis as the outcome variable and

cord PCT, GA, small for GA and length of labour as predicted showed cord PCT was

62

significantly higher in infants exposed to HCA (OR=2.49; 95%CI:1.45-4.28;

p=0.001).

Outcome N:n Model R2 R2 change p-value

GA, Small for GA, length of labour* 0.037 0.050

* + HCA (p<0.001) 0.130 0.092 <0.001

usCRP#

(mg/L)

194:17

* + HCA (p=0.586) + lnPCT (p<0.001) 0.545 0.416 <0.001

GA, Small for GA, length of labour 0.307 <0.001

* + HCA (p<0.001) 0.412 0.105 <0.001 PCT

(ug/L) 201:18

* + HCA (p=0.001) + lnCRP (p<0.001) 0.693 0.281 <0.001

N = Number of cases without HCA, n = number of cases with HCA GA = gestational age, HCA = histologic chorioamnionitis, R2 = Coefficient of Determination # = lnCRP +1

Table 3.6.1b Linear regression for the effect of HCA on cord usCRP and PCT

3.6.2 Cord and neonatal haematological parameters

Table 3.6.2a shows cord and neonatal haematological parameters in subjects with and

without HCA. No significant differences were seen between cord haematological

parameters in subjects with and without HCA. However, neonatal neutrophil count

trended towards significance (p = 0.054). For this reason, the relationship between

HCA and neonatal neutrophil count and neonatal WCC was examined using linear

regression modelling. These models were adjusted for GA, small for GA and length

of labour, as well as use of antenatal steroids.

63

Histologic Chorioamnionitis*

No Yes p-value

n=299 (92%) n=26 (8%)

Cord blood:

White cell count 8.41 (6.24-10.46)

[1.7-21.0]

8.16 (4.64-9.73) [3.0-21.5]

0.357

Platelet count 262 (220-322)

[5-549]

236 (196-294) [106-338]

0.201

Neutrophil count 2.05 (0.93-4.21) [0.2-13.1]

2.36 (1.04-3.29)

[0-10.6]

0.840

Nucleated red blood cell count 9 (4-23) [0-312]

9 (5.5-19.5)

[3-68]

0.839

Neonatal blood:

White cell count 9.2 (7.1-12.5) [2.1-24.5]

10.3 (6.2-18.0) [2.9-44.8]

0.478

Platelet count 248 (200-281) [27-500]

246 (198-310) [83-580]

0.751

Neutrophil count 3 (1.7-5.7)

[0.1-17.8]

4.5 (2.1-8.7)

[0.1-26.4]

0.054

Nucleated red blood cell count 9 (4-22) [0-416]

17 (3-33) [0-92]

0.326

*Median (Interquartile Range) [Range]

Table 3.6.2a Cord and neonatal haematologic parameters in subjects with and without HCA

64

Table 3.6.2b shows the results of linear regression analysis for neonatal WCC and

ANC. For neonatal WCC, GA, small for GA, length of labour and use of antenatal

steroids accounted for 8.6% of variation in the model. The addition of HCA to the

model accounted for a further 7.1% of variation and HCA was a significant variable

in the model (p<0.001). Logistic regression analysis showed that after adjusting for

GA, small for GA, length of labour and use of antenatal steroids, neonatal WCC was

significantly higher in neonates exposed to HCA (OR 1.12; 95% CI 1.03-1.22;

p=0.007).

Similarly for neonatal ANC, GA, small for GA, length of labour and use of antenatal

steroids accounted for 23.8% of variation in the model. The addition of HCA

explained a further 3.4% and HCA was a significant variable in the model (p=0.002).

Logistic regression analysis showed that after adjusting for GA, small for GA, length

of labour and use of antenatal steroids, neonatal neutrophil count was significantly

higher in neonates exposed to HCA (OR 2.21; 95% CI 1.20-4.09; p=0.011).

Outcome N:n Model R2 R2 change

p-value

GA, Small for GA, length of labour, AN steroids* 0.086 0.001 Neonatal

WCC

(x109/L)

195:23 * + HCA (p<0.001) 0.156 0.071 <0.001

GA, Small for GA, length of labour, AN steroids* 0.238 <0.001 Neonatal

ANC

(x109/L)#

218:23 * + HCA (p=0.002) 0.271 0.034 <0.001

N = Number of cases without HCA, n = number of cases with HCA GA = gestational age, HCA = histologic chorioamnionitis, R2 = Coefficient of Determination WCC = white cell count, ANC = absolute neutrophil count # = ln (neonatal ANC)

Table 3.6.2b Linear regression analysis for the effect of HCA neonatal WCC and ANC

65

3.6.3 Neonatal CRP

It is important to note that CRP was measured only in neonates where clinically

indicated. Table 3.6.3a shows the availability of CRP results in neonates during the

first 48 hours. Twenty-three of 26 (89%) of neonates exposed to HCA had CRP

measured within the first 48 hours of life compared to 191/299 (64%) of neonates not

exposed to HCA. Given the large proportion of missing data for CRP, particularly in

the non-HCA group, regression analysis was only performed for maximal CRP

recorded within the first 48 hours after delivery. This table also displays the number

of CRP measures that were less than the lower limit of detection at each time point

during the first 48 hours. Generally there were a higher proportion of undetectable

values on Day 0 than on Day 1 or Day 2.

% of total CRP measures available at that time point (i.e. Day 0, Day 1 or Day 2)

Table 3.6.3a Availability of neonatal CRP measures within the first 48 hours of delivery

Histologic Chorioamnionitis

Availability of CRP measures No Yes

n=299 n=26

Any CRP measures available:

191 (64%) 23 (89%)

Maximal CRP available:

191 (64%) 23 (89%)

CRP measures available:

Day 0 (birth-1 hr) 173 (58%) 22 (85%)

Day 1 (1 - 24hrs) 167 (56%) 19 (73%)

Day 2 (24 - 48hrs) 162 (54%) 21 (81%)

CRP measures less than 7mg/L:

Day 0 151 (87%)* 14 (64%)*

Day 1 92 (55%)* 7 (37%)*

Day 2 84 (52%)* 6 (29%)*

CRP measures less than 3mg/L:

Day 0 24 (14%)* 4 (18%)*

Day 1 15 (9%)* 0 (0%)*

Day 2 9 (6%)* 1 (5%)*

66

Table 3.6.3b describes Day 0, 1 and 2 neonatal CRP in neonates with and without

HCA analysed according to two different approaches for undetectable values. In the

first approach undetectable results (i.e. less than the reference range) were set to 0 if

the result was <3 mg/L or <7 mg/L. Using this approach, the maximal CRP level in

the first 48 hours, and CRP concentrations at Day 0, Day 1 and Day 2 were all

significantly higher in the group exposed to HCA. Median maximal CRP was 10

mg/L in the HCA group and 0 mg/L in the non-HCA group (p=0.014), median Day 0

CRP was 0 mg/L in both groups (p=0.009), median Day 1 CRP was 11 mg/L in the

HCA group and 0mg/L in the non-HCA group (p=0.001), and median Day 2 CRP was

10mg/L in the HCA group and 0mg/L in the non-HCA group (p=0.024).

In the second approach, undetectable results were set to 2.5 if <3 mg/L and 6.5 if the

result was <7 mg/L. With this approach, maximal CRP in the first 48 hours, and CRP

concentrations at Day 1 and Day 2, but not Day 0 were significantly higher in

neonates exposed to HCA. Median maximal CRP was 10mg/L in HCA group and 6.5

mg/L in the group without HCA (p=0.009), median Day 1 CRP was 11mg/L in the

HCA group and 6.5 mg/L in the group without HCA (p=0.001), and median Day 2

CRP was 10 mg/L in the HCA group and 6.5 mg/L in the group without HCA

(p=0.015). Median Day 0 CRP was 6.5 mg/L in both groups (p=0.226).

67

Histologic Chorioamnionitis Neonatal CRP No Yes p-value

n=299 n=26

Maximum CRP measure in first 48h postnatally #

0 (0-10) [0-108]

10 (0-32) [0-137]

0.014

Day 0 (Birth-1hr)# 0 (0-0)

[0-39]

0 (0-0)

[0-45]

0.009

Day 1 (1-24hrs)# 0 (0-8)

[0-108]

11 (0-34) [0-137]

0.001

Day 2 (24-48hrs)# 0 (0-9)

[0-53]

10 (0-17) [0-60]

0.024

Maximum CRP measure in first 48h postnatally ┼

6.5 (6.5-9)

[2.5-108]

10 (6.5-32) [4-137]

0.009

Day 0 (Birth-1hr) ┼ 6.5 (6.5-6.5) [2.5-39]

6.5 (6.5-6.5) [2.5-45]

0.226

Day 1 (1-24hrs) ┼ 6.5 (6.5-6.5) [2.5-108]

11 (6.5-34) [4-137]

0.001

Day 2 (24-48hrs) ┼ 6.5 (6.5-9) [2.5-53]

10 (6.5-17) [2.5-60]

0.015

#including undetectable values (set to zero) ┼ including undetectable values (set to 2.5 if <3mg/L or 6.5 if <7mg/L)

Table 3.6.3b Neonatal CRP levels in cases with HCA

Table 3.6.3c shows the results of linear regression analysis for maximal neonatal CRP

within the first 48 hours of delivery. GA, small for GA and length of labour accounted

for 1.0% of variation in the model. The addition of HCA to the model accounted for a

further 9.0% of variation and HCA was a significant variable in the model (p<0.001).

