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IMPERIAL COLLEGE
The roles of Prostanoid EP receptors in the
control of contractions of human myometrium
Shankari Arulkumaran
Institute of Reproductive and Developmental Biology
MD (Res)
2
TABLE OF CONTENTS
Declaration 4
Acknowledgements 5
Publications 6
Poster presentations 7
Abbreviations 8
Table of Figures 11
Abstract 13
1.1 Foreword 15
1.2 Clinical Aspects of Preterm Birth 21
1.3 The Anatomy of the Uterus and Cervix 47
1.4 The Endocrinology and Biochemistry of Labour 49
1.5 Myometrial Contractility 50
1.6 Hypothesis and Objectives 73
2.0 Materials and Methods 81
2.1 Contractility Studies 86
2.2 Drug Assessment 91
2.3 Data Analysis 93
3.1 Contractility in upper versus lower segment
myometrium
95
3.2 Contractility in prostaglandin stimulated upper and
lower segment myometrium
97
3.3 Discussion of contractility in upper and lower
segment myometrium
99
4.1 The effect of PGE2 on spontaneously contracting
lower segment myometrium.
101
4.2 The effect of PGF2α on spontaneously contracting
lower segment myometrium.
104
4.3 The effect of acetyl salicylic acid (ASA) on
spontaneously contracting lower segment myometrium
and PGE2 induced contractions
105
3
4.4 Discussion of the effects of PGE2, PGF2α and ASA
on contractility
111
5.1 The effect of an EP1 antagonist, ZD6416 upon
spontaneous and PGE2 stimulated contractions
113
5.2 The effect of an EP3 antagonist, L798106 upon
spontaneous and PGE2 stimulated contractions
119
5.3 Discussion of the role of EP1 and EP3 in human
lower segment myometrial contractility in relation to
their respective receptor antagonists
125
6.1 The effect of FPA, a PGF2α antagonist upon
spontaneous and PGF2α stimulated contractions
129
6.2 Discussion of the effect of FPA, a novel PGF2α
antagonist on contractility
133
7.1 The effect of combining the agonists, OT and PGE2
on spontaneously contracting lower segment
myometrium
135
7.2 The effect of combining the antagonists of OT and
PGE2 on spontaneously contracting lower segment
myometrium
138
7.3 Discussing the effect of combining the agonists, OT
and PGE2 and their respective antagonists on
spontaneously contracting lower segment myometrium
140
8.1 Discussion 142
References 154
4
DECLARATION
I declare that this thesis is my own work and is based on research that was
undertaken by me at Institute of Reproductive and Developmental Biology,
Imperial College Heathcare Trust from September 2005-2008. To the best
of knowledge, this thesis contains no material previously published or
written by another person, except where due reference has been made in the
text.
5
ACKNOWLEDGEMENTS
I would like to sincerely thank all the women at Queen Charlotte’s Hospital
who selflessly volunteered to partake in this project. This project would not
have been possible without their understanding of the bigger picture and
willingness to help other women and babies in need.
I have personal gratitude to my supervisor, Professor Bennett, for giving me
this opportunity. His unfailing patience and guidance has led not only to my
completion of this MD but he has been instrumental in my career
progression. I have him to blame for keeping me in Obstetrics and that is the
true definition of a mentor.
I am grateful to all the staff on the Queen Charlotte’s labour ward; past and
present, as well as my colleagues in the laborotary. In particular, to Yooni
Lee for her patience in trying to teach a clinician, basic science and to
Mandeep Kandola for her collaboration on this project.
I would like to thank Ian for his support, understanding and love throughout
my good and bad days.
I am most grateful to my parents, Arul and Gaytri for their love and support.
It is their solid foundation that has got me here and high standards that will
continue to spur me on. This MD is dedicated to them.
6
PUBLICATIONS
Arulkumaran S, Kandola MK, Hoffman B, Hanyaloglu AC, Johnson MR,
Bennett PR. The Roles of Prostaglandin EP 1 and 3 Receptors in the Control
of Human Myometrial Contractility. J Clin Endocrinol Metab. 2011.
Arulkumaran S, Bennett PR. Pathophysiology of Preterm birth in Fetal and
Neonatal Physiology. Polin, Fox, Abman (eds.) 4th
edition 2011.
Kandola MK, Almazidi M, Samarage M, Khanjani S, Arulkumaran S,
Bennett PR. The Role of EP2 and EP4 in Human Myometrium.
Reproductive Sciences 2010; (17): 81A-81A.
Taher SE, Chandiramani M, Soutter P, Arulkumaran S, Eliahoo J, Teoh T,
Hassan S, McIndoe A, Shennan A, Bennett PR. Ultrasound-indicated
cerclage for prevention of preterm delivery in primiparas following
treatment of cervical intraepithelial neoplasia. Ultrasound Obstet Gynecol
2009; 34(S1):144.
Arulkumaran S, Chollet A, Bennett PR. The role of PGF2 on myometrial
contractility studied with a selective FP antagonist. Reproductive Sciences
2008; (15): 3A-57A.
Arulkumaran S, Kandola M, McArthur Wilson RJ, Brooks DP, Bennett PR.
EP3 versus EP1 receptor antagonism in human term myometrial
contractility. Reproductive Sciences 2008; (15): 3A-57A.
Arulkumaran S, Kandola M, Wilson RJ, Brooks DP, Bennett PR. The Role
of Prostaglandin E2 Receptors in Human Myometrium During Pregnancy.
Reproductive Sciences 2007; (14): 227.
7
POSTER PRESENTATIONS
The role of PGF2 on myometrial contractility studied with a selective FP
antagonist.
Arulkumaran S, Chollet A, Bennett PR.
Society for Gynecologic Investigation International Conference, San Diego,
California, United States of America. March 2008.
EP3 versus EP1 receptor antagonism in human term myometrial
contractility.
Arulkumaran S, Kandola M, McArthur Wilson RJ, Brooks DP, Bennett PR.
Society for Gynecologic Investigation International Conference, San Diego,
California, United States of America. March 2008.
The role of prostaglandin E2 receptors in human myometrium during
pregnancy.
Arulkumaran S, Kandola M, Wilson RJ, Brooks DP, Bennett PR.
Society for Gynecologic Investigation International Conference, Las Vegas,
Nevada, United States of America. March 2007.
8
ABBREVIATIONS
AA arachidonic acid
ADH anti-diuretic hormone
ANOVA one-way analysis of variance
ASA acetyl salicylic acid
ASF/SF2 alternative splicing factor/splicing factor 2
ATP adenosine triphosphate
BkCa large conductance calcium-activated potassium (channel)
BMI body mass index
BV bacterial vaginosis
Ca Cl2 calcium chloride
Ca2+
calcium ion
cAMP cyclic adenosine monophosphate
CAP contraction associated protein
CI confidence interval
COX Cyclooxygenase
CPI-17 myosin phosphatase inhibitor phosphoprotein
CRH corticotrophin releasing hormone
CRP C-reactive protein
CX-43 connexin -43
DAPI 4',6-diamidino-2-phenylindole fluorescent stain
EP prostaglandin E receptor
fFN fetal fibronectin
Fig. Figure
FP prostaglandin F receptor
FPA prostaglandin F antagonist (Serono)
G protein guanine triphosphate-binding protein
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
GPCRs G-protein coupled receptors
GSK Glaxo-Smith Kline
GTN Glyceryl trinitrate
GTP guanosine triphosphate
GW796679X OT anatgonist (GSK)
Gαi / Gαq / Gαs heterotrimeric G protein subunit
IL-6 interleukin -6
IL-8 interleukin -8
InsP3 inositol 1, 4, 5,-triphosphate
IVH intraventricular haemorrhage
KCL potassium chloride
9
kDa kilo Dalton
KH2PO3 monopotassium phosphate
Ki dissociation constant
L- labour minus
L+ labour plus
L798106 EP3 antagonist (GSK)
LLETZ laser loop excision of the transformation zone
LPS Lipopolysaccharide
LS lower segment
MgSO4 Magnesium sulphate
MLCK myosin light chain kinase
MLCP myosin phophotase
MMP matrix metalloproteinase
MRC Medical Research Council
mRNA messenger ribonucleic acid
NaHCO3 sodium bicarbonate
NaCl sodium chloride
NF-κB nuclear factor kappa beta
No. Number
NSAID non-steroidal anti-inflammatory drug
OT oxytocin
OTR oxytocin receptor
p Probability
pc personal computer
PG prostaglandin
PGD2 prostaglandin D2
PGE2 prostaglandin E2
PGF2α prostaglandin F2 alpha
PGG2 hydroperoxy endoperoxide
PGH2 hydroxyl endoperoxide
PGHS prostaglandin G/H synthase
PGI2 Prostacylcin
pH power' of hydrogen ion
PKA cAMP dependent protein kinase
PPROM preterm premature rupture of membranes
PR Progesterone
PUFA unbalanced polyunsaturated fatty acids
RCOG Royal College of Obstetricians and Gynaecologists
RDS respiratory distress syndrome
RNA ribonucleic acid
10
RR relative risk
RU486 Mifepristone
SDS-PAGE sodium dodecyl sulphate polyacrylamide gel
electrophoresis
SNP single nucleotide polymorphism
SP-A surfactant protein A
STD sexually transmitted disease
TXA2 Thromboxane A2
TXB2 Thromboxane B2
TNFα tumour necrosis factor alpha
US upper segment
ZD6416 EP1 antagonist (GSK) oC degree Celcius
β Beta
γ Gamma
11
TABLE OF FIGURES
Chapter 1
Page
Fig. 1.1 Cerebral palsy rates according to gestational age,
infant gender and plurality 16
Fig. 1.2 Neonatal survival by gestational age and improvement
in survival by week 22
Fig. 1.3 Epidemiological and environmental risk factors for preterm
labour 25
Fig. 1.4 Chorioamnionitis 28
Fig. 1.5 Placental abruption 30
Fig. 1.6 Cervical length measurement 36
Fig. 1.7 Anatomy of the non-pregnant uterus 49
Fig. 1.8 Factors influencing preterm labour 51
Fig. 1.9 Pathway for action of placental corticotrophin releasing
hormone on parturition. 54
Fig. 1.10 Biosynthetic pathway for the formation of eicosanoids derived
from arachidonic acid 58
Fig. 1.11 Signaling pathways for EP1 and EP2/4 67
Fig. 1.12 Signaling pathways for EP3 isoforms 69
Fig. 1.13 Summary of the literature findings with regards to myometrial
expression and changes of EP1 and EP3 72
Fig. 1.14 Labour associated change of EP1 receptor protein expression
in human myometrium at term 75
Fig. 1.15 Labour associated change of EP3 receptor protein expression
in human myometrium at term 76
Fig. 1.16 EP1 receptor expression in matched LS and US segments of
term L+ and L- myometrium 78
Fig.1.17 EP3 receptor expression in matched LS and US segments of
term L+ and L- myometrium 79
Chapter 2
Fig. 2.1 Myometrial sample taken from the lower segment of human
myometrium at elective caesarean sections at term 82
Fig. 2.2 Myometrial sample taken from the upper segment of human
myometrium at elective caesarean sections at term 84
Fig. 2.3 A cervical punch biopsy 85
Fig. 2.4 The Myograph 800MS 87
Fig. 2.5 The effect of stretch on myometrial contractility 89
Fig. 2.6 A real time image of spontaneous contractions 90
Fig. 2.7 An illustration of the parameters used to calculate the data 94
12
Chapter 3
Fig. 3.1 Spontaneous contractility in upper versus lower segment
myometrium 96
Fig. 3.2 Contractility in prostaglandin stimulated upper and lower
segment myometrium 98
Chapter 4
Fig. 4.1 A pictorial representation of increasing concentrations of
PGE2 on lower segment myometrium 101
Fig. 4.2 The effect of PGE2 on spontaneously contracting lower
segment myometrium 103
Fig. 4.3 A pictorial representation of increasing concentrations of
PGF2α on lower segment myometrium 104
Fig. 4.4 The effect of PGF2α on spontaneously contracting lower
segment myometrium 106
Fig. 4.5 The effect of ASA on spontaneously contracting lower
segment myometrium 108
Fig. 4.6 The effect of ASA on PGE2 induced contractions 110
Chapter 5
Fig. 5.1 The effect of increasing concentrations of ZD6416 on
spontaneous contractions 114
Fig. 5.2 The effect of pre-incubating LS myometrium with ZD6416 on
spontaneous contractions 116
Fig. 5.3 The effect of ZD6416 on PGE2 stimulated contractions 118
Fig. 5.4 The effect of increasing concentrations of L798106 on
spontaneous contractions 120
Fig. 5.5 The effect of pre-incubating LS myometrium with L798106
on spontaneous contractions 122
Fig. 5.6 The effect of L798106 on PGE2 stimulated contractions 124
Chapter 6
Fig. 6.1 The effect of increasing concentrations of FPA on
spontaneous contractions 130
Fig. 6.2 The effect of FPA on PGF2α stimulated contractions 132
Chapter 7
Fig. 7.1 The effect of combining OT and PGE2 on spontaneous
contractions 137
Fig. 7.2 The effect of combining GW796679X and L798106 on
spontaneous contractions 139
13
ABSTRACT
The primary function of the uterus during pregnancy is to harbour the
growing fetus in a quiescent environment. Upon maturation of the fetus, the
uterus generates forceful contractions during labour. Prostaglandins are
central in the parturition process. PGE2 in particular, is produced in large
quantities by fetal membranes and possible roles include cervical ripening
and the stimulation of uterine contractions.
PGE2 exhibits a wide spectrum of physiological actions depending on the
distribution and subtypes of EP receptors. EP1 and EP3 mediate
contractions, but there is no consensus about their relative importance.
Preliminary experiments were undertaken to examine the possible
expression patterns of EP1 and EP3. The expression data showed there to be
no significant changes between the localisation in upper or lower segment
biopsies using both RNA and protein samples. However, there was a labour
associated change seen in EP3 isoform expression at mRNA level together
with changes in the protein expression of EP3 in the lower segment using
immunofluorescence. Given that the expression studies suggest that it is the
EP3 receptor, and not the EP1 receptor, that is most important in
myometrial contractility, I established an in vitro tissue bath to conduct
functional studies examining contractility in upper and lower segment
myometrial biopsies.
14
My data demonstrated that stretch of term human myometrium results in
spontaneous contractions. There was no difference between upper and lower
segment myometrium, so subsequent experiments were conducted on lower
segment biopsies. The addition of acetyl salicylic acid (a non-selective COX
inhibitor) did not completely stop spontaneous contractility but reduced the
total work done significantly. This could be reversed by add back of PGE2.
Prostaglandins are therefore significant but not crucial for spontaneous
contractility. Both PGE2 and PGF2α increased the total work done
significantly compared to spontaneous contractions; E2 more so than F2α.
The PGF2α antagonist did not inhibit spontaneous contractions, but did
inhibit PGF2α induced contractions. The EP1 antagonist did not inhibit
spontaneous contractions but EP3 antagonist did. These results suggest that
an EP3 antagonist is a better candidate as a new tocolytic compound
compared to FP or EP1 antagonist.
15
1.1 Foreword
For the most part of the 20th
century, preterm birth, defined as birth at less
than 37 completed weeks of gestation, was viewed as an unpredictable and
inevitable fact of life. Medical efforts thus focused on reducing the
consequences of prematurity, rather than preventing its occurrence. This
approach resulted in improved neonatal outcomes, but preterm birth remains
costly both emotionally and financially to the affected infants and their
families, and adds an economic burden to society.
The EPIPAGE study (Larroque B et al 2008) concluded that one in ten
children born before 33 weeks gestation had cerebral palsy by the age of
five years and 40% had some kind of disability. Fig 1.1 illustrates the
prevalence of cerebral palsy decreasing with increasing gestational age (i.e.
20% at <27 weeks, 12% at 27 to 28 weeks, 8% at 29 to 30 weeks, 7% at 31
weeks, and 4% at 32 weeks).
16
No.
Proportion With Cerebral
Palsy, % P
Total 1954 8.2
Gestational age at
birth
<.001
24–25 wk 62 19.4
26 wk 82 22.0
27 wk 146 12.3
28 wk 191 11.0
29 wk 196 8.2
30 wk 315 8.3
31 wk 424 6.8
32 wk 538 4.4
Infant gender .10
Male 1018 9.2
Female 936 7.1
Plurality .35
Singletons 1339 8.2
Twins 529 8.9
Triplets and
quadruplets
86 4.4
Fig 1.1 Cerebral Palsy Rates According to Gestational Age, Infant
Gender and Plurality. Cerebral palsy was slightly but not significantly
more frequent among boys than girls. There was however, no
difference in cerebral palsy rates according to plurality (Pierre-Yves
Ancel 2006).
