Melatonin levels and embryo quality in IVF patients with diminished ovarian reserve: a comparative study.

Eighty-five patients were recruited for this study and stratified into three groups based on age and ovarian reserve status: two experimental groups (DOR-Path, n  = 25 and DOR-Phy, n  = 33) and a control group (NOR, n  = 27). Detailed baseline characteristics are presented in Table  1 . Parameters such as average age, BMI, duration of infertility, serum hormone levels and infertility factors were compared among the groups. The results showed that, as expected, significant differences emerged between the DOR-Path and NOR groups in bFSH, AFC, AMH. However, no significant differences were noted in age, BMI, infertility duration, bLH, bE2, and P. Similarly, comparisons between the DOR-Phy and NOR groups highlighted significant differences in age, bFSH, AFC and AMH. Conversely, BMI, infertility duration, bLH, bE2, and P showed no significant differences. In the comparison between DOR-Path and DOR-Phy groups, significant differences were observed in age, bFSH, and AMH. However, no significant differences were found in BMI, infertility duration, bLH, bE2, P and AFC. The findings illustrate substantial variations in negative predictors (age, bFSH) and positive predictors (AFC, AMH) linked to physiological ovarian reserve hypoplasia in comparison to the NOR group. Likewise, considerable differences in negative predictors (bFSH) and positive predictors (AFC, AMH) were observed concerning pathological ovarian reserve hypoplasia. Table 1 The baseline characteristics of patients with NOR, DOR-Path and DOR-Phy Variables NOR ( n  = 27) DOR-Path ( n  = 25) DOR-Phy ( n  = 33) P -value Age (years) 29.85 ± 3.23 a 30.68 ± 2.81 a 42.85 ± 1.67 b < 0.001 BMI (kg/m 2 ) 21.92 ± 2.61 21.99 ± 1.91 22.48 ± 2.21 NS Infertility Duration (years) 3.33 ± 2.17 3.32 ± 2.21 5.12 ± 4.55 NS Primary infertility (%) 40.74% (11/27) 44.00% (11/25) 30.3% (10/33) NS Indications Tubal Factor (%) 55.56% (15/27) 40.00% (10/25) 36.36% (12/33) - Male Factor (%) 18.52% (5/27) 24.00% (6/25) 39.39% (13/33) - Mixed Factor (%) 25.92 (7/27) 36.00% (9/25) 24.25% (8/33) - bFSH (U/L) 7.11 ± 2.39 a 24.06 ± 3.35 b 14.41 ± 3.59 c < 0.001 bLH (U/L) 5.23 ± 2.25 6.39 ± 3.84 5.93 ± 2.75 NS bE 2 (pg/ml) 37.22 ± 16.98 35.40 ± 13.54 38.58 ± 28.47 NS P (ng/ml) 0.23 ± 0.10 0.25 ± 0.16 0.29 ± 0.23 NS AFC (n) 16.04 ± 4.96 b 4.48 ± 1.09 a 4.15 ± 1.54 a < 0.001 AMH (ng/mL) 4.10 ± 1.88 c 0.56 ± 0.32 a 0.63 ± 0.40 b < 0.001 DOR-Path, pathological diminished ovarian reserve; DOR, physiological diminished ovarian reserve; NOR, Normal ovarian reserve; BMI, Body mass index; Primary infertility : Refers to women who have never achieved pregnancy; Tubal Factor : Denotes patients who have issues with their fallopian tubes; Male Factor : Involves the spouses of patients who have infertility-related factors; Mixed Factor : Indicates that both male and female partners have infertility factors; bFSH, basic follicle-stimulating hormone; bLH, basic luteinizing hormone; bE2, basic estrogen; P, progesterone; AFC, antral follicle count; AMH, anti-Mullerian hormone; Different letters represent significant differences between groups The baseline characteristics of patients with NOR, DOR-Path and DOR-Phy DOR-Path, pathological diminished ovarian reserve; DOR, physiological diminished ovarian reserve; NOR, Normal ovarian reserve; BMI, Body mass index; Primary infertility : Refers to women who have never achieved pregnancy; Tubal Factor : Denotes patients who have issues with their fallopian tubes; Male Factor : Involves the spouses of patients who have infertility-related factors; Mixed Factor : Indicates