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choline) and neuropeptides (angiotensin II, arginine vasopressin and OT) are
involved in the stress-induced responses of the neuroendocrine system (Plotsky
et al., 1989). The release of CRH from cultured placental cells during the incubation
with norepinephrine, acetylcholine, angiotensin II and arginine vasopressin, or OT
suggests a possible in vivo interaction among these substances. In agreement with
the regulation of the hypothalamic CRH, although interleukin (IL)-1 stimulates
the release of CRH from cultured placental cells, on the contrary IL-2 has no effect
(Petraglia et al., 1989c). Since indomethacin prevents the CRH release induced by
IL-1, it has been suggested that the action of IL-1 is mediated by PGs (Petraglia
et al., 1987a) (Figure 1.2).
The CRH-binding protein (CRH-BP) is a 37-kDa protein of 322 amino acids,
mainly produced by the human brain and the liver (Petraglia et al., 1996b) that is
able to bind circulating CRH and urocortin, thus modulating their actions on
pituitary gland (Potter et al., 1992). Further, sources of CRH-BP during pregnancy
are placental trophoblast, decidua and fetal membranes (Petraglia et al., 1993a;
1996b). In detail, the syncytial layer of placental villi at term intensely expresses
CRH-BP mRNA and immunoreactivity, whereas rare positively hybridized cells
are observed within the cytotrophoblasts and mesenchymal cells. Large decidual
cells, amniotic epithelial cells and chorionic cytotrophoblasts stained positively for
CRH-BP mRNA and protein.
The CRH-BP is measurable in maternal plasma, and levels remain stable in non-
pregnant women and during gestation until the third trimester of pregnancy
(Petraglia et al., 1996a, b; Reis et al., 1999; Florio et al., 2002d). At this time, maternal
plasma CRH-BP concentrations signi¬cantly and rapidly decrease in the last 4“6
weeks before labor (Linton et al., 1993; Petraglia et al., 1996a, b; Reis et al., 1999;
Florio et al., 2002d), returning to approximately non-pregnant levels during the ¬rst
24 h postpartum. Thus, opposite changes in concentrations of CRH (higher) and
CRH-BP (lower) in maternal plasma occur at term, so that the availability of bio-
active CRH increases during the activation of labor. Cord blood CRH-BP levels are
higher (Petraglia et al., 1997a), while amniotic ¬‚uid levels are lower than in maternal
plasma and have a similar trend, decreasing until term pregnancy (Florio et al., 1997).
Recently, another component of the CRH family, urocortin, has been described.
Its sequence is similar to ¬sh urotensin (63%) and human CRH (45%) (Vaughan
et al., 1995). Placental and decidual cells collected at 8“11 weeks or 38“40 weeks
of gestation express urocortin mRNA and immunohistochemistry localized uro-
cortin staining in syncytial cells of trophoblast as well as in amnion, chorion and
decidua of fetal membranes (Petraglia et al., 1996c; Florio et al., 1999b). In detail,
24 F. Petraglia et al.


immunoreactive urocortin was then localized in syncytiotrophoblast cells and in
some extent in cytotrophoblast cells of placental villi at term, as well as in fetal
membranes and maternal decidua.
Urocortin levels are undetectable during pregnancy, with no rise with increasing
gestational age as is seen for CRH (Glynn et al., 1998). This lack of urocortin rise
throughout pregnancy is further supported by an absence of gestational age-
related changes in placental urocortin mRNA expression (Florio et al., 1999b).
Urocortin levels were higher at labor than those previously reported during preg-
nancy, but they did not change signi¬cantly at the different stages of labor when
evaluated longitudinally. Some patients displayed a trend towards increasing levels,
whilst others had variable concentrations (Florio et al., 2002b).


Placental control of ACTH secretion

Placental ACTH, also called chorionic corticotropin (hCC) is a product of the
proopiomelanocortin (POMC) gene and has the same structure and immuno-
genic and biologic activity as pituitary ACTH (Waddell and Burton, 1993).
Placental ACTH is localized to the cytotrophoblast in the ¬rst trimester and to the
syncitiotrophoblast in the second and third trimesters (Cooper et al., 1996). There
is a signi¬cant increase of POMC gene expression in the placenta with the advance
of gestation, which is manifested by increasing levels of POMC mRNA as well as
immunoreactive ACTH (Cooper et al., 1996). Among the possible local effects of
placental ACTH are the stimulation of placental steroidogenesis (Barnea et al.,
1986) and reduction of vascular resistance (Clifton et al., 1996).
The addition of CRH to primary trophoblast cell cultures stimulates ACTH
secretion in a dose-dependent manner (Petraglia et al., 1987c; 1999a). Moreover,
the addition of a CRH antagonist is able to block the CRH-induced ACTH release
from placental cells (Petraglia et al., 1987c; 1999a). The concentration of CRH
required for 50% of maximal stimulation of ACTH secretion is higher than the
concentration necessary to release ACTH from cultured anterior pituitary cells
(Petraglia et al., 1987c). CRH-induced ACTH secretion is mediated by cyclic
adenosine monophosphate (cAMP) as second messenger and evidence that this
intracellular mechanism operates in placenta comes from the observation that
dibutyryl cAMP and forskolin, a diterpene that stimulates adenylate cyclase activ-
ity, stimulate ACTH release from cultured trophoblast cells with the same intensity
of corticotropin-releasing factor (CRF) without potentiating the effect of CRH
(Petraglia et al., 1987c).
The CRH-BP reverses the CRH-induced ACTH release from placental cells
(Petraglia et al., 1993a; 1996b), as in the pituitary (Potter et al., 1992). These ¬nd-
ings indicate a similarity between pituitary and placental CRH-induced ACTH
25 Placental expression of neurohormones and other neuroactive molecules


