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\begin{document}

\title{Pathophysiological mechanisms of maternal pro-inflammatory mediators in
preterm labour}



\author[1]{Kwame Adu-Bonsaffoh}%
\author[1]{Fidelis Bayor}%
\affil[1]{University of Ghana Medical School}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
Available therapeutic interventions for managing preterm labour have not
been consistently successful due to controversies related to its
aetiology. However, multiple mechanisms including inflammation play a
significant role in the pathogenesis of preterm labour. The
extracellular matrix of the amniochorion contains collagen fibres that
maintain the tensile strength of the amniochorion, resisting mechanical
stress and preventing rejection of the foetal allograft. Expression of
pro-inflammatory mediators in the amniochorion triggers production of
prostaglandins in the uterus and enzymatic degradation of the resilient
extracellular matrix of the foetal membranes by matrix
metalloproteinases leading to uterine contractions, cervical remodelling
resulting in preterm labour.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Preterm labour is defined as the spontaneous onset of regular and
painful uterine contractions resulting in the dilatation of the cervix
or cervical effacement prior to 37 weeks of gestation(1,2). Preterm
labour may result in preterm birth which is generally classified based
on the gestational age at birth as: extremely preterm (before 28 weeks),
very preterm (between 28 and 32 weeks) and moderately preterm (after 32
weeks and before 37 weeks)(3,4). The moderate preterm birth can further
be described as late preterm if the onset of it occurs between 34 and 37
weeks of gestation(3,4). Etiologically, preterm birth can either be
spontaneous (natural onset of labour or preterm premature rupture of
membranes) or provider-initiated (medically induced labour or caesarean
delivery) depending on the clinical presentation(5--7).

The global incidence of preterm birth is 12\% which is equivalent to
approximately 15 million babies born preterm each year(8,9). The
incidence ranges from 5\% among the European countries to nearly 20\% in
some African countries(9--11). As the principal cause of increased
morbidities and mortalities among children less than five years old,
prematurity has become a major public health concern(9,12) and
compromises the emotional and psychological states of many families with
preterm infants born to them. This is attributable to the accelerated
economic burdens exerted due to increased cost of health care and
learning demands(5,10,11,13). Evidence has shown that infants who are
born at the extremes of gestational age are at increased risk of severe
longstanding health complications (11,13--15). The consequences of
preterm birth are common during the neonatal period and may persist into
adulthood. Studies have also shown that male infants born preterm have
poorer prognosis and are at increased risk of health complications
compared to their female counterparts(11,16,17).

The actual aetiology of preterm labour is not known, although multiple
mechanisms have been propounded in the pathogenesis of preterm labour.
For instance, intrauterine infections, inflammation, mechanical stress
or over distension of the uterus, intrauterine growth restriction (IUGR)
and uteroplacental hypoxia or haemorrhage are said to trigger the onset
of preterm labour (5,11,15,18). Additionally, maternal race (black
women, African-American or Afro-Caribbean) or ethnicity, younger
maternal age, advanced maternal age (\textgreater{}35 years), cigarette
smoking, low maternal weight, obesity, multiple pregnancy, use of
assisted reproductive techniques, maternal history of preterm birth and
the marginalised may also contribute to the onset of preterm
labour(11,19). Regardless of the predisposition, maternal
pro-inflammatory mediators play a central role in the initiation of
preterm labour.

Clinical management of preterm labour is extremely challenging with poor
outcomes especially when uterine contractions are established with
progressive cervical changes. The well-known unpredictability of the
clinical course of preterm labour results from the unresolved
pathophysiology of the condition. The major hypothesized
pathophysiological mechanisms of preterm labour include inflammation
resulting from multiple aetiological pathways. Improved understanding of
the mechanistic role of pro-inflammatory mediators in preterm labour is
critical in evidence-based clinical management especially inflammation
associated with chorioamnionitis and urinary tract infections(8). The
aim of this review was to appraise the pathophysiological mechanism of
pro-inflammatory mediators in spontaneous preterm labour and their
associations with multi-factorial etiological pathways. The paper also
recognises the discusses the pathophysiology of uterine contractions and
the phases of uterine smooth muscle activity in the non-pregnant state,
during pregnancy and parturition. In this paper, essential physiological
pathways and the biological basis of available therapeutic agents for
preterm labour are explored.

\textbf{Probable causes of preterm labour}

The role of genetics in the pathogenesis of preterm labour has been duly
recognised and well documented. Emerging evidence indicates that preterm
birth appears to be transmitted primarily in a matrilineal manner across
generations and is greatly influenced by mutual environmental factors.
Thus, the risk of a woman having a preterm delivery is heightened if her
maternal biological relations had preterm deliveries but appears not to
follow paternal lines(20,21). In human genomic studies, changes in
single gene sequence were found to be associated with disorders such as
polyhydraminos, myotonic dystrophy, cervical incompetence(10,21,22) and
twin gestation which increase the risk of preterm labour and may be
transmitted from one generation to the next(23,24). These observed
single nucleotide polymorphisms in the gene can respond to inflammatory
stimuli such as infection resulting in the production of
pro-inflammatory mediators and breakdown of extracellular matrix by
matrix metalloproteinases leading to preterm labour (figure 1) (23).
Also because of the genotypic variations among different individuals,
exposure to environmental pollutants put them at increased risk of
preterm labour(10,22).

