Melatonin improves fertilization rate in assisted reproduction: Systematic review and meta-analysis

Highlights • Melatonin had beneficial effects on outcomes in assisted reproductive technologies, but it had no influence on pregnancy.• Melatonin had beneficial effects such as the improvement in the fertilization rate.• Melatonin has a beneficial effect on the formation of mature oocytes (MII).


Introduction
Infertility affects millions of women worldwide and is one of the main causes of the unfulfilled dream of having a child. 1 Assisted reproduction techniques may be accompanied by complications such as Ovarian Hyperstimulation Syndrome (OHSS), which can be fatal. 2As many as 20 % to 33 % of In Vitro Fertilization (IVF) cycles are affected by mild OHSS, whereas moderate to severe OHSS reportedly occurs in 3 % to 8 % of patients. 3latonin is a hormone that is primarily produced by the pineal gland.−6 Melatonin plays a role in sleep and in physiological oocyte maturation. 7Both in vitro and in vivo studies, in animals and in humans, show the benefits of melatonin as a substance that reduces the oxidative stress of cells related to reproduction, even improving fertilization rates. 8wo recent works have demonstrated that melatonin not only alleviates reactive oxygen species, but also improves apoptosis and that it may clinically benefit women who have developed OHSS. 9,10Other works discuss the important role, still under study, of melatonin related to steroid sex hormones, in one of these works it was observed that melatonin plays a role in maintaining follicular function through the production of progesterone, 11 while in the most recent review, it is considered that melatonin plays a role not only in the production of progesterone but also in estrogen and spermatogenesis. 12Among the positive results of using melatonin for women on Assisted Reproductive Technology (ART) are increases in the number of mature oocytes, the fertilization rate, the number of high-quality embryos, and, in some cases, increased pregnancy rates. 13,14iven the above, this study aimed to evaluate the effects of melatonin on assisted reproductive technologies through a systematic review and a meta-analysis.

Materials and methods
For the systematic review, the authors drew on several articles and guidelines, including Berstock et al., 2019, 15 Hennessey et al., 2019, 16 and Page et al., 2021. 17The meta-analysis was conducted in accordance with Higgins et al., 2022. 18

Search strategy
The studies selected for this review were published between January 2008 and April 2023, and they are indexed in PubMed and Scielo (Fig. 1).The initial search yielded 127 articles.After applying the eligibility criteria, they were narrowed down to the 17 articles included in this systematic review.Fig. 1 shows the selection process in detail.
Drawing on Page et al., 2020, 17 the P (Population) in the P.I.C.O. of this systematic review is patients, i.e., the female research participants who were selected to take exogenous melatonin and who had or had not ovarian hyperstimulation syndrome, depending on the article; the I (Intervention) is the administration of exogenous melatonin; the C (Comparison) is the comparison of the control group and the experimental group (exposed to melatonin); the O (Outcomes) is the outcomes as described in Table 1.
This review was conducted following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) recommendations. 15

Inclusion and exclusion criteria
The exclusion criteria covered animal studies (n = 74), articles whose titles or abstracts did not fall within the scope of the present study (n = 11), and review articles (n = 8).An additional 16 articles were excluded for lack of access to the full text because they were not freely accessible.The remaining 24 articles were fully read.By applying a second round of exclusion criteria, 7 more studies were screened out.The 17 articles that remained met the inclusion criteria of this study (see Table 1 for details) and were thus included in the study (Fig. 1).The inclusion and exclusion criteria were based on Page et al., 2020. 17

Statistical analysis
For descriptive analysis, calculations were made for means, standard deviations, mean differences, and odd ratios with a 95 % Confidence Interval.Meta-analysis was carried out with the Review Manager 5.4.1 software program (Cochrane Collaboration, Oxford, UK).For the 95 % CI and the overall effect size, values of p ≤ 0.05 were assumed for significant differences.The work seals the concentrations of melatonin in the follicular fluids and their role in human reproduction We have demonstrated that higher folicullar fluid melatonin concentrations were related to better ART outcomes Ma et al. [24]  2018 Clinical Trial IVF ICSI ET This study was the only one that did not have the presence of melatonin, therefore it was important for the inclusion criteria in the present systematic review because it worked with studies of two fluids of two follicular follicles in women with ovarian hyperstimulation syndrome.
Follicle count measured on the day of hCG administration was the only predictive factor for the occurrence of OHSS

Results
Melatonin is involved in a number of the body's physiological processes, one of which is regulating fertility.The articles included in this study are detailed in Table 1, which also includes information about the authors, publication year, methods, melatonin treatment, rationale for article inclusion, and key results or outcomes.−28,32−34

