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The initiation of term labor has been related to an increased myometrial respon-
siveness to OT near or at the time of parturition and, a variety of mechanisms have
been invoked to initiate parturition. However, the involvement of the local OT
production and secretion within the uterus is the ¬nal pathway of the mechanism
leading to parturition linked to local PG release (Challis et al., 2000) and an imme-
diate in¬‚ux of Ca2 into the cytoplasm of myometrial cells from both extracellular
and intracellular sites (Challis et al., 2000). In fact, OT is not only a neurohormone
35 Placental expression of neurohormones and other neuroactive molecules


but also a locally produced substance (Chibbar et al., 1993; Petraglia et al., 1996d;
Challis et al., 2000) with possible paracrine actions, able to stimulate PG produc-
tion (Petraglia et al., 1996d; Challis et al., 2000). The PGs themselves may be uterotonic,
and drive the arachidonic acid metabolism toward cyclo-oxygenase products
rather than the less active lipoxygenase metabolites (Ticconi et al., 1998). In
explant culture of human choriodecidua, OT markedly increases the production of
PGF2 , PGE2 and leukotrienes in contrast to amnion where PGE2 is the primary
PG product (Pasetto et al., 1992). Therefore, OT might have a dual role in fetal
membranes: it could directly enhance PG production and also indirectly stimulate
the synthesis and release of cytokines involved in the regulation of PG output by
tissue (Zicari et al., 2002).
At the end of pregnancy in the human and in the rat, there is a large increase in
content of decidual mRNA encoding OT. The concentration of OT peptide for
these two species in the decidua is not greater than that in the circulation and
it would, if released, probably act on the endometrium rather than on the
myometrium. Indeed, in cyclic ruminants, PG production by the endometrium in
response to OT is the signal precipitating luteolysis, unless a blastocyst is present to
block OT receptor (OTR) expression (Challis et al., 2000). It is generally thought
that changing secretion of estrogen and progesterone towards the end of preg-
nancy is important in the regulation of OT peptide and receptor gene expression.
The content of OT mRNA in human decidua increases in vitro in response to
estrogen, with no effect of progesterone, while in the rat endometrium in vivo the
reported stimulatory action of estrogen is enhanced by progesterone (Chibbar et al.,
1995). Although there appears to be no change in OT metabolism around the time
of parturition, OTR gene expression is upregulated (Takemura et al., 1994), so that
the onset of labor coincides with an increase in the paracrine rather than systemic
release of OT. This local OT production seems to be regulated by other paracrine
factors, such as CRH, activin A and PGs (Florio et al., 1996) (Figure 1.5).

(B) Role of CRH
Labor and delivery are the main physiological stress conditions and among the
neuroendocrine factors which play a role in the maintenance of uterine quiescence
and involved in the onset of parturition, CRH has been one of the more investi-
gated in the last decade. In vitro data support a role for CRH at labor (Figure 1.5).
In fact, CRH and ACTH stimulate the release of PGF2 and PGE2 from cultured
amnion, chorion, decidual and placental tissues (Jones et al., 1989; Benedetto et al.,
1994; Petraglia et al., 1995b; 1999a). These effects are inhibited in presence of anti-
sera to CRH and to ACTH. Moreover, in placenta but not in amnion or decidua, the
stimulatory effect of CRH on PGF2 and PGE2 output is attenuated in presence of
an antibody to ACTH, thus supporting the possibility of paracrine stimulation by
36 F. Petraglia et al.