17
Despite diseases of pregnancy and the perinatal period contributing
considerably to the burden of ill health worldwide, there is an apparent lack
of initiative by pharmaceutical companies in this field. Fisk et al (2008)
found just 17 obstetric drugs being developed, compared with 660 for
cardiovascular disease and 34 for amyotrophic lateral sclerosis. Only 9 of
the 17 are completely new, and interestingly, these are mostly oxytocin
antagonists. The authors claim pregnant women are still being treated with
drugs belonging to a bygone age – magnesium sulphate, nifedipine and
hydralazine, and that the market must therefore be modified to allow
obstetric therapeutics to modernise.
A lack of detailed information on the mechanisms of preterm labour has
hindered the development of novel treatments and preventative measures.
Therefore, there is a huge unmet need for effective tocolysis. Tocolytics are
medications used to suppress premature labor (from the Greek tokos,
childbirth, and lytic, capable of dissolving). The use of a tocolytic drug is
associated with a prolongation of pregnancy for up to one week, but with no
significant effect on preterm birth or on perinatal or neonatal morbidity.
However, if the few days are put to good use, such as completing a course
of corticosteroids or in utero transfer, tocolysis should be considered.
Indomethacin, a non-selective prostaglandin G/H synthase (PGHS)
inhibitor, has been used widely in the prevention of preterm birth. King JF
et al (2005) assessed the effects on maternal, fetal and neonatal outcomes of
18
COX inhibitors administered as a tocolytic agent to women in preterm
labour when compared with (i) placebo or no intervention and (ii) other
tocolytics. The review included data from 13 trials with a total of 713
women. Indomethacin was used in 10 trials. When compared with placebo,
COX inhibition (Indomethacin) resulted in a reduction in birth before 37
weeks' gestation (one trial, 36 women), an increase in gestational age and
birthweight (two trials, 67 women). A comparison of non-selective COX
inhibitors versus any COX-2 inhibitor (two trials, 54 women) did not
demonstrate any differences in maternal or neonatal outcomes. Due to the
small numbers, all estimates were thought to be imprecise. However, it has
been its adverse effects, in particular effects on the fetal kidneys and the
ductus arteriosus, as well as associations with an increased risk of intra-
ventricular haemorrhage (IVH) and necrotizing enterocolitis in the neonate
that have limited the use of Indomethacin (Seubert De et al 1999).
Nimesulide, a PGHS-2 selective non-steroidal anti-inflammatory drug
(NSAID) has been used in women at high risk of preterm delivery but is
also associated with serious fetal side effects (Sawdy RJ, Lye S et al 2003).
Rofecoxib, a PGHS-2 specific inhibitor is associated with few fetal side
effects but does not reduce the incidence of preterm delivery and is
associated with an increased risk of late preterm birth (Groom KM et al
2005). The authors give a number of possible explanations for the
disappointing efficacy of prostaglandin synthesis inhibitors including the
possibility that the inhibition of COX-2 may activate feed-forward
19
mechanisms, therefore increasing the availability of arachidonic acid or
directly increasing COX-2 expression. Furthermore, it may be that once the
COX-2 pathway is inhibited, prostaglandins are synthesised via another
pathway, such as COX-1. Finally, it is possible that the inhibition of
prostaglandin synthesis alone is not sufficient to stop the inflammatory
cascade and therefore the process leading to labour continues via other
mechanisms.
Drugs which inhibit the various PG receptors are now available raising the
possibility that specific inhibition of PG receptors might be a strategy to
prevent preterm birth. PG receptors are coupled via G proteins to the second
messengers cAMP, inositol triphosphate or intracellular calcium release. PG
receptors have been classified pharmacologically into five main subtypes
(DP, EP, FP, IP, and TP) one for each of the major classes of PGs i.e. PGD,
PGE, PGF, prostacyclin and thromboxane respectively. Inhibition of FP has
been proposed as a strategy to prevent preterm delivery. FP antagonists have
been shown to inhibit contractions in isolated sheep and human
myometrium (Olson DM et al 2003) and to open BKCa channels (Doheny
HC et al 2007). FP antagonists also delay RU486 (Mifepristone) or
lipopolysaccharide (LPS) induced labour in mice, rat and sheep (Olson DM
et al 2003, Chollet A et al 2007). However PGE2 is a major PG product of
fetal membranes and decidua and is thought to act to induce labour partly
through diffusion to the myometrium. PGE2 binding to myometrium has
been shown to be 8-10 fold higher than PGF2α (Hoffman HJ et al 1984).
20
PGE2 binds to its myometrial receptor with higher affinity than does PGF2α
(Giannopoulos G et al 1985) to its receptor, and PGE2 has been shown to be
8-10 fold more potent than PGF2α in stimulating myometrial contractions
(Martin RL et al 2002). Targeting the PGE receptor alone, or in combination
with other receptor agonists/ antagonists, may therefore be a more effective
strategy for preventing or delaying preterm delivery.
21
1.2 Clinical Aspects of Preterm Birth
Epidemiology
Preterm birth refers to birth before 37 completed weeks of gestation. It
accounts for 6-10% of all births in Western countries and more than two-
thirds of all perinatal deaths. Preterm birth may result from preterm labour,
spontaneous or induced, or it may be by planned Caesarean section due to
maternal or fetal complications (Murphy DJ 2007). It is estimated that up to
30-40% of all cases are induced or elective and the remaining 60-70% occur
spontaneously (Khan KS et al 2007). Epidemiological studies of preterm
labour vary in terms of categorization, but generally, labour at <24 weeks is
considered pre-viable, <28 weeks is extremely preterm, 28-31 weeks very
preterm and 32-36 weeks mildly preterm (Moutquin JM 2003). Gross
outcomes after 34 weeks are generally considered to be as good as those
after 37 weeks gestation (Fig 1.2), though the risk of minor morbidities
remains (Seubert DE et al 1999). It is the 3-7% of preterm births that occur
before 34 weeks gestation that account for around 75% of neonatal mortality
and 50% of long term neurological impairment in children (Khan KS et al
2007).
22
Gestational
age
(weeks)
Approximate
survival
(%)
Approx
improvement in
survival per week
(%)
21 0 -
22 Rare -
23 25 25
24 50 25
25 70 10
26 80 6
27 86 5
28 91 3
29 94 1
30 95 1
31 96 1
32 97 1
33 98 1
34 99 1
35 99+ <1
36 99+ <1
Fig 1.2 Neonatal survival by gestational age and improvement in
survival by week. Gross outcomes after 34 weeks are generally
considered to be as good as those after 37 weeks gestation
(Seubert DE et al 1999).
23
Risk factors for preterm labour include race, age, socio-economic status and
body mass index (BMI). In the United Kingdom, the risk of preterm birth is
6% in white Europeans but 10% in Africans or Afro Caribbeans (Murphy
DJ 2007). Similarly, African-American women are over-represented in rates
of pre-term birth in the United States. A poorer socio-economic status in
such groups and other minority groups add environmental causes to the
plausible genetic causes of preterm birth (i.e. polymorphisms in tumour
necrosis factor alpha, TNF-α, a pro-inflammatory cytokine) (Macones GA et
al 2004, Engel SA et al 2005). Other environmental causes include nutrition,
substance abuse, and psychosocial factors (Murphy DJ 2007).
A strong association with preterm birth in women with a low BMI, poor
weight gain or excess weight gain and obesity was reported in eight primary
studies of 122,647 women (Dietz PM et al 2006). Alcohol consumption,
smoking and cocaine exposure all increase the risk of preterm birth. Dose-
response effects have been described in alcohol consumption in various
European studies, as well as increased risks from low to moderate and heavy
smoking (Albertsen et al 2004, Burguet et al 2004). Studies from the United
States confirm increased risks with cocaine exposure and other psychiatric
and substance use disorders (Bada HS et al 2005, Kelly RH et al 2002).
Maternal stress may increase susceptibility to preterm labour. Maternal
stress may act via a neuroendocrine pathway that activates the maternal-
placental-fetal endocrine systems that promote parturition via an immune
and/or inflammatory pathway (Wadhwa et al 2001). The complex
24
interactions between maternal demographics, environmental exposures and
genetic susceptibility of the mother and fetus require a coordinated research
approach that brings together the disciplines of epidemiology, laboratory
based science and clinical trials to offer the best hope of significant
advances in understanding preterm birth (Murphy DJ 2007) (Fig 1.3).
25
Maternal Demographic
African-American / Hispanic race
Low BMI/ excessive weight gain/ loss
Young maternal age
Obstetric
Previous early pregnancy loss
Previous preterm birth
Short inter-pregnancy interval (<12 months)
Medical
Procedures – LLETZ, amniocentesis
Fetal Male gender
Multi-fetal pregnancy
Assisted conception
Paternal Older paternal age
Environmental Infection
Bacterial vaginosis/ STD’s
Peridontal infection
Socioeconomic/ Psychosexual
Social inequality
Physical violence
Marital status – single/ cohabitation
Stressful/ traumatic life events/ depression
Substance use/ toxins
Excess alcohol - >3 drinks/ day
Smoking – particularly moderate/ heavy
Cocaine
Pollutants – sulphur dioxide/ particulate matter
Nutrition
Elevated homocysteine/ suboptimal B-12, B-6
Unbalanced polyunsaturated fatty acids (PUFA)
Genetic TNF-a pro-inflammatory cytokine – polymorphisms
Factor VII/XII – polymorphisms
Fig 1.3 Epidemiological and environmental risk factors for preterm
labour (Murphy DJ 2007).
26
Causes of Preterm Labour
Common associations with the onset of preterm labour in the human are
intrauterine infection, placental abruption, cervical incompetence, premature
membrane rupture, multiple pregnancy, intrauterine death, fetal and uterine
abnormalities and chorioamnionitis. However in many cases, there is no
discernable underlying pathology (Blanks AM et al 2007). Whilst current
antenatal screening relies on overt clinical and environmental signs and
symptoms, the clustering of preterm labour within families and recurrence
in susceptible women presents the case for investigating a possible
underlying genetic predisposition (Orsi NM et al 2007).
Infection
There is a strong correlation between infection within the uterus and the
onset of spontaneous preterm labour. Infection has the potential to activate
all of the biochemical pathways ultimately leading to cervical ripening and
uterine contractions; even more so in a cervix that may be shortened or
weakened. The bacterial species identified in the majority of cases of
congenital infections is usually identical to that in the maternal lower genital
tract. Following twin preterm delivery, chorioamnionitis is more common
and severe in the presenting twin than in the second twin. These factors all
suggest that ascending infection from the lower genital tract is the
commonest mechanism for chorioamnionitis (von Dadelszen P 2003).
27
There is also a recent hypothesis surrounding chronic periodontal infection
acting as reservoirs for bacterial products and/ or inflammatory mediators
that play a role in the development of preterm labour. Moderate to severe
periodontal disease identified in the antenatal period, is associated with an
increased risk of preterm delivery, as is periodontal disease progression
(Offenbacher S et al 2006). Several causative pathogens have been
identified, including the prevotella and pophyromomas species (Urban E et
al 2006).
Chorioamnionitis
Chorioamnionitis usually starts with overgrowth of potential pathogens in
the maternal lower genital tract. This is followed by the presence of the
organisms in the uterine cavity, particularly in the decidua of the lower
segment. A localized inflammatory reaction then occurs, leading to
deciduitis, chorionitis and extension through the amnion into the amniotic
cavity (Fig 1.4), before finally infection of the fetus itself, by aspiration and
swallowing of infected amniotic fluid (Romero R et al 1988). The most
common microbial isolates from the amniotic cavity of women in preterm
labour are ureaplasma, urealiticum, fusibacteria and mycoplasma hominis.
Bacterial vaginosis and trichomonas vaginalis are the two most studied
organisms associated with preterm birth (Carey JC et al 2000). However,
screening and treatment of bacterial vaginosis does not seem to influence
the increased risk of preterm birth (Klebanoff MA et al 2005).
28
Fig 1.4 Chorioamnionitis is defined by the overgrowth of potential pathogens in the
maternal lower genital tract followed by the presence of the organisms in the
uterine cavity, particularly in the decidua of the lower segment. A localized
inflammatory reaction then occurs, leading to deciduitis, chorionitis and extension
through the amnion into the amniotic cavity before finally infection of the fetus itself
occurs via aspiration and swallowing of infected amniotic fluid.
29
Abruption
Placental abruption (Fig 1.5) may lead to the onset of preterm labour. This is
thought to be through release of thrombin which stimulates myometrial
contractions by protease activated receptors. This may explain the clinical
impression that preterm labour associated with chorioamnionitis is often
rapid whereas that associated with placental abruption is less so, because in
placental abruption there is no pre-ripening of the uterine cervix.
Generation of thrombin may also play a role in preterm labour associated
with chorioamnionitis when it is released as a consequence of decidual
haemorrhage (Nath CA et al 2007).
30
Fig 1.5 Placental abruption is a complication of pregnancy, wherein the placenta
has separated from the uterus of the mother. It is the most common pathological
cause of late pregnancy bleeding. In humans, it refers to the abnormal separation
after 20 weeks of gestation and prior to birth.
31
Cervical Incompetence
The cervix maintains the fetus in situ during pregnancy and dilates during
labour to allow delivery of the baby. This distal component of the uterus is
primarily composed of connective tissue, of which the majority is collagen
arranged in fibres and embedded in proteoglycans. There is a small amount
of smooth muscle (10%) and fibroblast cells (Thomson A et al 2005). Prior
to and in early labour, the biochemical makeup of the cervix changes,
making it softer (i.e. ripens) and allowing for dilatation. These biochemical
changes include a decrease in collagen associated with an increase in
cervical prostaglandins, pro-inflammatory cytokines, leucocytes, cell
adhesion molecules and nitric oxide synthase production (Norman JE 2007).
Classical cervical incompetence may be diagnosed in women who have an
inherent weakness in the cervix or those who have a weakened cervix due to
previous medical or surgical treatments. Such treatments include mechanical
cervical dilatation at the time of a termination of pregnancy or surgical
evacuation of products of conception. During treatment for cervical
neoplasia, destruction of cervical tissue can occur due to laser treatment or
biopsies, and in the case of cervical carcinoma, the entire cervix can be
removed during a trachelectomy. It is now recognized that ‘cervical
competence’ is a continuum. In a woman with a shortened or damaged
cervix, bacteria may ascend into the lower segment of the uterus, stimulate
an inflammatory response, which itself leads to further cervical shortening
and dilation and so to a ‘vicious cycle’ ending with cervical dilatation and
32
then labour. The same syndrome may be seen where the cervix is of normal
length but the microbiological load is high. Variations in microbiological
load may lead to delivery at different gestational ages in different
pregnancies in the same woman.
Multiple pregnancies
The rate of preterm labour is high in clinical conditions such as multiple
pregnancy and polyhydramnios, which are associated with uterine stretch.
Over distension of the uterus leads to premature up regulation of contraction
associated proteins which mediate cervical ripening. Studies in sheep have
shown that stretch increases myometrial the expression of oxytocin
receptors (OTR) and COX-2 (Wu WX et al 1999); whilst in human models,
stretch of myometrial and amnion epithelial cells have been shown to
increase the expression of COX-2 and PGE2 synthesis (Mohan AR et al
2007). In addition, multiple pregnancies are associated with multiple
placentae, and therefore an earlier rise in placental CRH concentrations in
the circulation and subsequently higher rates of preterm birth (Warren WB
et al 1990).
Genetics
The most consistent risk factor in identifying preterm labour is a previous
pregnancy resulting in preterm birth. Most studies quote a risk of 15-18%
with one previous preterm birth and 28-32% with two previous preterm
deliveries (Hoffman HJ et al 1984, Carr-Hill RA et al 1985). This has been
33
suggested to be evidence for a genetic basis to preterm labour. However,
this is problematic, as preterm labour is a complex syndrome involving
various aetiologies and is masked by multiple confounding environmental
risk factors. To date, a single gene determinant for preterm labour inherited
in a conventional Mendelian manner has not been identified. Instead, it is
generally accepted that susceptibility to preterm labour is governed by
multigene effects (Orsi NM et al 2007). These effects would regulate
inflammatory, uteroplacental, endocrine, uterine contraction/ remodeling
and metabolic pathways (Berhman RE et al 2007).
Matrix metalloproteinases (MMPs) are proteolytic enzymes that cause
extracellular matrix degradation and are associated with tissue destruction
and remodeling. In preterm labour, activation is a cytokine-mediated
inflammatory process, resulting in the rupture of fetal membranes and
cervical ripening/ dilation. Single nucleotide polymorphisms (SNPs) which
may regulate MMP expression and activity have been studied. At present, a
vast number of polymorphisms have been identified in these proteases and
their tissue inhibitors (Orsi NM et al 2007, Fujimoto T et al 2002). In
addition to SNPs, complementary analytical approaches such as microarray
technology and chip arrays have now all been harnessed into determining
the susceptibility to preterm labour (Esplin MS et al 2005). Once these
genetic polymorphisms can be clearly related to physiological effects,
individual predispositions to preterm labour may enable a more targeted
approach for preventative care.