Evidence has shown that maternal psychological stress, anxiety,
financial difficulties and other life events increase endogenous
catecholamines and cortisol release which stimulate premature production
of placental corticotrophin-releasing hormone (CRH) thereby activating
the biological cascade leading to the onset of preterm labour(24).
Psychological stress also increases maternal vulnerability to infection
and inflammation due to weakened immune system (figure 1) (24).
Behavioural factors such as smoking and alcohol use are associated with
increased risk of preterm labour. Given that smoking and alcohol have
teratogenic effects and habitual engagement in these agents during
pregnancy puts them at high risk of sudden infant death syndrome (SIDS)
due to variability in autonomic nervous system function(25--27). The
Nicotine and carbon monoxide contained in smoke cause foetal hypoxia due
to carboxyhaemoglobin and vasoconstriction(27). They increase the risk
of low birth weight, foetal defects and placental complications and
compromise immune response to inflammation leading to preterm premature
rupture of membranes and preterm labour (figure 1) (24,25,27).
Consumption of alcohol during pregnancy has also proven to have
detrimental effect on foetal growth and development leading to
intrauterine growth restriction (IUGR) which poses a potential risk for
preterm labour (figure 1) (24,28).

Furthermore, pervasive indulgence in illegal drugs such as marijuana and
cocaine as well as the use of herbal medications in pregnancy have
severe embryotoxic and teratogenic effects increasing the risk of
small-for gestational age and foetal defects(29,30). Marijuana smoking
also increases the risk of carboxyhaemoglobin leading to foetal hypoxia.
On the other hand, cocaine causes elevated blood pressure due to
vasoconstriction since these psychoactive agents appear to interfere
with the reuptake of serotonin and catecholamines leading to compromised
immune response to inflammation (figure 1) (24,29).

Nutritional deficiencies (e.g. deficiencies in iron, folate or zinc, low
pre-pregnancy weight) have been implicated in preterm labour. For
instance, deficiency in iron causes iron deficiency anaemia which
increases the synthesis and hypersensitivity to endogenous
catecholamines leading to maternal and foetal hypoxia which in-turn
stimulates synthesis of foetal CRH and cortisol. The increased cortisol
production may activate the biological cascade leading to preterm
premature rupture of membranes, gestational hypertension, eclampsia IUGR
and preterm labour (24,31,32). In another pathway, iron deficiency
anaemia increases risk of infection and inflammation leading to preterm
birth (figure 1) (31,32). Physiologically, vitamin C stimulates the
synthesis of collagen fibres of connective tissue extracellular matrix
of the amnionchorion and deficiency in vitamin C depletes collagens
leading to preterm premature rupture of membranes and preterm
labour(figure 1) (24,33,34). Similarly, folate, zinc and pre-pregnancy
weight deficiencies increase the risk of restricted foetal growth and
birth defects(figure 1) (24,35). To buttress the importance of specific
nutrients, adequate maternal levels of fat-soluble nutrients provide
antioxidative, anti-inflammatory, and immunomodulatory health benefits
which are vital in preterm birth prevention. For instance, omega-3 fatty
acids facilitate increased production of specialized anti-inflammatory
mediators with resultant reduction in preterm birth risk (figure 1).
Combined effects of these nutrients support appropriate placental
organogenesis and function(36).

\section*{Uterine smooth muscle activity in the non-pregnant
state}

{\label{uterine-smooth-muscle-activity-in-the-non-pregnant-state}}

The uterus acts as a temporal receptacle which nurtures and nourishes
the implanted blastocyst while thwarting early expulsion of the product
of conception throughout pregnancy. It is a contractile smooth muscular
organ made up of fibrous connective tissue (37) that exhibits
spontaneous and rhythmic contraction and relaxation before, during and
after pregnancy(38,39). The non-pregnant uterus is not totally a
quiescent organ and exhibit intrinsic contractile properties. However,
contractions in the non-pregnant uterus vary physiologically from the
uterine contractile activities during pregnancy(40). Accordingly, both
non-invasive intrauterine pressure (IUP) and invasive ultrasound (US)
scan recordings have demonstrated that the non-pregnant uterus undergoes
patterns of phasic wave-like contractions throughout the menstrual
cycle(39,41--45) which are governed by ovarian steroid hormones (i.e.
oestrogen and progesterone)(38). Within the subendometrial layer of the
myometrium, cyclic patterns of oestrogen and progesterone receptors are
expressed which modulate the activity of the non-pregnant uterus(38,44).
These wave-like contractile patterns of the non-pregnant human uterus
vary depending on the phase of the menstrual cycle(figure 2) (42,44).
The rhythmic uterine contractions in the non-pregnant uterus favours
sperm transport and oocyte migration in the fallopian tubes,
fertilization and embryonic transport from the tubes into the uterine
cavity to facilitate embryonic nidation and implantation to
occur(39,41,42,44). Disruptions in this physiological uterine smooth
muscle property are associated with disorders such as dysmenorrhoea,
spontaneous and recurrent abortions, endometriosis, implantation
failures and infertility(42,44).