CRH and ACTH of PG production in intrauterine tissues (Jones and Challis,
1990). The CRH markedly stimulates the release of immunoreactive OT from cul-
tured placental cells in a dose-dependent fashion (Florio et al., 1996). Moreover,
the addition of CRH, but not of arginine vasopressin or NPY, increase the release
of immunoreactive OT three- to fourfold from placental cells.
Recent data indicated a role played by CRH directly on myometrial contractility,
due to the fact that CRH mediates its actions in the human myometrium via activation
of two distinct classes of CRH receptors, R1 and R2 (Hillhouse and Grammatopoulos,
2002). Contrasting data exists on the net role played by CRH, some suggesting
CRH as an important uterotonic, others as the main uterine quiescence factor
(Challis et al., 2000; Florio et al., 2002d; Hillhouse and Grammatopoulos, 2002). It
seems that different myometrial CRH receptors are recruited at labor, and that this
recruitment may be dynamically and differentially modulated by the great hor-
monal changes occurring at term pregnancy, so that CRH actions in vivo may dif-
fer from actions reported in vitro, according to different myometrial CRH receptor
expression and the induced af¬nity state.
For many years, investigators questioned whether there are fundamental differ-
ences between ovine pregnancy, in which the fetal adrenal gland plays a pivotal role
in the process of parturition, and human pregnancy, in which the role of the fetal
adrenal gland in this process is less clear (Challis et al., 2000). Human placental
CRH may directly and preferentially stimulate the fetal adrenocortical production
of DHEA-S to as great an extent as ACTH, while stimulation of cortisol by CRH
occurs to a much lesser degree than stimulation by ACTH (Smith et al., 1998)
(Figure 1.4). Further, placental CRH can stimulate production of proopime-
lanocortin and some of its derivatives in the placenta, including ACTH, -MSH
and -END in syncitiotrophoblast cells in vitro (Reis et al., 1999; Challis et al.,
2000; Florio et al., 2002d; Hillhouse and Grammatopoulos, 2002). Placental CRH
could, however, like fetal CRH, also stimulate fetal pituitary ACTH. Placental CRH
may stimulate fetal pituitary ACTH, which then stimulates fetal adrenal DHEA-S,
which is used by the placenta for conversion to estrogen by the process of aromati-
zation (Challis et al., 2000; Florio et al., 2002d). This increase in estrogen then could
serve as a trigger for the cascade of events leading to labor and parturition. In fact,
estrogens increase uterine contractility by increasing myometrial excitability, myome-
trial responsivity to OT and other uterotonic agents, as well as stimulate the syn-
thesis and the release of PGs by fetal membranes (Challis et al., 2000; Florio et al.,
2002d). Further, estrogens stimulate proteolytic enzymes in the cervix, such as col-
lagenase, which break down the extracellular matrix permitting the cervix to dilate.
Thus, consistent with the observation that CRH preferentially stimulates fetal
adrenal DHEA-S directly, was the observation that CRH increased the abundance
of mRNAs encoding the enzymes for the conversion of androgen to estrogen
37 Placental expression of neurohormones and other neuroactive molecules


(Smith et al., 1998). Thus, it was hypothesized that the rapid rise in placental CRH
which occurs at the end of gestation at the time when CRH-BP decreases, serves as
the inciting event leading to placental aromatization (Reis et al., 1999; Challis et al.,
2000; Florio et al., 2002d; Hillhouse and Grammatopoulos, 2002). The increasing
estrogen, then, would initiate the chain of events terminating in labor and delivery
(Challis et al., 2000; Florio et al., 2002d). Thus, there may be a feto-placental unit
which involves fetal glucocorticoids and placental CRH as well as that involving
fetal DHEA-S and placental estrogen.
Thus, among the possible processes governing the initiation of human parturi-
tion are the following:

(1) The rise in placental CRH at the end of pregnancy stimulates fetal pituitary
ACTH, which in turn stimulates increased fetal adrenal cortisol and DHEA-S
production. The increasing concentrations of cortisol, in addition to maturat-
ing enzymes in organs critical for postnatal existence, further stimulate pro-
duction of placental CRH by a feed-forward mechanism. The increasing
production of DHEA-S provides additional substrate for placental aromatiza-
tion to estrogen, which triggers the cascade leading to labor and delivery.
(2) The increasing production of placental CRH directly and preferentially stimu-
late fetal adrenal DHEA-S, which is then converted by placental aromatization
to estrogens which trigger the cascade leading to parturition.
(3) CRH exerts direct effect on the myometrium and fetal membranes to increase
myometrial contractility.