34
Prediction of Preterm Labour
As precise disease mechanisms behind preterm birth are not so well
understood, clinical predictive tests and preventative treatments remain
limited. However, if women who are at high risk of spontaneous preterm
birth in early pregnancy can be identified, they may be targeted for more
intensive antenatal care and prophylactic interventions. A history of
previous preterm birth, second trimester loss or induced abortion is a non-
invasive, simple, cost-effective method to identify women who may benefit
from more and intensive screening. Further assessment can be made by the
use of ultrasonographic cervical length measurement, cervical-vaginal fetal
fibronectin screening and the detection of uterine contractions.
Fetal fibronectin
Fetal fibronectin (fFN) is an extracellular matrix found in the decidua
basalis next to the placental intervillous space. It helps to attach the fetal
membranes to the uterine decidua and when mechanical or inflammatory
changes occur, is leaked into the cervico-vaginal fluid. It can be measured
quantitatively in cervico-vaginal secretions, obtained from the posterior
fornix when performing an internal examination with a speculum. A
commercial kit for use on-site has been produced, which gives a positive
result when concentrations are > 50ng/ml.
fFN is useful in women in threatened preterm labour with not only strong,
painful contractions, but also in those who are asymptomatic, where a
35
positive test is considered to reflect the consequences of intrauterine
infection (Shennan A et al 2006). At present, it is widely accepted as a good
test for excluding preterm birth in those presenting with threatened preterm
labour due to its strong negative predictive value: 0.5% deliver within 7-10
days after a negative test (Honest H et al 2002). Furthermore, a positive
result is associated with the greatest odds ratio for preterm delivery before
35 weeks gestation (Goldenburg RL et al 2001).
Cervical length measurement
Ultrasonographic cervical length measurement is a reproducible method of
assessing the cervix, with little inter- and intra-observer error (Fig 1.6). A
transvaginal probe is used to obtain the length between the internal and
external os. The risk of preterm delivery increases exponentially with
decreasing length, from <1% at 30mm to 80% at 5mm (Heath VC et al
1988). In women with symptoms of preterm labour, it is estimated that 50%
of women with a cervical length of less than 15mm deliver within 7 days
(Nicolaides KH et al 2004).
36
A.
B.
Fig.1.6 In ultrasonographic cervical length measurements, a transvaginal
probe is used to obtain the length between the internal and external os.
Cervical shortening is demonstrated above by A) a normal cervical canal
with a closed internal os and B) funneling and shortening of the cervix.
37
Prevention of preterm labour
Cerclage
If preterm labour does occur secondary to cervical incompetence, inserting a
suture around the cervix is commonly used to increase mechanical strength
to the cervix and thereby prevents premature opening in a subsequent
pregnancy. There are two approaches to the cerclage; abdominal and
vaginal. In the vaginal approach, the McDonald or Shirodkar methods are
employed. The McDonald approach involves a suture or tape placed around
the cervix at the junction between the vagina and the cervix. In the
Shirodkar suture, the supra-cervical vagina is incised and the bladder
reflected upwards, allowing the suture to be placed at the cervico-isthmic
junction. The vaginal epithelium is then repaired (Norman JE 2007).
The most common indication for an abdominal cerclage is a failed vaginal
cerclage. It requires a transverse incision in the lower abdomen and the
peritoneum over the cervix and bladder to be deflected down, in order to
place a suture above the cardinal and uterosacral ligaments. A caesarean
section is required to deliver the baby and the suture is often not removed,
unlike in the vaginal cerclage, where removal at 37-38 weeks gestation
enables vaginal delivery of the baby. Abdominal cervical cerclage may also
be placed laparoscopically, although published data is currently limited to
isolated case reports. Although cervical cerclage is widely used in clinical
practice, there is still limited evidence on its efficacy, whether it is
38
beneficial once cervical shortening has occurred and at what length to
intervene (Simcox R et al 2007). An international multicentre trial
conducted by the Medical Research Council (MRC) and Royal College of
Obstetricians and Gynaecologists (RCOG) recruited 1292 women with a
history of early delivery or cervical surgery. The findings showed 25
women needed to be treated to prevent one preterm delivery with no benefit
to the neonate. However, in women with three or more second trimester
losses, a cervical suture halved the incidence of preterm delivery prior to 33
weeks gestation, and in these cases a cervical cerclage was recommended
(MRC/RCOG 1984).
Progesterone
Progesterone is a pro-pregnancy hormone and withdrawal in many animal
models, have shown to lead to an onset of labour. In humans, a functional
withdrawal is more likely but supplementation of progesterone may
nevertheless be beneficial in preventing preterm labour. Progesterone has a
negative effect on many contraction associated proteins and on
prostaglandin activity (Patel FA et al 1999). In addition, it decreases the
uterine sensitivity to oxytocin (Grazzini E et al 1998). Placebo controlled
studies using 100mg progesterone or placebo as a vaginal suppository
between 24 and 34 weeks, found a significant reduction in preterm delivery
rates at <37 weeks and <34 weeks in high risk populations (da Fonseca EB
et al 2003). A study which also examined neonatal outcomes showed a
reduction in the incidence of birth weight <2500g, necrotizing enterocolitis,
39
intraventricular haemorrhage and the need for supplemental oxygen, but not
perinatal death or respiratory distress syndrome (Meis PJ 2003). Studies of
the clinical use of progesterone are ongoing. On the available evidence, it is
estimated that one in seven high risk women need to be treated with
progesterone prophylactically to prevent one case of spontaneous preterm
birth before 34 weeks gestation (Khan KS et al 2007).
Management
The management of preterm labour may be prophylactic to prevent the onset
of preterm labour, as discussed above, or reactive once preterm labour has
started. The primary aims of such measures are for the antenatal
administration of corticosteroids to the mother and for in utero transfer from
a peripheral unit to a hospital with neonatal intensive care facilities where
the outcome for the preterm neonate can be improved significantly. It is
therefore essential that a diagnosis of preterm labour should not be
overlooked. It is usual to define the onset of labour at term as being when
regular uterine contractions lead to cervical change or dilatation, but an
initial diagnosis of preterm labour is usually based upon contractions alone.
The majority of women diagnosed as being in preterm labour based upon
contractions alone will go on to deliver at term without intervention.
Therapy can be more accurately targeted if it is limited to women who are
either fibronectin positive or have a short cervix.
40
Currently used tocolytics
Tocolytics may delay delivery for short periods of time in a minority of
women. Worldwide the recommendation for their use is varied and the
preferred pharmacological agent differs significantly. They include non
steroidal anti-inflammatory drugs (NSAIDS), β-adrenergic agonists,
oxytocin antagonists, calcium channel blockers, magnesium sulphate and
nitric oxide donors.
β-mimetics, such as Ritodrine and Salbutamol have been used widely as
tocolytics. They stimulate β2-adrenergic receptors in smooth muscle, which
act via cAMP to reduce sensitivity and absolute levels of intracellular
calcium, causing myometrial relaxation (Groom KM 2007). They may be
successful in delaying preterm delivery for a 48 hour period, but have no
significant impact on improving neonatal morbidity or mortality.
Sympathomimetics are associated with significant and severe maternal side
effects. These include chest pain, dyspnoea, tachycardia, palpitations,
tremor and hypokalaemia (Giles W et al 2007).
NSAIDS such as Indomethacin, appear to be able to delay delivery for 7-10
days and reduce rates of delivery <37 weeks gestation (Keirse M 1995).
They inhibit the cyclo-oxygenase (COX) enzyme responsible for the
conversion of arachidonic acid to prostaglandins. Their use is limited
however, by fetal side effects. Premature closure of the ductus arteriosus can
occur in up to 10-50% of fetuses exposed to indomethacin and is more
41
prevalent in later gestations (>32 weeks) and if treatment occurs for
>48hours (Macones GA et al 2001). Other concerns include risks of
necrotizing enterocolitis, intraventricular haemorrhage and effects on the
fetal kidneys (Norton ME et al 1993, Kirshon B et al 1991). Selective COX-
2 inhibitors, such as Rofecoxib have been trialed, but unsuccessfully.
Rofecoxib has reversible negative side effects to the fetus, but does not
reduce preterm delivery rates <30 weeks and surprisingly, was shown to
increase deliveries < 37 weeks gestation (Groom KM et al 2005).
Magnesium sulphate, often used in the management of pre-eclampsia, is
widely used as a tocolytic in North America. It acts as a calcium antagonist
at the neuromuscular junction (Groom KM 2007). However, randomised
placebo controlled trials of magnesium sulphate have shown no significant
short term delay of delivery, increase in birth weight or difference in
perinatal mortality when compared to placebo (Lyell DJ et al 2007). With
limited access to more efficacious tocolytics with fewer side effects, it
remains the most commonly used tocolytic in the United States.
Nitric oxide donors, such as in the use of GTN patches are popular due to
the ease of administration and low side effect profile. However, they have
yet to be proven efficacious in randomised controlled trial and fail on an
intention-to-treat basis (Duckitt K et al 2002, Bisits A et al 2004).
42
Atosiban marketed as an oxytocin antagonist, is in fact a competitive
oxytocin and vasopressin antagonist. Oxytocin receptor blockade prevents
oxytocin induced contractions as well as a reduction in extracellular calcium
influx and release of calcium from intracellular stores and therefore an
inhibition of contractility (Groom KM 2007). Administration of Atosiban
results in a dose dependent inhibition of uterine contractility and oxytocin
mediated prostaglandin release. In pregnant women, Atosiban is 46-48%
plasma protein bound and appears to have limited cross-over into the fetal
circulation.
The randomised controlled trials comparing Atosiban to beta mimetics show
similar efficacy, but a superior safety profile in terms of maternal adverse
outcomes (Marsal K 2001, Moutquin JM et al 2001). There is still some
uncertainty over the preferred use of Atosiban over Nifedipine (a calcium
channel blocker) for tocolysis. In the absence of a direct comparison
between the two tocolytics, adjusted indirect comparisons with Nifedipine
have shown to significantly reduce rates of respiratory distress syndrome
(RDS) in the neonate but do not significantly decrease time to delivery
(Coomaraswamy A et al 2003).
Nifedipine is a calcium channel blocker originally developed almost fifty
years ago as a treatment for angina pectoris. It is now used widely for the
treatment of coronary heart disease, hypertension and chronic stable angina.
The effect of calcium channel blockers in relaxing the myometrium has also
43
been known for several years. Calcium channel blockers exert their effect
by binding to L-type channels and reducing intracellular levels of calcium.
This blocks the transmembrane influx of calcium ions into muscle cells. By
binding to intracellular calcium binding proteins, Nifedipine is therefore,
able to block the flow of extracellular calcium into myometrial cells and in
this way relaxes the myometrial smooth muscle.
There have been no trials comparing Nifedipine with placebo, but it has
been compared against a beta sympathomimetic, Ritodrine in a number of
randomized controlled trials. Nifedipine is preferable as it has a better side
effect profile and demonstrated a reduction in the number of preterm births
within seven days of starting treatment (King et al 2003). The RCOG
support the use of Nifedipine for tocolysis, but it is not licensed for use in
United Kingdom and there is no standardized administration dose or regime.
Corticosteroids
Corticosteroids in the management of preterm labour can be administered as
a single course of either betamethasone or dexamethasone. If given,
between 24 and 34 weeks gestation and up to 7 days before preterm
delivery, it has a significant effect upon neonatal morbidity and mortality.
This is principally due to a significant reduction in respiratory distress
syndrome (RDS) and intraventricular haemorrhage (IVH) rates. Antenatal
corticosteroids have a receptor mediator effect on all of the components of
the surfactant system in type-2 pneumocytes. They also however have
44
effects on the structural development of the lungs, lead to accelerated
maturation of the fetal intestine, have effects upon the myocardium and on
catecholamine responsiveness. This may explain the reduced incidences of
necrotising enterocolitis and intraventricular haemorrhage seen in extremely
preterm infants that appear to be independent of the effect upon RDS
(Roberts D et al 2006). The long term consequences of recurrent exposure to
high dose steroids, suggesting adverse effects on development and
behaviour, has lead to a policy of limiting administration of antenatal
corticosteroids to a single course if possible.
Antibiotics
The administration of antibiotics to prevent preterm labour is variable in
clinical practice. There have been many studies of different antibiotics and
routes of administration, but the results vary according to the pre-existing
risk of the population. Research has focused on bacterial vaginosis (BV),
where the normal vaginal lactobacilli are replaced by a mixed flora with
high concentrations of anaerobic bacteria. Other organisms known to
increase the risk of adverse outcomes are Trichomonas vaginalis and
Mycoplasma hominis (Leitich H et al 2003). The effects of metronidazole,
erythromycin and clindomycin have been shown in some studies to improve
success in symptomatic women, women at high risk and those with a
positive fFN (Hauth JC et al 1995, Lamont RF et al 2003). However, other
studies have shown an increase in rate of delivery before 37 weeks gestation
with the use of metronidazole; perhaps, due to the eradication of normal
45
flora and hence the proliferation of harmful bacteria (Klebanoff MA et al
2001). A recent meta-analysis looking at prophylactic antibiotics for the
prevention of preterm birth in women at risk included 17 trials and found no
reduction in preterm birth irrespective of the criteria used to assess risk (i.e.
abnormal vaginal flora, previous preterm birth or positive fetal fibronectin
status). This was regardless of the antimicrobial agent used or the
gestational age at time of treatment (Simcox R et al 2007). Similarly, the
ORACLE II study showed no evidence of benefit of antibiotics in
established preterm labour. A later analysis of the longer term outcomes of
the ORACLE II babies shows that antibiotic use increases the risk of
cerebral palsy (Kenyon S et al 2008).
46
Preterm prelabour rupture of membranes
Preterm prelabour rupture of membranes (PPROM) occurs in approximately
2% of all pregnancies and accounts for up to one third of preterm deliveries.
The consequence of PPROM is as many as 50% cases deliver within a
week, 75% within two weeks and 85% within a month. As with preterm
labour, postnatal survival following PPROM is directly related to
gestational age at delivery and birth weight. The retention of amniotic fluid
within the uterus is associated with a better outcome. The management of
PPROM continues to be controversial, however many obstetricians will
favour conservative management in preterm premature rupture of
membranes before 34 weeks and would induce labour relatively early in
women whose membrane rupture occurs subsequent to 34 weeks.
Conservative management should include clinical surveillance for signs of
chorioamnionitis including regular recording of maternal temperature and
heart rate and cardiotocography. A rising white cell count or a rising CRP
level may indicate the development of chorioamnionitis, though neither of
these is highly specific. Lower genital tract swabs are routinely taken and
positive cultures for potential pathogens are useful in directing antibiotic
therapy for both the mother and the preterm neonate. The potential benefits
of tocolytic drugs do not apply in the majority of cases of PPROM and
neither do the few case reports of amnio infusion.
47
1.3 The Anatomy of the Uterus and Cervix
The uterus is the organ of gestation, receiving the fertilised egg in its cavity,
retaining and supporting it during the development of the fetus and
becoming the principal agent in its expulsion at the time of parturition. It is
situated in the pelvis between the bladder and rectum; it is retained in
position by the round and broad ligaments on each side and the cardinal and
uterosacral ligaments below (Fig 1.7). The non-gravid uterus measures
about 7cm in length, 4cm in breadth at the fundus and almost 2cm in
thickness.
The uterus consists of two parts: (1) the corpus, with its broad upper
extremity, the fundus and (2) the cervix, which is partly above the vagina
and partly in the vagina. The division between the corpus and the cervix is
indicated externally by a slight constriction, and by the reflection of the
peritoneum from the anterior surface of the uterus on to the bladder, and
internally by a narrowing of the canal, the internal os.
The uterus is a muscular organ which increases in fibrous and elastic tissue
during pregnancy, together with the stretching and hypertrophy of
myometrial cells. These smooth muscle cells form functional contractile
bundles, covered in connective tissue and interspersed with an extensive
vasculature. In addition, there is a varied population of leucocytes that
secrete cytokines, including macrophages, neutrophils, large granular
48
lymphocytes and other descendents of T-cells as reviewed by Terzidou
(2007). Single myocytes may produce force in any direction thereby
allowing the uterus to generate a multidirectional force as a whole. During
active labour, the fundal region contracts to expedite delivery, whilst the
lower segment relaxes to allow passage of the fetal head. A fundo- cervico
gradient has been described with regards to contractile receptor proteins
such as oxytocin and connexin-43 (Fuchs AR et al 1984, Sparey C et al
1999).
The cervix comprises the lower, cylindrical portion of the uterus, containing
collagen fibrils, elastic connective tissue, blood vessels and some muscle
fibres. Macrophages and neutrophils infiltrate the cervix at term inducing
cytokines and also producing enzymes capable of digesting extracellular
matrix proteins, necessary for cervical ripening.
49
Fig 1.7 Diagram demonstrating the anatomy of the non-pregnant uterus.