Meta-analysis of assisted reproduction outcome variables
The comparison between melatonin intake and the clinical pregnancy rate of assisted reproduction yielded no statistical difference.
Only 6 studies included this variable and with the following results: p = 0.64, I 2 = 37 % (Fig. 2); risk ratio of 1.22 (0.71-2.09).Caution is needed in interpreting this outcome, for there are numerous other variables involved in a healthy pregnancy resulting from assisted reproduction techniques, including the physiological conditions necessary for achieving pregnancy.
The meta-analysis of the fertilization rate outcome in percentage (%) showed a positive effect of the melatonin treatment, as the difference between the melatonin treatment groups and the control groups was statistically significant (p ≤ 0.00001, I 2 = 88 %, Fig. 3; risk ratio = 0.84 [0.79, 0.90]).The fertilization rate is an important indicator of reproductive outcomes, and the group of women who took melatonin had improved results compared to the group of women who did not take it (Fig. 3).Fig. 2. Meta-analysis of clinical pregnancy rate in patients receiving and not receiving melatonin.Fig. 3. Meta-analysis of fertilization rate in patients receiving and not receiving melatonin.
In the only four studies addressing follicle count, melatonin had positive effects on the growth of follicles as shown by p ≤ 0.00001, I 2 = 85 % (Fig S2 ), which points to statistical significance.
The MII oocyte is the female germ cell in an ideal state of maturation for fertilization.
A meta-analysis of maternal age in years was performed in 11 studies, and no statistically significant difference was found (p = 0.64 and I 2 = 64 %).Body mass index (kg/m 2 ) was a sociodemographic variable analyzed in 8 studies, with no significant differences in outcome (p = 0.59, I 2 = 77 %).A third sociodemographic variable was women's infertility time, and it approached the significance level at best (p = 0.06, I 2 = 0 %).
In short, melatonin had beneficial effects as shown by the increased fertilization rate and other outcomes of the reproductive process.The clinical pregnancy rates, however, were not significantly different in the group comparison.

Discussion
The main findings were that women who took melatonin had an improved fertilization rate and reaped other benefits from assisted reproductive technologies.However, melatonin intake did not result in a higher clinical pregnancy rate.
Of the 17 studies selected for this systematic review, only two articles, those by Li et al., 2019 35 and Zhang et al., 2023, 34 deal directly with the subject of ovarian hyperstimulation and the way in which melatonin can be a hormone that aids reproductive results.Li et al., 2019, concluded that melatonin produced by the follicular follicle helps predict OHSS, while Zhang et al., 2020, took a deeper approach.They demonstrated how melatonin, in addition to having anti-apoptotic properties, can improve oxidative stress in OHSS and concluded that it can indeed prevent OHSS.In a recent study by Hu et al., 2020, 36 promising results were obtained, but they differ from those of this meta-analysis, in which the clinical pregnancy rate improved with the administration of melatonin to patients with an OR of 1.43.However, their study comprised only articles with an RTC design, an advantage offset mainly by the low-quality bias and the heterogeneity of the articles.In a recent meta-analysis by Mejihede et al. 2021 35 with 7 articles from randomized controlled trials, oral melatonin supplementation resulted in an increase in the number of mature oocytes, and a trend for increasing CPR, albeit not significant. 32A recent study also verified the relationship between melatonin and the increase in gene expression in rats of follistatin and of Inhibin Beta-A, substances necessary for good hormonal regulation of the ovary and oocyte maturation. 35One of the mechanisms may be through the melatonin receptor in ovarian follicles, but there are also other mechanisms that do not depend on the receptor and are related to antioxidant substances. 32,34,35,37ome recent studies demonstrate that melatonin either from follicular fluid, granulosa cells, or exogenous sources has important roles concerning the quality of oocytes.It can delay the aging of the ovaries and their functions and improve the antioxidant properties of the oocytes, leading to improved reproductive outcomes such as an improved fertilization rate. 5,28,32,34,35he strength of the present work lies in demonstrating through metaanalysis that variables analyzed at the onset of assisted reproduction techniques improved to benefit the women who used exogenous melatonin.On the other hand, in the author's judgment, an important limitation is that in nearly half of the studies, the risk of bias was unclear or was not mentioned, impairing the quality of the studies.Another limitation, and the most important one, is that there was no difference in the clinical pregnancy rate between the groups.Further, there were not enough studies among the selected articles to analyze the main variable of assisted reproduction, namely the rate of live births.

Limitations of the study
This study has two main limitations.First, it is a systematic review and as such there is no data collection.Also, the results are those of previously published articles.Second, with respect to the meta-analysis of the clinical rate of pregnancy variable, there is no statistical difference between the use and the nonuse of melatonin, despite its beneficial molecular and cellular effects as judged by the values presented in the articles.

Conclusion and future perspectives
Melatonin is not a substance that has been used frequently in assisted reproduction.However, it has the following advantages for use in clinical practice: it is low cost; it is commercially available; it is a hormone produced by our own body and thus has no side effects; it has, as one of its main physiological actions, the capacity to reduce the oxidative stress of oocytes, but due to the few existing studies, this feature is still being overshadowed by the main results of human reproduction, such as clinical pregnancy rate and live birth rate.Therefore, the authors suggest that this line of research into melatonin use in assisted reproductive technologies be expanded with double-blind randomized multicenter studies.Melatonin had beneficial effects such as the improvement in the fertilization rate, although the authors did not obtain significance in the clinical pregnancy rate.
Additional studies, such as double-blind randomized clinical trials with many participants, are needed, particularly as regards melatonin action on ovarian hyperstimulation syndrome.

Fig. 1 .
Fig. 1.Flowchart of the systematic review.* Consider, if feasible to do so, reporting the number of records identified from each database or register searched (rather than the total number across all databases/ registers).** If automation tools were used, indicate how many records were excluded by a human and how many were excluded by automation tools.From: Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, et al.The PRISMA 2020 statement: an updated guideline for reporting systematic reviews.BMJ 2021;372:n71.doi:10.1136/bmj.n71.For more information, visit: http://www.prisma-statement.org/.

Table 1
Exogenous melatonin in previous clinical studies.