This was further analysed according to whether cases of HCA had a maximal CRP

level on Day 0, Day 1 or Day 2. Results showed that there was a significant

relationship between HCA and maximal CRP on day 1 (p<0.001) and day 2 (p=0.03),

but no significant relationship with maximal CRP levels at birth (p=0.910).

68

Logistic regression analysis showed that after adjusting for GA, small for GA and

length of labour, maximal CRP levels were significantly higher in neonates exposed

to HCA (OR 3.26; 95% CI 1.73-6.14; p<0.001). This was largely accounted for by

cases in which the CRP peaked on Day 1 (OR 5.39; 95% CI 2.35-12.39, p<0.001).

Cases of HCA with maximal CRP on Day 0 or Day 2 did not have significantly

different CRP levels from cases without HCA.

Outcome N:n Model R2 R2 change

p-value

GA, Small for GA, length of labour* 0.010 0.542

* + HCA (p<0.001) 0.090 0.080 0.001

Neonatal

maximal

CRP

(mg/L)

214:23

* + HCA D0# (0.910) + HCA D1# (<0.001) + HCA

D2# (p=0.030) 0.154 0.144 <0.001

N = Number of cases without HCA, n = number of cases with HCA GA = gestational age, HCA = histologic chorioamnionitis, R2 = Coefficient of Determination WCC = white cell count, ANC = absolute neutrophil count # = HCA D0 is cases of HCA with maximal CRP on Day 0, HCA D1 is cases of HCA with maximal CRP on Day 1, HCA D2 is cases of HCA with maximal CRP on Day 2

Table 3.6.3c Linear regression analysis for maximal neonatal CRP levels within the first 48 hours after delivery

69

CHAPTER 4:

The reliability of the umbilical cord full blood picture as a measure of

haematological parameters in the immediate post-natal period

70

4.1 Introduction

In this study, haematological parameters were measured using an automated

haematology analyser in the clinical haematology laboratory at a tertiary perinatal

centre. As the measurement of haematological parameters in cord blood is not routine

at this institution, it was essential to establish the validity of results.

Umbilical cord blood parameters differ from those in later life. The three lineages

present in peripheral blood (red cells, white cells and platelets) are all affected by

conditions specific to fetal existence and birth. These factors have an impact on the

ability of routine haematology analysers to accurately measure haematological

parameters in cord blood samples.

4.1.1 Red cells

Fetal haematopoeisis differs from paediatric and adult haematopoiesis, and this affects

haemoglobin levels and red cell indices. In the first half of gestation, the majority of

erythrocytes in the fetal-placental circulation are immature erythrocytes, i.e. nucleated

red blood cells (NRBC) [158]. The fetal NRBC count gradually declines throughout

pregnancy [159, 160]. A normal count of 0-10 NRBC/100 WBC is present at birth,

whereas in healthy children and adults NRBC are not seen in the peripheral blood

[161]. During the third trimester, red cell production is 3-5 times that of an adult,

resulting in a progressively increasing red cell count, haemoglobin level, haematocrit

and MCV throughout gestation [162]. Hence, haemoglobin levels and red cell indices

at birth are strongly affected by gestational age (GA).

Fetal red cells present a particular challenge to routine haematology analysers. While

red cells in health adults and children display little variation in size and shape, cord

red blood cells show increased red cell heterogeneity [163, 164]. Furthermore, there

is a greater variation in osmotic resistance [165], with a subpopulation of “young” red

cells showing increased resistance to osmotic lysis due to higher surface area to

volume ratios [166, 167]. Since automated haematology analysers lyse red blood

cells to measure haemoglobin concentrations, these characteristics of cord blood

71

require use of aggressive lytic agents or extended lysis times to accurately measure

haemoglobin levels [167].

4.1.2 Leukocytes

The total WCC and ANC are significantly affected by both GA and mode of delivery.

Firstly, normal neutrophil counts are highly dependent on GA. Fetal granulopoiesis is

less compartmentalised than in adults such that there is a higher proportion of

immature neutrophils in the peripheral blood for the first 72 hours after delivery,

highest at the time of delivery [76]. Furthermore cord blood is a rich source of

haemopoietic stem cells [168]. Mature neutrophils are the last of the fetal blood cells

to appear in the circulation [169] and levels gradually increase throughout gestation

until birth [160]. Secondly, mode of delivery also has a significant influence on the

cord neutrophil count. In neonates whose mothers laboured, the neutrophil count is

significantly higher for the first 18 hours after delivery, regardless of whether the

infant was delivered by vaginal birth or caesarean section [76]. Thus, GA and mode

of delivery are important factors that influence the interpretation of total WCC and

ANC.

Automated measures of the total and differential white cell count are problematic in

cord and neonatal blood. Modern haematology analysers are capable of performing

five-part differentials only after lysis of the red cells. The subpopulation of lysis-

resistant red cells in cord blood may be counted as white cells leading to erroneously

high results. To overcome this fetal blood requires prolonged lysis times, use of an

aggressive lytic agent, or pre-dilution of samples [66]. Lysis-resistance, and the

presence of immature white cells and NRBC, causes the haematology analyser to flag

the sample for peripheral film review to verify results. Furthermore, NRBC are

included in the automated total white cell count. In newborns with very high NRBC

counts this can significantly over-estimate the total WCC. Thus, all cord and neonatal

blood samples require review of the blood smear and a manual white cell differential

to appropriately classify immature cells and correct for the presence of NRBC [170]

and are prone to intra-observer error.

72

4.1.3 Platelets

Fetal platelets are present in the same concentration as adults from approximately 18

weeks gestation [160]. However, the small volume of samples and potential

contamination with amniotic fluid make these samples particularly prone to clot

formation and platelet activation. In one study, up to 5% of cord samples were clotted

[171]. Further, large immature platelets may not be measured by techniques that rely

on particle size, a phenomenon commonly seen in high platelet turnover conditions,

such as immune thrombocytopaenic purpura (ITP) [172]. Thus, automated analyser

results from cord samples may give erroneously low measured platelet counts.

Review of the blood smear assists in the identification of artefactually low platelet

counts.

4.1.4 Summary

In summary unique factors significantly influence measurement of cord blood

haematological parameters. It was crucial to establish the validity of cord blood

haematological data measured in this study, before these data could be used to address

the main hypothesis: that there is a correlation between the presence of HCA and cord

blood haematological paramaters.

4.2 Methods

The three cell lines measured on a standard haematology analyser are red cells, white

cells, and platelets. For this thesis, the parameters of interest were corrected WCC,

ANC and I:T ratio. The two aspects of quality that were considered were accuracy

and precision.

4.2.1 Accuracy of red blood cell parameters and platelet counts

Cord blood full blood counts were measured in newborns, as per the study protocol,

and neonatal blood counts were measured where clinically indicated. Thus, while

cord blood counts were measured in most of the study cohort, neonatal blood counts

were only measured in a subset of the total cohort.

73

All available cord blood films from the cohort of newborns were reviewed (n=212).

Any blood films with platelet clumps, fibrin strands/clots, or poor staining were

excluded from further analysis (n=15, 7.6% of cord samples). One infant received a

red cell transfusion within the first 24 hours of life and was excluded from analysis.

Of the remaining, 132 newborns had a full blood count performed on a peripheral

blood sample within the first 24 hours of life. Paired cord and neonatal samples from

these 132 subjects were therefore used to assess the accuracy of measured blood

parameters. The accuracy of WCC, ANC, I:T ratio and NRBC were not assessed

directly. Total WCC and ANC increase markedly within hours of birth reaching a

maximum concentration at 18-24 hours [76]. Similarly, NRBC can change rapidly

within hours of birth as affected by many conditions associated with acute stress in

the immediate postnatal period [158], including labour and vaginal delivery, acute

hypoxia, infection, blood loss, and haemolysis [158]. Instead, accuracy was assessed

indirectly by comparing other parameters that were likely to be similar in the cord

sample and a neonatal sample collected within 24 hours of delivery. These parameters

were Hb, RCC, MCV, MCH, MCHC and platelet count.

Red cell parameters and platelet counts in cord and neonatal samples taken from the

same newborn were compared using Bland-Altman limits of agreement analysis

[173]. Using this method, the 95% limits of agreement is the interval defined by the

mean difference between the two measurements ± 1.96 standard deviations. These

limits are expected to contain the difference between measurements of 95% of pairs

of similar individuals.

4.2.2 Precision of corrected WCC, ANC, I:T ratio and NRBC

Forty-two (21.3%) blood films were randomly selected from the total number of valid

cord blood films (n=197). To evaluate the intra-rater reliability, three observers

independently performed a manual white cell differential on the selected subset of

cord blood films on two occasions. The second set of observations was made

independently of the first set of observations i.e. observations were performed on

different days and without reference to the first observations. To assess the inter-rater

reliability, three different observers scored the same subset of cord blood films

independently. The observers were a consultant paediatric haematopathologist, a

74

senior medical scientist with extensive experience in paediatric haematology, and a

haematology advanced trainee (the candidate). The parameters that were assessed

were corrected WCC, ANC, I:T ratio and NRBC.

The Intraclass Correlation Coefficient (ICC) was used to measure the absolute

agreement between individual blood parameters as scored by different observers. ICC

is calculated as the ratio of between-group variance to total variance. ICC will

approach 1 when there is no variance between raters and no residual variance left to

explain. In most circumstances ICC values of 0.7-0.8 are satisfactory, however for

clinical applications, a correlation of ≥0.90 is considered acceptable [174, 175], so

this threshold was used in the current study.