that both male and female partners have infertility factors; bFSH, basic follicle-stimulating hormone; bLH, basic luteinizing hormone; bE2, basic estrogen; P, progesterone; AFC, antral follicle count; AMH, anti-Mullerian hormone; Different letters represent significant differences between groups To comprehensively evaluate melatonin level variations among patients with DOR-Path, DOR-Phy, and NOR, we conducted ELISA assays on samples of patient serum (base serum and hCG day serum) and follicular fluid collected on the day of oocyte retrieval. Figure  1 illustrates the outcomes. Analysis of melatonin levels in base serum re-vealed a significant decrease ( P  < 0.05) in both the DOR-Path and DOR-Phy groups com-pared to the NOR group. However, no notable difference was observed between the DOR-Path and DOR-Phy groups. The values were 183.26 ± 43.90 (pg/mL) for the NOR group, 149.63 ± 43.78 (pg/mL) for the DOR-Path group, and 149.30 ± 37.88 (pg/mL) for the DOR-Phy group (Fig.  1 a). Examination of melatonin levels in hCG day serum indicated no significant differences among patients in the DOR-Path, DOR-Phy, and NOR groups (Fig.  1 b). Conversely, the analysis of melatonin levels in follicular fluid showcased the lowest levels in the DOR-Path group and the highest in the NOR group. Significant differences were evident between both the DOR-Path (145.43 ± 39.30 pg/mL) and DOR-Phy groups (162.30 ± 59.66 pg/mL) when compared with the NOR group (236.84 ± 55.26 pg/mL) ( P  < 0.001) (Fig.  1 c). Subsequently, we conducted an analysis of melatonin levels in volunteers categorized into distinct groups. Comparisons of melatonin levels in serum and follicular fluid at different stages indicated the highest levels in follicular fluid and the lowest in hCG day serum (Fig.  1 d–f). Noteworthy differences in melatonin levels were observed within the NOR group: significant disparities between base day serum and follicular fluid ( P  < 0.001), as well as between hCG day serum and follicular fluid ( P  < 0.001) (Fig.  1 d). However, no significant difference emerged in melatonin levels between serum on the base day and hCG day. The values were 183.26 ± 43.90 (pg/mL) for the base serum, 164.03 ± 55.78 (pg/mL) for the hCG day serum, and 236.84 ± 55.26 (pg/mL) for the follicular fluid (Fig.  1 d). In contrast, the melatonin levels within each sample in the DOR-Path and DOR-Phy groups exhibited no significant differences between them (Fig.  1 e, f). These findings indicate distinct melatonin level variations in the base serum and follicular fluid among patients with DOR-Path, DOR-Phy, and NOR. Particularly note-worthy are significantly lower melatonin levels in the DOR-Path and DOR-Phy groups compared to the NOR group in follicular fluid. Fig. 1 Melatonin Levels in NOR Group, DOR-Path Group, and DOR-Phy Group. ( a ) Compared with the control subjects ( n  = 27), base serum melatonin levels were reduced in patients with DOR-Path ( n  = 25) and DOR-Phy ( n  = 33); ( b ) There were no significant differences in serum melato-nin concentrations on hCG day among NOR Group, DOR-Path, and DOR-Phy patients; ( c ) Com-pared with the NOR Group, Melatonin levels in the follicular fluid were reduced in patients with DOR-Path and DOR; ( d ) The melatonin levels of different specimens in NOR Group; ( e ) The melatinin levels of different specimens in DOR-Path patients; ( f ) The melatonin levels of different specimens in elderly DOR-Phy patients; When the P  < 0.05, it indicates statistical significance Melatonin Levels in NOR Group, DOR-Path Group, and DOR-Phy Group. ( a ) Compared with the control subjects ( n  = 27), base serum melatonin levels were reduced in