Potential Utility of Melatonin in Preeclampsia,Intrauterine Fetal Growth Retardation,and Perinatal Asphyxia

Preeclampsia

Melatonin levels are altered in women with abnormally functioning placentae during preeclampsia and IUGR. ²³ Preeclampsia is a multisystem disorder that occurs in 5% to 10% of pregnancies, and it is commonly diagnosed in the latter half of pregnancy, but it may involve poor placentation during the earliest stage of pregnancy and cause increased maternal and neonatal mortality and morbidity. This disorder is a leading cause of premature delivery and IUGR. The role of OS and nitrosative stress in preeclampsia is well accepted, ²⁴ although the causes of generation of toxic oxygen derivatives, which contribute to molecular damage, are debated. Placenta plays a major role in the pathogenesis of preeclampsia that is characterized by abnormal placentation and reduced placental perfusion. The derived OS/nitrosative stress may result from permanent hypoxia or intermittent hypoxia/reoxygenation correlated with placental maldevelopment, for example, due to an inadequate attachment of the placenta to the uterine wall. ²⁵ In addition to anomalous interstitial trophoblastic invasion, abundantly documented in the literature, other possible etiopathogenic pathways have been proposed based on genetic, immunologic, inflammatory, and even related nutritional factors. ²⁶

Placental damage by ROS and RNS in preeclampsia might be facilitated by a reduction in local antioxidant defense, although it is not clear whether the problem consists of a compromised antioxidant defense system or the damage of critical enzymes secondary to free radical action. Furthermore, reduced activities of placental SOD and glucose 6-phosphate dehydrogenase in the placenta of women with preeclampsia ²⁷ are documented aims with a significant increase in placental lipid peroxidation levels compared with placentae from normal pregnant women. ²⁸ Negi et al ²⁹ also reported a significant elevation in the levels of 8-hydroxydeoxyguanosine, protein carbonyls, nitrite, and iron along with reduced levels of CAT and vitamins A, E, and C in the umbilical cord blood of preeclamptic and eclamptic pregnancies. These factors could influence the risk of oxidative damage in infants born to preeclamptic/eclamptic pregnancies. Many et al ³⁰ showed intensive immunoreactivity for nitrotyrosine in invasive cytotrophoblasts in placental biopsies and vascular endothelium in the floating villi obtained from women with preeclampsia. The presence of nitrotyrosine is suggestive of damage due to peroxynitrite and reflects enhanced production of the superoxide anion radical.

Reduced levels of night-time circulating melatonin have been found in pregnant women with severe preeclampsia when compared with those in women with a normal pregnancy or with mild preeclampsia, suggesting a role in the pathogenesis of this disease. ³¹ In preeclampsia, related to increased OS induced initially by a production of cytotoxic factors, ^28,32 a deficiency of melatonin would explain the suppressed antioxidant capacity. Furthermore, melatonin plays an important role in controlling blood pressure ³³ : administration reduces both systolic and diastolic pressure, and it has been shown that hypertensive participants have lower blood melatonin concentrations at night. Moreover, when patients were given supplemental melatonin, night-time blood pressure was reduced. ³⁴ Blood pressure normally undergoes a circadian fluctuation with high values during the day and lower levels at night, and these fluctuations are less marked in preeclampsia where there are also reduced levels of nocturnal melatonin. ³⁵ Also, pregnant women with lower melatonin levels have a higher risk of developing preeclampsia. ³⁶ Lanoix et al compared levels of melatonin, its precursor serotonin, its synthesizing enzymes AANAT and ASMT, and its membrane receptors MT1 and MT2 in preeclamptic placentae with those from gestation-matched normotensive control placentae. They observed that relative to control tissues, preeclamptic placentae had reduced AANAT gene expression and enzyme activity along with lower ASMT activity. Moreover, dramatically depressed concentrations of melatonin and elevated levels of its precursor, serotonin, were observed. Finally, preeclampsia was associated with a significant reduction in both the MT1 and the MT2 receptor in the placenta. In view of these outcomes, it seems clear that the markedly depressed level of melatonin in preeclamptic placentae is a consequence of impaired production of the indoleamine due to a major deficiency in the rate-limiting enzyme AANAT. ³⁷ In an animal model, melatonin reduces ischemia/reperfusion injury in the placenta and could potentially alleviate some of the signs of preeclampsia. ³⁸ In women, despite numerous interventions, including bed rest, betamimetics, use of calcium channel blockers, oxygen or nutrient supplementation, and antioxidants such as vitamins C and E, there is no sufficient evidence to support their use for poor placentation manifesting as preeclampsia. ^39,40 Melatonin, in addition to local production, readily crosses the placenta, and it is safe for both mother and fetus. ⁴¹ Administration during pregnancy is currently being investigated in the context of poor placentation, especially for its potential to reduce OS. In detail, Hobson et al ⁸ recently published a phase 1 pilot clinical trial investigating whether maternal melatonin administration could delay the interval to delivery following a diagnosis of preterm preeclampsia. They enrolled 20 women with preterm preeclampsia and took baseline measurements of maternal and fetal well-being (Table 1), levels of OS, ultrasound Doppler studies, and other biomarkers of preeclampsia. Women were given 10 mg oral melatonin 3 times daily until delivery. The primary outcome was time interval between diagnosis and delivery compared to historical controls. Secondary outcomes compared the baseline measurements mentioned previously with twice-weekly measurements during treatment and then 6 weeks postpartum. This study is the first human trial to assess the potential clinical and biochemical effects of melatonin in pregnancies complicated by preterm preeclampsia. The results have not yet been published.