4.3 Results

4.3.1 Accuracy of red blood cell parameters and platelet counts

Red blood cell parameters

To assess accuracy, the parameters from the cord blood count were compared to

results from the neonatal blood count within 24 hours of delivery. For MCV, MCH

and MCHC, there was very little difference between the two measurements. The

differences between the cord and neonatal measurements were 1.2 fL for MCV

(p<0.001), 0.3 pg for MCH (p=0.002), and 1.3 g/dL for MCHC (p=0.075). Although

the differences for MCV and MCH were statistically significant, the magnitudes of

these differences are not clinically significant. The Bland Altman plots (Figure 4.3.1)

for MCV, MCH and MCHC show that less than 5% of paired results were outside the

95% limits of agreement. These data are summarised in Table 4.3.1.

For Hb and RCC there was a clinically and statistically significant difference between

the cord and neonatal measurements. Hb was 15g/L higher in the neonatal sample

(p<0.001), while RCC was 0.44 x1012/L higher in the neonatal sample. Furthermore,

the Bland Altman plots show that 7 (5.1%) of the paired Hb results and 8 (6.1%) of

the paired RCC results were outside the 95% limits of agreement.

75

Platelet counts

For platelet count, there was no overall difference observed between the cord and

neonatal results. However, 9 (7.0%) of the paired results were outside the 95% limits

of agreement. Eight of these 9 pairs were below the lower 95% limit of agreement.

Mean cord result

Mean neonatal

result

Mean Difference (95% CI) 95% LOA Samples outside 95%

LOA*

Hb (g/L) 153 168 15 (11 – 18)‡ -53 - 23 7 (5.2%)*

RCC (x1012/L) 4.05 4.47 0.42 (0.32 – 0.51)‡ -1.49 – 0.65 8 (6.1%)*

MCV (fL) 113.6 112.4 1.2 (0.7 – 1.6)‡ -3.8 – 6.2 6 (4.5%)*

MCH (pg) 38.0 37.7 0.3 (0.1 – 0.4)† -1.6 – 2.1 5 (3.8%)*

MCHC (g/dL) 334.2 335.5 1.3 (-0.1 – 2.9) -18.0 – 15.4 5 (3.8%)*

Platelet count (x109/L) 252 252 0 (-12 – 12) -134 - 134 9 (7.0%)**

* n=132, ** n=129 LOA = limits of agreement † <0.01‡ <0.001

Table 4.3.1 Mean haemoglobin (Hb), Red Cell Count (RCC), Mean Cell Volume (MCV), Mean Corpsular Haemoglobin (MCH), Mean Corpuscular Haemoglobin Concentration (MCHC) and platelet counts in paired cord and neonatal samples

76

(a) (b)

(c) (d)

(e) (f)

Figure 4.3.1 Bland Altman plots comparing cord and neonatal results (a) Haemoglobin (Hb), (b) red cell count (RCC), (c) mean cell volume (MCV), (d) mean corpuscular haemoglobin (MCH), (e) mean corpuscular haemoglobin concentration (MCHC) and (f) platelet count (plt). The x axis shows the average of the cord and neonatal results. The y axis shows the difference between the cord and neonatal results. The solid line represents the mean difference between cord and neonatal results. The dashed line represents the mean difference between cord and neonatal results ± 1.96 standard deviations.

77

4.3.2 Precision of corrected WCC, ANC, I:T ratio and NRBC

Table 4.3.2 and Figure 4.3.2 summarise the results of analysis of intra-observer and

inter-observer reliability for corrected WCC, ANC, I:T ratio and NRBC. The intra-

observer and inter-observer reliability for corrected WCC, ANC and NRBC was

excellent, with intraclass correlation values above the minimum of 0.90. I:T ratios

were poorly reproducible. The ICC was 0.325 for inter-observer reliability (across 3

observers), and 0.480 and 0.120, respectively for the intra-observer reliability for

observer 1 and observer 2. The poor correlation of I:T ratio results within one

observer and between observers is demonstrated graphically in Figures 4.3.2a and

4.3.2b.

Intra-observer reliability* Parameter

Observer 1 Observer 2 Observer 3

Inter-observer

reliability**

Corrected WCC 0.972 0.989 0.968 0.968

ANC 0.963 0.960 0.936 0.974

I:T ratio 0.480 0.120 0.354 0.351

NRBC 0.907 0.961 0.965 0.976

Table 4.3.2 Intra-observer and inter-observer reliability for corrected WCC, ANC, I:T ratio and NRBC * = Intraclass correlation coefficient, calculated from results of observations on two separate occasions by the same individual ** = Intraclass correlation coefficient, calculated from results of Observer 1, 2 and 3 WCC = white cell count ANC = Absolute neutrophil count NRBC = nucleated red blood cell count I:T = Immature:Total ratio

78

(a) (b)

(c) (d)

Figure 4.3.2 Intra-observer correlation for observer 1 a. Corrected WCC b. ANC c. NRBC d. I:T ratio

4.4 Discussion

The two major aspects of quality in the cord blood that were assessed were accuracy

and precision. For this thesis, the primary parameters of interest were umbilical cord

corrected WCC, ANC and I:T ratio.

4.4.1 Accuracy of red cell parameters and platelet counts

Accuracy is the degree of closeness of a measured value to its true value. In this

study, the true value was defined as the value from a neonatal sample taken within the

first 24 hours of life. The rationale for this was that measurement of haematological

79

parameters in neonatal samples was routine practice at this institution, whereas

measurement of cord parameters was not. The neonatal results for this study were

validated by the usual quality control practices within the clinical laboratory.

The parameters that were assessed depended on the likelihood that the true value

would be the same in the cord and neonatal samples. As discussed previously, total

WCC, ANC, I:T ratio and NRBC may change rapidly within hours of birth. Thus, the

accuracy of these parameters could not be assessed by direct comparison of paired

cord and neonatal samples. Instead, accuracy was assessed indirectly by comparing

other parameters that were likely to be similar in the cord sample and a neonatal

sample collected within 24 hours of delivery. These parameters were Hb, RCC, MCV,

MCH, MCHC and platelet count.

MCV, MCH, and MCHC

For MCV, MCH and MCHC, the analysis shows the measurements were highly

reproducible between paired cord and neonatal samples. There were no clinically

significant differences between paired measurements for these parameters. In

addition, less than 5% of paired results for all three parameters were outside the 95%

limits of agreement, indicating that these parameters were measured accurately.

Hb and RCC

For Hb and RCC, there was a clinically and statistically significant difference

between the paired cord and neonatal samples. The neonatal Hb level was 15 g/L

higher than the cord Hb level, and the neonatal RCC was 0.44 x1012/L higher than the

cord RCC. These results cannot be explained by a true increase in red cell mass, as

this would have required production of new red blood cells from the bone marrow in a

very short period of time.

There are two possible explanations for the difference in results. First, a high number

of neonatal samples were capillary samples collected via heel prick. A number of

studies have shown that there may be differences in Hb and RCC results from

capillary samples. In a study of 30 children (aged 2 to 13 years) and 30 adults, Moe

80

[176] found that Hb, RCC and haematocrit results were all higher in venous versus

capillary samples collected at the same time; the reverse of findings from this study.

For children, the capillary results were 5 g/L lower for Hb, 0.18 x1012/L lower for

RCC and 1.7% lower for haematocrit when compared to the venous results. These

findings have not been replicated in other studies [177, 178]. For example, a more

recent study of 40 healthy adult volunteers, found that Hb, RCC and haematocrit were

all significantly higher in capillary collections than venous collections, whereas no

difference was seen in MCV, MCH and MCHC [177], consistent with the findings

from this study. The magnitudes of these differences were 3.2 g/L for Hb, 0.10

x1012/L for RCC and 1.2% for haematocrit. Another study of 30 one-year-old infants,

the microhaematocrit from a capillary (fingerstick) sample (mean 36.6%) was

significantly higher than the microhaematocrit measured from a venous sample

collected at the same time (mean 34.6%) [178]. Furthermore, while there was no

significant difference in mean Hb concentrations between capillary and venous

samples, the correlation of paired Hb results was 0.81, whereas the correlation for

MCV was 0.98. Thus there may be differences in Hb and RCC between capillary and

venous samples as a result of sampling issues. The investigators from these papers

report that the discrepancies between capillary and venous results may be caused by

sampling and dilutional error inherent in the capillary method. It is also apparent

from the literature that the magnitude of difference between capillary and venous

sampling is not as large as the differences observed in this study.

The second explanation for the difference between cord and neonatal samples is the

fluid redistribution that occurs in newborns soon after birth. Following delivery there

is a rapid decrease in extracellular fluid volume, mediated by atrial natriuretic peptide

[179-181]. As an adaptation to birth, pulmonary vascular resistance decreases,

resulting in increased pulmonary blood flow, increased left atrial venous return,

stretching of the myocardial muscle fibres in the left atrium and release of atrial

natriuretic peptide [179]. This may be further enhanced by rapid expansion of the

intravascular fluid compartment after birth as a result of placental transfusion and

reabsorption of fluid from the lung [179]. The effect of atrial natriuretic peptide is

attenuation of the renin-angiotensin-aldosterone system and promotion of natriuresis

and diuresis [182]. Very preterm infants may also sustain significant plasma volume

81

contraction through high insensible fluid losses [183]. These mechanisms result in a

reduction in circulating plasma volume and haemoconcentration [184].