50
1.4 The Endocrinology and Biochemistry of Labour
There is a challenge in predicting, diagnosing and managing preterm labour,
because despite extensive research, the mechanisms that control the length
of human pregnancy and signal the onset of labour have not yet been fully
determined. Throughout pregnancy the uterine cervix needs to remain firm
and closed whilst the body of the uterus grows by hypertrophy and
hyperplasia whilst remaining quiescent. During pregnancy, ‘pro-pregnancy’
factors such as progesterone and prostacyclin inhibit myometrial
contractility. Parturition involves a synchronisation of changes in the
structure of the cervix, myometrial activity manifesting as painful uterine
contractions and ruptured fetal membranes leading to cervical dilation and
effacement. At the end of the pregnancy, ‘pro-labour’ factors begin to
mediate remodeling of the cervix and the uterus is stimulated to begin co-
coordinated contractions (Terzidou V 2007). It has been suggested that
labour results from the activation of a ‘cassette of contraction-associated
proteins’ (CAPs), which include gap junction proteins, oxytocin and
prostanoid receptors, enzymes for prostaglandin synthesis and cell signaling
proteins (Lye et al 1994). However, the term ‘labour associated proteins’
may be more appropriate since these are also responsible for activating fetal
membrane prostaglandin and cytokine production, as well as cervical
remodeling and ripening (Fig 1.8).
51
Choriodecidual bacterial colonisation
(endotoxins and exotoxins)
Fetal tissue response Maternal response
Fetus Chorioamnion and placenta Decidua
Increased Decreased chorionic prostaglandin
Corticotrophin- dehydrogenase Increased cytokines
releasing hormone and chemokines
Increased adrenal Increased prostaglandins Neutrophil infiltration
Cortisol production
Increased metalloproteases
Myometrial Chorioamnion weakening
contractions and rupture Cervical weakening
Preterm delivery
Fig 1.8 Factors influencing preterm labour.
52
Inflammation
Inflammation is now considered to be integral to the pathophysiology
behind preterm labour. Microarray and suppression subtractive
hybridisation studies have demonstrated an up regulation of a vast number
of pro-inflammatory genes in fetal membranes, myometrium and the cervix
with the onset of labour (Charpigny G et al 2003). This is demonstrated by
an influx of inflammatory cells into the uterus and increased expression of
pro-inflammatory cytokines (Young A et al 2002). The transcription factor,
nuclear factor-kappa B (NF-κB) which is activated within the uterus at the
time of labour, regulates a number of inflammatory factors, including IL-6,
IL-8 and COX-2 (which leads to prostaglandin synthesis) (Allport VC et al
2003). At term, this inflammatory-type response may be triggered by the
maturing fetal lung which produces increased surfactant lipid and protein
(i.e. surfactant protein A, SP-A). This is associated with the activation of
macrophages in the amniotic fluid and an increased inflammatory response
in the uterus (Condon JC et al 2004).
Corticotrophin-releasing hormone
Corticotrophin-releasing hormone (CRH) is a peptide produced by the
hypothalamus in response to stress. CRH is also expressed abundantly in the
placenta and in maternal serum CRH has been shown to rise in the second
half of pregnancy, peak during labour and rapidly decline postpartum. In
patients who have gone on to deliver prematurely, elevated levels of CRH in
53
the maternal circulation have been identified as early as 18 weeks of
gestation (McLean M et al 1995). The exponential curve depicting the CRH
increase is shifted to the left in women who subsequently deliver preterm
and to the right in women who deliver post dates. This suggests that CRH
production is linked to a placental clock (Fig 1.9) which determines the
length of gestation (Smith R, Van Helden D et al 2007). The implications of
this clinically could mean that maternal plasma CRH concentrations could
help indentify women at high risk of preterm delivery and CRH antagonists
may be useful in preventing preterm labour (Smith R, Nicholson RC et al
2007).
54
Maternal Stress Fetal Stress
Cortisol
Angiotensin II +
Norepinephrine membranes enhanced fetal
ADH placenta lung maturity
Acetylcholine decidua
CRH
+
PG
Contractions Rupture of Cervical change
membranes
Fig 1.9 Pathway for action of placental corticotrophin releasing hormone on
parturition.
55
Progesterone
Progesterone is thought to act during pregnancy to inhibit labour-associated
gene expression and inhibit contractions. Unlike most mammalian
counterparts, human circulating progesterone levels and progesterone
receptors (PR) in the myometrium do not fall with the onset of labour.
However, clinically the administration of a PR antagonist RU486 can be
used to ripen the cervix and induce labour. In the absence of a demonstrable
fall in progesterone concentrations, it may be possible that a ‘functional
progesterone withdrawal’ precedes the onset of labour. This could be due to
changes in the different PR levels or isoforms (Mesiano S et al 2002),
specifically a reduction in myometrial responsiveness to progesterone by an
increased expression of PR-A relative to PR-B (Pieber D et al 2001). It may
also be due to the interaction between NF-κB and PR or changes in PR co-
activator and co-repressor expression.
NF-κB
NF-κB is a cytokine-inducible transcription factor activated in response to
infection and pro-inflammatory cytokines which regulates many pro-
inflammatory and labour associated genes (Baeuerle PA et al 1996). In most
cells, NF-κB exists in an inactive form in the cytoplasm, bound to an
inhibitory protein, such as IκBα. Three distinct NF-κB signaling pathways
have been identified to date (Terzidou V 2007). Labour is associated with an
increase in the baseline level of NF-κB DNA binding and transcriptional
56
activity in the human amnion and because NF-κB down regulates
progesterone receptor function, it seems to be related to a ‘functional
progesterone withdrawal’ in the human myometrium (Chapman NR et al
2004).
Prostaglandins and cyclo-oxygenases
It is now established that prostaglandins (PGs) play a central and essential
role in the biochemistry of human labour
(Albertsen K et al 2004).
Prostaglandins of the E and F series stimulate uterine contractions
throughout pregnancy and are used clinically to induce first and second
trimester abortion and labour at term. There is increased synthesis of PGs by
intrauterine tissues, particularly the fetal membranes, at the time of labour
which is associated with a rise in the concentrations of PGs and PG
metabolites in amniotic fluid and maternal plasma (Slater DM et al 1995,
1999).
Prostaglandins belong to the eicosanoid family of lipid mediators and are
derived from arachidonic acid (AA) found in membrane phospholipids of
the cell. The prostaglandin biosynthetic pathway (Fig 1.10) can be divided
into three main stages. The initial step involves the mobilization of AA from
membrane phospholipids by the action of phopholipase enzymes. In the
second stage, the free AA is converted to an unstable prostaglandin
57
intermediate, in a two-step reaction catalysed by the cyclo-oxygenase
(COX) enzymes. This reaction involves the conversion of AA to
hydroperoxy endoperoxide (PGG2), which in turn undergoes reduction to
form hydroxyl endoperoxide (PGH2). Since no pathway exists for the re-
conversion PGH2 to AA, the COX enzymes mediate the committing step of
prostaglandin biosynthesis. Finally, the PGH2 intermediate is converted to
the terminal prostaglandins PGE2, PGF2α, PGD2, prostacyclin (PGI2) and
thromboxane by their respective synthase enzymes (Kniss DA 1999).
In addition to mediating the committing step, the COX enzymes are also
thought to catalyse the principle rate-limiting step of prostaglandin
biosynthesis (Slater DM et al 1995). The COX enzymes exist as two main
isoforms: the constitutively expressed COX-1 and the inducible COX-2
isoenzyme. In human labour, there is substantial evidence for the greater
importance of the COX-2 enzyme rather than COX-1. Amnion is a major
source of prostaglandins and displays a marked increase in the synthesis of
PGE2 with labour onset and stretch. This is associated with the selective
induction of COX-2 expression (Slater DM et al 1999). Similarly, a labour
associated increase in COX activity in the chorion is attributed to the
upregulation of COX-2, but not COX-1 expression. Upregulation of COX-2
expression in lower segment myometrium has been shown to occur with
advancing gestational age and was maximal just prior to labour at term,
suggesting a role for myometrial COX-2 in the early phases of the
58
parturition cascade (Romero R et al 1988).
Arachidonic Acid
Epoxygenases Lipoxygenases
Cyclogenase reaction COX-1
COX-2
HETES Epoxides
Leukotrienes
PGG2
Peroxidase reaction COX-1
COX-2
PGI synthase PGH2 TXA synthase
PGI2 TXA2
TXB2
PGD PGF PGF
synthase synthase isomerase
PGD2 PGF2α PGE2
Fig 1.10 Biosynthetic pathway for the formation of eicosanoids derived from
arachidonic acid.
59
In relation to the role of prostaglandins in preterm labour, they are products
of the inflammatory process and also play a key role in the mechanisms of
infection associated premature labour. Infection increases prostaglandin
production by amnion, chorion and decidua through the activity of bacterial
products, cytokines, growth factors and other inflammatory mediators
(Gomez R et al 1995, Kelly RW 2002). Concentrations of PGE2 and PGF2α
in amniotic fluid are significantly higher in women with preterm labour who
have confirmed chorioamnionitis than women with preterm labour and no
evidence of infection (Romero R et al 1988). In addition, higher levels of
prostaglandins are produced from the amnion of patients with histologic
chorioamnionitis compared to amnion from patients without documented
chorioamnionitis (Lopez Bernal A et al 1987).
Interleukin 8
IL-8 acts a chemokine and is produced by human endometrium, chorio-
decidua and myometrium. IL-8 plays an important role in cervical ripening,
probably aids in the formation of the lower segment of the uterus in late
pregnancy and mediates the influx of inflammatory mediators, specifically
leukocytes, seen in the myometrium during labour (Mohan AR et al 2004).
60
Oxytocin
Oxytocin (OT) is a nonapeptide produced by the posterior pituitary, known
to have a potent contractile activity on the pregnant uterus. However, it does
not seem to be essential for normal parturition as there is no increase in
circulating oxytocin in the mother or fetus with the onset or during labour.
Moreover, normal parturition has been observed in cases of clinical pituitary
gland dysfunction (Phelan JP et al 1978). However, the expression of the
oxytocin receptor (OTR) does increase in the pregnant uterus. An up-
regulation by two orders of magnitude has been demonstrated leading to a
strong increase in sensitivity towards OT (Soloff MS 1975). Comparable
increases in OTR mRNA concentrations in the myometrium are associated
with this up-regulation of OT- binding sites (Jeng YL et al 2003). Post
delivery, myometrial OT-binding sites rapidly decrease, whereas OTR
expression remains high in the mammary gland throughout lactation (Soloff
MS et al 1979).
In many animals, OTR is regulated by changes in steroid hormone
concentrations (Soloff MS 1975). In humans, the lack of marked changes in
oestrogen concentrations or progesterone withdrawal suggests that OTR has
a more complex regulation (Gimpl G et al 2001). Further relationships in the
OTR system have been demonstrated, in particular with prostaglandins.
Treatment with naproxen, a prostaglandin-synthesis inhibitor, resulted in a
dramatic decrease in OTR in the rat uterus at term. Administration of PGF2α
61
restored the increase in OTR, implying the possibility of a positive feedback
loop between prostaglandins and OTR (Chan WY 1980).
62
1.5 Myometrial Contractility
Irrespective of the aetiology of preterm labour, the underlying mechanism
by which the myometrium contracts is thought to be the same. During
pregnancy, the uterus maintains a quiescent environment, whereas at the
point of fetal maturity, it generates forceful contractions, observed to
originate from the fundus to expel the conceptus, whilst the lower segment
remains relaxed in order to allow the fetal head to pass.
The basic functional unit of the myometrium is the smooth muscle cell,
which is in turn, arranged into bundles of approximately 300+/- 100µm in
diameter. Each bundle is surrounded by connective tissue interspersed with
microvasculature. The bundles are further organized into fasiculi, which are
covered by a dense collagen matrix and the major vasculature of the
myometrium (Young RC et al 1999). This therefore enables the uterus to
generate an expulsive force dependent on the orientation of these fibres
relative to the cervix. Uterine contractions are then initiated by the
propagation of action potentials within these defined vectors of muscle
bundles (Blanks AM et al 2007). The intracellular basis for contraction is via
the cyclical attachment and detachment of cross-bridges between myosin
and actin filaments, triggered via activation of the regulatory 20kDa chain
of myosin molecules. Myosin light chain is activated by phosphorylation by
myosin light chain kinase, which in turn, is activated by calmodulin (Word
RA et al 1993). It is through the actions of calmodulin, that during an action
63
potential, the calcium influx can be connected with a contraction; a process
known as excitation-contraction coupling (Blanks AM et al 2007). Once the
uterus has been activated, the myometrium is susceptible to stimulation by a
number of different agonists, primarily prostaglandins and oxytocin.
Myometrial cells maintain ionic gradients across the plasma membrane and
with the change of membrane permeability to calcium, sodium, chloride and
potassium, generate an action potential. In human myometrium, the
depolarising current is mainly mediated by calcium (via L-type calcium
channels) and repolarisation is mainly carried by potassium (Shmygol A et
al 2007). The process of contractility in myometrial smooth muscle is ATP-
dependent and is proportional to the activity ratio of myosin light chain
kinase (MLCK) to myosin phosphotase (MLCP). It is by altering the
activity ratio of MLCK to MLCP that oxytocin and PGF2α increase the
sensitivity of the myometrium to calcium (Woodcock NA et al 2004, 2006).
This sensitisation occurs after contraction has been initiated and is mediated
by a decrease in MLCP activity, which in turn is controlled by several
processes such as the activation of G-protein rhoA or the activation of CPI-
17 by protein kinase C (Kitazawa T et al 2000).
In order to propagate action potentials, myometrial cells are connected by
connexins (gap junction proteins) through which ions and some cellular
metabolites can pass. In the myometrium, an increase in the number of gap
junctions has been observed in both term and preterm labour, as well as
64
improved intracellular conductance and metabolic communication (Garfield
RE et al 1988, Sims SM et al 1982). Connexin-43 (CX-43) appears to be the
most important in gap junction formation as it is present in all gap junction
plaques and a delay in parturition has been observed in CX-43 knockout
mice (Kilarski WM et al 2001, Döring B et al 2006). Furthermore, in human
myometrium, the expression of CX-43 was found to be the greatest at the
fundus implicating a stronger contractile potential to the upper segment
(Sparey C et al 1999).
Physiological stimulation once myometrial activation has occurred is
principally via prostaglandins and oxytocin. Oxytocin increases contraction
frequency, duration and force by increasing the frequency of depolarisation,
sensitising the contractile machinery to calcium and prolonging the duration
of the action potential plateau (Nakao K et al 1997, Woodcock NA et al
2004). G-protein coupled oxytocin receptors release G-protein subunits
(from Gαi) which leads to the activation of phospholipase C, which is
responsible for the mobilisation of calcium from sarcoplasmic reticular
stores via inositol 1, 4, 5,-triphosphate (InsP3). This is sufficient to open L-
type calcium channels and allow full depolarisation (Phaneuf S et al 1996).
65
Prostaglandin receptors
Prostaglandins were originally thought to interact directly with the cell
membrane, thereby altering its characteristics and modulating cell
physiology. However, their different activity profiles and generation of
second messengers, principally cAMP, phosphatidylinositol turnover and
Ca2+
, suggests that they interact with discrete receptors and that different
receptors exist for each PG. Coleman et al (1994) used specific antagonists
to further characterize these receptors pharmacologically. They classified
the receptors as one each for PGs, D2, F2α, I2 and TXA2, as DP, FP, IP and
TP and four for PGE2 as EP1-4. The first receptor that was isolated and
cloned was the TP receptor. This is a G protein linked member of the
rhodopsin family of receptors with seven transmembrane domains.
Subsequently, homolgy screening using mouse cDNA libraries led to the
identification of the structures of all eight receptors, each encoded by a
different gene (Narumiya S et al 1999).
More recently, several splice variants have been identified for human EP3
(eight isoforms), sheep and bovine FP (two isoforms), rat EP1 (two
isoforms) and human TP (two isoforms) (Pierce KL et al 1998). They vary
largely in their cytoplasmic C-termonal regions where coupling with G
protein occurs. The amino acid sequences have been fully described for rat
and sheep. There is only 20% homology between amino acid sequences of
various receptors within a species, but 79-89% homology exists between
like receptors of different species (Olson DM 2003).
66
The EP receptor family is composed of four distinct subtypes; EP2 and EP4
stimulate adenylate cyclase and cAMP production via the stimulatory
subunit Gs, resulting in muscle relaxation (Fig. 1.11), whereas EP1 and EP3
mediate muscle contraction through increased phosphinositol turnover and
calcium mobilisation, or decrease adenylate cyclase and cAMP levels,
respectively (Coleman RA et al 1994).
67
Figure 1.11 Signaling pathways for EP1 and EP2/4. EP1 is coupled to Gq and
upon PGE2 binding there is a signal cascade that results in activation of
phospholipase C (PLC) and mobilisation of intracellular calcium (Ca2+
) stores. EP2
and EP4 are coupled with Gs. PGE2 binding results in activation of adenylyl
cyclase (AC) and cyclic adenomonophosphate (cAMP). This leads to activation of
protein kinase A (PKA) and subsequent phosphorylation (P) of CREB, which leads
to up-regulation of the cyclic AMP response element (CRE) (Kandola M 2008).
