Melatonin supplementation attenuates cuproptosis and ferroptosis in aging cumulus and granulosa cells: potential for improving IVF outcomes in advanced maternal age

Abstract

Background: Advanced maternal age is associated with decreased oocyte quantity and quality and in vitro fertilization (IVF) success rates. This study aimed to investigate whether melatonin supplementation can improve IVF outcomes in women of advanced maternal age by modulating cuproptosis and ferroptosis.

Methods: This prospective cohort study included 161 women aged 35-45 years undergoing IVF-frozen embryo transfer cycles. Participants were assigned to either melatonin (n = 86, 2 mg daily for ≥ 8 weeks) or control (n = 75) groups. Cumulus cells were analyzed for cuproptosis and ferroptosis-related gene expression. Additional experiments were conducted on the HGL5 human granulosa cell line to assess mitochondrial function and metabolic reprogramming.

Results: Melatonin supplementation significantly improved IVF outcomes in women aged ≥ 38 years, increasing clinical pregnancy rates (46.0% vs. 20.3%, P < 0.01), ongoing pregnancy rates (36.5% vs. 15.3%, P < 0.01), and live birth rates (33.3% vs. 15.3%, P < 0.05). In cumulus cells from patients, gene expression analysis revealed that melatonin modulated cuproptosis and ferroptosis-related genes, including ATP7B and GPX4, with more pronounced effects in the ≥ 38 years group. This suggests melatonin enhances cellular resilience against oxidative stress and metal-induced toxicity in the ovarian microenvironment. In vitro studies using HGL5 cells showed melatonin reduced oxidative stress markers, improved mitochondrial function, restored expression of glycolysis and TCA cycle-related genes and modulated cuproptosis and ferroptosis-related gene expression. These findings provide mechanistic insight into melatonin's protective effects against regulated cell death in ovarian cells, potentially explaining the improved IVF outcomes observed.

Conclusions: Melatonin supplementation significantly improved IVF outcomes in women of advanced maternal age, particularly those ≥ 38 years old, likely by modulating cuproptosis and ferroptosis and enhancing mitochondrial function in cumulus and granulosa cells. These results suggest that melatonin could be a promising adjuvant therapy for improving IVF success rates in older women.

Keywords: Antioxidant therapy; Assisted reproduction; Mitochondrial function; Oocyte quality; Oxidative stress; Reproductive aging.

Fig. 1

Comparison of clinical pregnancy rates, ongoing pregnancy rates, and live birth rates between the melatonin and control groups. Data are presented for women aged ≥ 35 years (A) and women aged ≥ 38 years (B) undergoing IVF-FET cycles. Data were assessed using Chi-square test. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. CPR, clinical pregnancy rate; OPR, ongoing pregnancy rate; LBR, live birth rate; MR, miscarriage rate

Fig. 2

Expression levels of cuproptosis and ferroptosis-related genes in human cumulus cells following melatonin supplementation across different age groups. mRNA expression levels of genes associated with cuproptosis (A) and ferroptosis (B) were measured in human cumulus cells from participants aged ≥ 35 years and ≥ 38 years after melatonin supplementation. The bars represent fold changes in gene expression relative to the control group for each age group. Cuproptosis-related genes include ATP7B, SLC31A1, FDX1, DLD, and DLAT, while ferroptosis-related genes include TFRC, GPX4, NCOA4, SLC3A2, and SLC7A11. Data were assessed using a t-test, with significant changes observed between melatonin-treated and control groups. Genes such as ATP7B and GPX4 showed particularly pronounced age-dependent responses. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 3

Melatonin attenuated ROS-induced mitochondrial dysfunction in aging HGL5 cells. HGL5 cells at early (P59) and late (P113) passages were treated with melatonin (25 µM) for 24 h to assess its effects on mitochondrial function. Mitochondrial function was evaluated using flow cytometry to measure four key parameters: (A) oxidative stress, (B) mitochondrial ROS levels, (C) hydrogen peroxide levels, and (D) mitochondrial membrane potential. Data are presented as percentage (%). The graph demonstrates how melatonin treatment affects these parameters differently in early versus late passage cells, highlighting its potential age-dependent effects. Statistical significance was analyzed using one-way ANOVA followed by a post hoc Tukey test to determine significant differences between groups and is denoted as *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 4

Melatonin influenced metabolic reprogramming in aging HGL5 cells. This figure illustrates the effects of melatonin supplementation on key metabolic pathways in aging HGL5 cells. The diagram presents simplified representations of glycolysis and the tricarboxylic acid (TCA) cycle, with key enzymes and metabolites highlighted. Metabolic changes were quantified, and significant differences between groups were assessed using one-way ANOVA followed by a post hoc Tukey test to determine the impact of melatonin on these metabolic pathways. Statistical significance is denoted as *p < 0.05, **p < 0.01

Fig. 5

Melatonin increased mitochondrial oxygen consumption in aging HGL5 cells. This figure demonstrates the effects of melatonin on mitochondrial function in aging HGL5 cells under oxidative stress conditions. (A) The line graph shows real-time oxygen consumption rate (OCR) measurements using a Seahorse Bioscience analyzer. The arrows indicate the injection points of various compounds: oligomycin (1 µM), FCCP (1 µM), and antimycin A (0.5 µM) with rotenone (0.5 µM). (B) Bar graphs represent quantified OCR values at key stages of mitochondrial respiration: basal respiration, maximal respiration, ATP production, spare respiratory capacity, proton leak, and non-mitochondrial respiration. Statistical significance was assessed using one-way ANOVA followed by a post hoc Tukey test. (C) Western blot analysis was performed to evaluate the levels of oxidative phosphorylation complexes and the antioxidant proteins NRF2 and KEAP1. Statistical significance is indicated by ***p < 0.001

Fig. 6

Expression levels of cuproptosis and ferroptosis-related genes after melatonin treatment in aging HGL5 cells. This figure illustrates the impact of melatonin treatment on the expression of genes involved in cuproptosis and ferroptosis pathways in aging HGL5 cells (passage 113). (A) Bar graphs show the relative mRNA expression levels of cuproptosis-related genes (ATP7b, SLC31A1, FDX1, DLD, DLAT) in control, H₂O₂-treated, and melatonin + H₂O₂-treated groups. (B) Similar representation for ferroptosis-related genes (TFRC, GPX4, NCOA4, SLC3A2, SLC7A11). mRNA levels were quantified using RT-qPCR analysis, with RNU6-1 as the reference gene. Statistical significance was assessed using one-way ANOVA followed by a post hoc Tukey test to determine differences between groups. Significance levels are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 7

Schematic representation of melatonin-mediated reprogramming of energy metabolism in cumulus cells and granulosa cells. This diagram illustrates the key pathways and molecular mechanisms through which melatonin influences energy metabolism in cumulus cells and granulosa cells to mitigate cuproptosis and ferroptosis. It highlights how melatonin exerts protective effects on germ cell health and functionality by modulating metabolic pathways and reducing oxidative stress