Sub Grouping Of Preterm Birth

Published Date: 02 Nov 2017

Table of Contents

ABSTRACT 1

1. INTRODUCTION Error: Reference source not found

1.1 Preterm Birth Error: Reference source not found

1.2 Aetiology of Preterm Birth Error: Reference source not found

1.3 Risk Factors for Preterm Birth Error: Reference source not found

1.4 Treatments for Prevention of Preterm Birth Error: Reference source not found

1.5 Processes of Human Labour and Delivery Error: Reference source not found

1.5.1 Cervical ripening Error: Reference source not found

1.5.2 Myometrial contractions Error: Reference source not found

1.5.3 Rupture of the fetal membranes Error: Reference source not found

1.6 Mediators of Labour Error: Reference source not found

1.6.1 Pro-inflammatory cytokines Error: Reference source not found

1.6.2 Cyclooxygenase-prostaglandin pathway Error: Reference source not found

1.6.2.1 Cyclooxygenases Error: Reference source not found

1.6.2.2 Prostaglandins Error: Reference source not found

1.6.3 Matrix metalloproteinases Error: Reference source not found

1.7 Transcriptional Factors Error: Reference source not found

1.7.1 Activator protein (AP-1) transcriptional pathway Error: Reference source not found

1.7.2 Nuclear factor kappa B (NF-κB) pathway Error: Reference source not found

1.8 Experimental Design and Rationale Error: Reference source not found

ABSTRACT

Studies using Saccharomyces cerevisiae revealed the existence of a unique family of

proteins named Sirtuins. Sirtuins are highly conserved all the way from bacteria to

humans. These proteins play important roles inside the cell, as nicotinamide adenine

dinucleotide-dependent protein deacetylases. Sirtuins are major regulators of multiple

cellular functions; they can modify the epigenetic landscape and are able to alter the

proteome of the cell. Several studies have analyzed the role of sirtuins as links between

cancer treatments, caloric restrictions and aging. However, recent discoveries demonstrate new roles for these proteins. Sirtuins have also caught the attention of the biotechnology industry, and

small molecule activators and inhibitors have been developed. The following review

describes key aspects of sirtuin biology, presents the current situation of sirtuins in research, and analyzes the emerging roles of sirtuins in cell metabolism, concentrating on the response from mammals in labour and delivery.

The mammalian sirtuins have emerging roles in a variety of important basic and

age-associated processes like cancer, diabetes, obesity, inflammation, muscle

differentiation, heart failure, and neurodegeneration, thereby attracting wide interest for

promising therapeutic intervention. More recently though are the studies of sirtuins in relation to the processes of human labour and delivery. However, many of the mammalian sirtuins remain

poorly characterized, even more so in relation to the inflammatory response in labour and delivery.

Preterm birth is a significant health issue is one of the most important obstetric complications, with approximately 8-9% born premature, with some countries up to 17%. Perinatal morbidity and mortality is highly associated with preterm birth. A key contributor is most cases is inflammation and the cascade of pathways following the inflammatory response, which then usually leads to initiating uterine contractions and rupture of fetal membranes. Pro-inflammatory cytokines induce the production of matrix metalloproteinase’s (MMPs) that degrade the extracellular matrix (ECM) within the cervix and fetal membranes, and prostaglandins which initiate uterine contractions that can lead to preterm birth. The pro-inflammatory transcription factors, nuclear factor-κB (NF-κB), etc., play a key role in promoting the formation of pro-labour mediators. In non-gestational tissues, sirtuins have been found to inhibit NF-κB, and in-turn its downstream targets, including various cytokines and prostaglandins. Therefore, the aim of this study is to determine if sirtuins may have any regulatory affect in infection-induced pro-labour mediators in human gestational tissues. This is summarized in 3 key aims;

To establish the tissue-specific expression of SIRT proteins at the time of spontaneous preterm labour in human gestational tissues.

To elucidate the effects of altering the expression of SIRT proteins on the effector pathways of labour and delivery in human gestational tissues.

To determine the effect of SIRT1 activators on preterm delivery and neonatal outcome in a model of preterm birth.

Include brief summary of samples to collect and tests to conduct? ie, pcr, ihc, wb?

Collectively, all previous biochemical and genetic studies will help to further explain sirtuin mediated mechanisms of homeostasis and inflammatory response in birth, thereby opening new routes for potential therapeutic intervention and for further studies in vivo.

With the aid of this study we will then further understand the processes of human labour. This may assist improvement on current models and intervention strategies to attempt to suppress preterm labour for more than a few days, though only where deemed appropriate. Using the sirtuins enzyme model and response pathway, this may present novel intervention point for the development of therapeutics to prevent preterm births and reduce incidence of related perinatal morbidity and mortality.

Preterm Birth

Sirtuins in mammals/humans

There are a total of seven mammalian sirtuin proteins, SIRT1-7, which vary

widely in their cell-specific function and localization in vivo (Dali-Youcef et al, 2007). Phylogenetic analysis, as shown previously in Figure 1.1, groups the mammalian sirtuins

into four Classes, I-IV: SIRT1-3 all fall within Class I (a group that also includes all of

the S. cerevisiae sirtuins); SIRT4 is in Class II (which includes nematode, insect,

bacterial, and protozoan sirtuins); SIRT5 is in Class III (together with many sirtuins from

prokaryotes); lastly, SIRT6 and SIRT7 group within Class IV.

