1.2.1 Corticotropin-Releasing Hormone (CRH)

The secretion of maternal HPA axis hormones increases throughout pregnancy and is related to placental function, as circulating maternal CRH originates almost entirely from the placenta (Reference Petraglia, Florio and Nappi6) (Figure 1.1). However, the responsiveness of the HPA axis to stressors is reduced during pregnancy, as shown by reduced ACTH and corticosteroids, in order to neutralize the impact of stress by minimizing stress-induced fetal exposure to maternal glucocorticoid (Reference Welberg and Seckl12). Unlike CRH, it is uncertain whether maternal plasma ACTH originates from the maternal pituitary and/or from the placenta. Maternal ACTH and cortisol, but not CRH, undergo a typical circadian rhythmicity in the maternal circulation, suggesting that CRH is mostly of placental origin (Reference Waddell13).

In the plasma of nonpregnant women, CRH concentrations are very low (around 15 pg/ml) or undetectable. The human placenta expresses large amounts of CRH (1000 times higher than in myometrium and choriodecidua) resulting in high CRH levels in maternal serum during pregnancy. Plasma CRH levels increase during the first trimester of pregnancy, then rise steadily until term (Reference Florio, Severi and Ciarmela14). CRH levels are approximately 800 pg/ml during late third trimester and peak (2000–3000 pg/ml) during labor, becoming undetectable within 24 hours after delivery. In the fetal circulation, a linear correlation exists between maternal and fetal plasma CRH levels: CRH concentrations in umbilical venous plasma are higher than in the umbilical artery, supporting the notion that placenta is a major source of fetal plasma CRH (Figure 1.1).

Mechanisms regulating placental CRH release are not the same as those regulating hypothalamic release: prostaglandins (PGs), neurotransmitters (norepinephrine, acetylcholine), neuropeptides, and cytokines (IL-1) all stimulate CRH, whereas progesterone (P4), nitric oxide, and estrogens decrease placental CRH production. In the mother, cortisol inhibits hypothalamic CRH and ACTH release, with a negative feedback. In striking contrast, cortisol stimulates CRH release by the decidua, trophoblast, and fetal membranes, and it drives maternal and fetal HPA activation with a positive feedback loop (Reference Challis, Bloomfield and Booking15). This positive feed-forward system is a unique feature of placental CRH, suggesting that it might not require such “safety switch-off” mechanisms, and the demand for the actions of CRH increases as pregnancy progresses toward term (Reference Petraglia, Imperatore and Challis4). In fact, at term and in labor, circulating levels of CRH, ACTH, and cortisol are increased (Reference Ochȩdalski, Zylinńska and Laudański16), although they are not necessarily indicative of maternal HPA axis activation.

Placental CRH has complex effects including a role in the onset of labor (Reference Vitoratos, Papatheodorou and Kalantaridou17, Reference Challis, Matthews and Gibb18), resembling the timer of a biological clock counting from the early stages of gestation and signaling the timing of parturition (Reference McLean, Bisits and Davies19). Interestingly, maternal CRH levels have been reported to be lower in women delivering post-term and higher in those who will deliver preterm compared to those in women delivering at term. These findings corroborate the important role of this neurohormone in regulating the placental “clock” of human pregnancy and in influencing its length (Reference Torricelli, Ignacchiti and Giovannelli20).

Regarding endocrine functions, CRH regulates implantation, trophoblast cell growth and invasion, tissue remodeling through modulation of secretion of matrix metalloproteinases, and vascular tone through activation of the nitric oxide pathway, as well as inflammation through PGs release (Reference Voltolini and Petraglia2).

CRH also plays a pivotal role in regulating estrogen and progesterone production in the third trimester (Reference Rainey, Rehman and Carr21), corroborating the hypothesis that placental CRH levels are linked to the length of gestation in humans. The rapid rise of CRH in late pregnancy is associated with estriol (E3) surge and critically altered progesterone (P)/E3 and estriol/estradiol (E3/E2) ratios that create an estrogenic environment predisposing to the onset of labor (Reference Smith, Smith and Shen22). CRH also regulates myometrial contractility, exerting diverse roles at different stages of gestation. In fact, it mediates both relaxation and contraction of myometrium, depending on different patterns of expression and biologic effects of CRH receptors.

CRH and Ucn exert their actions by activating CRH receptors, named CRHR1 and CRHR2 (Reference Grammatopoulos23). CRH has high affinity only for CRHR1, Ucn shows approximately the same affinity for both receptors, whereas Ucn2 and Ucn3 bind with high affinity only to the CRHR2. Nonpregnant human myometrium expresses three CRHR subtypes: 1α, 1β, and 2β. As pregnancy progresses, the myometrium starts to express CRHR2α.

