Melatonin dampens fetal membrane inflammation linked to preterm birth

A new ex vivo study reveals how melatonin may calm infection-triggered fetal membrane inflammation, offering fresh clues for future strategies against inflammation-related preterm birth.

Study: Melatonin modulates inflammation in human fetal membranes: an ex vivo approach. Image Credit: Pikovit / Shutterstock

In a recent study published in the journal Scientific Reports, a group of researchers evaluated whether melatonin reduces lipopolysaccharide-induced inflammation in human fetal membranes in an ex vivo model and determined whether the TLR4/MyD88/NF-κB signaling pathway is involved in this effect.

Background

Preterm birth is one of the leading causes of infant mortality and morbidity. Infection-driven inflammation of the fetal membranes is a major trigger of early labor because it stimulates the release of inflammatory molecules that weaken these protective tissues. The pineal gland and placenta produce melatonin, which regulates circadian rhythms, including sleep-wake timing, and inhibits inflammatory responses. During pregnancy, melatonin production increases significantly and may influence the developing fetal environment. To determine how melatonin affects the regulation of inflammation in human fetal membranes, further research is needed.

About the study

In the study, 12 healthy women who had uncomplicated term cesarean deliveries without any prior labor were selected, and fetal membranes were taken from each. The women did not have diabetes mellitus or high blood pressure (hypertension), obesity, or other metabolic diseases during pregnancy or a urinary or vaginal infection during the third trimester of their pregnancy. Fetal tissue samples were cultured in a two-chambered culture system that preserves the natural separation of the maternal-facing choriodecidual region and fetal-facing amniotic cavity. Each side of the fetal membrane was treated independently. Assays were performed in independent experimental subsets of these samples.

Before LPS exposure, researchers treated the compartments with melatonin at a physiological amniotic concentration (1 nM) for 24 hours, then dosed fresh melatonin every 6 hours due to its short half-life. To reproduce inflammation associated with bacterial infection, the choriodecidual side was then exposed to lipopolysaccharide (LPS; 500 ng/mL), a bacterial endotoxin, while melatonin treatment continued. In addition to LPS treatment, some samples were co-treated with luzindole, a non-selective melatonin receptor antagonist, to determine whether melatonin receptors were responsible for the observed effects.

Following treatment, researchers analyzed cytokine and chemokine levels using ELISA, including TNF-α, IL-1β, IL-6, IL-10, CCL2, CCL3, and CCL5. Additionally, researchers used quantitative polymerase chain reaction (qPCR) to measure gene expression, and ELISA and Western blotting to analyze TLR4, MyD88, and NF-κB proteins.

Study results

Exposure to LPS produced a strong inflammatory response in both the choriodecidual and amniotic regions of the fetal membranes. It increased the secretion of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, as well as the anti-inflammatory cytokine IL-10. Melatonin significantly reduced the secretion of all four cytokines, with pronounced reductions in TNF-α and IL-1β, indicating that it attenuates key early inflammatory signals in this ex vivo model. When tissues were treated with luzindole, many of melatonin’s anti-inflammatory effects were diminished, suggesting that these protective actions were at least partly mediated through melatonin receptors. The regulation of IL-6 differed between the choriodecidual and amniotic compartments, suggesting that IL-6 may also be regulated by mechanisms independent of melatonin receptors.

The expression of messenger ribonucleic acid (mRNA) for TNF-α and IL-1β genes increased significantly when tissues were treated with LPS, but was suppressed by melatonin and restored by luzindole. IL-6 was also increased in response to LPS, while melatonin did not have a significant effect on IL-6 mRNA, and IL-10 gene expression was not measured.

LPS stimulation increased the levels of CCL5, CCL2, and CCL3, chemokines involved in immune-cell recruitment during infection-associated inflammation. Melatonin decreased the production of CCL5 and CCL3 in fetal membrane compartments, with effects that varied by compartment. In contrast, there was no statistically significant effect of melatonin on CCL2 chemokine production. CCL5 mRNA expression increased, and both CCL2 and CCL3 showed a trend towards increased expression after LPS addition. Melatonin did not alter the expression of any of these three chemokines at the transcriptional level, suggesting that the observed secretion-level changes may involve post-transcriptional mechanisms, pre-formed chemokine release, or indirect effects of altered cytokine signaling rather than direct changes in chemokine mRNA expression.

Further analyses showed that LPS increased TLR4, MyD88, and NF-κB, key components of the inflammatory signaling pathway. Melatonin significantly reduced the levels of these proteins, whereas luzindole largely reversed these changes. Together, these findings indicate that melatonin suppresses infection-induced inflammation in human fetal membranes by limiting the production of inflammatory cytokines, reducing selected chemokines, and modulating the TLR4/MyD88/NF-κB signaling pathway.

Conclusion

The findings show that melatonin exerts significant anti-inflammatory effects in human fetal membranes exposed to LPS-induced inflammation. It reduced inflammation-associated readouts by lowering cytokine secretion, selected cytokine mRNA expression, and proteins involved in the TLR4/MyD88/NF-κB signaling pathway. These effects were partly mediated through melatonin receptors, although some responses appeared to involve additional mechanisms.

Overall, the results support the potential role of melatonin-related pathways in limiting infection-associated membrane inflammation and provide a foundation for future studies testing whether these pathways can be safely and effectively targeted in inflammation-related preterm birth. Because this was an ex vivo study using term, non-labor fetal membranes, the findings do not yet show that melatonin supplementation can prevent preterm birth in pregnant patients.

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