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Table 1.

Clinical Trials With Melatonin Involving Women with Preeclampsia, Fetal Growth Restriction, and Term Newborns With Perinatal Asphyxia.

Study	Trial Design	Population	Melatonin Dose and Route	Efficacy	Safety Problems
Hobson et al 8	Protocol for a phase 1 pilot clinical trial	20 women with preeclampsia	10 mg oral 3 times daily until delivery	Results not yet published
Alers et al 9	Protocol for a phase 1 pilot clinical trial	12 women with early-onset fetal growth restriction	4 mg oral twice daily until delivery	Results not yet published
Fulia et al 65	Randomized, blinded, placebo-controlled trial	10 term asphyxiated neonates (melatonin); 10 term asphyxiated neonates (placebo)	10 mg every 2 hours, 8 doses orally	Significant reductions in serum malondialdehyde and nitrite/nitrate levels at both 12 and 24 hours in the melatonin group. Three deaths in control group and none in study group	No
Aly et al 67	Prospective, randomized trial	15 term asphyxiated neonates (melatonin + hypothermia); 15 term asphyxiated neonates (hypothermia alone); 15 term healthy neonates	10 mg kg, 1 oral daily for 5 days	Survival without abnormalities at 6 months significantly increased in the melatonin/hypothermia group	No

Intrauterine Growth Restriction

It is well known that chronic fetal hypoxemia, most commonly caused by placental dysfunction, produces an adaptive reduction in fetal growth that complicates about 5% of pregnancies. ⁴² Intrauterine fetal growth retardation is associated with preterm birth and, among survivors, an increased likelihood of neurodevelopmental problems including motor and sensory deficits, cognitive and learning difficulties, and cerebral palsy. ⁴² It has been demonstrated that OS is correlated with IUGR, and this condition is often complicated by intrauterine hypoxia and impaired blood flow to the fetus. A chronic restriction in uterine blood flow elicits placental and fetal responses in the form of growth adaptation to hypoxia. Intrauterine hypoxia likely induces ROS generation and, therefore, fetal OS. ⁷

Lipopolysaccharide (LPS) from the cell membranes of gram-negative bacteria is a commonly used toxin in experimental situations to promote free radical-mediated oxidative damage. Chen et al ⁴³ demonstrated that melatonin protects the placenta from LPS-mediated oxidative toxicity and reduces the damage inflicted by LPS on the fetus. When mice were treated with LPS, the toxin increased intrauterine fetal death, caused IUGR, and induced biochemical changes consistent with elevated OS in both mothers and fetuses. ⁴³

