Although the role of melatonin on fetal development has been the subject of a number of studies, little is known about the function of melatonin in the placenta. We previously showed that melatonin receptors are expressed and are functional in JEG-3 and BeWo cell lines, both in vitro models of human trophoblast. Local synthesis of melatonin in placenta has been proposed, but the human placenta's ability to synthesize melatonin de novo has never been studied. The purpose of this study was to investigate the expression [reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis] and activity (radiometric assay) of melatonin synthesizing enzymes, and characterize the expression of the melatoninergic receptors in human term villous trophoblast. The results show that arylalkylamine N-acetyltransferase and hydroxyindole O-methyltransferase melatonin synthesizing enzymes are expressed and active in villous trophoblast as well as in JEG-3 and BeWo placental choriocarcinoma cells. In addition, immunohistochemical analysis demonstrated the presence of MT1, MT2, and retinoid-related orphan nuclear receptor alpha melatonin receptor proteins in both villous cytotrophoblast and syncytiotrophoblast (STB) as well as in endothelial cells surrounding the fetal capillaries and in the villous mesenchymal core. RT-PCR and western blot analysis in primary cultures of human term trophoblast confirmed the expression of all three melatonin receptors in villous cytotrophoblast and STB cells. This study demonstrates for the first time a local synthesis of melatonin and expression of its receptors in human trophoblasts and strongly suggests a paracrine, autocrine, and/or intracrine role for this indolamine in placental function and development as well as in protection from oxidative stress.

The melatonin system in preeclamptic pregnancies has been largely overlooked, especially in the placenta. We have previously documented melatonin production and expression of its receptors in normal human placentas. In addition, we and others have shown a beneficial role of melatonin in placental and fetal functions. In line with this, decreased maternal blood levels of melatonin are found in preeclamptic compared with normotensive pregnancies. However, melatonin production and expression of its receptors in preeclamptic compared with normotensive pregnancy placentas has never been examined. This study compares (i) melatonin-synthesizing enzyme expression and activity, (ii) melatonin and serotonin, melatonin's immediate precursor, levels and (iii) expression of MT1 and MT2 melatonin receptors in placentas from preeclamptic and normotensive pregnancies. Protein and mRNA expression of aralkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT), the melatonin-synthesizing enzymes, as well as MT1 and MT2 receptors were determined by RT-qPCR and Western blot, respectively. The activities of melatonin-synthesizing enzymes were assessed by radiometric assays while melatonin levels were determined by LC-MS/MS. There is a significant inhibition of AANAT, melatonin's rate-limiting enzyme, expression and activity in preeclamptic placentas, correlating with decreased melatonin levels. Likewise, MT1 and MT2 expression is significantly reduced in preeclamptic compared with normotensive pregnancy placentas. We propose that reduced maternal plasma melatonin levels may be an early diagnostic tool to identify pregnancies complicated by preeclampsia. This study indicates a clinical utility of melatonin as a potential treatment for preeclampsia in women where reduced maternal plasma levels have been identified.

Melatonin is highly produced in the placenta where it protects against molecular damage and cellular dysfunction arising from hypoxia/re-oxygenation-induced oxidative stress as observed in primary cultures of syncytiotrophoblast. However, little is known about melatonin and its receptors in the human placenta throughout pregnancy and their role in villous trophoblast development. The purpose of this study was to determine melatonin-synthesizing enzymes, arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT), and melatonin receptors (MT1 and MT2) expression throughout pregnancy as well as the role of melatonin and its receptors in villous trophoblast syncytialization. Our data show that the melatonin generating system is expressed throughout pregnancy (from week 7 to term) in placental tissues. AANAT and HIOMT show maximal expression at the 3rd trimester of pregnancy. MT1 receptor expression is maximal at the 1st trimester compared to the 2nd and 3rd trimesters, while MT2 receptor expression does not change significantly during pregnancy. Moreover, during primary villous cytotrophoblast syncytialization, MT1 receptor expression increases, while MT2 receptor expression decreases. Treatment of primary villous cytotrophoblast with an increasing concentration of melatonin (10 pM-1 mM) increases the fusion index (syncytium formation; 21% augmentation at 1 mM melatonin vs. vehicle) and β-hCG secretion (121% augmentation at 1 mM melatonin vs. vehicle). This effect of melatonin appears to be mediated via its MT1 and MT2 receptors. In sum, melatonin machinery (synthetizing enzymes and receptors) is expressed in human placenta throughout pregnancy and promotes syncytium formation, suggesting an essential role of this indolamine in placental function and pregnancy well-being.

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