During menstruation or early follicular phase, the uterus exhibits
primarily antegrade (from fundus to cervix) labour-like and expulsive
contractions (figure 2) involving all layers of the myometrium to
evacuate the content of the uterus (menses). Uterine contractions during
menstruation are often felt and can be associated with blunt pain
(dysmenorrhoea) requiring medications if contractions are
vigorous(44,45). This period of luteofollicular changeover is largely
under the influence of progesterone depletion due to spontaneous
degeneration of the corpus luteum and increased gene expression for
prostaglandins by uterine tissues (figure 2) (39,45). In both mid and
late follicular phase of the menstrual cycle, only the subendometrial
layer exhibits a progressive increased in wave-like uterine contraction
patterns which are retrograde (from cervix to fundus) (figure 2). This
retrograde uterine contractions, often not perceived by the woman,
coupled with proliferation of endometrial glands which aid the transport
of sperm towards the distal end of the fallopian tubes where
fertilization takes place and normally terminates at the pre-ovulatory
period(41,42,44--46). These wave-like contractions and glandular
proliferation during the follicular phase are controlled by increased
oestrogen (E2) levels (figure 2) (41,42,44,46) indicative of oestrogen
predominance in the proliferative phase of the uterine cycle.

The post-ovulation period is characterized by progressive uterine
quiescence facilitated by significant rise in progesterone levels
following the successful development of the corpus luteum from the
ruptured ovarian follicle which terminates at the middle of the luteal
phase (figure 2). This luteinisation associated uterine quiescence
promotes successful establishment of pregnancy characterised by
embryonic transfer from the fallopian tube into the endometruim
depending on the biochemical readiness of the endometruim for
implantation of the embryo and placentation(39,41,42,46). Following
implantation, the uterus is transitioned from a non-pregnant state to a
pregnant milieu (figure 2) which allows the developing blastocyst to
differentiate into the foetal membranes comprising rich in connective
tissues in both the amnion and the chorion(47). The connective tissue
extracellular matrix of the amniochorion contains collagen fibres that
maintain the tensile strength of the amniochorion; resisting mechanical
stress and prevents rejection of the foetal allograft. Specific collagen
fibres of the fibrous connective tissue within the amniotic compact
layer provide the tensile strength while collagen fibres of both
reticular and spongy layers of the chorion provide mechanical
support(47). The major types of collagen fibres include: type I, II,
III, IV, V and VI collagens which are embedded in the fibrous
tissue(47).

Typically, the inner layer of the placenta is made up of the amniontic
membrane which is composed of amnion and chorion. The amnion has five
separate layers; the epithelium, basement membrane, compact layer,
fibroblast layer and spongy layer(48,49). The epithelium which consists
of a single layer of epithelial cells is proximal to the developing
foetus. The basement membrane of the amnion is a thin layer composed of
collagens III and IV and noncollagenous glycoproteins laminin, nidogen,
and fibronectin. The compact layer is dense nearly without cells and
forms the main fibrous structure of the amnion. The fibroblast layer of
the amnion is the thickest and consists of fibroblasts embedded in a
loose collagen network with abundance of noncollagenous glycoproteins.
The outermost spongy layer forms the interface between the amnion and
chorion and composed of a nonfibrillar meshwork of collagen III and an
abundant content of proteoglycans and glycoproteins(48,49).

The chorion is made up of a reticular layer, basement membrane and
trophoblast layer which is adhered to the maternal decidua. The
reticular layer contacts the spongy layer of the amnion and forms a
majority of chorion's thickness. The reticular network is composed of
collagens I, III, IV, V, and VI. The basement membrane anchors the
trophoblasts to the reticular layer with collagen IV, fibronectin, and
laminin. The trophoblast layer is the deepest layer which attaches to
the decidua(48). Type I and III collagen fibres are known to provide
tissue support while type II, IV, V and VI provide scaffoldings in
maintaining tensile strength(47). Distortion in the nomenclature of
these collagen fibres diminishes the tensile strength of the
amniochorionic extracellular matrix which increases myometrial activity
and cervical remodelling leading to the onset of preterm labour(47).

\textbf{Physiological uterine activity during pregnancy and parturition}

During pregnancy and labour, the myometrium undergoes distinct molecular
changes from noncontractile phynotype (quiescence) to contractile
phynotype depending on the gestational age (37,50,51). This process of
uterine activity during pregnancy and parturition can be divided into at
least four separate phases(52--54). The four recognized phases of
uterine activity include quiescence, activation, stimulation and
involution (figure 2).