Correspondingly, studies have shown that haemoglobin levels do in fact increase

during the first three days of life [185]. Hence, it is possible that in this study

haemoconcentration resulting from fluid shifts within the first 24 hours of delivery is

the cause for discrepancy between paired cord and neonatal samples.

As there were sampling and physiological differences between cord and neonatal Hb

and RCC measurements, Hb and RCC were not considered suitable parameters to

assess the accuracy of measurement in cord samples.

Platelet count

For platelet count, although the mean cord and neonatal results was identical, there

were a high number of paired samples outside the 95% limits of agreement (9/132,

7%). Interestingly, 8 of these 9 paired samples the difference between cord and

neonatal results were outside the lower limit of agreement, rather than the upper limit.

This indicates that for these samples, the cord result was lower than the neonatal

result.

As discussed in the introduction to this chapter, cord samples are prone to clot

formation and platelet activation. Despite each sample being examined for overt clot

formation and each film being examined for platelet clumping and fibrin clot

formation, it is possible that these methods are not sensitive to platelet activation in

the sample. On the other hand, heel prick or capillary samples are also prone to

platelet activation. According to usual laboratory practice, all neonatal samples are

checked for clot formation and all neonatal samples have a blood film made which is

checked for platelet clumping and fibrin clot formation. If any clot formation is

detected, these results are not issued. It is possible that the quality control in normal

laboratory practice is more rigorous than it was for the cord samples in this study.

Regardless of the reason for these results, it appears that platelet counts were not

measured sufficiently accurately in the cord samples in this study (i.e. results were

outside the 95% limits of agreement). However, this did not cause a discrepancy

between the mean platelet count of the cord and neonatal samples.

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Overall findings

Overall, of the parameters that were assessed for accuracy, MCV, MCH and MCHC

were the most reliable. MCV, MCH and MCHC are determined during development

of the red blood cell and remain constant for the lifespan of the red cell. The results

for these values are not affected by physiologic changes within the first 24 hours of

delivery or by sample collection. Hb and RCC could not be assessed for accuracy due

to differences in the type of sample and physiologic changes in fluid balance in the

newborn. Cord platelet counts were likely affected by platelet activation, which

influenced the accuracy of platelet measurement.

So can these results be extrapolated to cord WCC and ANC? This is a difficult

question to answer. The major mechanism likely affecting the accuracy of cord

samples in this study was fibrin formation and platelet clumping. It is known that

platelets and white cells interact [186] and fibrin clots often incorporate white cells

[187]. Thus, the presence of fibrin deposits with a sample may lower the WCC and

ANC. Data from this study showed there was a degree of inaccuracy in cord platelet

measurement. It is possible that WCC and ANC may be similarly affected, however

whether this would have significantly changed the measured cord WCC and ANC is

not known.

4.4.2 Precision of corrected WCC, ANC, I:T ratio and NRBC

Precision is the degree to which repeated measurements give the same or similar

results. To assess precision of corrected WCC, ANC, I:T ratio and NRBC, manual

white cell differentials were determined multiple times to assess the inter-observer

and intra-observer reliability.

For both intra-rater and inter-rater reproducibility, the corrected WCC, ANC and

NRBC were shown to be reliable with an ICC of >0.90 for all comparisons.

However, I:T ratio was unreliable. The intra-observer ICC was 0.480 for one

observer and 0.120 for another observer. A similar, lack of concordance was seen for

inter-observer reliability (ICC 0.531 for three different observers).

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The lack of precision of the I:T ratio shown in the present study is consistent with

findings from other studies [63, 64]. There are two major sources of error that

account for this. First, although the appearance of band cells is well characterised

[57], the ability of technicians to consistently identify band cells is poor. This was

demonstrated by van der Meer et al [63] who surveyed hospitals in the Netherlands

with 100 PowerPoint slides of leukocytes from the same septic patient. Of the 100

cells, individual concordance for 19 segmented neutrophils and 13 band cells was less

than 75%. The second source of error is the inherently high statistical variability of

the 100-cell manual leukocyte differential [64, 188]. These inaccuracies are

compounded when the immature granulocyte count is divided by the total granulocyte

count. The conclusion from recent publications has been that I:T ratio is inaccurate,

imprecise, of limited clinical utility and should cease to be used in daily clinical

practice [57, 63, 64]. As I:T ratio was not demonstrated to be a precise measure, it

was omitted from further analysis.

4.4.3 Limitations of analysis

The main limitation of this analysis was the lack of a gold standard comparator for

neonatal and cord parameters. Accordingly, it was difficult to conclude whether cord

parameters were measured accurately. For the analysis, it was assumed that the

neonatal result represented the “true” value. However, it is clear that there have been

significant physiological and sampling issues that may affect the validity of this

assumption. In hindsight, it would have been helpful to know how many of the

neonatal samples were obtained by heel-prick collection. Unfortunately, this data was

not collected at the time and could not be ascertained retrospectively.

The primary parameters of interest for this thesis were cord WCC and ANC. The

rapid changes that occurred physiologically in the first few hours of birth meant that

the accuracy of cord parameters could not be assessed directly. Instead, accuracy was

assessed indirectly. From the data in this study it can be inferred that there may have

been some inaccuracy in measured cord WCC and ANC, but the magnitude of this

problem cannot be estimated.

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For precision, the inter-observer and intra-observer variability of three observers was

assessed. All three individuals had experience in the area of paediatric haematology,

however the three individuals assessed in this study were not the technicians working

day-to-day in the clinical laboratory. Whether there would be a lesser (or greater)

degree of precision in the cord WCC, ANC, and NRBC if the usual laboratory staff

were assessed remains unknown.

The observations from this study are a single laboratory’s experience with cord blood

results. It would be interesting to know whether other centres using different

analysers, different staining techniques and different observers would have similar

results.

4.4.4 Conclusion

For this thesis the main parameters of interest to correlate with HCA were corrected

WCC, ANC, and I:T ratio. For accuracy, MCV, MCH, and MCHC were highly

consistent in cord and neonatal samples. The accuracy of cord Hb and RCC could not

be assessed as a result of physiological and sampling differences between the two

specimens. There was a minor degree of inaccuracy shown between cord and

neonatal platelet counts and it is possible that the same mechanism could affect WCC

and ANC. It is assumed that this would not have significantly affected the overall

cord WCC and ANC, so these parameters were considered accurate for use in the

remainder of this thesis.

For precision, WCC, ANC and NRBC were measured precisely. However, I:T ratio

was demonstrated to be poorly reproducible between observers and even within the

same observer. Thus I:T ratio was not used in further analysis for the remainder of

this thesis.

85

CHAPTER 5:

General discussion

86

This study addressed the effect of HCA on inflammatory markers in the mother at the

time of delivery, and inflammatory and haematological markers in the cord blood and

neonate up to 48 hours following delivery. The key findings are summarised in

Section 5.1, including discussion of what new information this study brings to our

understanding of changes to inflammatory and haematological markers in the early

neonatal period. The limitations of this research and the significance of the findings

in the context of the wider literature are detailed in Section 5.2 and 5.3 respectively.

Finally potential areas of future research are addressed in Section 5.4, and concluding

remarks in Section 5.5.

5.1 Summary and discussion of findings

Intrauterine inflammation and the fetal inflammatory response syndrome plays an

important role in the pathogenesis spontaneous PTD, and is independently associated

with a number of adverse neonatal outcomes such as periventricular leucomalacia and

cerebral palsy, bronchopulmonary dysplasia, necrotising enterocolitis and early onset

neonatal sepsis. The hallmark of this inflammatory process is HCA [33]. This study

examined the impact of HCA on inflammatory markers, such as corrected WCC,

ANC, CRP and PCT, in the mother at the time of delivery, the cord blood, and the

neonate within 48 hours following delivery. These markers are used routinely in

clinical practice to diagnose or exclude sepsis in the newborn. One of the aims of this

study was to establish the extent to which these markers may be influenced by the

presence of HCA.

The key findings to emerge from this thesis were that HCA is associated with

significantly higher:

(1) maternal usCRP levels, but not PCT levels, on the day of delivery,

(2) umbilical cord usCRP and PCT levels at birth,

(3) neonatal corrected WCC and ANC within the first 24 hours of delivery, and

(4) neonatal CRP levels within the first 48 hours of delivery, in particular between 0

and 24 hours of delivery.

Each of these findings is discussed in detail in the remainder of this section.

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5.1.1 Maternal inflammatory markers

C-reactive protein

This study found a positive association between maternal CRP and the presence of

HCA (OR 2.86; 95% CI 1.47-5.57; p=0.002). This was an expected result and

confirms the findings from numerous previous studies [138-144]. CRP is an acute

phase reactant produced by the liver in response to pro-inflammatory cytokines,

particularly IL-6 and IL-1ß. CRP has a high molecular mass (106kDa) and does not

cross the placental barrier [189]. Hence the observed elevation of CRP is the result of

inflammation in the mother, in particular activation of monocytes, macrophages

and/or T cells. These data indicate that intrauterine infection is the one of the

significant drivers of maternal inflammation resulting in HCA and an elevated CRP

level. However, CRP is a non-specific marker of inflammation and other possible

sources of inflammation need to be taken into account.

The potential confounding factors that were considered in the analysis were PROM,

duration of labour, and intrapartum fever. PROM is an inflammatory condition that

often occurs in conjunction with intrauterine infection and HCA [190]. It may be

caused by maternal reproductive tract infection, but is associated with many other

initiating events that may occur independently of intrauterine infection and HCA.

