Melatonin as an antioxidant and anti-inflammatory agent in the placenta

The placenta is a multifaceted, temporary organ with numerous biological functions largely considered endocrinologic and immunologic (14). The critical roles of the placenta include facilitating embryonic implantation into the uterine wall, promoting fetal growth, and maintaining maternal–fetal tolerance (15). In addition, this organ removes harmful waste products and carbon dioxide from the fetal circulation, provides nutrients and oxygen, and envelopes the fetus in a protective immunologic membrane throughout gestation (15–17). The umbilical cord transfers blood through the placenta to supply the fetus with adequate oxygen and nutrients for survival throughout pregnancy (18). This exchange occurs without the fetal blood and the maternal blood intermingling (17, 18). Hormones produced by the placenta include estrogen and progesterone, which promote the expansion of the uterus to accommodate the growing fetus and placenta (19). Other key immunoprotective roles include facilitating the passive transfer of IgG from the maternal to the fetal circulation and affording protection against invasive pathogens in intrauterine and postnatal life (20, 21).

The formation of the placenta begins after the fertilized egg is implanted into the uterus, which occurs approximately 8 to 10 days after conception (15, 17). The placenta will gradually grow during the first 3 months of pregnancy and then increase in size corresponding to the uterus after 4 months (15, 17). There are several layers and sublayers of tissue that constitute the multifunctional placenta. This review will primarily focus on a sublayer in the chorion layer, namely, the trophoblast, and the decidual layer of the placenta.

The placenta comprises two distinct sides, the maternal and the fetal sides (Figure 1). The chorion is a highly vascular outer embryonic membrane layer surrounding the fetus (14, 22). The chorion has two sublayers: the trophoblast (cytotrophoblast and syncytiotrophoblast) and the extraembryonic mesoderm (Figure 1) (15, 20). The trophoblast layer is positioned on the fetal side of the placenta. Trophoblast cells are essential for implantation because they interact with the maternal uterine endometrium, which promotes syncytiotrophoblast and cytotrophoblast development (20). Trophoblast differentiation and implantation ensure adequate blood supply and limit fetal immune rejection (16, 20). Cytotrophoblast cells are progenitor stem cells of the syncytiotrophoblast that can differentiate into syncytiotrophoblast cells as finger-like projections and ensure that the fetus receives an adequate blood supply with nutrients and oxygen (15, 20, 23). Syncytiotrophoblast cells transport nutrients to the fetus and remove waste products (16). This requires the syncytiotrophoblast to have ample blood supply and blood flow. In addition, these cells must undergo apoptosis continuously throughout gestation for the appropriate channeling of blood (24, 25). Because the syncytiotrophoblast cells have this specialized function and undergo apoptosis, these cells must be regenerated regularly to ensure adequate and continuous blood transport between the maternal and fetal circulations (26). A defect in trophoblast differentiation may compromise the integrity of the placenta, resulting in pregnancy complications, such as preterm birth, preeclampsia, and IUGR (27).

On the maternal side, the primary tissue that comprises this side of the placenta is the decidua, which originates from the endometrium (14). The decidua develops after the blastocyst attaches to the uterine wall, which involves tissue remodeling that supports both residential and immune cells (28). It includes terminally differentiated endometrial stromal cells, maternal blood, and maternal vascular cells (14, 16, 28). There are three distinct sections of decidua, which are named relative to the embryo: the decidua capsularis that covers the implanted embryo, the decidua basalis which is the region between the embryo and the myometrium, and the decidua parietalis, which lines the fetal membrane and the remaining endometrium of the placenta (14, 28, 29). Anchoring villi hold the chorion and the decidua together by securing the decidua basalis to the cytotrophoblast layer (14, 30). Like the trophoblast, the decidua promotes immune tolerance of the semi-allogenic (half of genes from the mother and half of genes from the father) fetus by limiting recognition from maternal immune cells. The decidua also provides nutritional support before placenta formation (28). Defects in the development of the decidua or decidualization may result in implantation failure, pregnancy loss, or pregnancy complications later in gestation (28).