All seven mammalian sirtuins share a conserved enzymatic core of roughly 275

amino acids, as summarized in Figure 1.2, consisting of two important domains: a large

Rossmann fold for binding to nicotinamide adenine dinucleotide (NAD+) and a smaller

domain formed by two insertions of the large domain that binds onto a zinc ion. The

acetylated peptide substrate that is targeted by the sirtuin binds to the cleft between the

two domains. The common catalytic core is often flanked by unique sequences of

different length at the N- and/or C-terminal ends. It is thought that these variable

sequences contribute to sirtuin regulation and function in vivo.

The sub-cellular distribution of the mammalian sirtuins is intriguing and an active

area of study. Although human SIRT1 does play some role in the cytoplasm of the cell, it

is enriched in the nucleus, along with SIRT6 and SIRT7 (Michishita et al., 2005). In the

nucleus, however, these three sirtuins differ greatly in their distribution: SIRT1 binds to

both euchromatin and heterochromatin (Vaquero et al, 2007), SIRT6 binds to telomeric

chromatin (Michishita et al., 2008) and may also modulate transcription at other sites

(Kawahara et al., 2009), and SIRT7 is nucleolar with unknown function (Michan and

Sinclair, 2007). Moreover, SIRT2 is enriched in the cytosol, whereas SIRT3-5 localize to

the mitochondria (Cooper and Spelbrink, 2008; Michan and Sinclair, 2007; North et al.,

2005 & 2003).

The biochemical properties of these enzymes also vary. For example, SIRT1 has

robust deacetylase activity, yet SIRT5 has very weak activity (North and Verdin, 2004;

Vaziri et al., 2001). SIRT4 acts as an obligate mono-adenosine diphosphate (ADP)-

ribosyl tranferases (Haigis et al., 2006; Liszt et al., 2005), with no detectable deacetylase

activity, yet SIRT2, SIRT3, and SIRT6 (Kawahara et al., 2009; Michishita et al., 2008;

Liszt et al., 2005) have both biochemical activities. (An upcoming section explores the

biochemistry of sirtuins in greater detail, particularly modes of regulation in vivo.)

SIRT1, the human orthologue of the yeast Sir2 protein, remains the most widely

studied (and best-understood) mammal sirtuin. For example, SIRT1 has been shown to target several nuclear and cytoplasmic proteins with key roles in transcription, stress

response, and apoptosis, e.g., p53, Ku70, MyoD, forkhead box 03 protein (F0X03), and

nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB) (Porcu and

Chiarugi, 2005; Sinclair, 2005). Thus, regulation of SIRT1-dependent deacetylation may

contribute to a wide array of biological responses ranging from enhanced cellular

resistance and lowered morbidity to lifespan extension in mammals. Yet, it is important

to mention that SIRT1 can also promote tumorigenesis in certain biological

contexts/tissues; for example, there is interest to understand the potential role of SIRT1 as

a negative regulator of the tumor suppressor p53 (Pruitt et al., 2006; Chen et al., 2005b).

Unlike SIRT1, however, the other mammalian sirtuin family members are not well

understood, and many still remain poorly characterized.

SIRT2 is cytoplasmic and nuclear: it deacetylates tubulin, suggesting a potential

involvement in microtubule organization, cellular structure, and possibly motility (North

et al., 2003), and it can also regulate p300 in the nucleus (Black et al., 2008). The

remaining sirtuins, SIRT3-7, though broadly expressed in some human cells and tissues,

have little to no known function in vivo, and few protein-binding partners and substrates

have been identified, as is particularly the case for the mitochondrial sirtuin SIRT3.

1.2.3 The human sirtuins (SIRTs)

The human sirtuins comprise a family of NAD+-dependent deacetylases conserved evolutionarily

from bacteria to humans. Their ortholog in lower organisms – Sir2 (silent information regulator

2) protein deacetylase has emerged as an important regulator in extending the life spans of S.

cerevisiae, C. elegans, and D. melanogaster (Guarente and Picard 2005; Longo and Kennedy

2006). Although the life extending effects of sirtuins in mammals have yet to be demonstrated,

the ubiquitously expressed human sirtuins are critical regulators of many cellular pathways

including insulin secretion, the cell cycle, and apoptosis, and are associated with a variety of ageassociated

diseases such as Alzheimer’s disease and cancer (Kim, Nguyen et al. 2007; Firestein,

Blander et al. 2008; Luo and Altieri 2008; Wang, Sengupta et al. 2008). Sirtuins are classifi ed as

the Class III histone deacetylases (HDACs). In contrast to the "classical" Class I and II histone

deacetylases (HDACs), which use an eletrophilic Zn2+ ion to directly hydrolyse the amide bond

CHAPTER 1. GENERAL INTRODUCTION 8

with water, sirtuins transfer an acetyl group from the lysine side chains of a protein substrate to

the co-substrate NAD+ generating nicotinamide and OAADPr (2’-O-acetyl-ADP ribose) (Denu

2005) as shown in Figure 1.2. A detailed deacetylation mechanism involving the generation of

a peptidyl-imidate intermediate in the fi rst chemical step has been proposed (Sauve, Celic et al.