These include behavioural factors such as smoking, substance abuse, nutritional

factors, coitus, and obstetric complications such as multiple gestation,

polyhydramnios, cervical insufficiency, gestational bleeding, antenatal trauma [190].

As previously discussed, the onset of labour is an inflammatory event and is

associated with production of inflammatory cytokines including IL-1ß and IL-6. The

presence of intrapartum fever was used as a non-specific marker of other infections or

sources of inflammation that may not have been related to intrauterine infection. In

this study women with HCA had a significantly higher prevalence of PROM, longer

duration of PROM before deliver, higher prevalence of labour, and higher prevalence

of intrapartum fever (>38C). On regression analysis, the presence of PROM, duration

of labour and presence of intrapartum fever explained 17.3% of variation in the

maternal CRP result. After adjusting for these potential confounding factors on

regression analysis, the presence of HCA was found to be a significant variable in the

model of maternal CRP (additional 4.3% of explained variation of CRP, p=0.023).

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Clearly, while there are many factors that influence maternal CRP, HCA is a

significant variable.

In absolute terms, women with HCA had a modest elevation of CRP (median 26 mg/L

with HCA versus 5.6 mg/L without HCA). Clinically almost three quarters of women

with HCA in this study had no symptoms of chorioamnionitis. These observations fit

well with the concept that intrauterine infection causes low-grade inflammation in the

mother.

Procalcitonin

There was no difference between serum levels of PCT in mothers with and without

HCA. Moreover, the absolute levels measured in both groups were negligible

(median 0.046 ug/L with HCA and median 0.037 ug/L without HCA). These low

concentrations would not have been detected without the use of a sensitive assay for

PCT, such as the one utilised in this study. Given the minimal PCT concentrations

observed in both maternal groups, it is unlikely that the lack of difference between

groups is due to type II error. Almost certainly PCT is not significantly increased in

mothers with HCA.

There is little evidence from the literature to indicate whether or not HCA is

associated with elevated maternal PCT. Only one study has directly addressed this

question. Torbe et al. [146] studied 48 women with preterm PROM, 30 with PROM

at term, 31 healthy women who delivered preterm, and 33 healthy women at term. Of

these, 14 had inflammatory changes in the placenta, and 34 did not. The authors do

not specify the method of scoring of HCA, nor whether mothers with PROM were

more likely to have HCA. Their results showed that PCT concentrations were similar

across all groups and between those with and without HCA. These data are consistent

with the results from this study.

As previously discussed, PCT is a more specific marker for bacterial infection than

CRP. Further, it more accurately reflects real-time inflammation as it increases

rapidly in response to infection and similarly, quickly returns to baseline at the offset

of infection. Data from this study confirms that for mothers, intrauterine infection is a

minor insult. HCA is defined by the presence of an inflammatory infiltrate within the

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placenta. This process evolves over many hours or even days. There are two possible

explanations for the lack of association of PCT with HCA. First, HCA is a mild

stimulus that does not stimulate PCT production or second, HCA causes a minor rise

in PCT that resolves by the time delivery occurs. Regardless, intrauterine infection

does elicit a mild inflammatory response in the mother, as evidenced by increased

CRP concentrations.

An alternative to this hypothesis is that intrauterine infection does cause elevated

maternal PCT levels at the onset of infection, but this bacterial infection has largely

resolved at the time of delivery. This possibility seems less likely as the majority of

mothers are asymptomatic, and those with symptoms are symptomatic at the time of

delivery. Importantly, this study only has a single measurement for maternal PCT and

CRP i.e. on the day of delivery. In the absence of data in the days and weeks prior to

delivery, one can only speculate as to the time between the onset of maternal

infection, inflammation and delivery.

5.1.2 Cord inflammatory markers

C-reactive protein

This study found that cord usCRP concentrations are significantly higher in infants

exposed to HCA, after adjusting for GA, small for GA and length of labour.

Although the median CRP in both groups was the same, the group exposed to HCA

had a higher 75th centile (2.9 versus 0 mg/L) and maximum value (63.9 versus 45.9

mg/L). Mann-U Whitney analysis shows that this difference is statistically significant

(p<0.001).

This positive association was an expected finding and is consistent with the published

literature. There are five other studies that examined whether the presence of HCA is

associated with elevated umbilical cord serum CRP levels [100-104]. All of these

studies found that HCA or funisitis is associated with elevated umbilical cord CRP

levels when measured by high-sensitivity methods (lower limit of detection 0.2-1.0

mg/L). HCA is a biomarker for FIRS, and the definition of the FIRS entails elevation

90

in fetal IL-6 concentrations. As IL-6 is one of the major stimuli for CRP production,

it follows that cord CRP concentrations should be elevated in newborns with HCA.

This study confirms that higher umbilical cord CRP concentrations in neonates

exposed with HCA are a marker of a fetal systemic response to intrauterine infection,

through inflammation and the generation of fetal cytokines such as IL-6. Given that

CRP does not cross the placenta [189], the presence of umbilical cord CRP signifies

that the fetus and not the mother, has generated an inflammatory response.

Interestingly the median cord CRP in newborns with and without HCA was 0 mg/L.

As previously described, serum CRP levels are increased within 12 to 24 hours of the

onset of inflammation. This study has shown that in 50% of newborns exposed to

HCA the CRP is not elevated. There are several possibilities that may explain this

observation. First, the onset of the inflammatory response in a fetus with HCA occurs

less than 12 hours from the time of delivery such that there is insufficient time for IL-

6 to stimulate CRP production from the liver. Second, the inflammatory response in

the fetus is not very intense and leads to only minor elevations in CRP. Third, the

inflammatory response has mostly dissipated by the time of delivery. It is apparent

that cord CRP alone is a poorly sensitive clinical test for the presence of HCA, as

50% of newborns with HCA did not have an elevated cord CRP.

As previously discussed, CRP is a non-specific marker of inflammation and other

possible sources of inflammation need to be taken into account. In the analysis of this

study, potential confounding factors that were considered were GA, being small for

GA (< 10th percentile) and length of labour. Previous studies have shown that infants

born at very early GAs have high levels of pro-inflammatory cytokines, particularly

IL-6 [191]. Newborns that are small for GA experience chronic inflammation in

utero and it has been observed that CRP levels are significantly higher in small for

GA infants when compared to appropriate for GA infants [155]. Further, it is known

that environmental stressors in the perinatal period, such as the mode of delivery and

length of labour, result in elevated inflammatory cytokines in newborns [7, 152, 153,

192]. In this study, infants with HCA were born at significantly lower GAs than those

without HCA (p<0.001). There was no difference in the proportion of infants that

were small for GA. There was also no difference in the length of labour between

91

those with and without HCA). On regression analysis, GA, small for GA and length

of labour accounted for only 3.7% of variation in CRP. While it is clear that there are

many other factors that influence cord CRP concentrations, HCA remains a

significant variable accounting for 9.2% of the variation in this model (p<0.001).

Procalcitonin

This study found that cord PCT concentrations are significantly higher in infants

exposed to HCA, after adjusting for GA, small for GA and length of labour. In

absolute terms, median cord PCT in infants exposed to HCA was 0.293 ug/L

compared with 0.064 ug/L in unexposed infants. As discussed in the previous section,

maternal PCT concentrations were negligible, thus it seems that cord PCT must have

been generated by the fetus. This observation is in keeping with other data that have

shown it is unlikely that PCT crosses the placenta [117, 118]. Given the real-time

relationship between PCT and inflammation, it is likely that inflammation begins in

the fetus shortly before delivery.

There is very little data in the literature regarding umbilical cord PCT concentrations

and HCA. Janota et al. studied 8 neonates with HCA and found that HCA did not

significantly influence cord PCT levels, but was associated with a more pronounced

post-natal increase in PCT at 72 hours and 7 days [121]. Three studies [88, 118, 128]

have found that PCT levels are higher in newborn infants that subsequently develop

early onset neonatal sepsis compared with uninfected neonates, however these studies

did not correlate PCT levels with the results of placental histology.

Like CRP, PCT is a non-specific marker of inflammation. Although it has a higher

specificity for bacterial infection than CRP, elevated levels are seen in a variety of

clinical settings and this needs to be considered in the analysis. For cord PCT

analysis, a similar rationale with regard to cord usCRP and sources of inflammation

was used. In addition to evidence showing the relationship between inflammation and

small for GA, and perinatal cytokine levels and length of delivery, there is data that

demonstrate neonatal PCT levels are dependent on GA [114]. As such, regression

analysis was adjusted for GA, small for GA and length of labour. In this analysis,

these variables GA, explained 30.7% of variation in the model, indicating that

together these variables have a highly significant effect of cord PCT levels. HCA

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remains a significant variable accounting for a further 10.5% of the variation in the

model (p<0.001).

As discussed in the literature review, there are many factors that complicate the

interpretation of PCT concentrations in the early neonatal period, including GA,

perinatal asphyxia, intracranial haemorrhage and maternal pre-eclampsia. The data

from this study shows that HCA and FIRS are further variables that need to be

considered when using PCT in the diagnosis of early onset neonatal sepsis.