In the first phase (uterine quiescence) which occurs in pregnancy, there
is increased inhibition of uterine activity by either separate or
combined autocrine-paracrine signalling transduction stimulated by
pro-pregnancy factors such as progesterone, prostacyclin (PGI2),
relaxin, parathyroid hormone-related peptide (PTHrP), calcitonin
gene-related peptide, adrenomedullin, vasoactive intestinal peptide
(VIP), nitric oxide, and CRH, which maintains the uterus in a relatively
quiescent state (52,53,55). This allows angiogenesis and tissue
remodelling especially around the placentation site to improve adequate
placental circulation, foetal nutrition and intrauterine growth (figure
2) (56). Alteration in the production of these agents during late
gestation may lead to the onset of preterm or term labour, whereas
administration of these compounds or their analogues may help maintain
uterine quiescence. Uterine smooth muscle relaxants act by signalling
through binding and activation of G-protein stimulatory (G\selectlanguage{greek}α\selectlanguage{english}s) subunit of
the G-protein coupled receptor (GPCR) located on the surface of the
myocytes. Activated GTP bound G\selectlanguage{greek}α\selectlanguage{english}s activates adenylate cyclase or
guanylate cyclase causing increased intracellular concentrations of
cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate
(cGMP). These nucleotides interfere with intracellular calcium release
and reduce the activity of myosin light chain kinase (MLCK) which are
required for shortening of the myofilaments and smooth muscle
contractions (figure 4) (52,53,55).

As pregnancy approaches term, the myometrium transitions from its
quiescence state to activation. This second phase of uterine activity is
characterised by progesterone diminution to oestrogen and CRH dominance
associated with increased mechanical stretch or uterotrophic
sensitisation (figure 2) (51,53,55). This leads to increased expression
of contraction-associated proteins (CAPs) including connexion 43 (Cx43,
a key component of gap junctions), agonist receptors (prostaglandins and
oxytocin) as well as increased influx of calcium ions into the uterine
myocytes(51--53,55). During the second phase of uterine activity,
upregulation of GJA1/Cx43 gene mediate hypersensitivity of receptors to
uterotonic agonists such as PGs and oxytocin which generate high
intensity and coordinated phasic uterine contractions(55,57). Cx43 is a
gap junction channel formed from hexamers of connexion proteins that
plays a critical role in cell to cell coupling and generates synchronous
myometrial contractions(57,58). It allows direct exchange of macro
molecules, ions, and second messengers such as cAMP, cGMP, inositol
phosphate and Ca2+ between cells which enable channels control and
coordinate cellular activity (figure 4) (58).

Stimulation represents the third phase of uterine activity during
parturition and is typified by increased uterine smooth muscle tone
stimulated by uterotonins such as PGs, oxytocin, and CRH. This phase is
characterised by increased synthesis of pro-inflammatory mediators (i.e.
IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-8, TNF-\selectlanguage{greek}α \selectlanguage{english}etc.), prostaglandins and white cell (e.g. monocytes
and neutrophiles) infiltration of the uterus, foetal membranes and
cervix; activating a biochemical process that triggers inflammatory
reactions within the uterus. Production of pro-inflammatory mediators
causes release of matrix metalloproteinases which breakdown the
extracellular matrix and increased gene expression for prostaglandins in
uterine tissues (figure 2). This leads to uterine contractions, cervical
remodelling and ripening(51,59,60). Increased expression of
prostaglandins receptors and gap junctions also occurs in the third
phase of parturition. Prostaglandins stimulate functional fundal
contractions, lower segmental contraction of the uterus and facilitate
cervical ripening while gap junctions permit cell to cell communication
by allowing intercellular exchange of macro molecules such as calcium,
cAMPs and cGMPs (figure 4) (59,60).

The last phase of uterine activity is characterised by massive uterine
involution to resemble the pregnancy stage and tissue remodelling after
delivery of the foetus and placenta, and has been attributed primarily
to the release of neuroendocrine oxytocin(51--53,55,60). During this
phase, there is a rapid withdrawal of pro-pregnancy factors (e.g.
progesterone) that maintains uterine quiescence and increased
recruitment of pro-labour factors (e.g. gap junctions, calcium-sensitive
potassium channels, and connexions) that stimulate the biochemical
changes similar to those occurring in labour (figure 2) (59,61).