68
The EP3 receptor is unique in that eight different receptor isoforms have
been identified, encoded by nine transcripts: EP3-I(1a), EP3-I(1b), EP3-II,
EP3-III, EP3-IV, EP3-V, EP3-VI, EP3-c and EP3-f generated by alternative
splicing at the carboxy terminal tail (Schmid et al 1995, Kotani et al 1997,
2000). As the carboxy terminal end of the receptor is important for
mediating G protein coupling, changes in this region appear to alter the
coupling affinity. EP3 is mainly coupled to Gi and hence towards a
contractile phenotype. However, splice variants EP3-II and EP3-IV may
couple Gs and/or Gq leading to different cellular responses (Namba et al
1993, Kotani et al 1995) (Fig. 1.12).
69
Figure 1.12. Signaling pathways for EP3 isoforms. A. EP3 is coupled to Gq and
upon PGE2 binding there is a signal cascade that results in activation of
phospholipase C (PLC) and mobilisation of intracellular calcium (Ca2+
) stores. B.
EP3 is coupled to Gi. PGE2 binding causes inhibition of adenylyl cyclase (AC) and
therefore mobilisation of intracellular Ca2+
. C. EP3 is coupled with Gs. PGE2
binding results in activation of AC and cyclic adenomonophosphate (cAMP). This
leads to activation of protein kinase A (PKA) and subsequent phosphorylation (P)
of CREB, which leads to up-regulation of the cyclic AMP response element (CRE)
(Kandola M 2008).
70
PGF2α similar to PGE2 is a potent stimulator of myometrial tissue
(Parkington HC et al 1999), but there is no consensus on how. The FP
receptor is coupled to Gαq class of G-proteins and therefore stimulation of
PLC should release InsP3 (Carrasco MP et al 1996). However this has not
been consistently demonstrated in freshly isolated myometrial strips from
pregnant patients (Schery MP et al 1988). What has been demonstrated
however is the calcium sensitising effect of PGF2α is rho-kinase mediated.
This is preceded by depolarisation and results in a large increase in
intracellular calcium (Woodcock NA et al 2006). This effect may aid
delivery by producing a strong contraction, whilst increasing the recovery
period between contractions to reduce the risk of hypoxia and fetal distress
(Parkington HC et al 1999).
There is little consistency in the literature concerning the myometrial
expression of the two ‘contractile’ receptors EP1 or EP3 (Fig 1.13). Astle et
al (2007) found that EP1 expression is upregulated in human lower segment
myometrium at the time of labour but found no change in upper segment
myometrium. Conversely, Smith GC et al (2001) found that EP1 expression
was greatest in baboon upper segment myometrium and found no changes
with labour. Grigsby PL et al (2006) found that human myometrial EP1
expression increases with gestational age in both upper and lower segment
but found no regional differences. Ma X et al (1999) found no expression of
EP1 at all in sheep myometrium.
71
With respect to EP3, Astle et al (2007) found greater expression in upper
segment, but mostly in vascular smooth muscle cells not myocytes.
Moreover, down regulation of EP3 with labour was observed. Smith GC et
al (2001) found EP3 expression to be greater in upper segment myometrium
with no labour associated changes. Grigsby PL et al (2006) found no
regional differences and no changes in association with labour. Leonhardt
A et al (2003) found that there was no expression of EP3 in human pregnant
myometrium but did find expression of EP1 and so concluded that PGE
leads to contractions via EP1. Ma X et al (1999) found a labour associated
increase in EP3 expression in sheep myometrium.
72
Fig 1.13 Summary of the literature findings with regards to myometrial expression
and changes of the ‘contractile’ PG receptors, EP-1 and EP-3 with regards to their
presence in the upper segment (US) and lower segment (LS) and changes with
gestation and labour.
Author Myometrial
sample
EP
receptor
Presence
in US
Presence
in LS
Changes
with
gestation
Changes
with
labour
Astle Human 1 + + ↑ LS
3 ++ + ↓
Smith Baboon 1 ++ + Nil
3 ++ + Nil
Grigsby Sheep 1 + + ↑
3 + + Nil
Ma Sheep 1 - -
3 + + ↑
Leonhardt Human 1 + +
3 - -
73
1.6 Hypothesis and Objectives
Currently, there are no effective tocolytics free of side-effects. PGE2
receptors represent potential targets, but which EP should be targeted would
depend upon expression and functional data. EP1 and EP3 are said to
mediate contractions, but there is no consensus about their relative
importance. There is little consistency in the data regarding EP expression
in myometrium during pregnancy. To examine the roles of EP1 and EP3 in
causing myometrial contractility, we undertook expression studies and
contractility studies. Mandeep Kandola, a scientist in our group, examined
the expression of EP receptors by Western blotting, immunofluoresence and
QRT-PCR in the upper and lower segment myometrial samples that I had
obtained from the pregnant, non-labouring women, at term, at elective
Caesarean sections and from labouring women at emergency Caesarean
sections.
Expression of EP-R protein on the cell surface
In order to study the changes that occur between quiescence and active
labour, as defined by contractions, immunohistochemistry combined with
immunofluorescence is often used to decipher the expression of proteins at a
particular time point. Immunohistochemistry refers to the process of
detecting antigens (e.g. proteins) in cells of a tissue section by exploiting the
principle of antibodies binding specifically to antigens in biological tissues.
Immunofluroscence uses the specificity of antibodies to their antigen to
74
target fluorescent dyes to specific biomolecule targets within a cell, and
therefore allows visualisation of the distribution of the target molecule
through the sample. The advantage of this method is that it is quantitative as
opposed to qualitative. Therefore, the higher the immunofluroscence seen,
the more receptor is present. To quantitatively assess the presence of
proteins, Western blotting is also used.
Using these two techniques we were able to assess PGE2 receptor protein
expression in the upper and lower segment myometrium. In the lower
segment, EP1 expression in the lower segment (LS) was less than that in the
upper segment (US). There were no significant differences between labour
plus (L+) and labour minus (L-) samples (Fig. 1.14). Similarly, there were
no significant changes observed in EP3 expression in both US and LS, as
well as L+ and L- myometrial biopsies (Fig. 1.15).
75
Figure 1.14. No labour associated change of EP1 receptor
protein expression in human myometrium at term. EP1 receptor
expression was compared in matched lower segment (LS) and
upper (US) segments of term laboring (L+) and term non-labouring
(L-) myometrium. Whole cell extracts were subjected to Western
blotting and blots were scanned for densitometric analysis. Values
were normalised using the housekeeping gene GAPDH. Results
are the mean of 3-6 samples and not significant (p>0.05).
Representative Western blots are shown.
76
Figure 1.15. No labour associated change of EP3 receptor
protein expression in human myometrium at term. EP3 receptor
expression was compared in matched lower (LS) and upper (US)
segments of term laboring (L+) and term non-labouring (L-)
myometrium. Whole cell extracts were subjected to Western blotting
and blots were scanned for densitometric analysis. Values were
normalised using the housekeeping gene GAPDH. Results are the
mean of 3-6 samples and the differences were not significant
(p>0.05). Representative western blots are shown.
77
Both the EP1 and EP3 receptors were demonstrated in all the sample groups
using immunofluorescence. Co-localisation with the smooth muscle alpha-
actin stain confirmed their presence in the smooth muscle. In the upper
segment, EP1 receptor protein was not significantly affected by labour
status in the US. There was however, significant change in the labouring
lower segment versus the non-labour samples (p=0.05) (Fig.1.16). Similarly
the EP3 receptor increased significantly in the labouring LS myometrium
(p=0.02) (Fig. 1.17).
78
Figure 1.16. EP1 receptors are significantly upregulated in the lower
segment of the labouring myometrium. EP1 receptor expression was
measured in matched lower (LS) and upper (US) segments of term labouring
(L+) and non-labouring (L-) myometrium. Tissue was paraffin-embedded and (A)
the specificity of the antibody determined using immunohistochemistry with
polyclonal EP1 antibodies. Replacement of anti-EP1 antibodies with pre-immune
IgG and saturation of anti-EP1 antibodies with blocking peptides were used as
negative controls. (B) Immunofluorescent analysis for EP1 was visualized with
red-fluorescent Alexa Fluor 594 dye and α-actin was visualized with green
fluorescent Alexa Fluor 488 dye. Nuclei were stained with DAPI. (C) Intensity of
receptor expression was analysed by measuring levels of fluorescence and
applying statistical analyses. These graphs represent the intensity of expression
of each PGE2 receptor in the cytosol of myocytes (n=3).
79
Figure 1.17. EP3 receptors are significantly upregulated in the lower
segment of the labouring myometrium. EP3 receptor expression was
measured in matched lower (LS) and upper (US) segments of term labouring
(L+) and non-labouring (L-) myometrium. Tissue was paraffin-embedded and (A)
the specificity of the antibody determined using immunohistochemistry with
polyclonal EP3 antibodies. Replacement of anti-EP3 antibodies with pre-immune
IgG and saturation of anti-EP3 antibodies with blocking peptides were used as
negative controls. (B) Immunofluorescent analysis for EP3 was visualized with
red-fluorescent Alexa Fluor 594 dye and α-actin was visualized with green
fluorescent Alexa Fluor 488 dye. Nuclei were stained with DAPI. (C) Intensity of
receptor expression was analysed by measuring levels of fluorescence and
applying statistical analyses. These graphs represent the intensity of expression
of each PGE2 receptor in the cytosol of myocytes (n=3).
80
A fundo- cervico gradient has been described with regards to contractile
receptor proteins such as oxytocin and connexin-43 (Fuchs AR et al 1984,
Sparey C et al 1999). However, there is no clear consensus between EP1
and EP3 localisation in labouring and non-labouring myometrium, as
illustrated by Fig.1.13.
In summary, the expression data showed there to be no significant changes
between the localisation in upper or lower segment biopsies using both
RNA and protein samples. However, there was a labour associated change
seen with EP1 and EP3 in the lower segment using immunofluorescence.
Furthermore, there were changes in the intracellular localisation of the
receptors; EP1 showed a significant increase in the nuclei of labouring
upper and lower segment myometrial biopsies. EP3, however, was primarily
in the cytoplasm. To demonstrate the significance of this, I established an in
vitro tissue bath to conduct functional studies examining contractility in
upper and lower segment myometrial biopsies.
81
2.0 Materials and Methods
The research was conducted at the Institute for Reproductive and
Developmental Biology, Imperial College at the Hammersmith Hospital
site. Collection of tissue was approved by the local Ethics Committee and
written informed consent was given by all subjects. Experiments were
performed on human myometrial biopsies taken at elective caesarean
section at term. All caesarean sections were conducted at Queen Charlotte’s
Hospital and samples stored in at least 10mls of Eagle’s medium. In each
case, a single lower segment biopsy of about 2 x 2 x 1cm was taken from
the upper margin of the lower segment incision (Fig 2.1). In the laboratory,
this was divided into 8 strips approximately 1 x 0.5 x 0.5cm (Fig 2.2) and
mounted within the 8 individual baths in the myograph.
82
Fig 2.1 Myometrial sample taken from the lower segment of human
myometrium at elective caesarean sections at term. Only women having
elective caesarean sections for breech or a previous caesarean section were
consented. Following the delivery of the fetus and placenta, myometrial
tissue would be cut from the upper edge of the uterine scar. This did not
cause additional bleeding or add significant time to the operation.
83
In order to sample upper segment myometrial tissue, four biopsies
measuring 0.5 x 0.5 x 0.5cm (Fig 2.2), were taken by inserting a
cervical punch biopsy (Fig 2.3) through the lower segment incision
and obtaining tissue from the fundus. Limitations to this methodology
include a degree of damage to the upper segment myometrium by the
cervical punch biopsy apparatus as well as some endometrial lining
being included in the biopsy. Pre-labour samples were taken at
elective caesarean section for indications such as breech presentation,
previous caesarean section or maternal request. In-labour samples
were taken from women with spontaneous onset of labour who
reached at least 4cm cervical dilatation who had not received either
prostaglandin or oxytocin. All samples were used within three hours
of collection.
84
Fig 2.2 Strips of myometrial tissue; lower segment (on the left) divided
from a larger sample, as illustrated in Fig 2.1, and upper segment (on
the right), obtained using the cervical punch biopsy.
85
Fig 2.3 A cervical punch biopsy used for obtaining upper segment myometrium at
caesarean sections at term.
86
2.1 Contractility Studies
Contractility studies were undertaken using a Myograph 800MS (Danish
Myo Technology) (Fig.2.4), which can be used to study small myometrial
samples of about 0.5cm in length. The Myograph 800MS with smaller organ
baths (capacity 4mls) was ideal for the smaller upper segment biopsies.
However, smaller baths are also more sensitive to changes in temperature,
pH, volume and oxygenation. For example, concentrations of substrates
that were added to the bath had to be diluted into smaller volumes so as to
not upset the bath by huge changes in total volume.
Myometrial samples were mounted between metal clasps attached to
isotonic tension transducers in individual organ baths filled with 4ml of
modified Krebs solution; the concentrate was kindly provided by Glaxo
Smith Kline, Harlow, and made up in the lab to the following specifications:
NaCl 119mmol/l, CaCl2 16mmol/l, NaHCO3 25mmol/l, KCl 14.7mmol/l,
KH2PO3 1.18mmol/l, MgSO4 1.17mmol/l, Glucose 5.5mmol/l to a pH 7.4 at
a temperature of 37oC. This was then perfused with a 95% O2/ 5% CO2
ratio gas mixture. Transducers were calibrated once a month by balancing
known 2 g weights on inverted scales that applied force onto the
transducers. This was used as a reference in the Chart software version 5.0
and later upgraded to 5.2 (PowerLab, ADI Instruments).
87
Fig 2.4 The Myograph 800MS with smaller organ baths (capacity 4mls) which was ideal for the smaller upper segment biopsies. The muscle strip was placed between two clamps and dissection forceps were used to attach the strips in order to reduce handling of the myometrium and minimise damage. The two clamps, when pulled apart, recorded the tension produced by the myometrial strip through a transducer. This was connected directly to the computer software which processed the data.
88
Preliminary experiments showed that to measure both spontaneous and
stimulated contractions, myometrial samples needed to be placed under 4g
of tension (Fig 2.5). The choice of setting the initial tension to 4g was based
on the tension that the most number of strips responded to (i.e. 80%). As the
experiment progressed, the tension would settle to 2g. This is consistent
with current published data. A baseline period of one hour was observed and
experiments were only conducted on strips that contracted spontaneously
within 90 min of the set-up and had achieved a minimum peak tension of 1g
(Fig 2.6). The average life span for the tissue strip was for a total of 90
minutes following the initial 60 minutes of incubation. Most experiments
were conducted on spontaneous contractions, unless otherwise stated. The
contractions had been stable for a minimum of 15 minutes before additional
agents were added in 15 minute intervals. All the data obtained; rate per
contraction, peak tension per contraction, duration per contraction, work
done per contraction and total work done was recorded using the Chart
software and processed by a PC based Excel program.
89
Tension (g) No. of strips contracting at 1hr (%)
0 0
2 25
4 80
6 50
8 40
Fig 2.5 Preliminary experiments showed that stretch is essential for
spontaneous contractility in myometrial strips. When using the Myograph
800MS, myometrial samples needed to be under a minimum of 4g
tension to achieve the maximum of strips to contract.
90
(g)
(mins)
60 70 80 90
2
4
6
8
10
12
Fig 2.6 A real time image of spontaneous contractions resulting from the
stretch of a non-labouring, lower segment myometrial strip after an hour.
Co
ntr
acti
on
forc
e (g
)
Time (s)
91
2.2 Drug Assessment
The first series of experiments compared upper segment with lower segment
myometrium. The initial step was to compare spontaneous contractions, in
both the upper and lower segment. The next experiment explored the effect
of increasing concentrations of PGE2 on spontaneous contractions.
Cumulative concentrations of PGE2 (10-10
, 10-9
, 10-8
, 10-7
, 10-6
mol/l) were
added to the organ baths at 15 minute intervals. The effect of increasing the
concentration of PGE2 on myometrial contractility was assessed by
calculating an average of the preceding 15 minute interval as a ratio of the
average in the first 15 minutes of spontaneous contractions.
A novel OT antagonist, GW796679X with a molecular weight (MW) of 484
and a dissociation constant (Ki) of 1.5nM was provided by Glaxo Smith
Kline, and compared against equimolar concentrations of a known OT
antagonist, Atosiban. Cumulative concentrations of OT (10-10
,10-9
,10-8
,10-7
,
10-6
mol/l Syntocinon) were then added to the organ baths at 15 minute
intervals. Similar to the first design and in following experiments, the effect
of the antagonists and/or agonists on myometrial contractility was assessed
by calculating an average of the preceding 15 minute interval as a ratio of
the average in the first 15 minutes of spontaneous contractions.