release. However, in contrast to the corticosteroid negative feedback on pituitary
ACTH secretion, glucocorticoids stimulate placental CRH secretion and mRNA
expression (Petraglia et al., 1987c; 1999a), and dexamethasone does not inhibit the
effect of CRH on placental ACTH release (Petraglia et al., 1987c; Robinson et al.,
1988).
In addition to CRH and urocortin, OT (Box 1.2) also is a potent stimulator of
ACTH from cultured placental cells (Petraglia et al., 1987c; 1989c; Margioris et al.,
1988). The effect resembles the neuroendocrine ¬ndings showing OT active on
hypothalamic CRH and on pituitary POMC-related peptides, participating in the
stress-induced events. The similarity between placental ACTH regulation and the
brain CRH/ACTH system is also con¬rmed by the evidence that the addition of
neuropeptide Y (NPY), IL-1, arginine vasopressin, angiotensin II, norepinephrine,
or acetylcholine increase CRH release.


Box 1.2 Oxytocin

The OT is a neurophyseal hormone composed of nine amino acids, synthesized
in the hypothalamus and stored in the neurohypophysis, where it acts as a neuro-
transmitter involved in sexual and maternal behavior (Acher and Chauvet,
1995). The synthesis of OT has been demonstrated in peripheral sites including
the ovary, decidua, chorion and placenta (Mitchell and Schmid, 2001) and, with
respect to the biological actions, it acts in the breast and the intrauterine tissues
to modulate lactation and parturition, respectively (Uvnas-Moberg and Eriksson,
1996; Challis et al., 2000; Mitchell and Schmid, 2001).

Expression and localization
Northern blot analysis, ribonuclease protection assays and in situ hybridization
analysis indicated local production of OT mRNA in trophoblast, amnion,
chorion and decidua. The highest abundance was found in the decidua where
the transcript appeared to be slightly smaller than that in the hypothalamus and
ovary, considerably less in chorion and amnion and very low in trophoblast
(Chibbar et al., 1993). With respect to trophoblast localization a large quantity
of OT-like substance exists in human placental tissue, mainly in the syncy-
tiotrophoblast (Mitchell and Schmid, 2001).
By ribonuclease protection assays, a signi¬cantly higher amount of OT mRNA
has been detected in tissue obtained after spontaneous labor compared with those
obtained at term but before labor onset. This suggested that OT mRNA levels
increase around the time of parturition either through increased transcription of
the mRNA or increased stability of the mRNA, thus supporting a role for OT in
26 F. Petraglia et al.



the mechanism of labor onset. The OT peptide has been measured in human fetal
membrane tissues with signi¬cantly higher concentrations in the decidua com-
pared with the amnion or the chorion (Chibbar et al., 1993; Takemura et al., 1994;
Mitchell and Schmid, 2001). The content of immunoreactive OT in total placenta
extracts increases throughout gestation, in parallel to maternal blood levels
(Chibbar et al., 1993; Mitchell and Schmid, 2001 Blanks and Thornton, 2003).
Since placental content is approximately ¬vefold greater than in the posterior
pituitary lobe, the main source of OT in pregnancy is the placenta (Reis et al.,
2001). The OT is secreted from cultured placental cells (Florio et al., 1996), and in
vitro studies showed an effect of OT in stimulating CRF (CRH) secretion from
cultured placental cells (Petraglia et al., 1996d; Challis et al., 2000).

Receptors
The OT signaling is transduced to physiological actions via the OTR. The OTR
is a 389 amino acid polypeptide with seven-transmembrane domains and
belongs to the class I G-protein-coupled receptor (GPCR) family (Kimura et al.,
1992). The OTR gene is present in single copy in the human genome and was
mapped to the gene locus 3p25“3p26.2 (Inoue et al., 1994) and the human OTR
mRNAs shows two different sizes, being of 3.6 Kb in breast and of 4.4 Kb in
ovary, endometrium and myometrium (Mitchell and Schmid, 2001).
By in situ hybridization and immunohistochemistry OTR mRNA was
detected in decidual cells and in the trophoblast of the chorion laeve, but not
in the trophoblast into the placenta, suggesting differences in its expression in
the trophoblast, depending on the localization. Indeed, the expression of OTR
mRNA and protein in the amnion, the other fetally derived tissue at the feto-
maternal interface, is much lower than that of trophoblasts in the chorion leave
(Chibbar et al., 1993; Takemura et al., 1994; Mitchell and Schmid, 2001).
The promoter region of the human OTR gene contains several consensus
sequences that have been reported to be affected by cytokines, such as TNF, IL-1
and IL-6 (Takemura et al., 1994). The concentrations of these cytokines in human
amniotic ¬‚uid are increased at the time of parturition both in normal term or
preterm labor in the absence of clinical evidence of infection (Reis et al., 2002).
Thus, it is possible that the timing of human parturition is regulated to a large
extent by the in¬‚uence of the immune system on the OTR gene, not only in cases
associated with intrauterine infection but also in the normal physiological process.