2001). The requirement of NAD+ for activity suggested sirtuins may function as cellular energy

sensors and serve as a link between cellular metabolism and reverse acetylation mediated cellular

pathways. Since all classes of HDACs are capable of deacetylate many non-histone substrates, it is

more appropriate to use protein deacetylase as a general designation for these enzymes.

1.2.4 Biological functions of the human SIRT family members

There are seven members in the human sirtuin family, SIRT1-7 (Figure 1.3). Each of them has

Figure 1.2 Deacetylation/ADP-ribosyl transfer reaction scheme of SIRTs.

CHAPTER 1. GENERAL INTRODUCTION 9

distinct cellular targets and diverse cellular

localizations (Michishita, Park et al. 2005).

SIRT1 has been the most studied, as it shares

the highest sequence identity with the founding

member of the Sir2 family from yeast. SIRT1 is

localized either to the nucleus or the cytoplasm

depending on tissue and cell type (Tanno,

Sakamoto et al. 2007). Sirt1 transgenic mice

display benefi cial phenotypes similar to mice on

a calorie-restricted diet – they are leaner and more metabolically active (Bordone, Cohen et al.

2007), supporting an anti-aging role of SIRT1. SIRT1 is also linked to oncogenesis. It promoted

deacetylation of p53 and p73 as well as E2F1 which represses the expression of target genes

inhibiting apoptosis; it also deacetylates BCL-6, which increases its oncogenic activity (Vaziri,

Dessain et al. 2001; Bereshchenko, Gu et al. 2002). SIRT1 is also upregulated in human lung

cancer, prostate cancer, and leukemia (Yeung, Hoberg et al. 2004; Bradbury, Khanim et al. 2005;

Kuzmichev, Margueron et al. 2005).

SIRT2 is thought to be the only cytoplasmic sirtuin. It has been implicated in the process

of cell division via α-tubulin deaceylation (North, Marshall et al. 2003) and histone H4 lysine 16

deaceylation during mitosis (Vaquero, Scher et al. 2006). SIRT1 and SIRT2 cytoplasmic have both

been demonstrated to undergo nucleo-cytoplasmic shuttling (North and Verdin 2007).

SIRT3, SIRT4, and SIRT5 are located in mitochondria but the comparison of Sirt3-/- and

Sirt3+/+ mice has provided compelling evidence that endogenous Sirt3 is responsible for the

majority of protein deacetylation in mitochondria (Lombard, Alt et al. 2007). SIRT4 has exhibited

no deacetylase activity to date. However, SIRT4 is reported to inhibit glutamate dehydrogenase

through ADP-ribosylation and it plays a role in insulin secretion (Haigis, Mostoslavsky et al. 2006).

Although the mouse knockout of Sirt5 does not affect the bulk acetylation state of mitochondrial

proteins (Lombard, Alt et al. 2007), SIRT5 deacetylates and activates carbamoyl phosphate

Figure 1.3 Human SIRT family phylogenetic

tree. Human sirtuin protein classifi cation based

on primary amino acid sequence.

CHAPTER 1. GENERAL INTRODUCTION 10

synthetase 1 (CPS1), which mediates the fi rst step in the urea cycle (Nakagawa, Lomb et al. 2009).

SIRT6 and SIRT7 are chromatin-associated sirtuins. SIRT6 is linked with heterochromatic

regions and SIRT7 is found in nucleoli (Michishita, Park et al. 2005). SIRT6 has both ADP-ribosyl

transferase and deacetylase activity, and modulates telomeric chromatin (Michishita, McCord

et al. 2008) and NF-κB-dependent gene expression through histone H3 Lysine 9 deacetylation

(Kawahara, Michishita et al. 2009). Loss of Sirt6 leads to a shortened lifespan and premature

aging (Mostoslavsky, Chua et al. 2006). Neural-specifi c deletion of Sirt6 in mice leads to postnatal

growth retardation, but the animals reach normal size and ultimately become obese over time

(Schwer, Schumacher et al. 2010). SIRT7 is involved in the activation of RNA polymerase I

transcription (Ford, Voit et al. 2006). Sirt7-knockout mice have shortened life spans with enhanced

infl ammatory cardiomyopathy (Vakhrusheva, Smolka et al. 2008).

1. General introduction

Preterm birth (PTB) remains one of the main causes of perinatal mortality and long-term

morbidity (1). The US incidence of PTB in

2000, 11.6 % (2). There has been a slight increase

during the last decades in the USA (1, 2) and

Canada (4). In the USA, the PTB rate has risen

steadily from 9.4 % in 1981, to 10.6 % in 1990

and 11.6 % in 2000 (2).

INTRODUCTION

14

Preterm children are also at increased risk of

developing cognitive and behavioral

abnormalities and of achieving poorly in

educational situations (12), problems which

persist until adulthood (13, 14). Nonretinopathy-

related visual abnormalities are

also seen in extremely preterm children (15,

16).