The human placenta is complex and unique, allowing for an intimate contact between maternal and fetal cells throughout gestation (31). The developing fetus has both maternal and paternal antigens, to which the mother’s immune system recognizes the paternal antigens as foreign and leads to the activation of the maternal immune system (31). Therefore, a highly regulated immune system is needed between mother and child to create a beneficial immunological environment that protects the growing fetus from maternal–fetal tolerance, inflammation, and invading pathogens, such as viral infections (32, 33). The placenta is composed of various immune cells, such as natural killer T (NKT) cells, decidual natural killer (dNK) cells, T cells, dendritic cells, B cells, and macrophages (Hofbauer cells and decidual) (31–34). An immense network of cellular connections is formed in the placenta via the interaction of these immune cells, trophoblast cells, and decidual stromal cells to form the immune system in the placenta. An imbalance in this network allows pathogens to infect the placenta and cross the placental barrier to infect the fetus, along with pregnancy complications, such as preterm birth, preeclampsia, spontaneous abortion, and IUGR (33, 35).

Every living organism and nearly every organ has a circadian clock that governs the daily rhythmicity of several physiological and biological processes (55). Core clock genes control the circadian rhythm in the suprachiasmatic nucleus (SCN), the master central pacemaker in the hypothalamus (34, 56). The SCN regulates the photoperiodic programming of the daily circadian clock and coordinates the clock machinery in peripheral tissues (57, 58). The cellular clock oscillates through core clock genes depending on the time of day. Through a transcriptional/translational feedback loop, the heterodimer BMAL1/CLOCK activates the heterodimer Per/Cry expression that suppresses the transcription of BMAL1 and CLOCK (59–61). Another short feedback loop involves the participation of BMAL1/CLOCK to activate the expression of REV-ERBα and RORα through binding to the nuclear orphan receptor (ROR) at the promoter site (34, 62).

The uterus and the placenta utilize circadian rhythm to carry out certain physiological functions, e.g., hormone release, parturition, and immune function, and entrain the fetus’ circadian rhythm (2, 63–66). Studies have shown that melatonin can synchronize the clock machinery in healthy and damaged cells to upregulate or downregulate specific clock genes to sustain optimal physiology in cells (34, 67, 68). Moreover, the placenta employs a circadian rhythm for the rhythmic release of melatonin. Melatonin is a ubiquitous molecule that conducts many functions. It is mainly known for its role as one of the circadian rhythm regulators, primarily synthesized in the pineal gland by pinealocytes under dark conditions (34, 69). During darkness, melatonin is rhythmically produced by the pineal gland—concentrations peak between the hours of 2 a.m. and 4 a.m (8).

Melatonin (N-acetyl-5-methoxytryptamine) is synthesized from serotonin (5-hydroxytryptamine) through a two-step reaction (Figure 2). In the first step, serotonin becomes acetylated by arylalkylamine N-acetyltransferase (AANAT) to become N-acetyl serotonin, the rate-limiting step. AANAT is the rate-limiting enzyme of melatonin because it controls the circadian rhythm of melatonin production via the pineal gland. The second step involves N-acetyl serotonin becoming methylated by acetyl serotonin o-methyltransferase (ASMT) to become melatonin (70–72). Given that serum melatonin concentrations are higher in pregnant than in non-pregnant women, studies have indicated that the primary source of this melatonin is the placenta (8–10). How the placenta produces melatonin without the need for the pineal gland or whether the circadian rhythm plays a role in this production has to be studied. The need for melatonin in the placenta is crucial for both the mother and the fetus because it depends on placental melatonin and maternal serum melatonin to provide photoperiodic information to control the internal rhythms of the fetus (34, 64). The placenta exposes maternal melatonin to the fetus at daily rhythmic intervals, with low concentrations during the day and high concentrations at night (8). Disrupting this rhythm during pregnancy may lead to detrimental outcomes for the mother and the growing fetus in utero and in adult life (8, 56, 61, 73). The development of the embryo, uterine implantation, placentation, and delivery may be regulated by the clock molecular machinery in the circadian rhythm (73). Circadian disruption or chronodisruption in pregnant women can lead to adverse outcomes in the offspring (61). Maternal chronodisruption is caused by mistimed eating, shift work, traveling across time zones, and immoderate artificial light exposure at nighttime (61). The impairment of the circadian rhythm during pregnancy compromises melatonin production, inhibiting the rhythm of melatonin release (74, 75). Chronodisruption may promote chronic illnesses in postnatal life, including diabetes, obesity, and cardiovascular disease (61).