Levels in biological ¬‚uids
A number of technical dif¬culties have been found in measuring plasma OT in
humans, due to the pulsatile OT secretion, the presence of oxytocinase, which
27 Placental expression of neurohormones and other neuroactive molecules



metabolize OT (Tsujimoto et al., 1992) and, to the antibodies used to measure
OT. The OT is measurable in maternal plasma during pregnancy with a gradual
rise of its levels with advancing gestation, but levels do not differ between early
labor and late pregnancy (Fuchs et al., 1981; 1991). It is secreted in discrete pulses
and the frequency of these pulses is signi¬cantly higher during spontaneous
labor than before the onset of labor (Fuchs et al., 1991). After spontaneous VD,
umbilical arterial plasma levels of OT are consistently higher than those in the
umbilical vein, whilst the fetal arterio-venous difference is less pronounced at
ECS section. At spontaneous VD, plasma levels from the umbilical cord artery
are signi¬cantly higher than the maternal levels, and signi¬cantly higher than at
elective abdominal delivery. Therefore, it is concluded that the human fetus can
be an important source of OT (De Geest et al., 1985) and this indirectly supports
the hypothesis that locally produced OT may act without being re¬‚ected in
maternal circulation.



CRH and pathologies of pregnancy

Several lines of evidence underlie the link between placental CRH and stress of
parturition in humans. In fact, during spontaneous labor maternal plasma CRH
levels progressively rise (Figure 1.3), reaching the maximum values at the most
advanced stages of cervical dilation (Petraglia et al., 1990a; Reis et al., 1999; Florio
et al., 2002d). In addition, subjects who underwent elective Cesarean (ECS) deliv-
ery had plasma and amniotic ¬‚uid CRH levels signi¬cantly lower than patients
after spontaneous vaginal delivery (VD) (Petraglia et al., 1990a; Reis et al., 1999;
Florio et al., 2002d). Moreover, the amount of CRH in placental extracts obtained
at term after spontaneous VD is signi¬cantly greater than the amount of extracted
from placentas obtained after Caesarean delivery (Petraglia et al., 1990a). In addi-
tion, during spontaneous physiological labor a signi¬cant decrease in CRH-BP
levels in maternal plasma (Linton et al., 1993; McLean et al., 1995), cord blood
(Petraglia et al., 1997a) and amniotic ¬‚uid (Florio et al., 1997) has been observed.
Women with preterm labor have maternal plasma CRH levels signi¬cantly
higher than those measured in the course of normal pregnancy (Korebrits et al.,
1998), but also in those who later develop preterm labor (McLean et al., 1995)
(Table 1.2). Taken together, this ¬nding suggests that the increase in CRH levels in
patients with preterm labor is not due to the process of labor itself, but indeed may
be part of the mechanism controlling the onset of labor.
Maternal plasma CRH is higher in women with threatened preterm labor who
give birth within 24 h from admission compared to those delivered after 24 h or
with normal women at the same gestational age (Petraglia et al., 1996a). However,
28 F. Petraglia et al.


Table 1.2 Levels of placental neurohormones in gestational diseases

Preterm labor PIH PE IUGR

GnRH
Activin A
Inhibin A n.e.
CRF
CRF-BP n.e.
NPY n.e. n.e.
CGRP n.e. n.e. n.e.
PTHrP n.e. n.e. n.e.
SST n.e. n.e.

: increased; : reduced; : unchanged levels; n.e.: not evaluated.



180 CRF
ACTH
160
NPY
Relative increase (%)




140
Cortisol
120
β-END
100
Catecholamines
80
60
40
20
0
Phase 1 Phase 2 Delivery
Figure 1.3 Stress hormones in maternal circulation at parturition. The sharp increase in the
concentrations of CRF, cortisol and NPY around the time of labor re¬‚ects acute placental
release. The placenta seems to participate in the stress response of human parturition



the continued elevation of CRH preceding clinical evidence of uterine contraction
suggests that CRH secretion is not suf¬cient to induce initiation of labor, and other
factors are required in this event (McLean et al., 1995; Reis et al., 1999; Florio et al.,
2002d). Maternal and fetal plasma CRH-BP levels are low in preterm labor
(Berkowitz et al., 1996; Petraglia et al., 1997a) resembling the physiologic pattern
observed at term. As CRH-BP modulates CRH actions on target organs, the preco-
cious fall in CRH-BP levels has been suggested to be involved in the pathophysiol-
ogy of preterm labor.
The PE, de¬ned as hypertension associated with proteinuria, complicates 2“8%
of pregnancies, and is an important cause of maternal and neonatal mortality

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