Placental melatonin system is present throughout pregnancy and regulates villous trophoblast differentiation.

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 ß-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.

Melatonin: the watchdog of villous trophoblast homeostasis against hypoxia/reoxygenation-induced oxidative stress and apoptosis.

Human placenta produces melatonin and expresses its receptors. We propose that melatonin, an antioxidant, protects the human placenta against hypoxia/reoxygenation (H/R)-induced damage. Primary term villous cytotrophoblasts were cultured under normoxia (8% O2) with or without 1mM melatonin for 72h to induce differentiation into the syncytiotrophoblast. The cells were then cultured for an additional 22h under normoxia or subjected to hypoxia (0.5% O2) for 4h followed by 18h reoxygenation (8% O2) with or without melatonin. H/R induced oxidative stress, which activated the Bax/Bcl-2 mitochondrial apoptosis pathway and the downstream fragmentation of DNA. Villous trophoblast treatment with melatonin reversed all the negative effects induced by H/R to normoxic levels. This study shows that melatonin protects the villous trophoblast against H/R-induced oxidative stress and apoptosis and suggests a potential preventive and therapeutic use of this indolamine in pregnancy complications characterized by syncytiotrophoblast survival alteration.

Placental melatonin production and melatonin receptor expression are altered in preeclampsia: new insights into the role of this hormone in pregnancy.

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.

Quantitative PCR pitfalls: the case of the human placenta.

Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is a rapid and high throughput gene expression quantification technology. In order to obtain accurate results, several key experimental design and standardization steps must be rigorously followed as previously described in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. This study investigates the effect of reference gene normalization and the impact of RNA degradation on gene expression of 8-oxoguanine DNA glycosylase in human placenta from pregnancies complicated by preeclampsia and gestational diabetes mellitus and their gestation-matched controls. The data presented here show how RNA quality and appropriate reference gene selection is not only important to obtain accurate and reproducible RT-qPCR data but how different and even opposite results can be reported if the key steps outlined in the MIQE guidelines are not followed. The procedures and associated results presented in this study provide the first practical application of the MIQE guidelines to placental analysis in normal and pathological pregnancies.

Melatonin: the smart killer: the human trophoblast as a model.

Melatonin has both the ability to induce intrinsic apoptosis in tumor cells while it inhibits it in non-tumor cells. Melatonin kills tumor cells through induction of reactive oxygen species generation and activation of pro-apoptotic pathways. In contrast, melatonin promotes the survival of non-tumor cells due to its antioxidant properties and the inhibition of pro-apoptotic pathways. In primary human villous trophoblast, a known pseudo-tumorigenic tissue, melatonin promotes the survival through inhibition of the Bax/Bcl-2 pathway while in BeWo choriocarcinoma cell line melatonin induces permeabilization of the mitochondrial membrane leading to cellular death. These findings suggest that the trophoblast is a good model to study the differential effects of melatonin on the intrinsic apoptosis pathway. This review describes the differential effects of melatonin on the intrinsic apoptosis pathway in tumor and non-tumor cells and presents the trophoblast as a novel model system in which to study these effects of melatonin.

Human placental trophoblasts synthesize melatonin and express its receptors.

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.

Expression of melatoninergic receptors in human placental choriocarcinoma cell lines.

BACKGROUND: Melatonin crosses the placenta and enters the fetal circulation. Moreover, experimental data suggest a possible influence of melatonin on placental function and fetal development in humans. To date, the expression and role of melatonin receptors in human placenta choriocarcinoma cell lines and in human term placental tissues remain to be elucidated. METHODS AND RESULTS: Results from RT-PCR, western blotting and confocal microscopy demonstrated that the MT1, MT2 and RORalpha1 melatonin receptors are expressed in the human term placental tissues and in choriocarcinoma cell lines JEG-3 and BeWo. Furthermore, enzyme-linked immunosorbent assay showed that 6-chloromelatonin (a melatonin agonist) inhibits, in a dose-dependent manner, forskolin-stimulated hCG-beta secretion in JEG-3 (P < 0.001) and BeWo (P < 0.05) cells but had no effect on basal human chorionic gonadotrophin (hCG-beta) levels. This effect of 6-chloromelatonin on forskolin-stimulated HCG-beta secretion was abolished by pertussis toxin (PTX), suggesting that melatonin regulates hCG-beta production by an action involving an inhibitory Gi/o protein. In PTX-treated BeWo cells, 6-chloromelatonin stimulated basal hCG-beta secretion (P < 0.001). CONCLUSION: These results demonstrate, for the first time, the expression of melatonin receptors in human term placental tissues and in choriocarcinoma cells and suggest a possible paracrine/autocrine function for melatonin in human placenta.