\textbf{Pro-inflammatory mediators and spontaneous preterm labour}

Inflammation plays a crucial role in the process of parturition(11,62).
Most conditions that lead to spontaneous preterm labour are associated
with preterm premature rupture of membranes (PPROM). In preterm labour,
pathological mechanisms involving the cervix, foetus, foetal membranes,
placenta, and myometrium activate prematurely one or more of the
components of the pathway of parturition(62). During preterm labour,
uterine activity is changed from a state of quiescence to a
pro-inflammatory milieu in a three-step process characterized by uterine
contractility, cervical remodelling and membrane activation/rupture
which is modulated by pro-inflammatory mediators/cytokines(11,62,63).
Intrauterine infection is one of the most frequent conditions leading to
preterm labour and the pathogenic mechanisms are related to activation
of receptors (toll-like receptors) on the innate immune system(62). The
significant components of the innate immune system are pattern
recognition receptors. Toll-like receptors (TLRs) are a class of pattern
recognition receptors that detect pathogen-associated molecular patterns
(PAMPS) derived from microorganisms and damage-associated molecular
patterns (DAMPS) released from immune cells, stressed and dying cells.
This stimulates intracellular signalling cascades leading to the
expression of pro-inflammatory mediators/cytokines by innate immune
cells resulting in normal or preterm labour (figure 3) (64,65).

Pro-inflammatory mediators are structurally classified into 4 groups:
the 4\selectlanguage{greek}α \selectlanguage{english}helix family members - interleukin 2 (IL-2), interferon gamma
(IFN-\selectlanguage{greek}γ\selectlanguage{english}) and IL-10; IL-1 family; IL-17 family; and chemokines.
Functionally, these pro-inflammatory mediators/cytokines are grouped
into T helper type 1 (Th1) cell reactions (cell-mediated immunity) and T
helper type 2 (Th2) reactions (humoral immunity)(62). Inflammatory
cytokines are produced by T Lymphocytes (CD4+) which modulate immune
response to inflammatory stimuli. Primarily, Th1 cells produce IL-1,
IL-2, IL-6, IL-12, IL-15, IL-18, IFN-\selectlanguage{greek}γ, \selectlanguage{english}and tumor necrosis factor alpha
(TNF-\selectlanguage{greek}α\selectlanguage{english}) and Th2-cells produce IL-4, IL-5, IL-10, IL-13, and granulocyte
macrophage colony stimulating factor (GM-CSF)(62,66).

The presence of intrauterine infection causes infiltration of
circulating monocytes and neutrophils into the myometrium and cervix
leading to significant gene expression for interleukins (IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6,
IL-8) and Tumour Necrosis Factor alpha (TNF-\selectlanguage{greek}α \selectlanguage{english})(11,62). Myometrial
contractions are stimulated by the increase of IL-1 and TNF-\selectlanguage{greek}α \selectlanguage{english}which
promote influx of calcium into myometrial smooth muscle cells(11,67).
Pro-inflammatory mediators also cause increased gene expression for
prostaglandin synthase coupled with cyclooxygenase-2 gene expression
leading to up-regulation of prostaglandins and collagenases in uterine
tissues and cervix (65). Prostaglandins, PGF2 and PGE2 are also involved
in the stimulation of myometrial contractions and cervical
ripening(68--70). Cervical shortening and softening is due to the
progressive increased in the amount of hyaluronidase and other enzymes
that break down the glycosaminoglycans (GAGS) while collagenases break
down the cervical collagen. This leads to increased water absorption by
the cervix. IL-4, IL-5, IL-10 and IL-13 produced by Th2-cells play a
rather potential protective role in the feto-maternal relationship and
prevent preterm labour (figure 3)(62).

In early stage of inflammation, activation and maturation of neutrophils
and macrophages occur by stimulatory effect of pro-inflammatory cytokine
such as IL-6 in addition to increased differentiation of natural killer
cells resulting in invasion of the cervical and endometrial tissue with
subsequent stimulatory effect on the uterine contractility(71).
Similarly, IL-6 causes increased oxytocin receptors expression on
myometrial cells with further enhancement of their responsiveness to
oxytocin. In another pathway, IL-6 causes increased production of
prostaglandins from activation of the hypothalamic-pituitary-adrenal
axis and this result in initiation of uterine contraction and
progressive cervical dilatation(72).

Pro-inflammatory mediators also stimulate matrix metalloproteinases
(MMPs) such as collagenase, gelatinase and stromelysin, hyaluronidases
and prostaglandins expression which promote extracellular matrix
degradation, uterine contractions and cervical ripening leading to
preterm labour (figure 3)(47,62,73,74). Major proteinases associated
with this mechanism are collagenases: 1, 2, 3 and 4, also known as
MMP-1, MMP-8, MMP-13 and MMP-18; gelatinases A and B (MMP-2 and MMP-9,
respectively); and stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10) are
normally expressed during labour(47,75).

\textbf{Physiological pathways and biological mechanisms of uterine
contraction}

Series of physiological events occur before, during and after pregnancy
which modulate myometrial contractility throughout the menstrual cycle
in the non-pregnant uterus, maintenance of pregnancy, promotion of child
birth and enhance involution(40,73,76,77). These events may include both
maternal and foetal characteristics which generate signalling molecules
necessary for the stimulation of myometrial contractions during
labour(73,76). Knowledge about the biological mechanisms and pathways
that control myometrial contraction and relaxation and how these
pathways can be regulated is paramount for clinical practice.
Conventional trials in animal models showed that parturition is
determined by activation of the foetal hypothalamic-pituitary adrenal
(HPA) axis with increased foetal cortisol secretion. Following
mechanical stress, activation of HPA pathway leads to reduction in
maternal progesterone levels and increased levels of oestradiol. This
endocrine imbalance promotes increased intrauterine production of
prostaglandins, cervical softening and the onset of myometrial
contractions (78,79).