Other compounds that were tested include acetyl-salicylic acid (ASA)
(Sigma-Aldrich, Poole, UK), PGF2α, a novel FP antagonist (FPA) donated
92
by Serono and two novel EP1 and EP3 antagonists donated by Glaxo Smith
Kline (GSK). FPA has a Ki of 6nM for the prostaglandin F receptor (FP)
and is 10-100 folds selective for FP compared with other prostanoid
receptors.
The EP1 (ZD6416) and EP3 (L798106) antagonists are both small
molecules. ZD6416 shows fpKi for EP1 8.4 +/-0.6 and for EP3 6.3 +/-0.4.
L798106 shows fpKi for EP1 6.0+/-0.4 and for EP3 7.9 +/-0.4. Schild
analysis has shown that each is a competitive antagonist devoid of agonist
activity up to 10µM. The effective concentrations of each against their
respective receptors are therefore 2 orders of magnitude apart. A
concentration of 100nM of each antagonist was calculated to ensure
complete and specific inhibition of the relevant receptor.
93
2.3 Data Analysis
Data was calculated by taking the average of each 15 minute interval and
expressing it as a ratio of the average in the first 15 minutes of spontaneous
contractions. In the case, where there were no spontaneous contractions
after the baseline one hour, due to the pre-incubation of an antagonist, the
data was not normalized and will be stated as such.
The first parameter analysed was rate per contraction. This looked at the
number of contractions that occurred within the set time period in seconds
(in the majority of cases, 900s = 15 minutes). The second parameter was the
peak tension, i.e. the maximum tension achieved by each strip in grams. The
third parameter was duration per contraction. This illustrated how long a
contraction lasted in seconds. The final parameters were the average work
done by each contraction and the total work done by all the contractions
within a set time frame. This was a calculation of the area under the curve
[height (gram) x width (second) x ½]. Statistical significance was
determined by ANOVA, where the p value was less than 0.05 (Fig 2.7).
94
Duration per contraction (s)
Peak tension (g)
Work done per contraction (g.s)
Rate per contraction = Number of contractions within a time framePeak tension (g) = Maximum tension achieved by the contractionDuration per contraction (s) = Time take per contraction from start to finishWork done per contraction (g x s) = Energy generated by each contraction (area under the curve)Total work done = Energy generated by all the contractions within the specified time frame (900s)
Fig 2.7 An illustration of the parameters used to calculate the results: rate
per contraction, peak tension per contraction, duration per contraction, work
done per contraction and total work done.
95
3.1 Contractility in upper versus lower segment myometrium
Two series of experiments were undertaken to compare contractility
between upper and lower segment myometrium. In the first series matched
(i.e. from the same patient) upper and lower segment biopsies from term
non-laboured myometrium were established in the myograph and the
absolute contractility parameters were compared (Fig 3.1). There were no
differences between lower and upper segment samples in the duration taken
for contractions to begin, or in any of the contraction parameters: rate per
contraction, peak tension per contraction, the duration per contraction, work
done per contraction and therefore in the total work done (p=0.149) at 90
minutes.
96
A B
C D
E
Fig 3.1 (A) Contraction rate, (B) peak tension, (C) duration per contraction, (D)
work done per contraction and (E) total work done in isolated myometrial strips
taken from upper and lower segment, non-labouring myometrium at term over 90
mins. No significant difference in any of the parameters was demonstrated
between matched upper and lower segment human myometrium (n=8) as
demonstrated by the total work done at 90 mins (p=0.149).
97
3.2 Contractility in prostaglandin stimulated upper and lower
segment myometrium
In the second series of experiments comparing upper and lower segment
myometrium, PGE2 was added at concentrations of 10-10
, 10-9
, 10-8
, 10-7
and
10-6
at 15 minute intervals, after the baseline period of spontaneous
contractions had been established (Fig 3.2). The effect of PGE2 upon
contractility was expressed as a ratio to spontaneous contracting
myometrium.
PGE2 caused a dose dependent increase in contractility in both upper and
lower segment myometrium at concentrations above 10-8
(p=0.042) versus
spontaneously contracting myometrium. However, there was no difference
in the total work done between PGE2 stimulated upper and lower segment
myometrium (p=0.177).
98
A B
C D
E
Fig 3.2 (A) Contraction rate, (B) peak tension, (C) duration per contraction, (D)
work done per contraction and (E) total work done in isolated myometrial strips
taken from upper and lower segment, non-labouring myometrium at term with
increasing concentrations of PGE2 added over 90 mins. No significant difference in
any of the parameters was demonstrated between matched upper and lower
segment human myometrium (n=6) as demonstrated by the total work done at
concentrations of 10-6
(p=0.177). However, PGE2 caused a dose dependent
increase in contractility in both upper and lower segment myometrium at
concentrations above 10-8
(p=0.042) versus spontaneously contracting
myometrium.
99
3.3 Discussion of contractility in upper and lower segment
myometrium
The body of the uterus comprises of two parts, the uterine corpus (upper
segment) and the isthmus (lower segment). A concept of functional
differences has been proposed both physiologically and at a protein
expression level. Various studies have shown differences in mRNA
expression between upper and lower segment, leading to criticism that
experiments on the lower segment may not truly represent the uterine body.
A fundal to cervical gradient has been described with regards to
prostaglandin receptors by Wikland et al (1983), to oxytocin receptors by
Fuchs et al (1984) and splicing factors SF2/ ASF by Pollard et al (2000).
This study was initiated in the first instance to identify, if any, a difference
between upper and lower segment, term, myometrial tissue. This study and
that of Lucas et al (2000) are the only studies to have directly compared
upper and lower segment myometrium from the same patient. Some studies
have suggested that PGE2 might contract the upper but have no effect, or
relax the lower segment (Gordon-Wright et al 1980). It is possible that
differences between studies may be due to the differences in the site from
which the myometrial biopsy is taken. If a lower segment myometrial
sample is taken from very close to the cervix then there may be more
cervical than myometrial smooth muscle cells present and the effect of
PGE2 may be different.
100
The first set of data demonstrated the effect of PGE2 on both upper and
lower segment myometrium, and agrees with similar data from Lucas at el
(2000). There were no differences in spontaneous contractility between
upper and lower segment myometrium (p=0.149). Furthermore, PGE2
consistently caused contractions of upper and lower segment tissue and
again there was no difference between the corpus and isthmus (p=0.177). In
addition, our own expression studies of EP1 and EP3 expression also show
no difference in upper versus lower segment myometrium. Recent evidence
using trans-abdominal magneto-myographic recording has demonstrated the
synchronisation of electrical burst activities, which are essential for
effective uterine contractions, are identified not only at the fundus but over
the entire uterus as well (Eswaran et al 2005). Furthermore, no specific pace
maker activity seems to have been identified over any specific area of the
uterus. This would correlate with anatomical findings by Lucas et al (2000)
and Word et al (1993) that demonstrate similar proportions of smooth
muscle in the uterine muscle in both upper (65.4%) segment and lower
(69.8%) segment myometrium. Given the ease to obtain lower segment
myometrium and my findings, subsequent experiments used only lower
segment myometrium.
101
4.1 The effect of PGE2 on spontaneously contracting lower
segment myometrium.
Fig 4.1 illustrates the effect of PGE2 on spontaneously contracting human
lower segment uterine tissue taken in real time from the myograph.
pge2+ep3,1
Channel 3 (
g)
4
6
8
10
12
14
16
pge-1
0
pge-9
pge-8
pge-7
pge-6
1:06:40 1:15:00 1:23:20 1:31:40 1:40:00 1:48:20 1:56:40 2:05:00 2:13:20 2:21:40 2:30:001 2 3 4 5
03/11/2005 4:16:33.906
PGE-10 PGE-8 PGE-7 PGE-6PGE-9
(g)
Time (s)
Fig 4.1 A pictorial representation of increasing concentrations of PGE2 on an
isolated myometrial strip taken from the lower segment of non-labouring
myometrium at 15 min intervals.
Increasing concentrations of PGE2 over time (s)
Contr
action f
orc
e (
g)
102
PGE2 added at concentrations of 10-10
, 10-9
, 10-8
, 10-7
, 10-6
at 15 minute
intervals, after the baseline period of spontaneous contractions had been
established caused a dose dependent increase in contractility in
concentrations above 10-8
versus spontaneously contracting myometrium
(p=0.042). The effect of PGE2 upon contractility was expressed as a ratio to
spontaneous contractility in the preceding 15 minutes and then as a
percentage of maximally stimulated contractility (Fig 4.2).
103
A B
C D
E
Fig 4.2 (A) Contraction rate, (B) peak tension, (C) duration per contraction, (D)
work done per contraction and (E) total work done in isolated myometrial strips
taken from lower segment, non-labouring myometrium at term with increasing
concentrations of PGE2 added over 75 mins. PGE2 caused a dose dependent
increase in contractility in lower segment myometrium at concentrations over 10-8
(p=0.042) versus spontaneously contracting myometrium as demonstrated by the
total work done (n=7).
104
4.2 The effect of PGF2α on spontaneously contracting lower
segment myometrium.
Fig 4.3 illustrates the effect of PGF2α on spontaneously contracting human
lower segment uterine tissue taken in real time from the myograph.
pgf2
Channel 1 (
g)
0
5
10
15
20
25
pgf-
10
pgf-
9
pgf-
8
pgf-
7
pgf-
6
1:06:40 1:15:00 1:23:20 1:31:40 1:40:00 1:48:20 1:56:40 2:05:00 2:13:20 2:21:40 2:30:00 2:38:201 2 3 4 5
02/11/2005 14:01:12.003
PGF-10 PGF-9 PGF-8 PGF-7 PGF-6
(g)
Time (s)
Fig 4.3 A pictorial representation of increasing concentrations of PGF2α on
an isolated myometrial strip taken from the lower segment of non-labouring
myometrium at 15 min intervals.
Increasing concentrations of PGF2 α over time (s)
Contr
action f
orc
e (
g)
105
There was a statistically significant increase in the total work done when
PGF2α at a concentration of 10-7
was added to spontaneously contracting
lower segment myometrium (p=0.014), (Fig 4.4). It appears to be less potent
than PGE2 as a higher concentration of PGF2α is required to significantly
increase the total work done by spontaneously contracting myometrium.
106
A B
C D
E
Fig 4.4 (A) Contraction rate, (B) peak tension, (C) duration per contraction, (D)
work done per contraction and (E) total work done in isolated myometrial strips
taken from lower segment, non-labouring myometrium at term with increasing
concentrations of PGF2α added over 75 mins. PGF2α caused a dose dependent
increase in contractility in lower segment myometrium at concentrations over 10-7
(p=0.014) versus spontaneously contracting myometrium as demonstrated by the
total work done (n=10).
107
4.3 The effect of acetyl salicylic acid (ASA) on spontaneously
contracting lower segment myometrium and PGE2 induced
contractions
Pre-incubation of lower segment human myometrial strips with ASA, an
inhibitor of both COX-1 and COX-2, for one hour prior to the experiment
led to a 3-fold reduction in peak tension and therefore overall work done,
from 15 minutes into the experiment (t15) (p=0.022). Due to the design of
the experiment (i.e. pre-incubation of the myometrium), the data could not
be normalised and is represented as such (Fig 4.5).
108
A B
C D
E
Fig 4.5 (A) Contraction rate, (B) peak tension, (C) duration per contraction, (D)
work done per contraction and (E) total work done in isolated myometrial strips
taken from lower segment, non-labouring myometrium at term with a pre-incubation
of 1μl of ASA for 60 mins prior to the experiment commencing (n= 10). This led to a
3-fold reduction in peak tension and therefore a significant reduction in the total
work done from 15 minutes into the experiment (p=0.022).
109
In the next experiment, PGE2 was added in increasing concentrations from
10-10
to 10-6
to myometrium pre-treated with ASA, and the effects were
compared with spontaneously contracting myometrium and ASA pre-treated
myometrium (Fig 4.6). The addition of PGE2 returned peak tension and
overall work done per contraction to control values.
As above, inhibition of contractility was again demonstrated by ASA, as the
total work done by the controls was significantly greater (p=0.007). The
total work done by the ASA pre-treated strips that had the addition of PGE2,
was significantly greater than the strips with ASA alone (p=0.001) but not
the controls (p=0.311).
110
A B
C D
E
Fig 4.6 (A) Contraction rate, (B) peak tension, (C) duration per contraction, (D)
work done per contraction and (E) total work done in isolated myometrial strips
taken from lower segment, non-labouring myometrium at term with increasing
concentrations of PGE2 added to pre-incubated strips of myometrium of ASA for an
hour, against strips pre-incubated with ASA alone and against controls (n=7).
There was statistical significance in the reduction of total work done by the strips
with ASA alone in comparison to the controls (p=0.007) and those strips that had
increasing concentrations of PGE2 (p=0.001). There was no statistical difference
between the controls and the ASA pre-incubated strips with the addition of PGE2
(p=0.311).
111
4.4 Discussion of the effects of PGE2, PGF2α and ASA on
contractility.
The second set of experiments quantified the effect of known agonists,
PGE2 and PGF2α. There was a statistically significant increase in the total
work done by the myometrial strips following the addition of either agent.
This was observed at concentrations above 10-8
(p=0.042) and 10-7
(p=0.014) for PGE2 and PGF2α respectively. This confirms results of
previous studies which demonstrated that PGE2 binds to myometrium 8-10
fold more than PGF2α (Hoffman HJ et al 1984). Furthermore, Giannopoulos
G et al (1985) have demonstrated that PGE2 binds to its myometrial receptor
with a higher affinity than PGF2α, and Martin RL et al (1995) have shown
PGE2 to be 8-10 fold more potent than PGF2α in stimulating myometrial
contractions.
Incubation of strips with acetyl salicylic acid (ASA) an inhibitor of both
COX-1 and COX-2 for one hour prior to the experiment led to a 3-fold
reduction in peak tension and therefore overall work done, from 15 minutes
into the experiment (p=0.022).
Furthermore, the addition of PGE2 (from concentrations of 10-10
) to pre-
incubated myometrium containing ASA, returned peak tension and overall
work done per contraction to normal values. The total work done by the
ASA pre-treated strips that had the addition of PGE2, was significantly
112
greater than the strips with ASA alone (p=0.01) but not the controls
(p=0.311). ASA (as in the previous experiment) again demonstrated
inhibition of contractility against spontaneously contracting myometrium
(p=0.07). These data suggest that stretch and synthesis of prostaglandins is
essential for spontaneous contractility in human myometrial strips and that
PGE2 is more important in stimulating contractions than PGF2α.
Having established that the synthesis of PGs is key for spontaneous
contractility, further experiments were conducted to discover a method of
blocking the effects. An established approach has been to block the pathway
in the synthesis of PGs, essentially by blocking the enzyme pathways (i.e.
COX), but NSAIDs (e.g. Rofecoxib, Indomethacin) are associated with
harmful side effects for both mother and fetus (Groom KM et al 2005,
Sawdy RJ, Lye S et al 2003). An alternative approach would be to allow the
production of PGs, but to specifically block the receptors instead.
113
5.1 The effect of an EP1 antagonist, ZD6416 upon spontaneous
and PGE2 stimulated contractions.
Three series of experiments were undertaken to assess the effects of the EP1
antagonist ZD6416 upon spontaneous and PGE2 stimulated contractions. In
the first series, ZD6416 was added at increasing concentrations of 10, 100
and 1000nM (i.e. the optimal concentration and one order of magnitude
either side) to spontaneously contracting myometrial strips at 15 minute
intervals (Fig 5.1). The EP1 antagonist had no effect upon any individual
aspect of contractility or upon total work done at any of the above
concentrations (p=0.117, p=0.103, p=0119) respectively.
114
A B
C D
E
Fig 5.1 (A) Contraction rate, (B) peak tension, (C) duration per contraction, (D)
work done per contraction and (E) total work done in isolated myometrial strips
taken from lower segment, non-labouring myometrium at term with increasing
concentrations of ZD6416 added over 45 mins (n=6). No significant difference in
any of the parameters was demonstrated as illustrated by the total work done at
the following concentrations, 10nM, 100nM or 1000nM (p=0.117, p=0.103, p=0119)
respectively.
115
In the second series of experiments, ZD6416 at 100nM was added to the
Kreb’s solution at the start of the experiment (Fig 5.2). Pre-incubation with
ZD6416 at 100nM for an hour did not inhibit any aspect of contractility nor
the total work done (p=0.104). Due to the nature of the experiment (i.e. pre-
incubation of the myometrium), the data could not be normalised and is
represented as such.
116
A B
C D
E
Fig 5.2 (A) Contraction rate, (B) peak tension, (C) duration per contraction, (D)
work done per contraction and (E) total work done in isolated myometrial strips
taken from lower segment, non-labouring myometrium at term with a pre-incubation
of 100nM of ZD6416 for 60 mins prior to the experiment commencing (n= 14). Pre-
incubation with ZD6416 did not inhibit any aspect of contractility including the total
work done up to 45 mins following the pre-incubation period (p=0.104).