CRH-R1 and CRH-R2 stimulate divergent signaling pathways. CRH-R1 contributes to the maintenance of myometrial relaxation during pregnancy through activation of the adenylyl cyclase/cAMP pathway. In chorion trophoblast cells, CRH exerts a tonic stimulatory effect on 15-hydroxyprostaglandin dehydrogenase (PGDH) activity, an effect that may help to maintain a metabolic barrier that prevents bioactive PGs passing from the chorioamnion to the myometrium preventing myometrial activation during pregnancy (Reference McKeown and Challis24). In contrast, at term, CRH induces phosphorylation of CRH-R2 variants, with subsequent stimulation of the phospholipase C/inositol triphosphate pathway and increase of myosin light chain phosphorylation, promoting myometrial contractility (Reference Grammatopoulos and Hillhouse25, Reference Simpkin, Kermani and Palmer26). Furthermore, CRH induces stimulation of placental and membrane PGs output, and the inhibition of progesterone production (Reference Jeschke, Mylonas and Richter27).

CRH binding protein (CRH-BP) binds circulating CRH with high affinity, inhibiting its functions (Reference Zhao, Hoheisel and Schauer28); in fact their interaction results in the dimerization of the protein and clearance of CRH from the circulation. Maternal serum CRHBP levels do not change significantly during most of gestation, but they fall during the final weeks of normal pregnancy and still further with the onset of labor (Reference Florio, Woods and Genazzani29). Significantly lower CRH and higher CRHBP levels are present in post-term pregnancies, suggesting that the lack of the reciprocal changes in CRH and CRHBP levels may delay the onset of labor.

Fetal stress responses are independent from those of the mother (Reference Gitau, Fisk and Teixeira30): The fetal hypothalamus releases CRH, together with placental CRH, inducing fetal pituitary ACTH secretion and the synthesis of cortisol by the fetal adrenal gland and maturation of the fetal lungs. In turn, the rising cortisol concentrations in the fetus stimulate placental CRH production through a positive feedback mechanism (Figure 1.1). ACTH in turn controls adrenocortical functional development, including angiogenesis and expression of steroidogenic enzymes.

The maturation of the fetal lungs as a result of increasing cortisol concentrations represents a fundamental aspect of fetal adaptive mechanisms to extra-uterine life activated in part by the stress of delivery. Moreover, fetal lung maturation is associated with increased production of surfactant protein A and phospholipids, both pro-inflammatory factors, that may stimulate myometrial contractility through increased production of PGs by fetal membranes and the myometrium itself. Thus, the fetus herself contributes to the onset of labor (Reference Challis, Bloomfield and Booking15, Reference Mesiano, DeFranco and Muglia31).

Given its multiple effects on a number of placental functions, the role for CRH as a marker of pregnancy pathology has been proposed. CRH is abnormally secreted when obstetric complications occur, such as in pre-term birth (PTB), pre-eclampsia (PE), pregnancy-induced hypertension (PIH), and fetal growth restriction (FGR). In the case of miscarriage, CRH peptide has been found to be more expressed in the placenta in spontaneous abortion than in elective abortion of the same gestational age. In early pregnancy, high glucocorticoid secretion is associated with miscarriage compared with women with ongoing gestation, suggesting that maternal stress in the first trimester is associated with a higher risk for spontaneous abortion (Reference Nepomnaschy, Welch and McConnell32). Furthermore, CRH expression is significantly higher in preterm than in term placentas (Reference Torricelli, Ignacchiti and Giovannelli20) and maternal CRH levels at mid-gestation are higher in women who subsequently would have spontaneous PTB (Reference Hobel, Dunkel-Schetter and Roesch33). Interestingly, chorioamnionitis associated with PTB activates the placental CRH pathway in vivo (Reference Torricelli, Novembri and Bloise34). In hypertensive disorders of pregnancy, maternal plasma and cord blood CRH levels are higher in women affected by PIH and PE compared to healthy women (Reference Florio, Imperatore and Sanseverino35). Additional data confirm that pregnancies complicated by PE and FGR are associated with abnormal placental vascular resistance and abnormally high umbilical vein CRH levels.

1.2.2 Urocortins (Ucns)

Urocortin (Ucn) is synthesized and secreted by placental and fetal membranes, similar to CRH. However, urocortin is undetectable in maternal plasma during pregnancy, with no rise with increasing gestational age (Reference Glynn, Welton and Rodriguez-Linares36). Plasma levels are higher at labor, but they do not change significantly at the different stages of labor. Ucn has similar effects as CRH, augmenting matrix metalloproteinase, ACTH, and PGs secretion from cultured human placental cells, enhancing myometrial contractility. Ucn stimulates placental ACTH secretion, via a CRHR1-dependent mechanism, without any significant difference from CRH-induced ACTH release (Reference Petraglia, Florio and Benedetto37). Ucn has high affinity for CRHR1 and CRHR2 families and the CRH binding protein; particularly, Ucn exhibited greater affinity for CRHR2, acting as its natural ligand. Human myometrium expresses Ucn (Reference Clifton, Qing and Murphy38), which activates a number of intracellular signaling pathways that contribute to the activation of myometrial contractility.