Against this background, it is understandable

that the economic costs for PTB are

considerable, related both to the initial

hospitalization in the neonatal intensive care

unit and to the long-term consequences of PTB.

However, comparatively little research has

been devoted to measuring the economic costs

of preterm delivery, especially the combination

of the short-term and long-term costs (17). One

study has estimated the magnitude of the

additional costs attributable to low birth weight

at 35 % of all health care costs for infants (< 1

year) and 10 % of all health care costs for

children (18). This is equivalent to the cost of

unintentional injuries, the leading cause of

death for children after infancy (17). It has been

shown that a collaborative quality improvement

of the neonatal care of very preterm infants can

quickly and substantially reduce the costs of

care (19). This implies that preventive measures

aimed at reducing the incidence of PTB will

immediately release economic resources, and

2. Preterm birth

2.1 Sub-grouping of preterm birth

The possibility that the clinical consequences

of prematurity for the infant could vary

according to causal mechanism has led to a

subdivision of PTB into sub-groups (20). One

classification of PTB is based on the manner

in which the patients presents, i.e. spontaneous

preterm labor (PTL), preterm prelabor rupture

of membranes (pPROM) and elective induction

or cesarean section (CS) (usually due to a

complication of pregnancy), respectively (20).

The first two groups are often combined and

called spontaneous PTB, in contrast to

indicated PTB. This subdivision has been

questioned but is still the one in most common

use (21). All studies related to this issue are

hospital-based.

The proportion of preterm deliveries resulting

from PTL varies in different studies from 18 to

64 % (22-24), while that resulting from

pPROM is reported at between 7 and 51% (25-

27). The proportion of preterm deliveries

classified as indicated varies between 18 and

38% (23, 25, 28).

2.2 Spontaneous preterm birth

Since the beginning of the eighties a compelling

amount of evidence has been presented,

suggesting a strong association between

microbial invasion of the amniotic cavity

(MIAC), PTL and pPROM. MIAC and the

presence of pro-inflammatory cytokines in the

amniotic fluid (AF), the intra-amniotic

inflammatory response, are known to induce

the release of uterotonic substances such as

prostaglandins from gestational tissue, thereby

causing uterine contractions and labor (29).

Infection may cause up to 30% of PTL cases

and may either be clinically evident or, more

often, subclinical (30). At any rate, the etiology

of PTB is multifactorial and the exact cause in

each case can rarely be identified.

Evidence of activation of the fetal

hypothalamic-pituitary-adrenal axis in relation

to preterm delivery has also been presented

(31). Spontaneous parturition in primates is

characterized by fetal adrenal synthesis of C19

androgens which, in turn are aromatized by the

placenta into estrogens, including estrone,

estradiol and estriol. Among non-human

primates, a rise in AF concentration of estrone

precedes or coincides with increases in AF

prostaglandin concentrations, which begin to

rise several days before parturition (32). In

humans, placental corticotropin releasing

hormone (CRH) (33, 34), maternal salivary

estradiol (35) and maternal plasma estradiol

concentrations all increase before spontaneous

preterm parturition (31). In contrast to

infection-associated parturition, spontaneous

parturition at term is associated either with no

increase or minor increases in AF

concentrations of cytokines (36, 37).

2.3 Animal models for studying

spontaneous preterm birth associated

with infection and inflammation

Several animal models have been developed

in order to study further the role of infection

and inflammation in PTB. Some investigators

have used a mouse model for studies of

infection- and inflammation-associated PTB

(38-41). Lipopolysaccharide (LPS) and proinflammatory

cytokines have been injected in

pregnant mice (day 12-14 of 19-day pregnancy)

and preterm delivery has been observed within

24 hours.

Others have used a rabbit model (42-47);

Dombroski et al have, for instance, shown that

bacterial inoculation via the vagina results in

PTB (46). There is also a primate model;

pregnant rhesus monkeys (36, 48) can be

infected (day 130 of 167 day pregnancy) which

will cause PTB (36).

4. Infectious and inflammatory mechanisms

in preterm birth

4.1 General inflammatory mechanisms

4.1.1 Inflammatory cells

Maternal and fetal inflammatory responses

include the involvement of cytokines,

chemokines, adhesion molecules, extracellular

matrix proteins with adhesive properties and

matrix metalloproteinases. All five of these

groups facilitates the transendothelial migration

of cells out of the circulation and into adjacent

organ parenchyma, which is essential for the

inflammatory process (79). When it comes to

PTB, nearly all of the disparate maternal and

fetal cell types in the uteroplacental unit are

integrated into the cytokine network. The

highly versatile macrophage, abundant in

uteroplacental tissues, has turned out to be a

potential pivotal cell type in this context (80).

The polymorphonuclear leukocytes found in

AF are of fetal origin in the majority of cases

(81). These data have raised the question of how

the fetal leukocytes might gain access to the

AF; via extravasation from the umbilical cord,

from chorionic vessels, from the chorionic plate

of the placenta or from fetal endothelium in

vessels in the alveolar space of the lung (81).