patients with DOR-Path ( n  = 25) and DOR-Phy ( n  = 33); ( b ) There were no significant differences in serum melato-nin concentrations on hCG day among NOR Group, DOR-Path, and DOR-Phy patients; ( c ) Com-pared with the NOR Group, Melatonin levels in the follicular fluid were reduced in patients with DOR-Path and DOR; ( d ) The melatonin levels of different specimens in NOR Group; ( e ) The melatinin levels of different specimens in DOR-Path patients; ( f ) The melatonin levels of different specimens in elderly DOR-Phy patients; When the P  < 0.05, it indicates statistical significance Upon comparing IVF outcomes among the three groups, we noted that patients in the DOR-Path and DOR-Phy cohorts with low melatonin levels experienced inferior IVF outcomes. As illustrated in Table  2 , there were significant disparities between the DOR-Path and NOR groups in variables such as the number of oocytes, oocytes/AFC, 2PN embryos, cleavage, day 3 usable embryos, and day 3 good quality embryos ( P  < 0.001). Similarly, the DOR-Phy group demonstrated comparable outcomes to the NOR group ( P  < 0.001). However, no statistically significant difference was found between the DOR-Path and DOR-Phy groups. Table 2 Comparisons of IVF outcomes among patients with NOR, DOR-Path and DOR-Phy Variables NOR ( n  = 27) DOR-Path ( n  = 25) DOR-Phy ( n  = 33) P -value No. of oocytes (n) 15.26 ± 5.40 b 3.36 ± 1.30 a 3.00 ± 1.75 a < 0.001 No. of oocytes/AFC 0.94 ± 0.09 b 0.75 ± 0.22 a 0.67 ± 0.24 a < 0.001 No. 2PN (n) 10.04 ± 3.36 b 2.52 ± 1.56 a 1.91 ± 1.59 a < 0.001 No. of cleavage (n) 8.74 ± 3.12 b 2.36 ± 1.38 a 1.91 ± 1.59 a < 0.001 No. of day 3 useable embryos (n) 5.63 ± 4.13 b 1.48 ± 1.33 a 1.03 ± 0.85 a < 0.001 No. of day 3 good quality embryos (n) 3.93 ± 3.13 b 0.48 ± 0.92 a 0.42 ± 0.71 a < 0.001 No. of oocytes, The number of oocytes retrieved from the ovaries; No. of oocytes/AFC, Ratio of oocyte yield to antral follicle count; No. 2PN, the number of zygotes that have two pronuclei; No. of cleavage, the number of embryos that have undergone cleavage; No. of day 3 useable embryos, the number of embryos that have developed to a stage by day 3 post-fertilization where they are considered viable for transfer back into the uterus or for cryopreservation; No. of day 3 good quality embryos, the number of embryos that not only are useable by day 3 but also meet certain quality criteria; Different letters represent significant differences between groups Comparisons of IVF outcomes among patients with NOR, DOR-Path and DOR-Phy No. of oocytes, The number of oocytes retrieved from the ovaries; No. of oocytes/AFC, Ratio of oocyte yield to antral follicle count; No. 2PN, the number of zygotes that have two pronuclei; No. of cleavage, the number of embryos that have undergone cleavage; No. of day 3 useable embryos, the number of embryos that have developed to a stage by day 3 post-fertilization where they are considered viable for transfer back into the uterus or for cryopreservation; No. of day 3 good quality embryos, the number of embryos that not only are useable by day 3 but also meet certain quality criteria; Different letters represent significant differences between groups To delve deeper into the predictive role of melatonin in serum and follicular fluid regarding IVF outcomes, we examined the correlation between melatonin levels in serum and follicular fluid and the parameters of IVF outcomes. Our observations revealed a significant positive correlation between melatonin levels in serum and follicular fluid and the parameters of IVF outcomes (Fig.  2 ). The most robust correlations in follicular fluid melatonin levels with IVF outcome parameters (Fig.  2 k-o) include the number of oocytes (r s =0.506, P  < 0.001), cleavage (r s =0.552, P  < 0.001), 2PN (r s =0.551, P  < 0.001), day 3 usable embryos (r s  = 0.483, P  < 0.001), and day 3 good quality embryos (r s =0.586, P  < 0.001). A relatively strong correlation was observed between melatonin levels in base serum and IVF outcome parameters (Fig.  2 a-e), including the number of oocytes (r s =0.291, P  = 0.007), cleavage (r s =0.338, P  = 0.002), 2PN (r s =0.329, P  = 0.002), day 3 usable embryos (r s =0.276, P  = 0.010), and day 3 good quality embryos (r s =0.356, P  = 0.001). Additionally, melatonin levels in hCG day serum exhibited a less pronounced correlation with IVF outcomes, but they remained significantly different ( P  < 0.05) (Fig.  2 f-j), involving the number of oocytes (r s =0.232, P  = 0.033), cleavage (r s =0.273, P  = 0.012), 2PN (r s =0.258, P  = 0.017), day 3 usable embryos (r s =0.241, P  = 0.027), and day 3 good quality embryos (r s =0.316, P  = 0.003). Based on the aforementioned results, melatonin levels in base serum, hCG day serum, and follicular fluid exhibit a significant positive correlation with IVF outcomes. Fig. 2 Correlation between the melatonin levels in base serum and follicular fluid and IVF out-comes ( n  = 85). ( a-e ) Correlation between IVF outcomes and melatonin levels in base Serum; ( f-j ) Correlation between IVF outcomes and melatonin levels in hCG Day Serum; ( k-o ) Correlation between IVF outcomes and melatonin levels in follicular fluid; When the P  < 0.05, it indicates statistical significance Correlation between the melatonin levels in base serum and follicular fluid and IVF out-comes ( n  = 85). ( a-e ) Correlation between IVF outcomes and melatonin levels in base Serum; ( f-j ) Correlation between IVF outcomes and melatonin levels in hCG Day Serum; ( k-o ) Correlation between IVF outcomes and melatonin levels in follicular fluid; When the P  < 0.05, it indicates statistical significance

In conclusion, our study demonstrates that melatonin in serum and follicular fluid, as a highly correlated indicator of ovarian reserve, oocyte number, and embryo quality, has great potential to be a predictive marker of ovarian reserve and IVF outcome. However, further large-scale prospective studies are needed to validate our results.

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 (Table S1 ). 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. 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 [ 26 ]. 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 [ 11 , 27 ]. Evidence suggests that melatonin enhances ovarian function in infertile patients through various roles, including antioxidant, anti-apoptotic, and endocrine modulation [ 14 , 28 ]. Consistent with the research findings of Tamura et al. [ 15 ], 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 [ 29 ]. 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 [ 4 , 30 , 31 ]. 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 [ 19 ]. 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 [ 32 ]. 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 [ 33 – 35 ]. 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 [ 36 ]. Additionally, our findings revealed a negative correlation between melatonin levels in serum and follicular fluid and bFSH levels, aligning with prior research [ 18 , 37 ]. 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. [ 38 ]. 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 [ 39 – 41 ]. 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 [ 42 ]. 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 [ 43 ]. 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.