Ucn2 and Ucn3 are localized in syncytiotrophoblast and extravillous trophoblast cells, while Ucn2 is also localized to blood vessel endothelial cells, leading to the suggestion of a role of Ucn2 in regulating the placental vascular endothelial behavior. In the fetal membranes, Ucn2 is distributed only in amnion, while Ucn3 is found in both amnion and chorionic cells (Reference Imperatore, Florio and Torres39). Ucn2 and Ucn3 modulate HPA axis activity at the hypothalamic level in a paracrine or autocrine fashion, but unlike CRH and Ucn, peripheral Ucn2 or Ucn3 administration does not increase either pituitary or placental ACTH secretion (Reference Imperatore, Florio and Torres39). Hypothalamic Ucn2 expression is increased by glucocorticoids and modulates basal HPA-axis circadian amplitude (Reference Hashimoto, Nishiyama and Tanaka40). Ucn2 plays a major role in the control of myometrial contractility during human pregnancy, involving the binding with CRHR2 (Reference Karteris, Hillhouse and Grammatopoulos41).

Maternal plasma and cord blood Ucn levels have been found to be higher in women delivering pre-term compared to those delivering at term. Furthermore, Ucn levels in arterial cord blood are higher than in venous cord blood and in maternal plasma, suggesting a fetal rather than an exclusively placental source of the peptide at preterm parturition (Reference Florio, Torricelli and Galleri42). Similarly, maternal plasma and cord blood Ucn levels are higher in women affected by hypertensive disorders of pregnancy associated with FGR compared with healthy women (Reference Florio, Torricelli and De Falco43). Interestingly, early PE placental samples show stronger immunoreactivity for Ucn2 than for Ucn3, while Ucn3 immunostaining was stronger in late PE samples (Reference Imperatore, Rolfo and Petraglia44). Ucn has been found to contribute to the pathogenesis of FGR possibly through negative regulation of placental system A activity, which represents a placental amino acid transporter whose normal activity is fundamental for maintaining fetal growth (Reference Giovannelli, Greenwood and Desforges45).

1.8.1 Neuropeptide Y (NPY)

Neuropeptide Y (NPY) is a sympathetic neurotransmitter discovered in 1982 and has been shown to be involved in increased blood pressure, angiogenesis, and vasoconstriction. Sympathetic nerves and chromaffin cells of the adrenal glands have been hypothesized to be the major source of circulating NPY outside of pregnancy. In pregnant women, acidic extracts of human placental tissues collected at term contain high immunoreactive NPY (Reference Sahay, Kale and Joshi66).

NPY is present in cervical tissue from both early pregnant women and women at term, but no difference in intensity or distribution of NPY-containing nerves was demonstrated between early and term pregnant women or between women in labor and women before onset of labor. In pregnant women, maternal plasma NPY levels are higher than in nonpregnant women, without significant changes throughout gestation (Reference Petraglia, Coukos and Battaglia67). Plasma NPY levels fall within a few hours after delivery, supporting the placental origin of the circulating NPY in pregnancy.

A role of NPY during labor at term and preterm is yet to be defined, but a functional association between the increase of NPY during labor and vaginal delivery is in accordance with the spontaneous labor-induced increase of other stress-related hormones such as catecholamines, cortisol and ACTH. It has been suggested that NPY is implicated in PE and PIH, as elevated maternal plasma NPY levels have also been reported in women affected by hypertensive disorders (Reference Khatun, Kanayama and Belayet68), whereas a reduced placental expression of NPY was observed, suggesting the involvement of the receptors in the abnormalities of placental angiogenesis (Reference Sahay, Kale and Joshi66).

1.8.2 Relaxin

Relaxin is a peptide hormone that is a member of the insulin family. In women, circulating relaxin is a product of the corpus luteum of pregnancy, but is also synthesized by other reproductive organs such as the uterus, decidua, and placenta, where relaxin acts locally. Circulating relaxin is secreted in a pattern similar to that of hCG.