A primate model has been developed to study

this (82) but results have not yet been published.

The brain was previously thought to be

protected by the blood-brain-barrier, but it has

been established that the brain can no longer

be described as an immune-privileged organ

devoid of the capacity to initiate an

inflammatory response (83). The blood-brain

barrier is relatively ineffective in preterm

infants (84). It has been hypothesized that

cytokines and chemokines activate endothelial

cells and leukocytes in the circulation, diminish

the effectiveness of the blood-brain barrier (85)

and gain access to and activate microglia and

astrocytes, which in turn produce chemokines

and more cytokines (79). The chemokines

function as chemo-attractants for activated

inflammatory cells, including those in the

circulation (79).

The immediate response of the brain to a variety

of insults is characterized by the proliferation

and hypertrophy of microglia cells (the resident

inflammatory cells). The process of gliosis is

accompanied by the infiltration of activated

inflammatory cells derived from the periphery,

the inflammatory products of which act in

consort with centrally derived mediators in

eliciting a response to the injury (83). Although

some monocytes, macrophages and microglia

in the brain are derived from resident cells,

others are derived from the circulation (86).

4.1.2 Pro-inflammatory cytokines

The concept of pro-inflammation is based on

the genes coding for the synthesis of small

mediator molecules that are up-regulated

during inflammation. Type II phospholipase

A2, cyclooxygenase (COX)-2 and inducible

nitric oxide (NO) synthase are examples of

these genes. These genes code for enzymes that

increase the synthesis of platelet activating

factor and leukotrienes, prostanoids and NO.

Pro-inflammatory cytokine-mediated

inflammation does not normally occur in

healthy individuals (87). Interleukin (IL)-1 and

tumor necrosis factor (TNF)-α are considered

to be the prototypic pro-inflammatory

cytokines. Although inflammatory products

such as endotoxins act as triggers, the cytokines

IL-1 and TNF-α are particularly effective and

act synergistically in the process of activating

the inflammatory genes (87). Interferon (IFN)-

γ, IL-6 and IL-18 are other cytokines

considered to possess pro-inflammatory

properties. Genes coding for chemokines are

also considered to be pro-inflammatory (87).

18

Infectious and inflammatory mechanisms in preterm birth and cerebral palsy

IL-6

IL-6 is a protein consisting of 185 amino acids

and glycosylated at positions 73 and 172. It is

synthesized as a precursor protein consisting

of 212 amino acids. Monocytes express at least

five different molecular forms of IL-6 with

molecular masses of 19-26 kDa. It maps to

human chromosome 7p21-p14.

The IL-6 gene promoter contains many

different regulatory elements allowing the

induction of gene expression by various stimuli,

including glucocorticoids and cAMP. The

nuclear factor kappa B (NF-κB) binding site is

responsible for the induction of IL-6 production

in non-lymphoid cells, so NF-κB is considered

to be an important transcription factor for IL-6

(88).

IL-6 is a cytokine with pro-inflammatory and

immunomodulatory properties, produced by

many cell types including activated phagocytes,

macrophages, monocytes and endothelial cells.

IL-6 functions in both innate and specific

immunity and stimulates the synthesis of acute

phase proteins by hepatocytes as well as the

growth of antibody-producing B lymphocytes

(88). TNF-α and IL-1 stimulate the

transcription of IL-6 which acts synergistically

with these two cytokines in many situations

(87). IL-6 is known to be a multifunctional

cytokine that regulates immune response,

hematopoesis, the acute phase response and

inflammation (88).

IL-18

IL-18 is a recently described member of the

IL-1 cytokine family (due to its structure,

receptor family and signal transduction

pathways) and was initially defined as an IFN-

γ inducing factor (89, 90). IL-18 is synthesized

as a precursor, requiring caspase-1 for cleavage

into the active form (90). Human pro-IL-18

contains 193 amino acids and has a molecular

mass of 24 kDa (91). Activated IL-18 cytokine

has a molecular weight of 18kDa (92). It maps

to human chromosome 11q22 (90). The

intracellular signal pathway includes the

transcription factor NF-κB which up-regulates

transcription of the IL-18 gene (92).

IL-18 is a cytokine with pro-inflammatory and

pro-apoptotic properties. It is mainly

synthesized by macrophages, monocytes and

keratinocytes, but can also be produced by

epithelial cells (93-97). IL-18 enhances the

inflammatory process by stimulating the

production of INF-γ and TNF-α and IL-1β (91).

IL-12 can act synergistically with IL-18 to

provoke a T helper 1 response (97, 98).

4.1.3 Chemokines

Chemokines (short for chemoattractive

cytokines) are small (8-10 kDa) inducible

proteins (99, 100). Chemokines have the

capacity to activate leukocytes and mediate

inflammatory reactions. These proteins are

involved in diverse immune responses to which

they recruit various leukocytes (99). Four

classes of chemokines have been described,

based on the conserved cysteine residues on

the mature protein: α-chemokines, β-

chemokines, γ-chemokines and fractalkine or

neurotactin (101). The two major sub-families

are: C-X-C chemokines (α-chemokines) which

have an amino acid between two cysteines, and

C-C chemokines (β-chemokines), which have

two adjacent cysteines. α-chemokines possess

potent chemoattractive properties, mostly for

neutrophils; β-chemokines exert their effect on

monocytes, lymphocytes, eosinophils and mast

cells, but not on neutrophils (88, 99, 100).