Melatonin’s effects are mediated by two receptors, MT1 (Mel1a) and MT2 (Mel1b) (Figure 3) (76, 77). The MT1 receptor is approximately 350 amino acids long, while the MT2 receptor is 362 amino acids long (78). The molecular weight of both receptors is about 39–40 kDa (78). MT1 and MT2 are G-protein coupled receptors. G-protein coupled receptors are essential membrane proteins that form the fourth-largest superfamily in the human genome (79). The G-protein receptor has three subunits: α, β, and γ. When a ligand binds to the extracellular receptor, this initiates a signal transduction cascade that induces a conformational change, promoting the activation of the heterotrimeric GTP-binding protein (G-protein) (79). As the G-protein coupled receptor becomes activated, the α subunit disassociates from the β and γ subunits and releases guanine diphosphate (GDP). It binds to guanine triphosphate (GTP), which results in the conformational changes that later trigger the signal transduction of different G-proteins: inhibitory G-protein (G_i), stimulatory G-protein (G_s), and guanine-nucleotide binding protein (G_q) (79). Each of these proteins has different functions that activate distinct signaling pathways. The G_s protein stimulates the increase of cyclic AMP (cAMP) levels by activating adenylyl cyclase, while G_i inhibits cAMP levels (79). Moreover, G_q induces the expression of phospholipase C, which stimulates protein kinase C (PKC) and calcium (79). Consequently, the MT1 receptor homodimer and MT2 homodimer bind and activate the Gi–G protein and inhibits adenyl cyclase pathway signaling, decreasing forskolin-stimulated cAMP and protein kinase A signaling (60, 77, 80, 81). The MT1 and MT2 receptor heterodimer is associated with G_q, which enables the production of protein kinase C (PKC) and the increase of calcium associated with IP₃ (60, 77, 80, 81). MT1 and MT2 are widely distributed throughout the body and are present in almost all human cells (72, 82, 83). The primary responsibility of the MT1 receptor involves regulating the circadian cycle, while MT2 receptors control the body temperature through the peripheral tissue. In both the cytotrophoblast and the syncytiotrophoblast, studies have detected the presence of melatonin receptors in both primary isolated cells of the placenta and placental trophoblast cell lines (JEG-3 and BeWo) (34, 72, 82). The placenta constantly synthesizes melatonin receptors to promote the survival of the cytotrophoblast by decreasing free radicals and angiogenesis (34). Moreover, when disease (preeclampsia and preterm birth) is present in the placenta, melatonin receptor activity and melatonin levels are both decreased (34, 84). The binding of melatonin on MT1 homodimers and MT2 homodimers and the activation of the receptors indicate autocrine and paracrine activity needed for placental homeostasis and fetal development (34).