In a rat model of IUGR, where maternal nutrient restriction from 15 days postconception (dpc) significantly reduced pup birth weight, maternal administration of melatonin from 15 dpc was associated with increased birth weight, comparable with that of the control pups, and increased expression of the antioxidant enzymes magnesium superoxide dismutase and CAT. ⁴⁴ This effect was not reproducible in a maternal nutrient restriction sheep model of IUGR, where maternal melatonin treatment was associated with a nonsignificant reduction in fetal weight compared with control untreated nutrient-restricted dams and no difference in MDA, as a marker of OS. ⁴⁵ However, in the same study, maternal melatonin administration was associated with a significant rise in umbilical artery blood flow, both in adequately fed and nutrient-restricted dams, suggesting that it might have a beneficial effect on placental resistance. ⁴⁵

The use of melatonin as an antenatal antioxidant therapy in IUGR has been proposed as a promising and safe option to protect the fetal brain from injury. Miller et al ⁴⁶ evaluated the effects of oral melatonin administration on women with an IUGR fetus and showed that maternal melatonin, well tolerated by both mother and fetus, increased fetal melatonin levels and reduced OS in the placenta, as evidenced by reduced MDA levels, compared to IUGR pregnancies untreated with antenatal melatonin. Intrauterine fetal growth retardation in human infants has been associated with a reduction in melatonin secretion during the first 3 months of life. ⁴⁷ Furthermore, 6-sulfatoxymelatonin (6SaMT), a urinary melatonin metabolite, is impaired in adults who were growth restricted prenatally or were born after 40 weeks of gestation. ⁴⁷ Urinary excretion of 6SaMT suggests that there is a relationship between melatonin production and body size at birth. Lanoix et al ⁴⁸ evaluated the protective role of melatonin in multinucleated STB cell apoptosis, which is elevated in preeclampsia and IUGR. ^49,50 In this in vitro study, STB cell apoptosis was due to 4-hour hypoxia followed by 18 hours of reoxygenation. The hypoxia/reoxygenation procedure clearly caused OS in the STB, which led to the activation of Bax/Bcl-2 mitochondrial apoptosis pathway and DNA fragmentation. In this model, melatonin markedly reduced both apoptosis and associated DNA damage, supporting the possibility that it may be useful in vivo to limit complications of pregnancy that involve damage to STB cells and their premature loss. ⁴⁸

There are multiple mechanisms by which maternal melatonin supplementation could enhance umbilical blood flow. It would appear that increased NO bioavailability may account for elevated umbilical blood flow for its antioxidant properties. ⁵¹ Several studies elucidated the acute effects of melatonin on vascular tone, demonstrating complex effects, both vasorelaxant and vasoconstrictor. ⁵² Additionally, maternal nutrient restriction and decreased scotophase concentrations of melatonin have been associated with severely compromised pregnancies. ⁵¹ Shukla et al ⁵³ hypothesized that dietary melatonin supplementation during a compromised pregnancy enhances the bradykinin-induced relaxations of placental arteries, thereby ensuring sufficient umbilical blood flow to the developing fetus, and in addition, this treatment increased the sensitivity of cotyledonary fetal placental arteries to bradykinin-induced relaxation. This underlies, at least in part, the melatonin-induced increase in umbilical artery blood flow. ⁵³ Recently, Alers et al ⁹ published a phase 1 pilot clinical trial investigating whether maternal melatonin administration in pregnancies affected by fetal growth restriction could act on markers of OS in the placenta, and maternal and fetal circulations, on the clinical outcomes of pregnancies, and on brain injury and neurodevelopment in growth-restricted newborn babies (Table 1). They enrolled 12 women with a singleton growth-restricted fetus between 23 + 0 and 34 + 0 weeks. Women were given 4 mg oral melatonin twice daily until delivery. The results have not yet been published.

In conclusion, melatonin would appear to be fundamental for healthy pregnancy, and it acts not only as a regulator of circadian rhythms but also as an endocrine, paracrine, and autocrine modulator, immunomodulator, scavenger of free radicals, and indirectly as an antioxidant agent and cytoprotector.