Haematological variables

This study found no significant differences in cord WCC or ANC in newborns with

and without HCA. There is little published evidence with regard to the effect of FIRS

on cord WCC and ANC. Gotsch et. al. suggest that these parameters are significantly

higher in neonates with FIRS although they do not specify whether this observation is

from cord blood or peripheral blood samples in the early neonatal period [84]. There

is no published data on the direct relationship between umbilical cord ANC and WCC

and the presence of HCA. Interestingly, granulocyte colony stimulating factor (G-

CSF), the major cytokine that drives neutrophil production and maturation, is

significantly higher in fetuses with FIRS (median 714.4 pg/mL versus median

55.7pg/mL; p<0.01) [193].

In order to interpret these results, it is important to have an understanding of the

processes that regulate neutrophil concentration in the peripheral blood. Neutrophil

homeostasis is dependent on production and maturation, release from the bone

marrow, tissue infiltration, and apoptosis [194-196]. In animal studies, at resting

conditions it takes approximately 50 hrs to produce a neutrophil from its progenitor

cell and a further 65 hours before the mature neutrophil is released into the circulation

[197]. During sepsis, both neutrophil production time and storage time in the bone

marrow are significantly shorter (36 hrs and 34 hours, respectively) [197]. There is

also evidence that myeloid cells may skip mitotic steps during the proliferation phase

[197]. While G-CSF is the major cytokine that stimulates granulopoiesis and

enhances neutrophil mobilisation from the bone marrow, knock-out mouse data show

that G-CSF is not essential for stress granulopoiesis. Other pro-inflammatory

cytokines, including IL-6 and IL-3, also play an important role. Experimental models

93

have shown that under stress conditions, neutrophilia develops within 5-8 hours of the

onset of the inflammatory stimulus [198].

The observed lack of difference between the cord WCC and ANC in HCA may be

explained by delivery occurring less than 8 hours following the onset of fetal

inflammation, leaving insufficient time for a neutrophilia to develop. Evidence from

other studies has shown that FIRS involves increased concentrations of G-CSF and

IL-6 [84]. These cytokines produce an effect on neutrophil concentration in the

peripheral blood within 8 hours. If the fetal inflammatory process results in delivery

within this time-frame, it is possible that there has been insufficient time for an

increased cord WCC and ANC to develop. If this were true, then one would expect

an increased WCC and ANC on subsequent measurement within the first 24 hours

following delivery as observed in the post-natal haematological data (discussed in the

next section). Alternatively, cord WCC and ANC are relatively insensitive to low

level inflammation and are therefore not significantly affected by HCA.

5.1.3 Neonatal inflammatory markers

The impact of HCA may be seen up to 48 hours after delivery. In this section, I will

discuss the significant impact that HCA has on neonatal ANC and CRP

concentrations within the first 48 hours of delivery.

Neonatal white cell count (WCC) and absolute neutrophil count (ANC)

This study found that WCC and ANC measured within 24 hours of delivery are

significantly higher in neonates exposed to HCA, after adjusting for GA, small for

GA, length of labour, and treatment with antenatal steroids. While on univariate

analysis significant differences were not seen (WCC median 10.3 x109/L with HCA

versus without HCA 9.2 x109/L ; p=0.478, and ANC median 4.5 x109/L with HCA

versus 3.0 x109/L without HCA; p=0.054), the comparison for neonatal ANC

approached significance so multivariate analysis was undertaken. On regression

analysis, it was clear that HCA was a significant predictor for both neonatal WCC and

ANC. Whilst statistically significant differences in neonatal WCC and ANC exist

between infants in the HCA and non-HCA groups, the data show marked overlap

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between groups. This severely limits the clinical utility of neonatal WCC and ANC in

diagnosing in utero exposure to HCA.

There are very little published data on the effect of HCA on neonatal WCC and ANC.

Only two studies have investigated the strength of relationship between neonatal

neutrophil counts and HCA. De Dooy et al. [52] found that HCA is associated with a

significantly higher WCC on Day 0, and higher WCC and ANC on the first and the

second postnatal days. Jackson et al. [71] found that 99% of asymptomatic neonates

exposed to clinical chorioamnionitis had at least one abnormal neutrophil count on the

first day of life. The findings from this study are consistent with their observations.

Overall, these results give an insight into the timing of fetal inflammation relative to

delivery. Following from the discussion in the previous section on cord WCC and

ANC, pro-inflammatory cytokines and G-CSF are increased in the neonate with FIRS

at the time of delivery. These cytokines stimulate granulopoiesis and neutrophil

release from the bone marrow, resulting in neutrophilia. However, findings from this

study show that at the time of delivery there is no increase in the cord blood WCC and

ANC of newborns with HCA. Subsequent peripheral blood samples within the first

24 hours of delivery show increasing WCC and ANC. This observation adds weight

to the hypothesis that delivery occurs rapidly (within a few hours) after the onset of

fetal inflammation. At the time of delivery inflammatory cytokines are increased, but

the consequences are not seen on haematological parameters until the first 1-24 hours

following delivery.

As with CRP and PCT, WCC and ANC are non-specific markers of inflammation and

other possible sources of inflammation need to be considered. Using the same

rationale with regard to pro-inflammatory stimuli in the newborn, potential

confounding factors that were included in the analysis were GA, being small for GA

(< 10th percentile) and length of labour. Furthermore, there is evidence that GA and

length of labour directly affect neonatal neutrophil counts [76]. The use of antenatal

steroids is also known to cause de-margination of neutrophils from the endothelial

wall resulting in neutrophilia in newborns [157]. For neonatal WCC, GA, small for

GA, length of labour and use of antenatal steroids accounted for 8.6% of variation in

the model. The addition of HCA to the model accounted for a further 7.1% of

95

variation in neonatal WCC (p<0.001). Similarly for neonatal ANC, GA, small for

GA, length of labour and use of antenatal steroids accounted for 23.8% of variation in

neonatal ANC. The addition of HCA explained a further 3.4% of variation in ANC

(p=0.002). After considering these potential confounding factors, the adjusted OR

was 1.12 (95% CI 1.03-1.22; p=0.007) for neonatal WCC and was 2.21 (95% CI 1.20-

4.09; p=0.011) for neonatal ANC.

Neonatal CRP

In addition to the effect of HCA on neonatal WCC and ANC in the first 24 hours

following delivery, this study gives an indication that ongoing inflammation in the

baby results in increased CRP levels up to at least 48 hours following delivery.

Before these results are discussed in detail, it must be borne in mind that CRP was

measured in the neonates on Day 0 (birth-1hr), Day 1(1-24 hrs) and Day 2 (24-48 hrs)

only where clinically indicated. 89% neonates with HCA had CRP measures

compared with 63% neonates without HCA. Thus, while the results for neonatal CRP

are intriguing, they are not necessarily reflective of the entire population.

A further limitation of the data with respect to neonatal CRP was the large proportion

of results that were less than the lower limit of detection (i.e. <3mg/L or <7mg/L

depending on when the result was measured). It will be recalled that the data was

analysed in two different ways. In the first approach all undetectable results were set

to 0mg/L. In the second approach results were set to 2.5 if <3mg/L and 6.5 if <7mg/L.

Using the first approach all CRP measures in the first 48 hours after birth were

significantly higher in neonates exposed to HCA. Using the second approach,

maximal CRP in the first 48 hrs, CRP concentrations on Day 1 and Day 2, but not

Day 0 were significantly higher in the neonates exposed to HCA. The second

approach was considered more conservative as it did not inflate the differences

between groups. Similar proportions of CRP data were available on Day 0, Day 1 and

Day 2 (Table 3.6.3a), thus these results cannot be explained by differences in

availability of data at these time points. Thus, while there may be deficiencies in the

neonatal CRP data, this data supports the hypothesis that in utero exposure to HCA

has a significant impact on CRP concentrations in the first 48 hours following

delivery.

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The cord and neonatal CRP data also shows that there is a rapid increase in CRP

during the first 48 hours following delivery that may occur in all newborns, but

particularly in neonates exposed to HCA. Using the first approach to undetectable

CRP data, the median maximal CRP was 10mg/L in HCA group and 0mg/L in the

non-HCA group, median Day 1 CRP was 11mg/L in the HCA group and 0mg in the

non-HCA group and median Day 2 CRP was 10mg/L in the HCA group and 0mg/L in

the non-HCA group. Using the more conservative approach for analysis, results were

the same as previous but median CRP concentrations in the non-HCA group was

6.5mg/L at all time points. In contrast, median cord CRP in both groups was 0 mg/L.

This indicates that there may be a rapid increase in CRP following delivery in all

newborns, but that the increase is more dramatic in neonates exposed to HCA.

These results are consistent with previous studies in this area. There are two studies

that provide evidence that there may be a relationship between HCA and neonatal

CRP. De Dooy et al. [52], found that neonatal CRP levels on Day 0 and Day 1 were

significantly higher in neonates with HCA. Skogstrand et al. [51], showed that

neonatal cytokine levels measured from dried blood spots collected at mean 5 days

following delivery were significantly higher in preterm and very preterm infants.

This study did not correlate cytokine levels with the presence of HCA, but provides

evidence that the elevated cytokine levels associated with preterm birth are sustained

into early neonatal life.

HCA is the histological hallmark of FIRS. This syndrome is characterised by

systemic inflammation in the fetus, and is a risk factor for short-term perinatal

morbidity and mortality, as well as long-term sequelae such as bronchopulmonary

dysplasia and brain injury [84]. The mechanism by which these long-term sequelae

occur is the focus of current research. The findings from this study give an indication

that neonates with HCA may have a more exaggerated inflammatory response in early

neonatal life. Importantly, none of the neonates included in this study had early onset

noenatal sepsis, meaning that the differences that were observed in neonates with

HCA were not the result of early neonatal infection.