The contractile state of the myometrium is determined by the interaction
of the two major muscle proteins, actin and myosin. This interaction of
actin-myosin is influenced by myometrial signalling pathways which are
broadly categorised into signalling cascades regulating intracellular
calcium (Ca2+) concentration and those controlling the contractile
apparatus itself(40,77). Abundant in the plasma membrane of the uterine
myocytes are L-type calcium ion (Ca2+) channels which are ubiquitous,
large conductance, voltage-gated calcium channels (VGCC) (76,80).
Binding of an agonist (e.g. oxytocin) to specific receptor causes
depolarisation of the myocyte's membrane potential and opening of the
L-type calcium channels leading to rapid influx of extracellular calcium
ions and dramatic rise in intracellular calcium ion
concentration(73,76). The plasma membrane of the myocyte also contain
other types of calcium channels (i.e. T-type calcium channels) which
exhibit faster kinetics and greater conductance than the L-type (figure
4) (76).

Within the myometrium, agonist interaction with GPCR on the plasma
membrane (PM) of myocytes leads to activation of the G\selectlanguage{greek}α\selectlanguage{english}q subunit of the
trimeric G-protein. Activated G\selectlanguage{greek}α\selectlanguage{english}q subunit also binds and activates
membrane-bound phospholypase C\selectlanguage{greek}β \selectlanguage{english}which hydrolyzes phosphatidylinositol
bisphosphate (PIP2) into inositol-triphosphate (IP3) and diacylglycerol
(DAG)(73,76,77). IP3 interact with IP3-sensitive receptors (IP3-Rs) on
sarcoplasmic reticulum (SR) which causes release of calcium from its
storage sites in the SR into the cytosol. Increased cytosolic calcium
concentration also stimulates the rynodine receptors to cause
Ca2+-induced Ca2+ release (CICR)(40,73,76). Another mechanism, called
store-operated Ca2+ entry (SOCE), also regulates Ca2+ flux occurs when
the intracellular Ca2+ stores in the SR are exhausted, it stimulates the
PM to permit influx of extracellular Ca2+ into the cytosol(76).
Increased concentration of Ca2+ in the cytosol leads to binding of
calcium to calcium-sensitive protein, Calmodulin. The Calcium-Calmodulin
complex activates the enzyme Myosin Light Chain Kinase (MLCK) which in
turn causes increased phosphorylation of Myosin Light Chain (MLC)
leading to actin-myosin cross-bridge formation and activation of the
contractile machinery (figure 4)(40,73,76).

Relaxation of uterine smooth muscles occur when removal of cytosolic
Ca2+ occurs through closure of PM L-type Ca2+ channels, efflux of Ca2+
into the extracellular compartment through Ca2+-ATPase pumps on plasma
membrane and into intracellular Ca2+ stores in SR via SR/ER Ca2+-ATPase
(SERCA) pumps(40,73,76,77). Also, Myosin Light Chain Phosphatase (MLCP)
causes dephosphorylation of the myosin light chain which is regulated by
signalling through the small G-protein rhoA--rho-associated kinase and
protein kinase C (PKC) pathways. Dephosphorylation of the myosin light
chain inhibits actin-myosin cross-bridge formation leading to smooth
muscle relaxation (figure 4) (76).

Conversely, in postterm pregnancy although the actual aetiology is not
yet known, genetics and maternal and fetal factors are implicated in its
pathogenesis(73,81,82). Unlike preterm labour, fetal hypothalamic
pituitary adrenal (HPA) insufficiency and a disorder in placental
sulphatase activity (an X-linked recessive gene disorder) result in
reduced production of eostriol (E3) which plays a fundamental role in
the pathogenesis of postterm pregnancy(81,82). Subsequently, placental
CRH production declines with diminution in the positive feedback
mechanism on the production of foetal adrenal dehydroepiandrosterone
(DHEA). Decreased production of foetal eostriol and cortisol ensue which
interferes with the biological pathways for spontaneous onset of
labour(81).

\textbf{Biological basis of therapeutic agents for preterm labour}

In recent times, the prevention, prediction and treatment of preterm
labour has remained a

major focus for contemporary research in obstetrics (83). Generally, the
use of tocolytics has become a fundamental pharmacological therapy in
the clinical management of preterm labour(84). The biological basis for
the use of tocolytics is to delay delivery for at least 48hours to allow
for corticosteroids and magnesium sulphate administration to enhance
foetal lung maturation and prevention cerebral palsy respectively and
transfer to a tertiary healthcare facility with neonatal intensive care
facilities(83,84).