The escalating rates of reproductive infertility in both sexes have intensified as a consequential global public health issue [ 1 , 2 ]. Epidemiological studies report a substantial 50% surge in infertility prevalence over the past six decades [ 3 ]. Remarkably, ap-proximately 35% of cases involving couples unable to conceive are linked to ovarian failure [ 4 ]. While ovarian function naturally wanes around the age of 40, recent clinical data underscore a rising trend in diminished ovarian reserve occurring at earlier stages [ 5 ]. This divergence from the expected decline in ovarian function within the same age group categorizes ovarian aging into two distinct types: physiological diminished ovarian reserve correlated with advanced age (DOR-Phy) and pathological diminished ovarian reserve unrelated to age (DOR-Path) [ 6 , 7 ]. Contemporary research throughout this year has elucidated melatonin’s pivotal role in preserving the physiological and molecular facets of both normal and aging ovaries [ 8 ]. Melatonin, chemically known as N-acetyl-5-methoxytryptamine, is an indoleamine syn-thesized by diverse cells and rhythmically released by the pineal gland [ 9 ]. Its secretion follows a distinct circadian rhythm, predominantly occurring at night and regulated by the suprachiasmatic nucleus of the hypothalamus, underscoring its integral role in the circadian system [ 10 ]. Engaging in multifaceted physiological functions, melatonin acts as a potent antioxidant, manifesting protective effects against oxidative stress and in-flammation [ 11 , 12 ]. Additionally, it operates as an immune-active agent and mitochon-drial modulator, contributing to immune modulation, cardiovascular regulation, neuro-protection, and more [ 13 ]. Melatonin’s presence within ovarian follicular fluid and oocytes acts as a shield against oxidative damage and confers supplementary benefits by enhancing oocyte maturation, fertilization, and embryo development [ 4 ]. Demonstrating the capability to impede ovarian aging, melatonin regulates ovarian biological rhythms, fosters follicle formation, and augments oocyte quality and fertilization rates [ 14 ]. Furthermore, melatonin significantly facilitates oocyte maturation and embryo development by regulating the hypothalamic-pituitary axis and acting directly as an antioxidant. In clinical treatments for infertile women, melatonin has shown potential in reducing oxidative damage and improving fertilization rates [ 15 ]. Research has indicated that the combined use of melatonin and metformin can effectively restore infertility symptoms in polycystic ovary syndrome mice, including morphological changes in the ovaries and uterus, as well as improvements in hormone levels [ 16 ]. Additionally, studies by Feng et al. using animal models have found that melatonin can effectively prevent ovarian insufficiency induced by chemotherapy, thereby protecting ovarian reserve and fertility [ 17 ]. This evidence unequivocally underscores melatonin’s pivotal role in the context of ovarian aging. Understanding that follicular fluid, as the microenvironment for oocytes, profoundly influences oocyte development and developmental potential [ 18 ], recent re-search has proposed melatonin levels in follicular fluid as markers for in vitro fertilization (IVF) outcomes and predictors of ovarian reserve [ 19 ]. Nonetheless, the elucidation of melatonin levels in the follicular fluid and serum of patients with various types of diminished ovarian reserve remains unresolved. Therefore, this study endeavors to scrutinize melatonin levels in patients with DOR-Path, DOR-Phy, and NOR, aiming to delineate their impact on oocyte parameters and embryo quality. The resultant findings will significantly contribute to a nuanced comprehension of the role of melatonin in premature ovarian failure.