IL-8

IL-8 is the prototypic α-chemokine, previously

called neutrophil attractant/activating peptide-

1. IL-8 is produced by the processing of a

precursor protein consisting of 99 amino acids.

Processing of this precursor by specific

proteases yields N-terminal variants of IL-8.

IL-8 contains 72-77 amino acids (no

glycosylated sites) and has a molecular weight

of 8-11 kDa. The IL-8 gene maps to human

chromosome 4q12-q21. NF-κB is an important

transcription factor for IL-8 as well (88, 99).

IL-8 is produced by monocytes, macrophages,

fibroblasts, endothelial cells, keratinocytes,

melanocytes, hepatocytes, chondrocytes, and

a number of tumor cell lines in many different

cell types (88, 99). The synthesis of IL-8 is

strongly stimulated by IL-1 and TNF-α in many

cell types (88, 99). IL-8 is thought to be the

19

Bo Jacobsson

primary regulatory molecule of acute

inflammatory states, because of its potent

neutrophil chemotactic effect (99) . Neutrophil

migration into the inflamed peritoneal cavity

is severely inhibited in IL-8 transgenic mice

(102).

4.2 Infectious and inflammatory

mechanisms in preterm delivery

One reason for the lack of progress in reducing

PTB is the poor understanding of the

pathophysiological process related to PTL and

pPROM (30). Pathologic processes implicated

in the etiology of the preterm labor syndrome

include infection, uteroplacental ischemia,

uterine overdistension, abnormal allograft

recognition, allergic phenomena and cervical

disease (105, 106). Over the past two decades,

the important concept has emerged that PTL

most likely represents a heterogeneous

syndrome characterized by uterine contractility,

cervical ripening, and/or membrane rupture

(105). Inflammatory responses can be initiated

by any foreign stimulus, from foreign bodies

to specific bacterial pathogens (105). As only

a minority of PTL cases can be attributed to a

clinically evident intra-uterine infection, most

PTL cases secondary to infection are likely to

be the result of an inflammatory response in

the maternal and fetal gestational tissue

generated in response to these pathogens. This

inflammatory process is mediated by cytokine

production and ultimately results in

prostaglandin production, an important cascade

in the onset and propagation of myometrial

contractility and subsequent labor and birth

(107).

4.2.1 Microbial invasion of the amniotic

cavity

Microorganisms may gain access to the

amniotic cavity by the following pathways: 1)

ascending from the vagina and cervix; 2)

hematogenic dissemination through the

placenta (trans-placental); 3) retrograde seeding

from the peritoneal cavity through the fallopian

tubes; 4) accidental introduction at the time of

invasive procedures such as amniocentesis,

percutaneous fetal blood sampling or chorionic

villous sampling (107). Several lines of

evidence support the idea of the ascending route

as the most common (29, 106, 107):

histological chorioamnionitis is more common

and severe at the membranes rupture sites than

in other parts of the membranes (108), bacteria

identified in cases of congenital infections are

similar to those found in the lower genital tract

(106, 107, 109), inflammation of the chorionic

membranes is present in most cases of

congenital pneumonia (stillbirth or neonatal),

the membranes of the first twin more often has

signs of inflammation in twin gestations and

signs of inflammation have not been observed

exclusively in the second twin in the absence

of such signs in the first twin (106, 107, 110).

A four-stage process leading to intrauterine

infection has been proposed (111). Stage I: an

overgrowth of facultative organisms or the

presence of pathological organisms in the

vagina. Bacterial vaginosis may be one of the

manifestations at this stage. Stage II: the

microorganisms ascend, gain access to the

uterine cavity and then reside in the decidua.

Stage III: a localized inflammatory reaction

20

Infectious and inflammatory mechanisms in preterm birth and cerebral palsy

leads to deciduitis which extends into

chorionitis, leading to choriovasculitis (fetal

vessels inflammation) and, via the amnion

membrane (amnionitis), into the amniotic

cavity resulting in intra-amniotic infection.

Stage IV: once in the amniotic cavity, the

bacteria may gain access to the fetus by

different routes of entry: aspiration, otitis,

conjunctivitis and omphalitis. Seeding from

any of these sites to the fetal circulation leads

to bacteremia and sepsis (111, 112).

The bacteria isolated from the upper genital

tract among women who have had PTB are all

microorganisms generally believed to be of low

virulence, typically representative of the normal

microbial flora in the cervix and vagina (113,

114). The timing of the upper genital tract

infection is not completely understood but most

evidence indicates that bacterial ascension from

the lower to the upper genital tract occurs earlier

rather than later in gestation (114, 115).