Proinflammatory cytokines such as IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, and IL-8 have been
strongly associated with cervical dilation in preterm labour and
medications the decrease the concentration of these mediators
constitutes potential preventive interventions(72). The common
therapeutic agents which are currently used to abort uterine
contractions or maintain uterine quiescence include beta adrenergic
receptor agonists (ritodrine, isoxsuprine, salbutamol, terbutaline),
prostaglandin -Synthase inbibitors {[}including NSAIDS (e.g
indomethacin) and COX-2 inhibitors (e.g Celecoxib){]}, oxytocin receptor
antagonists (atosiban), calcium channel blockers (nifedipine) and
Magnesium sulphate. Tocolytics are usually administered as a monotherapy
although a combination of more than one agent maybe required in some
cases. However, a recent systematic review by Vogel et al did not
support the use of a polytherapy of tocolytics over monotherapy and this
is partly due to lack of trials of combination regimens of commonly used
tocolytics(85).

Beta agonists (betamimetics) bind to beta 2 adrenergic receptors on
surface membranous myometrial smooth muscle cells which activate adenyl
cyclase leading to increased intracellular levels of cAMPs and
activation of protein kinase A. Increased cAMPs levels decrease
intracellular calcium which specifically suppresses spontaneous and
oxytocin-induced uterine contractions. Three different subtypes of beta
adrenergic receptors are located in the uterus and beta 2 adrenergic
receptors constitutes approximately 80\%(86). However, the use of beta 2
adrenergic receptors is associated significant maternal side effects
such as palpitations, chest pain, breastlessness and pulmonary oedema in
severe cases which limits their clinical use for extended duration(86).

Atosiban is a nona-peptide, desamino-oxytocin analogue and a competitive
vasopressin/oxytocin receptor antagonist(87). Atosiban blocks oxytocin
receptors and prevents oxytocin-induced hydrolysis of
phosphatidylinositol 4,5-bisphosphate (PIP2) to IP3 and calcium efflux
into the cytoplasm(88). Thus, it inhibits signalling transduction on the
uterine smooth muscle leading to uterine relaxation (83,89). Atosiban
has minimal side effects with higher safety profile as compared to the
other tocolytics(90).

Selective COX-2 inhibitors such as celecoxib and non-specific COX
inhibitors such as indomethacin which are prostaglandin synthase-2
inhibitors inhibit the synthesis of prostaglandins (e.g. PGF2\selectlanguage{greek}α \selectlanguage{english}\& PGE2)
but do not affect the production of pro-inflammatory mediators (e.g.
IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, and TNF\selectlanguage{greek}α\selectlanguage{english}) because the action of pro-inflammatory mediators
is upstream of COX-2 expression(91,92). Prostaglandins induce uterine
contraction via facilitation of myometrial gap junction formation and
increasing the concentration of intracellular calcium concentration.
Therefore, prostaglandin synthase inhibitors are considered effective
tocolytics. However, their usage is limited because they cause premature
constriction of the ductus arteriosus, inhibit platelets aggregation and
impair renal function and decreased urine production resulting in
oligohydramnios. Therefore, the duration of indomethacin use should be
restricted and limited to gestational age of 32 weeks(93).

Magnesium sulphate is used broadly as a tocolytic and proven to be more
effective in fetal neuroprotection and preventing cerebral palsy(94,95).
Magnesium sulphate competitively blocks intracellular calcium influx and
activation of myosin light chain kinase leading to decreased myometrial
contractility. It also inhibits acetyl choline release by competing with
calcium at the motor end plate of the neuromuscular junction. Perhaps,
its usage is not primarily intended to delay delivery but to prevent
cerebral palsy via neuroprotection(94--97). The common side effects
include flushing, respiratory depression and cardiac arrest(88). Based
on our review, all the tocolytic agents in recent use appear to act on
areas that interfere with either calcium entry or prostaglandins
production which are downstream of pro-inflammatory mediators'
production.

\textbf{Conclusion}

Pro-inflammatory mediators play a central role in the pathophysiology of
preterm labour and may constitute the main targets for therapeutic
interventions in the prevention and clinical management of preterm
labour. Imbalances in the pro- and anti-inflammatory pathways maybe
responsible for preterm labour and postterm pregnancies respectively.
Increased expression of pro-inflammatory mediators such as IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6,
IL-8 and TNF-\selectlanguage{greek}α \selectlanguage{english}are associated with increased risk of preterm birth.
Although the etiology of preterm labour remains elusive, alteration in
multiple maternal and foetal physiological mechanisms are implicated
including genetic predisposition, psychological and environmental
influences.

