In this study, we measured melatonin levels in basal serum, hCG day serum, and follicular fluid among patients with DOR-Path, DOR-Phy, and NOR. The results indicated that, in comparison with the NOR group, the DOR group exhibited significantly lower levels of melatonin in both basal serum and follicular fluid. Subsequently, the study analyzed the correlation between these melatonin levels and clinical parameters as well as IVF outcomes. It was discovered that melatonin levels in serum and follicular fluid were strongly associated with age, bFSH, AFC, and AMH—established predictors of ovarian reserve. Furthermore, melatonin levels played a pivotal role in IVF outcomes; patients with higher melatonin levels retrieved more oocytes. The number of 2PN, cleaved zygotes, available embryos on day 3, and good-quality embryos on day 3 significantly increased with melatonin levels from the DOR group to the NOR group. Consequently, melatonin levels in serum and follicular fluid may serve as reliable predictors of ovarian reserve and IVF outcomes.
Ovarian reserve, correlating with both the quantity and quality of oocytes, signifies a woman's reproductive potential and, to some extent, mirrors IVF treatment outcomes. Despite the highly variable and challenging-to-predict outcomes of IVF treatment, ovarian reserve assessment is now a routine procedure for women undergoing IVF treatment cycles. The pre-treatment evaluation of ovarian reserve enables individuals to anticipate ovarian responsiveness and tailor treatment regimens to optimize IVF success. AMH, AFC, and bFSH are now acknowledged as reliable serum markers of ovarian reserve [24]. Nevertheless, AMH and AFC are predominantly utilized for predicting the number of oocytes and are not indicative of embryo quality; their predictive efficacy is too restricted for clinical significance [25]. Elevated bFSH levels reflect a quantitative rather than qual-itative decline in ovarian reserve [26]. Regrettably, there are no ovarian reserve markers that precisely predict the quality of oocytes or embryos.
Research has demonstrated significantly higher levels of ROS in the follicular fluid of patients with DOR compared to relatively healthy women. Excessive ROS further compromises oocyte quality by disrupting the follicular microenvironment [27]. Despite the critical role of ROS in cellular signal transduction and internal equilibrium, an excess of ROS can induce changes in genetic material, signaling pathways, transcription factors, and the ovarian microenvironment. This process accelerates the aging of oocytes and ovarian granulosa cells [7]. Melatonin, functioning as a broad-spectrum antioxidant and a potent free radical scavenger, can collaboratively counteract excessive intracellular ROS by initiating a cascade reaction and regulating the transcription of antioxidant enzyme genes [28, 29]. Evidence suggests that melatonin enhances ovarian function in infertile patients through various roles, including antioxidant, anti-apoptotic, and endocrine modulation [30, 31].
Consistent with the research findings of Tamura et al. [32], the concentration of melatonin in the follicular fluid of relatively healthy women is higher than that in their peripheral blood. This occurrence might be attributed to the capability of granulosa cells and oocytes in follicles to absorb melatonin from peripheral blood [33]. Therefore, we posit that melatonin, acting as the primary antioxidant in follicular fluid, could shield oocytes from oxidative damage by neutralizing ROS, thereby positively in-fluencing oocyte quality. Nevertheless, in the DOR-Path and DOR-Phy groups, melatonin concentrations in peripheral blood (basal serum, hCG day serum) and follicular fluid were lower than those in the NOR group. Notably, there were no significant differences in melatonin concentrations in peripheral blood and follicular fluid between the DOR-Path and DOR-Phy groups. Consequently, the elevated ROS in the follicle induced by age or pathological conditions cannot be offset by high melatonin concentrations, ultimately impacting oocyte quality [34–36].
As widely recognized, the quality of oocytes plays a pivotal role in influencing IVF outcomes. In this study, we observed a positive correlation between melatonin levels in base serum, hCG day, and follicular fluid, and IVF outcomes. Individuals with elevated melatonin levels exhibit heightened production of oocytes, 2PN-fertilized oocytes, zygotes cleaved, and D3 good-quality embryos. Importantly, this correlation experiences a substantial increase from the DOR group to the NOR group. This finding aligns with the results reported in the study conducted by Tong et al. on melatonin levels in follicular fluid [18].
When analyzing the correlation between melatonin levels and patients' clinical characteristics, a good correlation emerged between melatonin levels in both serum and follicular fluid and current clinical indicators of ovarian reserve function, including age, bFSH, AFC, and AMH. The amount of melatonin produced by human pineal gland di-minishes with advancing age [37]. Melatonin production by the human pineal gland di-minishes with age. Several studies suggest that this decline may be attributed to increased calcium deposition in the pineal gland, a reduction in pineal N-acetyltransferase, or a gradual alteration in the concentration of melatonin α1 receptors in the hypothalamus [38–40]. According to our findings, this age-related decline in melatonin, the same trend was found in serum and follicular fluid from the NOR group to the DOR-phy group. In mouse experiments, melatonin deficiency intensifies follicle activation and atresia, hastening the age-related decline in fertility. Conversely, the presence of melatonin in the body hinders follicle activation, growth, and atresia via the PI3K-AKT pathway, consequently retarding ovarian aging [41]. Additionally, our findings revealed a negative correlation between melatonin levels in serum and follicular fluid and bFSH levels, aligning with prior research [18, 42]. Moreover, our results demonstrated a significant and positive correlation between melatonin levels in both serum and follicular fluid with AFC and AMH, in agreement with the findings reported by Min et al. [43]. Hence, melatonin levels in both serum and follicular fluid emerge as biochemical markers strongly correlated with age, bFSH, AFC, AMH, oocyte number, and embryo quality. These markers exhibit promising potential as excellent predictive factors for both ovarian reserve and IVF outcomes.
In contrast to alternative markers of ovarian reserve, melatonin is a stable endoge-nously produced compound that can also be acquired through in vitro uptake. Research indicates that melatonin plays a pivotal role in mitigating oxidative stress and preventing oocyte apoptosis, while concurrently enhancing mitochondrial function. These effects contribute to the improvement of oocyte quality and enhance pregnancy outcomes in infertile patients [44–46]. In a study conducted by Song et al., ovarian senescence was significantly reduced in mice following the administration of melatonin for 6–12 months. This was substantiated by melatonin's ability to inhibit age-related declines in follicle number, litter size, and blastocyst rate [47]. Moreover, Bao et al. demonstrated that the addition of melatonin to the culture medium increased the percentage of high-quality Day 3 embryos in patients with recurrently poor-quality embryos. Additionally, it enhanced the blastocyst rate of vitrified-thawed cleavage embryos [48]. Therefore, the efficacy of melatonin supplementation in both embryo cultures and oocyte maturation medium for enhancing embryo quality in patients with DOR could be substantiated through additional clinical trials.
In addition, it is crucial to acknowledge the limitations of our study. Notably, we refrained from analyzing the implantation and pregnancy rates. This decision was in-fluenced by the cancellation of fresh embryo transfers in certain patients with DOR due to factors such as thin endometrium or inadequate embryo availability.