Regardless of when or how they arrive in the

upper genital tract, these bacteria may reside

in the chorio-decidual interface, where they are

associated with chronic low-grade

inflammation for many weeks before resulting

in spontaneous labor or rupture of the

membranes (114). Activation of the host

response as early as mid-trimester by elevated

levels of AF IL-6 at amniocentesis in a subset

of women that later had a late pregnancy loss

or an early spontaneous preterm delivery serves

as evidence of this process (116, 117). There is

also data available to support the possibility that

upper genital tract microbial colonization and

inflammation may be present prior to

conception (118). If Ureaplasma urealyticum

is detected at a mid-trimester amniocentesis,

the patient is likely to give birth at around 24

weeks of gestation (119, 120). Viral infections

seem to be of less importance related to late

fetal loss or early preterm delivery (116).

In summery, it is an established fact that

clinically silent upper genital tract infection and

inflammation are strongly associated with an

increased risk of spontaneous PTB (29, 106,

107, 111, 114, 120). The proportion of women

with MIAC is much greater in women whose

pregnancies have been the object of sampling

and delivery at earlier gestational age (115, 121,

122). However, although infection may only

account for perhaps 20–25% of all PTB and

although intra-amniotic inflammation (IAI)

may account for a similar proportion. Infection

and inflammation may account for the majority

of the early spontaneous PTB and thus the

majority of infant morbidity and mortality (115,

122).

4.2.2 The inflammatory process within the

uterine cavity

The main hypothesis on how ascending bacteria

can lead to spontaneous PTB is as follows:

bacterial invasion of the chorio-decidual space

activates monocytes both in the decidua and

the fetal membranes to produce a number of

pro-inflammatory cytokines. Some

combinations of these cytokines stimulate

prostaglandin synthesis and release and initiate

a sequence of neutrophil chemotaxis,

infiltration, and activation, culminating in the

synthesis and release of a number of

metalloproteases. The cytokines and

prostaglandins stimulate contractions, while the

proteases attack the chorioamniotic

membranes, leading to rupture of membranes.

Various proteases also remodel cervical

collagen, resulting in cervical ripening (120).

An overview is presented in Fig. 3.

Macrophages seem to play a pivotal role in the

defense against microbial invasion of the

uterine cavity by phagocytosis and by synthesis

and secretion of large amounts of cytokines and

other regulatory molecules. Macrophages are

a major cell type in both the maternal and fetal

compartments of the uteroplacental unit. They

are abundant in the decidua and in fibrous

tissues near the placenta. In the placenta,

macrophages can be isolated in the

mesenchymal stroma. In the extraplacental

membranes, fetal macrophages populate the

mesenchymal stroma between the amnion and

chorion layers (80).

The bacteria invading the choriodecidual

interface release endotoxins and exotoxins that

activate the decidua and the fetal membranes.

Several studies have shown that phospholipase

A2 and C and LPS are capable of stimulating

production of prostaglandins by the amnion and

21

Bo Jacobsson

decidua in vitro (123). Phospholipase A2 can

cleave and release arachidonic acid, which can

be metabolized to prostaglandins, leukotrienes

and epoxides. Substantial evidence supports the

critical role of prostaglandins in the preterm

and term labor process by inducing

myometrical contractility, as well as, changes

in the extracellular matrix metabolism

associated with cervical ripening; they are also

presumed to be involved in decidual and fetal

membrane activation (114, 123).

Until recently it was thought that bacteria alone

were responsible for various clinical and

metabolic derangements seen with infection,

but today we know that host immune responses

and endogenous products precipitated by

bacterial endotoxins are, to a large extent

responsible for the deleterious effects of the

infection (123). There is a growing body of

evidence that PTB may, in the presence of

bacterial invasion or infection, be caused by

the host-mediated response through activation

of the macrophage-monocyte system by

bacterial products and tissue injury. Proinflammatory

cytokines and chemokines (TNF-

α, IL-1α, IL-1β, IL-6, IL-8 and granulocytecolony

stimulating factor (G-CSF)) are released

on activation of these cells by microbial

products (114, 123). The release of these proinflammatory

cytokines and exotoxins create

a positive feedback loop which recruits more

monocytes and macrophages to the infected

area, sustaining the host-mediated response

(123).

Other pathways may play a role as well. In

chorionic tissue, prostaglandin dehydrogenases

normally inactivate the prostaglandins

produced in the amnion, preventing them from

reaching the myometrium and causing

contractions. If the chorionic membrane is

infected the dehydrogenase activity decreases,

allowing increasing quantities of

prostaglandins to reach the myometrium (124,

125) and uterine contractions.

Infectious and inflammatory mechanisms in preterm birth and cerebral palsy

Recent evidence suggests that infection may,

in many cases, involve the fetus. A fetal hostresponse

is induced with increased levels of

plasma cytokines. This is the background of

the concept of fetal inflammatory response

syndrome (FIRS) (123, 126, 127). In fetuses

with infection, increases in both the fetal

hypothalamic and placental production of

corticotropin-releasing hormone cause an

increase in fetal corticotropin secretion. This

results in fetal adrenal production of cortisol,

which in turn increases the production of

prostaglandins (114).

An alternative hypothesis has recently been

suggested, i.e. that the main event actually

happens in the choriodecidual interface and that

localized inflammation here is actually a

sufficient cause of preterm delivery.