Further, as discussed in the literature review, CRP is a widely used tool that assists in

the diagnosis of sepsis in the neonate and guides the duration of antibiotic therapy.

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The findings from this study provide evidence the inflammatory events in utero can

produce a sustained and clinically significant effect on CRP concentrations for at least

48 hours following delivery. Given that the high negative predictive value of serial

CRP results (<10 mg/L) within the first 8-24 hours is used to guide the cessation of

antibiotic therapy, it is possible that neonates with HCA have longer exposure to

unnecessary antibiotic therapy because of the persistent elevation in serum CRP.

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5.2 Clinical and diagnostic implications

Implications for researchers

Overall, this thesis brings intriguing insights into the degree and timing of

inflammation that occurs in mothers and infants with histological chorioamnionitis.

The three main ideas to come from the findings of this study were that

(1) for the mother, HCA is a minor inflammatory insult;

(2) in the cord blood, HCA results in inflammatory changes that are detected by

sensitive markers of fetal inflammation, such as usCRP and PCT,

(3) fetal inflammation has sustained effects on CRP and haematological parameters in

early neonatal life.

For the mother, intrauterine infection is the one of the significant drivers of maternal

inflammation resulting in HCA and an elevated CRP level. However, PCT, a more

accurate marker of real-time inflammation and a more specific marker for bacterial

infection than CRP, is not elevated in mothers. This may be because, for mothers,

intrauterine infection is a minor insult and does not stimulate production of PCT.

This concept concurs with the observations of others that it is fetal, not maternal,

serum cytokine levels that correlate with the degree of histological inflammation in

the placenta [199]. Importantly, this study only has a single measurement for

maternal PCT and CRP i.e. on the day of delivery. In the absence of data in the days

and weeks prior to delivery, one can only speculate as to the time between the onset

of maternal infection, inflammation and delivery.

At the time of birth, sensitive markers of inflammation, PCT and CRP, are

significantly higher in infants exposed to HCA. Less sensitive markers of

inflammation, such as WCC and ANC, were not significantly different. Subsequent

peripheral blood samples within the first 24 hours of delivery show that increased

WCC and ANC do occur in neonates exposed to HCA, adding weight to the

hypothesis that the inflammatory consequences are not seen on haematological

parameters until the first 1-24 hours following delivery.

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The findings from this study support the hypothesis that acute inflammation in the

fetus exposed to HCA is sustained into early neonatal life. For newborns, higher

umbilical cord CRP and PCT concentrations in neonates exposed with HCA are a

marker of a fetal systemic response to intrauterine infection, through the generation of

inflammatory cytokines such as IL-6. Interestingly, although a significant difference

was detected, the median cord CRP in newborns with and without HCA was 0 mg/L.

In the ensuing 48 hours after delivery, CRP increases rapidly in all newborns, but the

increase is more exaggerated in neonates with HCA.

Implications for clinicians

This study has also shown that CRP, WCC and ANC in the early neonatal period may

be influenced by HCA. This is important information as CRP, WCC and ANC are

diagnostic tools that assist in the diagnosis of early onset neonatal sepsis. In the

absence of other clinical signs, an elevated CRP, WCC and/or ANC can be used as

evidence for early onset neonatal sepsis. In particular, three serial CRP measurements

over the first 72 hours from initial evaluation have a sensitivity of 90-98% for the

diagnosis of neonatal sepsis. [91]. The data from this study show that this tenet may

be confounded by the presence of HCA. The study reinforces that there is no single

test that will diagnose neonatal sepsis, rather a combination of factors must be taken

into account.

Evidence from this study clearly shows that PCT in the cord blood is elevated in

newborns with HCA. This means that PCT levels in the early neonatal period are

confounded by the presence of HCA. As the diagnosis of HCA may not be known for

several days following delivery, this greatly impacts of the usefulness of PCT levels

as a predictor for the presence of early onset neonatal sepsis.

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5.3 Limitations of the study

The present study had several limitations. These were the reduced power resulting

from low numbers of cases of HCA, limitations in the neonatal CRP data, and the

cross-sectional nature of this study.

5.3.1 Study cohort, reduced statistical power

This was a large study of mothers and infants (n=325), predominantly born

prematurely (n=224, 69%). Within the study population, there were relatively few

cases of HCA (n=26, 8% of study population). In comparison, published data show

that HCA is seen in 66% of spontaneous PTD at less than 27 weeks gestation, 51% at

27-31 weeks, 23% at 32-35 weeks, and approximately 10% at 35-36 weeks [1, 35].

There are two likely explanations for the relatively low number of cases of HCA in

the study cohort. First, within the study population there was a relatively high

proportion of non-spontaneous PTD. This arose from a recruitment bias towards PTD

by elective caesarean section, with a high proportion of these being multiple gestation

pregnancies (64% elective caesarean section, 39% multiple gestation pregnancy). In

contrast, 18 of the 26 cases (69%) of HCA were born spontaneously either by vaginal

delivery or non-elective caesarean section. If cases of non-spontaneous PTD are

excluded, HCA accounts for 19% (18/94) of cases of spontaneous PTD, in keeping

with the published literature. Second, published evidence shows that HCA is more

seen more frequently at very early GAs [1]. While the study recruited subjects that

were predominantly born preterm, 66% were born at >32 weeks gestation (Table

4.1.1). If there had been a greater number of subjects recruited from the very

premature age group (<32 weeks), it is likely there would have been a greater number

of cases of HCA.

Nonetheless, despite low numbers of cases of HCA, the clinical characteristics of

these cases are consistent with findings from the literature. This study confirms the

observation that HCA is more common at early GAs. The proportion of subjects with

HCA was 37% (7/19) at <28 weeks gestation, 12% at 28-32 weeks gestation, 4%

(5/113) at 32-36 weeks gestation and 3% (3/98) at >37 weeks gestation. Published

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studies have reported that the incidence of HCA and PTD is influenced by

race/ethnicity [1, 9, 34, 200], and associated with PROM [1, 9]. This study confirms

these findings with a higher prevalence of HCA in mothers with Aboriginal/TSI

ethnic origin, and with PROM. While maternal smoking is a known risk factor for

PTD [1], the role of HCA in smoking-related PTD has not been investigated [1]. This

study showed a higher prevalence of HCA among mothers who smoke, however this

result was not analysed for potential confounding effects, such as differences in GA

between groups. Published studies have shown approximately 10% of women with

HCA have clinical symptoms of chorioamnionitis. In this study, 27% cases had

clinical symptoms of chorioamnionitis, compared with 1% in those without HCA.

The most common pathogens isolated from cases of HCA were Mycoplasma hominis

(n=5), and Ureaplasma urealyticum (n=2), consistent with cases described in the

literature [9]. These observations indicate that the 26 cases of HCA were typical of

those described in the literature.

The consequence of the low numbers of cases of HCA was a reduction in the

statistical power of the study to detect differences between subjects with and without

HCA i.e. type II error. This means that for variables where no association with HCA

was found, true differences between cases with and without HCA may not have been

detected. In addition, the low number of cases of HCA limited the number of

variables that could be used in linear regression modelling. If there had been a large

number of cases, other potentially confounding variables could have been included in

the models.

5.3.2 Neonatal CRP data

Measurement of neonatal CRP was not part of the original study protocol. Rather,

this information was collected opportunistically as part of the clinical data on enrolled

subjects. Thus, CRP was measured only where clinically indicated. This impacted on

the results in several ways.

First, a significant proportion of neonates did not have CRP measured in the neonatal

period. This was more likely to be the case in neonates that were not exposed to HCA

(89% with HCA had any CRP measure). While this may be an indicator that neonates

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exposed to HCA are more likely to be unwell in the early neonatal period, there are

several other confounding factors that also need to be considered. The lack of CRP

values in infants not exposed to HCA impacted on the ability to draw strong

conclusions from this data.

Second, a large number of neonatal CRP values were undetectable. Using a

conventional (non-high sensitivity) assay, the threshold for detection was 7 mg/L for

the initial part of the study, and 3mg/L when an improved technique was introduced.

For values less than these thresholds, we defined the CRP as 6.5 mg/L and 2.5 mg/L,

respectively. This was a conservative treatment of this problem and reduced the

power of the study to find a difference in neonatal CRP levels in infants with and

without HCA. Despite this, our study found a difference adding strength to the

argument that neonatal CRP levels are higher in infants exposed to HCA. The use of

a more sensitive assay for CRP in the neonatal period would have been more precise,

however it is also considerably more costly.

Third, the limitations of the neonatal CRP data impacted on the statistical analysis.

The large numbers of missing and undetectable values, combined with the relatively

low numbers of cases of HCA, restricted the regression analysis such that post-natal

variables could not be taken into account.

Ideally, neonatal CRP would be measured prospectively in a similar cohort of

newborns using a sensitive assay for CRP. This would give more complete data on

the cohort, tighter timing on specimen collection and overcome the issue of

undetectable CRP results due the insensitivity of the assay.

5.3.3. Limitations of the study design

Finally, this is a cross-sectional study. As such, while an association was

demonstrated between HCA and maternal usCRP, cord CRP and PCT, and neonatal

CRP, WCC and ANC, the mechanism by which this occurs cannot be inferred from

this study.

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5.4 Future directions

There are a number of potential avenues of future research which follow on from

these data. This study has demonstrated that the neonate exposed to HCA experiences

a major inflammatory response that manifests not just at delivery, but during at least

the first 48 hours following delivery. Among the key issues that should be explored

in future longitudinal studies are:

Does the effect on the newborn extend past the first 48 hours?