Perinatal Asphyxia

Oxidative stress contributes to the severity of several newborn conditions to the extent that Saugstad coined the phrase “oxygen radical diseases of neonatology.” ⁵⁴ The peculiar perinatal susceptibility to OS indicates that prophylactic use of antioxidants such as melatonin could help prevent or reduce oxidative injury related to these diseases in newborns. ¹¹

Fetal melatonin is of maternal origin and undergoes circadian fluctuations in synchrony with the day–night rhythm. ⁵⁵ Melatonin levels increase gradually from 24 weeks of gestation and reach the highest levels during the third trimester, returning to baseline levels by the second day of puerperium. ⁵⁶ In the last trimester of pregnancy, the fetus develops a biological clock that is responsive to maternal circadian rhythms with fluctuations in hormonal levels, behavior, and sleep. ¹² The supposed modulator of these rhythms is melatonin, which readily crosses the placenta without being altered. ⁸ After birth, the full-term neonate does not produce melatonin for 3 to 5 months, leading to transient melatonin deficiency. ⁵⁷ Prematurity itself does not hasten the maturation of the neurological network controlling melatonin secretion, rather the onset of pineal melatonin secretion is even more delayed when there is exposure to neurological insults. Thus, in premature neonates, the melatonin deficiency is more prolonged. ⁵⁸

The discovery that melatonin crosses the placenta could be beneficial to prevent free radical damage in the fetus at risk of asphyxia. ⁴¹ Melatonin may be considered an efficient neuroprotective when given shortly before or after birth. Acute in utero asphyxia in the late-gestation fetal sheep results in a significant biphasic increase in hydroxyl radical formation in the gray matter of the fetal brain, consistent with primary and secondary phase OS. When melatonin is administered as prophylaxis to the ewe, both primary and secondary increases in hydroxyl radicals are abolished. ⁵⁹ It has been demonstrated in rats that a rise in lipid peroxidation products and cerebral injury are also prevented with the preinsult administration of melatonin. ⁶⁰ Welin et al ⁶¹ demonstrated that postasphyxia melatonin treatment at a dose of 20 mg/kg attenuated the rise in activated microglia and levels of 8-isoprostane, a marker of lipid peroxidation, and reduced the number of apoptotic cells in the cerebral white matter in midgestation fetal sheep. Furthermore, Kaur et al ⁶² found that the protective effects of melatonin on various parameters in the periventricular white matter of hypoxic neonatal brains were due to its antioxidant properties. Maternal, placental, and fetal melatonin are integral to normal neurodevelopment. The developing brain is highly vulnerable to OS, and it has been shown that melatonin achieves powerful neuroprotective effects though diverse antioxidative mechanisms ⁶³ and by modulating inflammation and promoting brain repair and development in experimental models. ⁶⁴ In 2001, Fulia et al ⁶⁵ first administrated melatonin to 10 asphyxiated newborns. They found that serum MDA and nitrite/nitrate levels at both 12 and 24 hours (Table 1) in the melatonin group were significantly lower than that in the 10 infants of the control placebo group. Currently, hypothermia is recognized as an efficient treatment modality for neonatal hypoxic ischemic encephalopathy (HIE). The use of synergic strategies, such as the association between hypothermia and melatonin supplementation, may lead to a wider neuroprotective effect on the brain, thus improving neonatal outcome. In this regard, ⁶⁶ melatonin administration to newborn piglets has been shown to augment hypothermic neuroprotection by improving cerebral energy metabolism and by reducing brain damage. Recently, in 2015, Aly et al ⁶⁷ have first studied the potential efficacy of 10 mg/kg of melatonin when administered consecutively orally for 5 days, in combination with hypothermia, to infants with HIE. Less seizure activity detected by electroencephalography (Table 1), reduced white matter abnormalities at the brain magnetic resonance imaging examination, and better neurological outcome at 6-month follow-up have been noticed in the melatonin–hypothermia group compared to the hypothermia alone group. Although this study presented a few limitations, including a small sample size (15 neonates for each group) and short-term neurodevelopmental follow-up, it represents a promising starting point to support the combined use of melatonin and therapeutic hypothermia in infants with HIE. However, larger and specific studies to resolve problems of dosage, formulations, and length of treatment are desirable.