117
In the third series of experiments involving ZD6416, 100nM was added to
the Kreb’s solution after the baseline period of spontaneous contractions had
been established. Following which, at fifteen minute intervals, increasing
concentrations of PGE2 was added at concentrations from 10-10
to 10-6
(Fig
5.3).
As seen previously PGE2 caused a dose dependent increase in contractility
at concentrations above 10-8
(p=0.048), but there was no inhibitory effect of
ZD6416 at this concentration of PGE2 (p=0.899).
118
A B
C D
E
Fig 5.3 (A) Contraction rate, (B) peak tension, (C) duration per contraction, (D)
work done per contraction and (E) total work done in isolated myometrial strips
taken from lower segment, non-labouring myometrium at term with increasing
concentrations of PGE2 added over 75 mins to spontaneously contracting
myometrial strips and to strips pre-incubated with a 100nM of ZD6416 (n=7). PGE2
caused a dose dependent increase in contractility in lower segment myometrium at
concentrations over 10-8
(p=0.048) versus spontaneously contracting myometrium
as demonstrated by the total work done. There was no inhibitory effect of ZD6416
at this concentration of PGE2 (p=0.899).
119
5.2 The effect of an EP3 antagonist, L798106 upon spontaneous
and PGE2 stimulated contractions.
An identical series of experiments were undertaken to assess the effects of
the EP3 antagonist L798106 upon spontaneous and PGE2 stimulated
contractions. In the first series, L798106 was added at increasing
concentrations at 10nM, 100nM and 1000nM to spontaneously contracting
myometrial strips at 15 minute intervals (Fig 5.4). A significant decrease in
contractility at concentrations of 100nM (p=0.029) and 1000nM (p=0.020)
was demonstrated.
120
A B
C D
E
Fig 5.4 (A) Contraction rate, (B) peak tension, (C) duration per contraction, (D)
work done per contraction and (E) total work done in isolated myometrial strips
taken from lower segment, non-labouring myometrium at term with increasing
concentrations of L798106 added over 45 mins (n=7). A significant decrease in the
total work done was demonstrated at concentrations of 100nM (p=0.029) and
1000nM (p=0.020) respectively.
121
In the second series (Fig 5.5), pre-incubation of the lower segment
myometrium with L798106 at 100nM caused a significant inhibition, but
not a complete abolition, of spontaneous contractility (p=0.040) from thirty
minutes into the onset of spontaneous contractions (t30).
122
A B
C D
E
Fig 5.5 (A) Contraction rate, (B) peak tension, (C) duration per contraction, (D)
work done per contraction and (E) total work done in isolated myometrial strips
taken from lower segment, non-labouring myometrium at term with a pre-incubation
of 100nM of L798106 for 60 mins prior to the experiment commencing (n= 10). Pre-
incubation with L798106 caused a significant inhibition, but not a complete
abolition, of spontaneous contractility from thirty minutes into the onset of
spontaneous contractions (p=0.040).
123
In the third series of experiments involving L798106, 100nM of the EP3
antagonist was added to the Kreb’s solution after the baseline period of
spontaneous contractions had been established. Following which, at fifteen
minute intervals, increasing concentrations of PGE2 was added at
concentrations from 10-10
to 10-6
(Fig 5.6).
At a concentration on 100nM, L798106 completely inhibited the dose
dependent increase in contractility caused by PGE2 from 10-9
(p=0.028).
124
A B
C D
E
Fig 5.6 (A) Contraction rate, (B) peak tension, (C) duration per contraction, (D) work done
per contraction and (E) total work done in isolated myometrial strips taken from lower
segment, non-labouring myometrium at term with increasing concentrations of PGE2 added
over 75 mins to spontaneously contracting myometrial strips and to strips pre-incubated
with a 100nM of L798106 (n=8). As previously demonstrated, PGE2 caused a dose
dependent increase in contractility in lower segment myometrium at concentrations over
10-8
. The myometrial strips pre-incubated with L798106 significantly reduced the increase
in total work done caused by PGE2 from concentrations of 10-9
(p=0.028).
125
5.3 Discussion of the role of EP1 and EP3 in human lower
segment myometrial contractility in relation to their respective
receptor antagonists.
There are four PGE2 receptor subtypes (EP1, EP2, EP3, and EP4), each
encoded by a separate gene (Astle et al 2005). EP1 and EP3 are excitatory
(EP1 is coupled to Gαq and EP3 to Gαi) whereas EP2 and EP4 relax
myometrial smooth muscle (both couple to Gαs) (Bastien L et al 1994). In
the contractility experiments that I conducted, I concentrated on two novel
EP1 and EP3 receptor antagonists, ZD6416 and L798106 respectively.
Three series of experiments were undertaken to assess the effects of EP1
antagonist ZD6416 upon spontaneous and PGE2 stimulated contractions. In
the first series, ZD6416 added at increasing concentrations of 10, 100 and
1000nM (i.e. the optimal concentration and one order of magnitude either
side) to human, lower segment myometrial strips at fifteen minute intervals.
The EP1 antagonist had no effect upon any individual aspect of contractility
or upon total work done at any of the above concentrations (p=0.117,
p=0.103, p=0119 respectively).
In the second series of experiments involving ZD6416, 100nM was added to
the Kreb’s solution at the start of the experiment. Pre-incubation of the
myometrial strips with ZD6416 at 100nM did not inhibit any aspect of
contractility including the total work done (p=0.104).
126
In the third series of experiments, 100nM of ZD6416 was added to the
Kreb’s solution following the establishment of spontaneous contractions.
Increasing concentrations of PGE2 was added from 10-10
to 10-6
at 15 minute
intervals. As seen previously PGE2 caused a dose dependent increase in
contractility at concentrations above 10-8
, but there was no inhibitory effect
of ZD6416 at this concentration of PGE2 (p=0.899).
An identical series of experiments were undertaken to assess the effects of
the EP3 antagonist L798106 on spontaneous and PGE2 stimulated
contractions. L798106 was added at increasing concentrations of 10, 100
and 1000nM (i.e. the optimal concentration and one order of magnitude
either side) to spontaneously contracting myometrial strips at fifteen minute
intervals. Unlike the EP1 antagonist, a significant decrease in contractility
was demonstrated at concentrations of 100nM and 1000nM (p=0.029,
p=0.020) respectively.
In the second series of experiments involving L798106, pre-incubation of
the myometrium at 100nM caused a significant inhibition, but not complete
abolition, of spontaneous contractility (p=0.040), thirty minutes from the
onset of spontaneous contractions.
In the third series of experiments involving the EP3 antagonist, following
the onset of spontaneous contractions, increasing concentrations of PGE2,
were added to myometrium pre-incubated with a 100nM of L798106. The
127
EP3 antagonist successfully inhibited the dose dependent increase in
contractility caused by PGE2 from concentrations of 10-9
(p=0.028).
Two conclusions can therefore, be drawn from the preceding. Firstly, as the
reduction in contractility caused by ASA (COX inhibitor) was more
significant than that caused by either of the PGE2 receptor antagonists (EP1
or EP3), it is likely that PGE2 plays a vital but not exclusive role in
myometrial contractility.
Secondly, given that L798106 demonstrated a significant reduction in
myometrial contractility in comparison to ZD6416 (which demonstrated
none); it is likely that the stimulating effect of PGE2 acts predominantly via
the EP3 receptor. This provides evidence that the antagonism of the EP3
receptor (rather than the EP1 receptor) may prove a suitable strategy in
pharmacological tocolysis.
The contractility data correlates with our expression studies on EP1 and EP3
receptors in non-labouring, term, lower segment myometrium
(unpublished). It was demonstrated that EP1 becomes more nuclear
localised in association with labour. This data is also consistent with that of
Grigsby et al (2006), who found no regional differences in expression of
EP1 at term or changes in association with labour and also found that
expression of EP1 was localized to the nucleus in association with labour.
Astle et al (2007) also showed that EP1 is nuclear localized, but they
128
identified a significant increase in EP1 expression at mRNA level in the
lower segment in association with labour. However this increase, which was
not demonstrated at a protein level was only of the order of 25% and no
statistical correction was made for multiple testing. There is considerable
inter-subject variation in expression levels of EP and we have concluded
that, taken together, the available data suggests that there are no regional
differences or significant changes in EP1 expression with the onset of
labour, but that at the time of labour the receptor is localised to the nucleus.
This suggests that the function of EP1 at term may be less to mediate
intercellular signaling and more to mediate the nuclear effects of
endogenous, intracellular PGE2. EP1 is therefore unlikely to be the principal
receptor which mediates PGE2 stimulated myometrial contractions.
Furthermore, at a protein level it was demonstrated that EP3 expression
remains cytosolic in myometrium with the onset of labour, and like Grigsby
et al (2006), but unlike Astle et al (2007) or Leonhardt et al (2003) we found
significant expression in myocytes. We have concluded that the available
data suggests that there are no regional differences or significant changes in
EP3 expression with the onset of labour, but that EP3 is probably principally
contractile in its action and remains cytosolic or on the cell membrane. EP3
is therefore able to be liganded by extracellular, exogenous PGE2 and is
likely to be the receptor which mediates PGE2 stimulated myometrial
contractions.
129
6.1 The effect of FPA, a PGF2α antagonist upon spontaneous
and PGF2α stimulated contractions.
Increasing concentrations (100-1000nM) of a novel and selective FP
antagonist (FPA) were added to isolated, lower segment myometrial strips.
In comparison to the non-treated controls, FPA did not reduce the total work
done by spontaneous contractions, even at the highest concentration of
1000nM (10-6
) (p=0.50).
130
A B
C D
E
Fig 6.1 (A) Contraction rate, (B) peak tension, (C) duration per contraction, (D)
work done per contraction and (E) total work done in isolated myometrial strips
taken from lower segment, non-labouring myometrium at term with increasing
concentrations of FPA added over 45 mins (n=6). No significant difference in any of
the parameters was demonstrated as illustrated by the total work done even at the
highest concentration of 1000nM (10-6
) (p=0.5).
131
In the next experiment, 10-8
PGF2 was added to spontaneously contracting
strips of lower segment myometrium, following which increasing
concentrations of FPA (10-8
to 10-5
) were added at 15 minute intervals. The
total work done by the myometrial strips which had the cumulative addition
of FPA is decreased five-fold (Fig 6.2). This is in comparison to the PGF2
pre-treated strips alone (p=0.044).
132
A B
C D
E
Fig 6.2 (A) Contraction rate, (B) peak tension, (C) duration per contraction, (D)
work done per contraction and (E) total work done in isolated myometrial strips
taken from lower segment, non-labouring myometrium at term with increasing
concentrations of FPA (10-8
to 10-5
) added over 45 mins following an initial addition
of 10-8
PGF2 (n=12). Increasing concentrations of FPA, significantly decreased the
total work done by strips treated with PGF2 in comparison the PGF2 - treated
strips alone and at concentrations of 10-5
, total work done was reduced up to five-
fold (p=0.044).
133
6.2 Discussion of the effect of FPA, a novel PGF2α antagonist on
contractility.
The inhibition of FP has been proposed as a strategy to prevent preterm
delivery. FP antagonists have been shown to inhibit contractions in isolated
sheep and human myometrium (Olson DM et al 2003) and to open BKCa
channels (Doheny HC et al 2007). FP antagonists also delay RU486 or LPS
induced labour in mice, rat and sheep (Olson DM et al 2003, Chollet A et al
2007). Therefore, I attempted to demonstrate the inhibition of human
myometrial contractility with a novel and selective FP antagonist (FPA).
Pre-incubation of the baths with concentrations up to 1uM (10-6
) did not
affect the total work done by spontaneous contractions in comparison to the
non-treated controls (p=0.50).
However, in myometrium pre-treated with a concentration of 10-8
of PGF2α,
increasing concentrations of the FPA (from 10-7
) decreased the contractility
by five-fold. This was in comparison to PGF2α pre- treated myometrial
strips alone (p=0.044).
This demonstrates that FPA is able to block FP receptors, but does not
inhibit spontaneous contractions. It is likely therefore, that PGF2α has no
role in spontaneous myometrial contractility. This is in keeping with data
previously published; i.e. PGE2 binds to myometrium 8-10 times more than
PGF2α (Hoffman HJ et al 1984), PGE2 is 8-10 times more potent than PGF2α
in stimulating myometrial contractions (Martin et al 1995) and PGE2 binds
134
to its myometrial receptor with higher affinity than does PGF2α to its
receptor (Giannopoulos G et al 1985).
135
7.1 The effect of combining the agonists, OT and PGE2 on
spontaneously contracting lower segment myometrium.
Having successfully demonstrated the effects of the EP1 and EP3
antagonist, GSK were eager to investigate the effects of a novel OT
antagonist, GW796679X in combination with the aforementioned EP3
antagonist, L798106 on spontaneous contractions. The first step was to
investigate the effect of combining OT and PGE2 on spontaneously
contracting lower segment myometrium.
To determine if OT and PGE2 combined produce a synergistic effect, first a
concentration of 10-8
OT was added to spontaneously contracting
myometrium. At fifteen minute intervals, increasing concentrations of
PGE2 from 10-10
to 10-7
were added to the organ baths. These strips
containing both agonists were compared against myometrium with either
agonist alone or spontaneously contracting controls (Fig 7.1).
OT alone at a concentration of 10-8
increased contractility significantly in
comparison to spontaneously contracting controls (p=0.001). A similar
effect was demonstrated by PGE2 alone at a concentration of 10-9
(p=0.03).
Furthermore, in combination, the PGE2 and OT significantly increased the
total work done by the lower segment myometrium (p=0.035).
136
However, the combination of the two agonists caused a significant increase
in total work done in comparison to the myometrium with the OT alone
(p=0.041) but not by the PGE2 alone (p=0.32).
137
A B
C D
E
Fig 7.1 (A) Contraction rate, (B) peak tension, (C) duration per contraction, (D)
work done per contraction and (E) total work done in isolated myometrial strips
taken from lower segment, non-labouring myometrium at term with increasing
concentrations of PGE2 added over 60 mins following an initial addition of 10-8
of
OT; compared against PGE2 alone, OT alone and spontaneously contracting
myometrium (n=8). Both the agonists individually, 10-8
OT (p=0.001), 10-9
PGE2
(p=0.03) and in combination (p= 0.035) significantly increased the total work done.
Furthermore, the combined effect of the agonists on total work done was
statistically greater than the strips with OT alone (p=0.041) but not PGE2 alone
(p=0.32).
138
7.2 The effect of combining the antagonists of OT and PGE2 on
spontaneously contracting lower segment myometrium.
To determine if there is a synergistic effect when combining antagonists,
equimolar concentrations the OT antagonist, GW796679X and the EP3
antagonist L798106, lower segment myometrial strips were pre-incubated
with either the EP3 antagonist alone, the OT antagonist alone or a bath with
both the OT and the EP3 antagonists in combination against control strips of
spontaneously contracting myometrium.
There was a statistically significant inhibition of contractility in the
myometrial strips with L798106 alone at 30mins (p=0.029), but no
inhibition by GW796679X alone (p=0.547), nor by a combination of both
the antagonists (p=0.683).
A synergistic effect of combining both an EP3 and an OT antagonist was
not demonstrated in this experiment.
139
A B
C D
E
Fig 7.2 (A) Contraction rate, (B) peak tension, (C) duration per contraction, (D)
work done per contraction and (E) total work done in isolated myometrial strips
taken from lower segment, non-labouring myometrium at term pre-incubated with
equimolar concentrations of L798106, GW796679X and of the two antagonists
together for a period of 60 mins (n=10). There was a statistically significant
reduction in total work done by L798106 alone at 30mins (p=0.029), but no
inhibition by GW796679X alone (p=0.547), nor a combination of the two
antagonists (p=0.683).
140
7.3 Discussing the effect of combining the agonists, OT and
PGE2 and their respective antagonists on spontaneously
contracting lower segment myometrium.
Oxytocin is known to have a potent contractile activity on the pregnant
uterus. In many animal models, OTR is regulated by changes in steroid
hormone concentrations (Soloff MS 1975). In humans, the lack of marked
changes in oestrogen concentrations or progesterone withdrawal suggests
that OTR has a more complex regulation (Gimpl G et al 2001). Further
relationships in the OTR system have been demonstrated, in particular with
prostaglandins. Treatment with Naproxen, a prostaglandin-synthesis
inhibitor, resulted in a dramatic decrease in OTR in the rat uterus at term.
Administration of PGF2α restored the increase in OTR, implying the
possibility of a positive feed- back loop between prostaglandins and OTR
(Chan WY 1980).
In the final series of experiments, 10-8
OT increased total work done by
sponataneously contracting lower segment myometrium significantly
(p=0.001) as did 10-9
PGE2 (p=0.03). Unsurprisingly, a combination of OT
and PGE2 demonstrated an increase in total work done on spontaneous
contractions (p= 0.035). This combination of OT and PGE2 also increased
the total work done significantly over myometrial strips with 10-8
OT alone
(p= 0.041). This effect however, was not demonstrated in relation to
myometrial strips with PGE2 alone (p=0.32). This experiment therefore,
demonstrates that whilst PGE2 is a potent stimulator of spontaneous
141
contractions (more so than OT), no synergistic effect between PGE2 and OT
was observed.