This investigation enrolled 85 female volunteers who underwent IVF/ICSI treatment at the Reproductive Center of Luohu District People’s Hospital in Shenzhen between September 2022 and August 2023. The recruited volunteers were divided into three groups based on their ovarian re-serve and age: (1) patients with normal ovarian reserve (NOR group, denoted as the control group). Inclusion criteria were as follows: age ≤ 35, anti-Müllerian hormone (AMH) ≥ 1.1 ng/ml, antral follicle count (AFC) > 10, and requiring assisted reproductive treatment due to male factors. (2) Pathological diminished ovarian reserve (DOR-Path group). Inclusion criteria were: age ≤ 35, AMH < 1.1 ng/ml, and AFC  40, AMH < 1.1 ng/ml, and AFC < 6 [ 20 ]. Exclusion criteria for all recruited volunteers included autoimmune diseases, chromosomal abnormalities in both partners, acute infectious diseases, reproductive system tumors, ovarian endometriomas, adenomyosis, hyperprolactinemia, pelvic inflammatory disease, and related disorders. The protocol for ovarian stimulation (OS) was determined individually according to standard practice and the patient’s characteristics, including age, BMI, basal folli-cle-stimulating hormone (FSH), and antral follicle count (AFC), anti-Müllerian hormone (AMH), basal luteinizing hormone (LH), and basal estradiol (E2). Most patients were treated with a long Gonadotropin-releasing hormone (GnRH) agonist or a GnRH an-tagonist protocol [ 21 ]. For women with diminished ovarian reserves, the mild ovulation protocol [ 22 ] or luteal phase ovarian [ 23 ] stimulation was attempted. In these protocols, 4000–10,000 IU human chorionic gonadotropin (hCG) was administered when more than 60% of follicles were > 16 mm in diameter. Transvaginal ultrasound-guided oocyte re-trieval was performed 36–37 h after hCG injection, followed by IVF or ICSI based on sperm parameters. Embryos were scored according to the morphology assessment de-scribed by the Istanbul Consensus [ 24 ]. Serum samples from the volunteers were collected on two specific occasions: the third day of menstruation before the IVF-ET cycle (referred to as base serum) and on the day of human chorionic gonadotropin (hCG) injection (referred to as hCG day serum). Following blood extraction from the patients’ veins, it underwent centrifugation at 3500 g for 10 min. The resulting supernatant was gathered and stored at -20 °C for subsequent experiments. The follicular fluid was collected concurrently with oocyte retrieval [ 25 ]. Briefly, transvaginal ultrasound-guided oocyte retrieval was conducted 36–37 h following hCG injection, coinciding with the collection of follicular fluid. Subsequently, the gathered follicular fluid underwent centrifugation at 4 °C and 1000 g for 10 min. The resultant supernatant was then collected and stored at -80 °C for subsequent experiments. Melatonin levels were quantified using an MT Enzyme-Linked Immunosorbent Assay (HM10652, Bioswamp, Wuhan, China) following the manufacturer’s guidelines. Initially, 50 µL/well of standard substances at various concentrations (0, 25, 50, 100, 200, and 400 pg/mL) or 40 µL/well of serum and follicular fluid, along with 10 µL/well of bi-otin-labeled anti-melatonin antibody, were added to 96-well plates. Subsequent steps involved adding 50 µL/well of the enzyme label reagent, followed by a 30-minute incu-bation at room temperature. Post-incubation, the plates underwent five washes with a wash buffer and were developed with 100 µL of reagent. The reaction was terminated using a stop solution, and absorbance readings were taken at 450 nm. The concentration of MT in each sample was determined based on standard substance calibration. Each treatment was analyzed in triplicate and repeated independently at least three times. Data analysis was conducted using SPSS (Statistical Package for Social Sciences IBM Corporation, Armonk, NY, USA) version 24.0. Quantitative datas were presented as mean ± standard deviation ± s. Count datas were expressed as percentages (%). For quantitative datas within each group, One-Way ANOVA was applied, followed by pairwise comparisons using Fisher’s Least Significant Difference method for equal vari-ance parameters and Tamhane T2 for unequal variance parameters. The association be-tween melatonin levels and other parameters was explored using the Spearman rank correlation method. Statistical significance was set at P  < 0.05 for all tests.

Below is the link to the electronic supplementary material. Supplementary Materials 1: Table S1: Correlation between the melatonin levels in base serum and follicular fluid and BMI, bLH, bE2 and P levels ( n  = 85) Supplementary Materials 1: Table S1: Correlation between the melatonin levels in base serum and follicular fluid and BMI, bLH, bE2 and P levels ( n  = 85)