Choriodecidual inflammatory syndrome

implies that this inflammatory process and its

effects, rather than a silent infection in the

amniotic cavity, are the main issues in the

pathogenesis of PTL (128). This hypothesis

does not explain the strong association between

intra-amniotic cytokines and preterm delivery.

4.2.4 Sources of cytokines in the amniotic

fluid

The suggested main possible sources of

cytokines within the AF are decidua, fetal

membranes, maternal circulation and possibly

the fetus itself. The relative contribution of the

maternal and fetal compartments to the overall

inflammatory response is unknown (114).

There are different cell types within the decidua

that can act as sources of different cytokines,

e.g. activated resident macrophages located

throughout the reproductive tract, or

macrophage-like decidual cells released in

response to bacterial products (80, 111, 166).

The idea of an extra-amniotic source of intraamniotic

cytokines, is contradicted by a study,

the findings of which indicate that, AF does

not reflect the cytokines produced by the

decidua in case with intact and non-inflamed

fetal membranes (80). The fetal membranes

have been shown to be important producers of

cytokines, which are probably released into the

AF to some extent. Different layers in the

membranes are involved in cytokine production

in different ways (Table I). IL-6 and IL-8 are

produced by both layers of the membrane (167-

170). IL-18 is produced by the chorion

membrane but not by the amnion (171). IL-1β

is probably produced mostly in the chorion

(168).

Maternal circulation can be another source but

few reports have studied this issue. Researchers

in one study proposed that IL-8 does not cross

the placenta in either direction by simple

diffusion. Likewise, no evidence of active

transfer processes for IL-8 was found (172).

This implies that activation and chemotactic

effects of chorionic and amniotic IL-8 on

neutrophil granulocytes appears to be limited

to the fetal compartment (172).

The fetus itself could be a source of the

cytokines in the AF. Many different cytokines

and chemokines have been identified in

umbilical blood from newborns, e.g. IL-6, IL-

8 and IL-18. IL-6 has been identified in

umbilical cord blood obtained by cordocentesis

and in some of those cases with high intraamniotic

levels the blood levels are also

increased (127).

4.2.5 Cytokines in amniotic fluid

The traditional view on the onset of parturition

in the presence of infection has been that the

bacteria or their products (endotoxins) directly

stimulate prostaglandin biosynthesis. As

knowledge has accumulated and understanding

of the concept has changed, it has become

obvious that there was an overlap between labor

and non-laboring groups regarding the presence

of endotoxins; a link was missing (173). After

re-consideration the concept emerged that

infection (but also other triggering agents)

induced stimulation of immune cells to produce

cytokines and the subsequent synthesis and

release of prostaglandin E2 and prostaglandin

F2α, which have been shown to induce cervical

ripening, uterine contractions and labor at term

(174-177). Cytokines also have been implicated

in the physiological mechanisms of term labor

and pathophysiological mechanisms of PTL

(173). The association between several AF

cytokines has been evaluated in relation to

different preterm outcome variables (173). It

has been proven that several cytokines in AF

are increased in cases of MIAC e.g. TNF-α

(176, 178), IL-1 (176, 179, 180), IL-6 (176,

179-189), IL-8 (190, 191), IL-18 (192),

granulocyte colony stimulating factor (G-CSF)

(191), macrophage inflammatory protein-1α

(MIP-1α) (193) and growth related protein

(GRO-α) (194, 195).

4.2.6 Intra-amniotic inflammation

The gold standard for the diagnosis of infection

in clinical medicine is the isolation and

identification of the microorganism from body

fluid or tissue. However, microbial culture may

take days, and results are often not available in

time for important clinical decisions. This is in

contrast to the diagnosis of inflammation that

is more easily arrived at (e.g. blood cell count,

cytokine determination). Therefore the

prevalence and outcome of IAI has attracted

interest. IAI is more common than MIAC and

is associated with adverse maternal and

neonatal outcome (122). The maternal and

neonatal outcome of patients with only IAI

does not differ from that of MIAC (122). IAI

seems to be twice as common as MIAC (122,

185). These studies have been performed in

patients with PTL. IAI in women with pPROM

has not been studied to the same extent as in

women in PTL, but there is one study that has

provided indirect information regarding this

issue (196). Without calling the condition IAI,

the authors actually defined the condition

similarly by drawing a receiver-operationcharacteristic

(ROC) curve for both cervical

fluid and AF IL-6 in relation to MIAC and they

found the same cut-off level (0.35 ng/mL) for

both fluids. IAI in pPROM is also related to

delivery within 7 days and significant neonatal

morbidity (196). IAI seems to have different

characteristics in women with pPROM (197).

IAI without MIAC, may nonetheless be a

bacterial reason for the inflammatory reaction.

Studies using polymerase chain reaction (PCR)

to detect bacteria in the AF have been able to

demonstrate that the sensitivity for detecting

MIAC is much higher with this technique than

with standard procedures (198, 199). Another

subgroup of IAI without MIAC is cases with

extra-amniotic (between the amniotic and

chorionic membranes) detectable bacteria.

Previous studies have demonstrated that AF IL-

6 may be elevated in these patients (185), but

still IAI might nonetheless also have a nonbacterial

cause.