Available medical therapies for preterm labour have not been
consistently effective in aborting uterine contractions due to its
ill-defined pathophysiology. However, therapeutic interventions
primarily targeting the pro-inflammatory pathway seem promising as
inflammation plays a central role in the pathogenesis. In-depth
understanding of the mechanistic role of proinflammatory mediators
remains vital to the development of more efficacious therapies and
interventions for prevention and treatment of preterm labour. Further
research of high methodological quality is recommended to provide
adequate understanding of the pathophysiological mechanisms of maternal
pro-inflammatory mediators in preterm labour.

\textbf{Disclosure of interest}

The authors of this article declare that, no conflict of interest
exists.

\textbf{Contribution to Authorship:}

FB gathered, analyzed and compiled data sources and literature regarding
the pathophysiological mechanisms of maternal pro-inflammatory mediators
in preterm labour and was the major contributor in writing the
manuscript. KAB edited the manuscript for its intellectual content and
coherence. All authors read and approved the final manuscript.

\textbf{Funding}

Not applicable

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\textbf{Figure 2: Physiological uterine activity during the non-pregnant
state, pregnancy and parturition:} During early follicular phase or
menses there is increased labour-like uterine contractions modulated by
increased prostaglandins and progesterone withdrawal(38,44,45);
follicular phase is characterised by progressive increased in wave-like
uterine contractions which terminate at pre-ovulation influenced by rise
oestrogen levels(41,42,44--46); there is low amplitude contractions
during ovulation; luteal phase is characterised by uterine quiescence
which favours fertilisation, implantation and placentation. Angiogenesis
and tissue remodelling controlled by release of anti-inflammatory
mediators (IL-3, IL-4, IL-5, and IL-10) by trophoblast cells occur
during the luteal phase. Fertilisation results in increased uterine
relaxation controlled by rise in the levels of progesterone. There is
profound inhibition of uterine activity during the first phase of
pregnancy influenced by pro-pregnancy factors such as progesterone,
relaxin, prostacyclin, vasoactive intestinal peptide (VIP), nitric oxide
(NO) etc.; second phase of pregnancy is characterised by uterine
activation stimulated by progesterone withdrawal to oestrogen and
corticotrophin-releasing hormone (CRH) leading to gene expression for
contraction-associated proteins (CAPs); third phase is characterised by
increased responsiveness to uterotonins and uterine contractions with
progressive cervical dilation and effacement; fourth phase is
characterised by uterine involution and tissue remodelling governed by
withdrawal of pro-pregnancy factors (e. g progesterone, etc.) and
recruitment of pro-labour factors (e. g oxytocin) and gap junctions such
as connixion43 (cx43)(51,55,59)

\textbf{Figure 4} : \textbf{Physiological pathways and biological
mechanisms of uterine activity} : Binding of pro-pregnancy factors
(progesterone, relaxin vasoactive intestinal peptide etc.) to GPCR
especially G-protein stimulatory receptor (Gs-R) on plasma membrane (PM)
activates adenylate cyclase resulting in rise in cytosolic concentration
of cyclic adenosine monophosphate (cAMPs). Increased cAMPs inhibit Ca+
entry and Myosin light chain kinase (MLCK) which leads to uterine muscle
relaxation. Pro-labour factor (oxytocin, prostaglandin etc.) binding
activates trimeric G-protein coupled receptor (GPCR) results in opening
of voltage-gated Ca+ channels (VGCC) in PM. Activated G-protein Gq
subunit stimulates phospholypase C\selectlanguage{greek}β \selectlanguage{english}(PLC\selectlanguage{greek}β\selectlanguage{english}) to hydrolyze PIP2 into DAG
and IP3, IP3 activates IP3-sensitive receptor at the level of the
sarcoplasmic reticulum (SR) to induce calcium ions (Ca2+) release from
internal stores. These result in an increased in cytosolic Ca2+ levels
which ultimately activates MLCK through the intermediary activation of
Ca2+-Calmodulin (Ca2+-CM) complex. MLCK phosphorylates myosin light
chain (MLC) resulting in cross-bridge formation and muscle contraction.
Pro-labour factors inhibit cAMPs production by activating the G-protein
inhibitory receptor (Gi-R) on PM. Ca2+ signals are terminated by
extrusion of Ca2+ from the cytosolic compartment or sequestration into
internal stores via PM Ca2+ ATPases (PMCA) and SR Ca2+ ATPases (SERCA),
respectively. Activation of Ca2+-sensitive K+ channels serves to
repolarise the myocytes membrane and induces closure of VGCCs, limiting
further Ca2+ entry. Dephosphorylation of MLC by MLC Phosphatase (MLCP)
when specific GPCR activation stimulates RhoA-rho kinase results in
resetting of the contractile system and relaxation at the level of the
tissue. Nitric oxide binds to membranous Gq subunit of the GPCR
resulting in increased cyclic guanosine monophosphate (cGMPs) levels
which stimulates MLCP leading to smooth muscle relaxation (76).

\textbf{Hosted file}

\verb`Figures- Review.docx`  available at \url{https://authorea.com/users/576099/articles/709750-pathophysiological-mechanisms-of-maternal-pro-inflammatory-mediators-in-preterm-labour}

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