Relaxin binds to its receptor RXFP1, which has been localized to a wide variety of reproductive and non-reproductive tissues. Relaxin has many uterotropic effects including stimulating uterine growth and vascularization, remodeling extracellular matrix components, and regulating vascular endothelial growth factor in preparation for implantation. Evidence also supports a role for relaxin in the systemic maternal vascular adaptations required for a healthy pregnancy. Diminished relaxin levels in early pregnancy are linked with increased risks of miscarriage and the development of preeclampsia (Reference Marshall, Senadheera and Parry69). Furthermore, it has been shown that women who are more likely to deliver preterm have increased circulating relaxin levels (Reference Goldsmith, Weiss and Steinetz70).

1.8.3 Parathyroid Hormone-Related Protein (PTHrP)

Parathyroid hormone-related protein (PTHrP) is an oncofetal protein that is expressed in many mammalian tissues. There is substantial evidence to support its role in pregnancy and labor as a placental calcium transport activator, potent vasorelaxant, and inhibitor of uterine contractions (Reference Clemens, Cormier and Eichinger71).

Human amnion is a major source of PTHrP and PTHrP mRNA abundance in amnion decreases during labor at term. Similarly, there is a dramatic decrease in amniotic fluid PTHrP concentrations associated with term labor, which is not evident in amniotic fluid obtained from preterm labor. These results suggest that at term, the dramatic decrease in PTHrP activity in tissue and amniotic fluid might be part of the physiological mechanism involved in the onset and/or progression of labor (Reference Ferguson, Gorman and Bruns72).

1.8.4 Opioids

Three families of opioid peptides are recognized: endorphin (END), enkephalin (ENK), and dynorphin (DYN). They derive from three pre-hormones: pro-opiomelanocortin (POMC) for β-END, proenkephalin (P-ENK) for ENK and prodynorphin (P-DYN) for DYN. Mu (μ), kappa (κ), and delta (δ) are the main opioid receptor types, but κ receptors are the more important type present in the placenta. Placental content of κ receptors increases with gestational age, and term placental content of κ receptors correlates with type of delivery.

CRH stimulates pro-opiomelanocortin hormone (POMC) release from human cultured placental cells, suggesting placental regulation of POMC secretion, somewhat similar to that occurring in pituitary cells. However, in contrast to the negative feedback activity of glucocorticoids, dexamethasone is not effective either in suppressing ACTH or b-END release from placental cells or in reducing the CRH-induced release of placental ACTH/b-END release. Indeed, there is abundant evidence to show that corticosteroids increase rather than decrease expression and synthesis of CRH from trophoblasts.

Many studies agree on a large increase of plasma b-END during labor and at parturition, although they do not indicate whether the peptide has pituitary or placental origin. Maternal plasma levels of ENK are not significantly different from those of nonpregnant women and do not change throughout pregnancy, supporting a local role of the peptide. On the contrary, maternal plasma DYN levels in the third trimester and at delivery are higher than in nonpregnant women and high levels are also detectable in amniotic fluid and in umbilical vein plasma (Reference Ahmed, Cemerikic and Agbas73).

1.8.5 Neurosteroids

The placenta is a source of several neurosteroids comprising progesterone itself, its derivatives 5a-pregnane-3a-ol-20- one (allopregnanolone) and 5a-dihydroprogesterone, and its precursor pregnenolone sulfate (Reference Le Goascogne, Eychenne and Tonon74). Apart from progesterone, the role of placental neurosteroids in the physiology of pregnancy is largely unknown. These hormones may contribute to the neurochemical and behavioral changes of pregnancy and puerperium because they interfere with GABAergic circuits and have anxiolytic effects (Reference Dombroski, Casey and MacDonald75).

The levels of allopregnanolone in maternal serum increase progressively during gestation and, in contrast to progesterone, are augmented in hypertensive complications of pregnancy (Reference Luisi, Petraglia and Benedetto76). In PE, there is an increased activity of tyrosine hydroxylase in placental tissue, and this is likely to contribute the higher levels of catecholamines in the maternal circulation (Reference Manyonda, Slater and Fenske77). It is also a neuroactive hormone and may contribute to the psychological adaptation associated with pregnancy and puerperium. In fact, serum allopregnanolone levels were detectable postpartum and were significantly decreased in women with maternity “blues” (Reference Nappi, Petraglia and Luisi78).

A recent study showed that in healthy pregnant women, maternal serum allopregnanolone and progesterone levels significantly increased throughout gestation. No major changes were found at delivery, with the exception of a significant decrease of maternal and cord serum allopregnanolone levels during emergency cesarean section (Reference Luisi, Petraglia and Benedetto76). This suggests the critical role of these steroids in maternal brain adaptation during pregnancy and perhaps in the development of the fetal brain. It has been suggested that allopregnanolone is brain protective during PTB or FGR, reducing the impact of hypoxia (Reference Pluchino, Ansaldi and Genazzani79).