(C) Role of opioids
Opioids (Box 1.4) could play a role in the initiation of parturition. As parturition
approaches, a central opioid inhibitory mechanism is activated that restrains the
excitation of OT cells by brainstem inputs. In fact, OT secretion from the posterior
pituitary gland is increased during parturition, stimulated by the uterine contrac-
tions that forcefully expel the fetuses. Opioid is the predominant damper of OT
cells before and during parturition, limiting stimulation by extraneous stimuli,
and perhaps facilitating optimal spacing of births and economical use of the store
of OT accumulated during pregnancy (Russell et al., 2003). In fact, -END levels
are elevated, approximately twofold higher than circulating plasma levels, in the
colostrum and transitional milk of mothers who were vaginally delivered. Therefore,
it was hypothesized that -END may contribute to postnatal fetal adaptation, to
overcoming birth stress of natural labor and delivery, and at the same time to the
postnatal development of several related biologic functions of breast-fed infants
(Zanardo et al., 2001). The -END appears to be related with glucorticoid release,
energy balance and the stimulation of lipolysis (Petraglia, 1991; Ahmed et al., 1992;
38 F. Petraglia et al.



Box 1.4 Opioid peptides

Opioid peptides have a morphine-like activity. Three families are recognized:
END, ENK and DYN. They derive from three precursors of similar molecular
size and sequence homology. The POMC is the precursor of ACTH, -melanocyte-
stimulating hormone ( -MSH), -END and lipotropin. Proenkefalin (P-ENK)
is the precursor of ENK and prodynorphin (P-DYN) of DYN, rimorphin, leu-
morphin and neo-endorphins (Kieffer and Evans, 2002).


Expression and localization
The -END, methionine enkephalin (M-ENK) and DYN 1“8 and 1“13 are the
main opioid peptides identi¬ed in placental extracts. The DYN 1“8 seem to be the
predominant opioid peptide present in placental villus tissue (Agbas et al., 1995).
The -END was the ¬rst detected endogenous opioid peptide in the human
placenta (Ahmed et al., 1992). Immunohistochemical staining of placental tis-
sue for -END immunoreactivity is positive in the syncytiotrophoblast in both
early and term pregnancy (Odagiri et al., 1979). Cultures of human placenta
cells collected at term, release -END (Liotta and Krieger, 1980) and it is mea-
surable in homogenates of human amnion, chorion and decidua collected
throughout gestation (Facchinetti et al., 1990).
The M-ENK is the major representative of the other family of opioid peptides,
the ENK. Syncytial and cytotrophoblast cells contain immunoreactive M-ENK,
and its de novo syntesis in culture villi at term has been shown (Sastry et al., 1980).
The third family of opioids is represented by DYN, and human placenta is
also the source of the multiple forms of DYN despite dynorphins A(1“8) is the
major opioid present in the placental extracts (Agbas et al., 1995).


Receptors
Mu, kappa and delta are the main opioid receptor types. Each opioid exhibits
distinct binding activity towards each type of opioid receptor. The mu-receptor
has high af¬nity for M-ENK and -END (as well as morphine and dynorphin
A); the delta-receptor for leu-enkephalin; the kappa-receptor is the main target
for the DYN (Kieffer and Evans, 2002).
Receptor subtypes for the various endogenous opioid peptides are present on
placental cell membranes (Porthe et al., 1981; 1982), but kappa receptors is the
more important type present in the placenta, in fact the order of potency in cells
in vitro from term trophoblast tissue was kappa mu delta (Cemerikic
et al., 1992). Placental content of kappa receptors increases with gestational age
39 Placental expression of neurohormones and other neuroactive molecules



and term placental content of kappa receptors correlates with route of delivery
(Ahmed et al., 1992).