Pregnancy is associated with immunological changes that result in the fetus avoiding immune rejection by the mother. Increasing evidence associates immunological changes in pregnancy with melatonin and the circadian clock and synchronous programming of the immune system (34, 85). Melatonin possesses immunomodulatory properties (85) that use circadian regulation to stimulate cytokine production and lymphocyte proliferation and enhance phagocytosis (85–90). In both the luteal phase and early gestation, uterine NK cells represent most of the lymphocyte population. The decidual NK cells CD56^dim and CD56^bright comprise most of the leukocyte population in the decidua (91). Decidual cells activate thymocytes, macrophages, neutrophils, and NK cells (34, 92). At the same time, prostaglandins aid in uterine contractions and amplify TNF-α and IL-6 release (93). Alterations in NK cells, T regulatory cells (Tregs), Th17, and Th1/Th2 ratio may be associated with pregnancy-related complications, pregnancy loss, implantation failure, and preeclampsia (92). Melatonin affects the T cell subpopulation in gestation by increasing the differentiation of Th17 and Tregs (94, 95). Melatonin rhythm may be synchronized with Th1/Th2 cell populations in protecting and maintaining fetal survival (96).

Melatonin levels have a significant role in immunity during gestation by enhancing innate and cellular immunity. In the first trimester of pregnancy, macrophages are the second most abundant leukocyte population in the decidual layer of the placenta (33). They have the prominent role of sensing pathogens and immune effector cells, which suggests a central role in the inflammatory response of both placental and decidual infections (97). Melatonin activates the progenitor cell production of macrophages, NK cells, and granulocytes (98). Furthermore, melatonin promotes immune homeostasis during late and early pregnancy (34, 97). It can modify many transcription factors in macrophages, including hypoxia-inducible factor (HIF-1 α), nuclear factor-k-B binding (NF-kB), and interferon regulatory factors (IRFs) (99). In addition, melatonin inhibits cytokine expression in cells treated with lipopolysaccharide (LPS) (100).

Melatonin has a wide array of functions due to its ubiquitous nature. In reproductive physiology, melatonin exerts endocrine, autocrine, intracrine, and paracrine effects in the placenta (1, 8, 101). Melatonin can pass through the placental barrier and enter the fetal circulation to promote fetal growth and entrain the circadian rhythm of the fetus (34, 69). During pregnancy, melatonin levels increase throughout gestation and peak at term, promoting placental trophoblast cell survival and homeostasis and regulating hormonal production (102). Besides being a circadian-regulating hormone, melatonin is a powerful antioxidant and anti-inflammatory that can offset the effects of free radicals and scavenge ROS (103, 104). Melatonin can act directly on the free radicals or indirectly by stimulating antioxidant enzymes through its receptors (103, 105). Melatonin is both hydrophilic and lipophilic, which means that it can maneuver between cellular compartments easily and has access to ROS produced by the mitochondria (106, 107). In the presence of oxidative stress, melatonin can protect placental trophoblast cells against damage (71, 108). Melatonin decreases ROS levels by neutralizing and increasing the antioxidant enzymes’ (superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase) expression and activity (104, 109, 110). Moreover, melatonin can protect trophoblasts from apoptosis when oxidative stress occurs (108, 111), preventing pregnancy complications. In immunocompromised pregnancies that have rendered pregnancy complications, a supraphysiological dosage of melatonin has been shown to reverse the adverse effects of these complications for both the mother and the fetus (2, 34, 86).