The next step was to determine if a synergistic effect could be demonstrated
between an OT antagonist and a PGE2 antagonist. Control strips of lower
segment myometrium were set up in parallel with strips pre-incubated with
either the EP3 antagonist alone, the OT antagonist alone and a bath with
equimolar concentrations of both the OT antagonist and the EP3 antagonists
in combination. There was a statistically significant inhibition of the
myometrial strips by the EP3 antagonist alone at 30mins (p=0.029), but no
inhibition by the OT antagonist alone (p=0.547), nor the combination of
both antagonists (p=0.683). A synergistic effect of combining both an EP3
and an OT antagonist was not demonstrated in this experiment.
Given that 1) PGE2 is more powerful than OT in stimulating contractions
and 2) the OT antagonist is not effective as the EP3 antagonist in inhibitng
spontaneous contractions, these set of experiments support the argument in
favour of a new tocolytic.
142
8.1 Discussion
The primary function of the uterus during gestation is to harbour the
growing fetus in a largely quiescent environment. Upon maturation of the
fetus, the uterus must remodel itself sufficiently to generate forceful
contractions during labour. During preterm delivery, the process of
remodelling of the myometrium occurs early due to a number of causes,
although the underlying basis of myometrial contraction remains the same.
What is still unknown is to what degree various signalling factors and
hormones affect myometrial contractility and certainly myometrial
contractility in preterm labour. Due to the multi-factorial origin of preterm
labour, treatment options generally target the inhibition of uterine
contractility, rather than the underlying mechanisms of labour.
Tocolytics, used in Europe for the last 30 years, are the backbone of
treatment for the prevention of preterm labour. Tocolytic drugs are indicated
to prolong pregnancy in women with acute risk of preterm birth; in some
cases they are also administered for prophylaxis. The main rationale for use
of these drugs is to delay delivery for at least 48 hours in order to allow
treatment effect of corticosteroids or transfer of the pregnant woman to a
specialised high-risk obstetric unit. Both actions have been show to
markedly reduce neonatal morbidity and mortality (Husslein P et al 2003).
As most randomised trials of tocolytic agents lack power to provide definite
answers, and use delay in delivery as a surrogate endpoint for implied
143
improvements in neonatal outcome related to the administration of steroids,
systemic reviews and meta-analyses have been conducted to more
adequately study drug efficacy and safety. Several Cochrane reviews have
been published that compare different tocolytic agents to each other or to
placebo for women in preterm labour (King JF et al 2003, 2005,
Anotayanonth S et al 2004, Whitworth M et al 2008).
Many trials included maintenance treatment if and after contractions
stopped. Some trials excluded women with ruptured membranes, but in
others they were included. The most frequently evaluated agent was
Ritodrine, which has predominantly β2- receptor effects; relaxing muscles
in the uterus, arterioles and bronchi. Other tocolytics evaluated in these
trials included Isoxuprine, Terbutaline, Magnesium sulphate, Indomethacin,
Nifedipine and Atosiban. Overall tocolytics were associated with a
reduction in the odds of delivery within 24 hours (OR 0.47; 95% CI 0.29-
0.77), 48 hours (OR 0.57; 95% CI 0.38-0.83) and seven days (OR 0.60; 95%
CI 0.38-0.95). For beta agonists, Indomethacin, Nifedipine and Atosiban
these effects were statistically significant, but not for Magnesium sulphate.
However, there was no statistically significant reduction in births before 30
weeks (OR 1.33; 95% CI 0.53-3.33), before 32 weeks (OR 0.81; 95% CI
0.61-1.07) or 37 weeks of gestation (OR 0.17; 95% CI 0.02-1.62) (RCOG
2006). The choice of tocolytic agent depends mainly on the availability
following regulatory approval in the country, the drug efficacy and
144
effectiveness, the fetal and maternal safety profiles and the cost of
treatment.
Beta-mimetics have until the recent past, been the most commonly used
tocolytic agent. It is certainly the most thoroughly evaluated, but as their
mechanism of action is organ non-specific, they notoriously lead to
numerous adverse events in multiple organ systems, which are largely
cardiovascular in origin. Common adverse effects when beta-agonists are
compared with no treatment or placebo include: palpitations (48% beta-
agonists vs. 5% control), tremor (39% vs. 4%), nausea (20% vs. 12%),
headache (23% vs. 6%) and chest pain (10% vs. 1%) (Gyetvai K et al 2009).
Rare but serious and potentially life threatening adverse effects have been
reported following the use of beta-mimetics and there have been a small
number of maternal deaths associated with the use of these drugs.
Pulmonary oedema is a well documented complication, often associated
with aggressive intravenous hydration.
For the other tocolytic drugs, fewer types of adverse effects were reported,
and they occurred less frequently (Gyetvai K et al 2009). With Magnesium
sulphate, 7% discontinued treatment compared with none on placebo and
none of the women allocated Indomethacin discontinued treatment
compared to the control. For Atosiban, reported side effects were nausea
(11% Atosiban vs. 5% placebo), vomiting (3% vs. 4%), headache (5% vs.
7%), chest pain (1% vs. 4%) and dyspnoea (0.4% vs. 3%).
145
Systemic reviews of Nifedipine versus beta-mimetics have come to similar
conclusions (Oei SG et al 1999, Tsatsaris V et al 2001). In both, there has
been insufficient evidence for any conclusion, in particular with regards to
the effect on neonatal mortality. Nifedipine, is however, associated with a
greater chance of delaying labour for more than 48 hours (RR 1.13; 95% CI
1.01-1.267), a lower risk of respiratory distress syndrome and admission to
the neonatal unit. In addition, there were fewer maternal adverse effects
with Nifedipine in comparison to Ritodrine (16% vs. 45%). Nifedipine has
the advantage of oral administration and is economically sounder.
Trials comparing Indomethacin with beta-agonists have shown not much
difference in delay to delivery, but the former have fewer maternal side-
effects. However, the risk of premature closure of the ductus, renal and
cerebral vasoconstriction and necrotising enterocolitis associated with a
high dose and prolonged exposure of Indomethacin has led to concern and
reluctance of its use in the clinical arena (Eronen M et al 1991, Norton ME
et al 1993).
Atosiban has been compared with at least three different beta-agonists;
Ritodrine, Salbutamol and Terbutaline. The delay in delivery was not
statistically significant at 48 hours (88% Atosiban vs. 89% beta-mimetics)
(RR 0.99%; 95% CI 0.94-1.04) or at 7 days (80% vs. 77%) (RR 1.03%;
95% CI 0.95 – 1.11) (The Worldwide Atosiban vs. Beta-agonists Study
Group 2001). The side effect profile of Atosiban is significantly better than
146
beta-agonists, including chest pain (1% Atosiban vs. 5% beta-agonists),
palpitations (2% vs. 16%), tachycardia (6% vs. 76%), hypotension (3% vs.
6%), dyspnoea (0.3% vs. 7%), nausea (12% vs. 16%), vomiting (7% vs.
22%) and headache (10% vs. 19%). The financial implications of using
Atosiban are significantly greater however (£498.30 Atosiban vs. £0.15
Nifedipine), assuming complete 48 hour treatment regardless of in-utero
transfer (Siassakos D et al 2009).
Therefore, if a decision is made to use a tocolytic agent, Ritodrine is no
longer seems to be the drug of choice, given the most serious side effect is
pulmonary oedema (Tan TC et al 2006). Atosiban does not affect the
maternal heart rate or blood pressure (Goodwin TM et al 1994) and can be
used safely in women with cardiac disease and diabetic women. When
compared with placebo in a randomised trial (Romero R et al 2000),
Atosiban resulted in lower birth weight and an increase in infant deaths. In
this trial, there was an imbalance in randomisation that resulted in more
women under 26 weeks gestation and fewer over 32 weeks assigned to the
Atosiban group. Considering the significantly higher morbidity and
mortality of babies born under 26 weeks, compared with those born over 26
or 32 weeks, this imbalance can be argued to invalidate the findings of the
study (Word RA et al 2003, 2005). Other randomised trials without such
problems have not replicated these findings (Valenzuela GJ et al 2000).
With regards to the long term follow-up of infants of mothers who received
147
Atosiban, it has been reported that developmental outcomes at 6, 12 and 24
months are the same when compared to placebo (Papatsonis DN et al 2005).
Nifedipine, unlike Atosiban, is not licensed for use as a tocolytic in the
United Kingdom but given its relatively safe side-effect profile, its similar
efficacy to Atosiban in delaying labour and definite cost effectiveness, it
used in most obstetric units around the country. However, Nifedipine is
contraindicated in women with cardiac disease and should be used in
caution in women with diabetes. Cardiac failure has been reported with the
use of Nifedipine (van Geijn HP et al 2005, Tan TC 2006) and in cases of
maternal hypertension, where Nifedipine has been administered, there has
been an association with a redistribution of fetal blood flow (Vojcek L et al
1988). Brown MA et al (2002) have concluded that signs of fetal distress
during fetal heart rate monitoring under these circumstances, have increased
the rate of caesarean sections It is unknown whether these estimates can be
applied to Nifedipine when used for tocolysis.
In summary, it seems further evidence is required regarding the effects of
both Nifedipine and Atosiban on more substantive outcomes such as
neonatal mortality and morbidity, and on safety and long-term outcome for
the child. It is not therefore unreasonable to consider the need for a new
tocolytic.
148
Prostaglandins have long been postulated to play a role in the parturition
process, mainly as their synthesis is dramatically increased in association
with labour (Challis JR et al 2002, Olsen DM et al 2003). PGE2 in
particular, is produced in large quantities by fetal membranes and possible
roles include cervical ripening and the stimulation of uterine contractions
(Challis JR et al 2000). Therefore, these desired effects lend them to use in
obstetric practice.
PGE2 exhibits a wide spectrum of physiological actions depending on the
distribution and subtypes of EP receptors present (Breyer RM et al 2001).
All four PGE2 receptors EP1-4 are present in human myometrial tissue
during pregnancy, with distinct cellular patterns of expression being
observed. Expression of EP1-4 receptor proteins are localised to vascular
smooth muscle, vascular endothelium, glandular epithelium and decidual
components of myometrial tissue. However, there is no consensus on
whether there is a definite up-regulation of excitatory EP1 and EP3 or a
down-regulation of EP2 or EP4 in labour.
Functionally, EP1 and EP3 couple to G proteins, Gqα or Giα, and promote
calcium influx or the inhibition of adenylate cylase respectively (Breyer RM
et al 2001, Bos CL et al 2004). The stimulation of EP1 and EP3 would,
therefore, result in the contraction of smooth muscle or an increase in
inflammatory outputs. By contrast, EP2 and EP4 couple to Gsα to increase
adenylyl cyclase activity and intracellular cAMP levels, leading to smooth
149
muscle relaxation and repressed expression of many inflammatory cytokines
(Regan JW 2003).
The only consistency in the literature is that both contractile and relaxatory
EP receptors are simultaneously expressed in myometrial tissue. Thus,
pharmacological studies using isolated myometrial strips and EP receptor
agonists and antagonists is a more systematic approach to an understanding
of the mechanisms by which PGE2 exerts its effects. This may lead to the
pharmacological isolation of pro-labour actions of PGE2 from pro-
pregnancy actions and therefore to the improved management of obstetric
patients from current management.
In summary, our data demonstrates that the stretch of term human
myometrium results in spontaneous contractions. The addition of ASA (a
non-selective COX inhibitor) did not completely stop spontaneous
contractility but reduced the total work done by up to five-fold. This
indicates that prostaglandins play a significant role but not exclusive role in
spontaneous contractility. The PGF2α antagonist did not have an inhibitory
effect on spontaneous contractions, but only those contractions induced by
the addition of PGF2α prior to the experiment. The EP1 antagonist similar to
the PGF2α antagonist did not prove to be efficacious in inhibiting
spontaneous contractions. The only significant reduction in total work done
on spontaneously contracting lower segment myometrium was by the EP3
antagonist.
150
Schild analysis has shown that each is a competitive antagonist devoid of
agonist activity up to 10µM. The effective concentrations of each antagonist
against their respective receptors were two orders of magnitude apart. A
concentration of 100nM of each antagonist was calculated to ensure
complete and specific inhibition of the relevant receptor. The EP3 receptor
antagonist proved to cause significant reduction in spontaneous and PGE2
induced contractility in term, non-labouring myometrium. The EP1 receptor
antagonist, however, was not effective. These results therefore support the
use of the EP3 antagonist in further clinical trials with regards to a new
tocolytic compound.
The advantages of targeting the EP3 antagonist alone, centre in that it would
be more specific than knocking out PGE2 in general, and therefore
potentially have fewer side effects. EP3 has been described in the literature
as having other specific roles in the body. Inflammatory breast cancer (IBC)
is the most aggressive subtype of locally advances breast cancer with the
worst prognosis and shortest overall survival of any variant of this type of
disease (Dawood S et al 2008). Current observations indicate that COX-2,
EP3 and EP4 are all upregulated in IBC cells. Robertson FM et al (2010)
have suggested that agents directly or indirectly targeting the signalling
pathways of PGE2, EP3 or EP4 receptors may be important in inhibiting the
aggressive phenotype of IBC.
151
However, in the gastrointestinal tract, PGE2 appears to play an important
role in the modulating the mucosal integrity and various functions of the
alimentary tract. In the duodenum, acid perfusion produces damage in
animals lacking EP3 receptors (Takeuchi K 2010). As with all receptors in
the body, there are very few that are exclusive to one organ system in the
body. Ultimately one would have to work out a concentration of EP3
antagonist to effectively knock off myometrial contractility whilst avoiding
significant adverse effects globally in the body.
Whilst there has been considerable literature concerning EP receptor
function, expression and regulation in myometrium, there is little
information on key issues such as cellular distribution of EP receptor
expression and regulation in the uterine cervix. Several lines of evidence
support a pivotal role for PGE2 in the regulation of the cervical ripening
process. Firstly, PGE2 has been used clinically for at least 25 years to induce
softening of the cervix successfully at term human pregnancy (Hughes EG
et al 2001). Furthermore, COX-2 expression and PGE2 synthesis has been
found to increase at the time of parturition in the rat and sheep cervix
(Mitchell MD et al 1978, Dong YL et al 1996). In addition, PGE2 increases
human cervical collagenolytic activity and GAG synthesis in the rat cervix
(Cabrol D et al 1987), induces vasodilatation of human cervical arteries
(Allen J et al 1988) and thus promotes subsequent oedema and leukocyte
infiltration (Kelly RW 2002).
152
Schmitz T et al (2006) demonstrated the presence of all four EP receptor
proteins in all tissue components of the ovine cervix: the endothelium and
smooth muscle of the cervical blood vessels, the epithelium of the cervical
canal, the circular and longitudinal muscle layers and the stroma. The
localisation of the four EP receptors in blood vessels emphasises
components of the vascular system as possible targets for PGE2. EP1 and
EP3 receptors induce vasoconstriction, whereas EP2 and EP4 receptors
cause vasodilatation (Wright DH et al 2001). Furthermore, the effects of
steroid hormones on EP receptor expression in the cervix, demonstrate that
whilst progesterone has no effect on any of the four EP receptors, estradiol
significantly decreased EP1 and EP3 receptor protein expression in the
blood vessel media of the cervix (Schmitz T et al 2006).
Estradiol, by decreasing EP1 and EP3 receptor expression, would facilitate
EP2 and EP4 receptor dependent vasodilator effects of PGE2 and therefore,
promote cervical ripening. In other studies, EP4 receptor agonists have been
shown to increase pregnant guinea-pig cervical compliance (Kanayama N et
al 2004). In summary, by altering the balance of positive and negative
influences on the cervix, reducing EP1 or EP3 receptor expression, may as a
result increase the EP2 or EP4 receptor expression and cause cervical
ripening. The regulation of PG synthesis and action occurring in the
myometrium and cervix in association with labour appear to differ markedly
between the two tissues, indicating tissue- specific roles for prostaglandins.
153
It may be that the classical approach of using tocolytics, with the sole
purpose of arresting uterine contractions for the treatment of preterm labour
is not the correct approach. Preterm labour should be viewed as a syndrome
(Romero R et al 2006), where a number of pathological processes including
intrauterine infection/ inflammation, uterine ischaemia, uterine over-
distension, abnormal allograft reaction, allergy, cervical insufficiency and
hormonal disorders (progesterone related and corticotrophin-releasing factor
related) may provide a trigger which stimulates the complex cascade
culminating in uterine contractility, cervical dilatation and activation of the
membranes/ decidua all of which initiate labour. Therefore, a multi-factorial
approach in the diagnosis and management of preterm labour would appear
to be the best solution in the treatment of preterm birth.
154
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