Levels in biological ¬‚uids
Placenta and membranes contribute to the secretion of -END in the different
¬‚uid compartments. Concentration of -END in maternal plasma during preg-
nancy have been reported as unchanged (Cemerikic et al., 1992), decreased
(Goebelsmann et al., 1984), or progressively increasing (Newnham et al., 1983;
Panerai et al., 1983; Facchinetti et al., 1990), so that the question of a possible pla-
cental contribution to the circulating pool remains unsolved. However, the ¬nd-
ings that -END concentrations are higher in the placental tissue than in the
maternal or cord plasma (Petraglia, 1991; Petraglia et al., 1996d; Reis et al., 2001);
that immunoreactive -END in placental homogenates in the ¬rst is signi¬cantly
higher than in the second trimester; that at delivery the -END content is greater
than in the second trimester and that, in tissues collected at term, in the absence
of labor, -END levels are higher in tissues collected after VD (Facchinetti et al.,
1990) suggest that gestational tissues are important sources and that stress of
delivery greatly stimulates the placental secretion (Figure 1.5). On the contrary,
amniotic ¬‚uid -END concentrations have a completely different gestational
pattern, with no changes at parturition (Genazzani et al., 1984; Ko¬nas et al.,
1987; Mauri et al., 1990), suggesting a different source in this compartment.
Moreover, -END is elevated, approximately twofold higher than circulating
plasma levels, in the colostrum and transitional milk of mothers who were
vaginally delivered. Therefore, it was hypothesized that -END may contribute
to postnatal fetal adaptation, to overcoming birth stress of natural labor and
delivery, and at the same time to the postnatal development of several related
biologic functions of breast-fed infants (Zanardo et al., 2001).
Maternal plasma levels of M-ENK are not signi¬cantly different from those of
non-pregnant women and do not change throughout pregnancy (Sastry et al.,
1980), supporting a local role of the peptide. DYN was measured in maternal
blood, umbilical vein and amniotic ¬‚uid. No signi¬cant change was observed in
the plasma level of DYN in the ¬rst and second trimester of pregnancy as com-
pared with plasma obtained from non-pregnant women. However, a 2.2-fold
increase in DYN plasmatic levels was observed during the third trimester as well as
at delivery. High levels of DYN were also found in the amniotic ¬‚uid and the
umbilical vein plasma. Levels of DYN in the maternal plasma at the third trimester
of pregnancy and at delivery increase, therefore, a placental contribution to this
phenomenon has been speculated (Valette et al., 1986). High DYN levels are also
detectable in amniotic ¬‚uid and in umbilical vein plasma (Valette et al., 1986).
40 F. Petraglia et al.


Petraglia et al., 1996d; Reis et al., 2001; Russell et al., 2003). Another function
attributed to -END is the inhibition of painful sensations in women during
childbirth (Russell et al., 2003). Stress during delivery has been associated with ele-
vated umbilical cord plasma -END levels. Multiple regression modeling showed
that forceps delivery, maternal -END concentration, bradycardia, VD, and birth
weight each made independent contributions to elevated cord -END. Level of
cord -END independent of delivery stress exerted the primary in¬‚uence upon
child motor development and higher levels of stress-independent -END may play
a direct role in motor development (Rothenberg et al., 1996).

(D) Role of NPY and CGRP
The NPY (Box 1.5) synthesis by cytotrophoblastic cells, amnion, chorion and
decidua has been suggested to be involved in the mechanism leading to parturi-
tion. NPY stimulates the placental release of CRH (Petraglia et al., 1989a) and, it is
also able to modulate myometrial contractility (Stjernquist and Owman, 1987;
Tenmoku et al., 1988) (Figure 1.5). On the contrary, calcitonin gene-related pep-
tide (CGRP; Box 1.6) may have a role in maintaining uterine quiescence during preg-
nancy, from early to term gestation (Samuelson et al., 1985), as it is a potent



Box 1.5 Neuropeptide Y

Human NPY is a peptide of 36 amino acid residues (Grove and Smith, 2003)
belonging to a family of regulatory peptides that also includes peptide tyrosine
(PYY). By ¬‚uorescence in situ hybridization the NPY gene has been mapped
to chromosome 7p15.1 and exists in single copy (Grove and Smith, 2003). The
NPY expression is abundant and widespread in the central and peripheral nervous
systems, in particular in brain, in sympathetic neurons innervating cardiovas-
cular and respiratory systems, gastrointestinal and genitourinary tracts (Grove
and Smith, 2003). Physiological effects attributed to NPY include the stimula-
tion of food intake and inhibition of anxiety in the central nervous system
(CNS) (Grove and Smith, 2003; Pedrazzini et al., 2003); presynaptic inhibition
of neurotransmitter release in the CNS and the periphery; vasoconstriction
(Michel and Rascher, 1995); inhibition of insulin release; regulation of gut
motility; gastrointestinal and renal epithelial secretion (Grove and Smith, 2003;
Pedrazzini et al., 2003). Moreover, there is evidence that NPY is involved in the
regulation of anterior pituitary hormone secretion: in particular, NPY plays a
critical role in stimulating the basal pattern of luteinizing hormone (LH) release
(Grove and Smith, 2003; Pedrazzini et al., 2003).

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