What does elevated CRP levels do to the newborn?

Does inflammation in the early neonatal period have a negative impact in adult life?

What is the impact on immune function and innate immunity?

This study has shown that intrauterine exposure to HCA may influence surrogate

diagnostic markers for early onset sepsis in newborn infants. Future research to

investigate novel diagnostic markers, such as CD64 [201, 202] and soluble triggering

receptor expressed on myeloid cells (TREM-1) [203], or enhanced microbiological

molecular diagnosis, will help distinguish true invasive infection from HCA-driven

inflammation in the newborn infant.

5.5 Concluding comments

Overall this thesis brings new insights into the degree and timing of inflammation that

occurs in mother and infants with HCA. For infants fetal inflammation has sustained

effects on CRP and haematological parameters in early neonatal life. This study has

provided evidence that CRP, WCC and ANC are significantly higher in newborns

exposed to HCA, peaking at approximately 24 hours following delivery. This effect

is of sufficient magnitude to confound the interpretation of these common diagnostic

tests for early onset neonatal sepsis. Intrauterine exposure to HCA should be

considered in the neonatal intensive care unit when prescribing antibiotic therapy for

suspected early onset sepsis.

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Appendix 1 – Preliminary information sheet for parents

‘The immune responses of premature infants and susceptibility to infection early in life’

An information sheet for expectant parent(s)

Dear expectant parent(s) We are conducting a research project based at King Edward Memorial Hospital for Women looking at how babies who are born early (prematurely) fight infection (i.e. their ‘immune system’). We know that premature babies are prone to serious infection and this research looks at why the immune system of premature babies might make them more prone to serious infections. We are interested in babies who are delivered before 36 weeks gestation, as well as babies born on time, so that we can compare the two groups. As we cannot predict whether your baby will be born early, so we are letting all pregnant women know about this study antenatally. The study has been approved by the Ethics Committee at KEMH and PMH.

What does the study involve? We will only ask you to consider being part of the study if you happen to deliver before the end of the 35th week of pregnancy. We plan to take a blood sample (20mL – 1 tablespoon) from the umbilical cord (not from the baby) after the baby is delivered. This is blood that is usually discarded and so taking the sample will not affect your baby’s health. We will also take a small blood sample (30mls – 1½ tablespoons) from the baby’s mother at the time of birth, either when she is having blood taken as part of her routine care, or through a drip (‘IV line’). We will also take a sample of umbilical cord and placenta, which would normally be discarded. A small sample of blood (about five drops) may be taken when the infant is admitted to the neonatal nursery should he/she show signs of infection. After the delivery we will take a 5ml (1 teaspoon) sample of breast milk.

Having a baby prematurely can be a stressful experience. We will therefore not ask for the mother’s written consent to take part in the research at the time of delivery. This will be sought one or two days later, when you are more able to concentrate on the study. At this time, we will give the mother more detailed information about the study to read. One of the researchers will be available to answer any queries you may have. However, to answer some of the research questions, we need to process the blood and other samples immediately after collecting them. We will therefore process all samples all samples and store them in a freezer. If you choose not to take part in the study, all samples from you and your baby will be discarded. The decision not to take part in the study will not affect the care that you or your baby receives. You are free to withdraw from the study at any time.

I give my initial permission for samples to be collected from the umbilical cord, placenta, together with a sample of my blood, at the time of delivery. I understand that further details relating to this research will be discussed with me at the earliest opportunity and I may withdraw from the study at any point. Signed…………………………………………………………………………………………………………. Date…………………….……………………… Print mother’s name………….……………………………………………………………………..

Mother unable to sign Verbal assent given Mother’s name…………………………………………….. Date…………………………………………... Investigator’s

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Name………………………………………………………………… Signature….……………………………………………….

Appendix 2 – Full information sheet for parents and consent form

‘The immune responses of premature infants and susceptibility to infection early in life’

An information sheet for parent(s)

Dear parent(s) Thank you taking the time to read this information at this stressful time. This information sheet gives you some details about a research study on premature infants. Please take the time to read the information carefully before deciding whether you would like to take part in the research. Please feel free to ask any questions you may have about the study; contact details for the research team are given at the end of the sheet. Once you are happy that all your questions have been answered and you understand the research, you will be asked to sign a consent form. If you decide not to take part in the research, the care you and your baby receive will not be affected in any way. You can decide to withdraw from the study at any time. The KEMH/PMH Ethics Committee has approved this study. Background. Although most babies born prematurely (early) survive without undue problems, some babies are particularly susceptible to infection immediately after birth. Infection is an important cause of death and disability in newborn babies, particularly those born prematurely. This study will look at the response of premature infants to infection. We have some knowledge of the normal newborn babies’ response to infection (their immune response), but very little about how this is affected by other factors, such as being premature. We are particularly interested in a bug (germ) called Group B streptococcus (or GBS). This germ is the commonest cause of infection in newborn babies, with premature babies being most at risk. About 1 in 5 women carry GBS harmlessly in their birth canal (vagina) or gut, but only about 1 in 50 of their babies are infected with GBS. This study will look at the reasons why so few babies are infected with GBS, and how GBS is passed to infants following delivery, and will help us in the future prevention and treatment of these infections. We are also interested in the longer-term health of babies with infections, as this may be involved in lung damage and the need for long-term oxygen therapy, and also with disabilities such as cerebral palsy. Why has my baby been chosen to take part in the study? We are asking mothers of all babies born at KEMH before 36 weeks gestation to consider taking part in the study. We hope to enroll 500 premature babies over a 1-2 year period. We also want to enroll 200 babies born on time so that we can compare there immune responses with the preterm babies. It is important for the study that the samples are taken from the umbilical cord, placenta and from the mother at birth. We will try to discuss the study in detail with all women before they deliver, but sometimes premature labour progresses quickly and we may therefore be discussing this study with you after your baby is born. If you have already delivered, and if you gave initial permission, the samples have already been collected and stored. If you decide not to be part of the research, we will discard these samples. Your decision whether to participate in this research will not affect the care of you or your baby in anyway. You are free to withdraw from the study at any time. If you have twins or triplets, we would like to collect samples from the umbilical cords of all babies. We will therefore ask you to sign two (or three) consent forms, depending on how many babies you have.

What will the study mean for you and your child? We will collect samples of the umbilical cord and placenta, a 20 ml (1 tablespoon) sample of blood from the umbilical cord after birth (i.e. NOT directly from your child), a 30ml (1½ tablespoon) sample of your (the mother’s) blood, and a 5ml (1 teaspoon) sample of breast milk at a later stage. These samples will be processed and stored. We will then analyze the samples we’ve taken from yourself and the umbilical cord. If your child subsequently develops signs or symptoms of an infection whilst in the neonatal nursery, we would ask for your consent to collect a 1 ml blood sample (about five drops) at the time of routine blood sampling If you have received your antenatal care at another hospital, we will ask for your consent to contact that hospital for information regarding your pregnancy. All information will be anonymised, and stored securely and confidentially, so that only the researchers can access your personal details. We will ask for your permission to contact you in the future to discuss the possibility of taking part in follow-on research studies. What analysis will be performed on the samples? We are interested in you and your baby’s immune function and response to infection. We will look at the immune function of the blood cells that fight infection and some of the chemicals they produce, and at breast milk for the protective antibodies against common germs. We will also look at some of the genetic controls of the immune system; this part of the study is outlined on a separate information sheet and consent form that you will be given and asked to sign if you are willing to take part. As this is a research study, the significance of the results is unknown and the results will not affect the immediate care of you or your baby. We will therefore not release individual results to you or your family, unless it clearly will influence you or your family’s health and we are advised to release the information by the Ethics Committee. Are there any disadvantages involved in taking part? Neither your or your child’s care will be affected whether you decide to take part or not. All blood tests required for your or your child’s medical care would take priority over those needed for the study. Most of the blood taken will not be from your child but from the umbilical cord. We would only take a sample directly from your baby if s/he develops signs of infection and this is a small amount of blood that will not affect your child’s health. We will try and take the mother’s blood sample at the time that other blood tests are being taken. However, as it is important that we collect blood at the time of delivery, we may need to take a separate sample from the mother if she is not having any other blood tests. As you will know, blood sampling may be uncomfortable and may cause minor bruising. The benefits of taking part in research. Improvements in medical care depend on well run research studies, and these in turn depend on the participants. Taking part in studies such as ours will help us understand more about premature babies’ response to infection, and whilst not directly benefiting your child, may improve our ability to care for babies in the future. In the unlikely event that we discover that your baby has a clinically significant problem with their immune system, we will arrange specialist referral. Should you decide not to take part, this will not interfere with your or your child’s medical care. You may decide to withdraw from the trial at any time. If you decide not to take part all collected samples would then be safely discarded. All information will be recorded confidentially and stored under current data protection guidelines. The participating researchers will abide by the terms of the Code of Practice for the Use of Name-identified Data -NHMRC guidelines and all data will be retained for a minimum of 5 years. Please contact the study organisers Dr Peter Richmond (Pager No.7037) or Dr David Burgner (Pager No. 7062) if you have any concerns or queries. Hospital staff will help you contact the researchers via the hospital switchboard. If you have any complaints regarding the conduct of this study, please contact the Director of Clinical Services on 9340 8222. Many thanks for taking the time to read this information leaflet

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