Dysfunction of the placenta and oxidative stress contribute to unfavorable conditions in pregnancy, such as pre-eclampsia and preterm birth (2, 4, 24, 50, 112). Preterm birth is characterized as parturition that occurs before 37 weeks of gestation and is commonly associated with intrauterine infection and inflammation (2, 3, 113). A recent study found that melatonin treatment offsets placental inflammation in offspring exposed to LPS-induced maternal inflammation (84). Preeclampsia occurs after 20 weeks of pregnancy and is associated with hypertension and proteinuria, which induces oxidative stress in placental cells (3, 5, 114, 115). A study noted reduced serum melatonin levels and MT1 receptors in preeclamptic women (116). This reduction may have been due to the decreased placental secretion of melatonin induced by preeclampsia (116). Moreover, another study by Sagrillo-Fagundes and his group found that melatonin significantly regulated autophagy and inflammation in primary trophoblast cells that were exposed to a hypoxic/reoxygenation environment, mimicking preeclamptic pregnancy, thus reducing apoptosis in these cells. This study suggests that the placenta’s endogenous melatonin production is inhibited during preeclampsia, thus limiting melatonin in maternal blood and the placenta (111). Additionally, melatonin has been shown to reverse other complications during pregnancy and maintain a healthy pregnancy. In a study in mice, melatonin improved the efficiency of the placenta by increasing the expression of antioxidant enzymes in undernourished pregnancies (117). In a similar study, gestating sows were supplemented with melatonin, which indicated that melatonin could improve the redox status of the mother, fetus, and placenta and amplify the placental growth and function. This may be primarily due to melatonin’s ability to enhance the placenta’s antioxidant status and inflammatory response and activation (118). In all of these studies, melatonin had the potential benefit of acting as a preventative therapeutic agent for preterm birth, preeclampsia, and other pregnancy complications (84, 101, 116). In future studies, it will be essential to understand how melatonin uses its anti-inflammatory and antioxidant properties to target inflammation and oxidative stress in the placenta.

The induction of oxidative stress and inflammation at the maternal–fetal interface leads to activation of the NLRP3 inflammasome (119–121). The NLRP3 inflammasome complex is an intermediary in innate immune signaling that contributes to the pathogenesis of several diseases (122). Therefore, inhibition of the NLRP3 inflammasome pathway is a potential therapeutic target for inflammatory-related disorders (120, 122). The formation of the NLRP3 inflammasome is triggered by both internal and external factors, such as pathogen-associated molecular patterns (PAMPs), danger/damage-associated molecular patterns (DAMPs), and ROS (121, 123). An inducer of NLRP3 activation is the disassociation of thioredoxin-interacting protein (TXNIP), an intracellular redox regulator that inhibits antioxidants, from TRX (thioredoxin), a redox protein, via ROS or oxidative stress (122, 124). TXNIP and TRX form a complex with a redox relationship, which neutralizes the inhibitory properties of TXNIP, such as over-accumulation of ROS. After TXNIP disassociates and binds to NLRP3, it activates inflammasome (122, 124). The activation of the inflammasome complex entails two consecutive signals, namely (1): the priming signal is established by the activation of TLR receptors (PAMPs) through (for example) LPS, which leads to the secretion of inflammatory cytokines by the nuclear translocation of NF-kb (122, 123, 125, 126) and (2) the activation signal occurs due to the stimulation and binding of several factors that the complex recognizes, such as ROS and TXNIP/TRX disassociation, leading to the generation of caspase-1 (122, 123, 125, 126). Melatonin induces the inhibitory function of the NLRP3 inflammasome by either inhibiting or activating several different proteins and pathways (122). One of the several proteins and pathways melatonin uses to inhibit NLRP3 activation is NF-kb signaling, a master regulator of the priming phase of NLRP3 inflammasome activation (34). Melatonin also reduces TXNIP, which leads to the suppression of ROS and NLRP3 (122). Lastly, melatonin upregulates s Nrf2, an antioxidant protein that promotes ROS scavenging and elimination to facilitate protection against NLRP3 inflammasome activity through ROS scavenging and elimination mediated by Nrf2 (102).

Although there is no strong evidence currently to corroborate melatonin as a powerful antioxidant and anti-inflammatory in the placenta during oxidative stress, inflammation, and viral infection, future work should address whether melatonin’s antioxidant properties in the placenta are due to direct scavenging of ROS (supraphysiological) or receptor-mediated (physiological). Moreover, future work should address whether melatonin increases Nrf2 to deactivate the NLRP3 inflammasome during a viral infection at the maternal–fetal interface. Lastly, potential studies should determine safe and effective dosages that can be given to pregnant women during adverse pregnancy events like preeclampsia.