Importance of Melatonin in Assisted Reproductive Technology and Ovarian Aging

1Melatonin is an indoleamine (molecular weight 232.3) produced by all cells including those in the pineal gland, located in the posterior wall of the third ventricle. Melatonin is rhythmically released by the pineal gland such that it is mostly secreted during the night. This rhythm is controlled by the light–dark cycle, with light inhibiting melatonin synthesis and release. The circadian cycle of melatonin plays major roles in the regulation of body temperature, the secretion of various reproductively active hormones, and the circadian sleep–wake cycle. Melatonin, which is both fat-soluble and water-soluble, is distinctive in that it can easily pass through cell membranes. It is not only present in the blood but also in body fluids, including cerebrospinal fluid, follicular fluid, and seminal vesicle fluid. Melatonin receptors are found in numerous organs, and in addition to their own biorhythms, they determine, via several signaling mechanisms, secretion of various hormones, immune functions, lipid and glucose metabolism, as well as bone metabolism. These rhythms are involved in aging, carcinogenesis, and a number of diseases. Clinical research is underway in various fields including the application of melatonin in treating biological rhythm abnormalities, glucose and lipid metabolism disorders, immunoregulation, anticancer effects and aging-related diseases (Parkinson’s and Alzheimer’s disease).

2In addition to its receptor-mediated actions, melatonin’s ability to act as a direct free radical scavenger and as an indirect antioxidant has greatly broadened understanding of the mechanisms by which melatonin benefits reproductive physiology. Melatonin has potent antioxidative capacity to remove both reactive oxygen species (ROS) and reactive nitrogen species (RNS). As already noted, the scavenging actions are a direct effect and require no receptor mediation.

3In 1993, the discovery of melatonin as an antioxidant was achieved. Melatonin is a direct free radical scavenger with more powerful antioxidant potential than conventional antioxidants such as vitamin C and E, mannitol, and glutathione. Melatonin is both fat and water soluble, and can pass easily through cell membranes. Therefore, it exists in the cytosol as well as in mitochondria and nuclei. Melatonin, through direct scavenging and indirect antioxidant actions, limits oxidative stress in all cells and protects DNA and other components from damages. The antioxidative actions of melatonin and its metabolites are extremely vast, including the ability to neutralize superoxide anion (O2•−), hydroxyl radical (•OH), single oxygen (1O2), hydrogen peroxide (H2O2), hypochlorous acid (HOCl), nitric oxide (NO), and peroxynitrite anion (ONOO−). Melatonin is a valuable substance that particularly detoxifies the hydroxyl radical (•OH), which is highly reactive and toxic. When melatonin eliminates free radicals, it is converted to metabolites including cyclic 3-hydroxmelatonin (C3OHM), N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK). These metabolites, like melatonin, have potent antioxidative actions. Melatonin also indirectly increases antioxidant enzyme activity including superoxide dismutase (SOD) and glutathione peroxidase (GPx), and increases both antioxidant enzyme activity and mRNA expression via the membrane receptors of melatonin (MT1, MT2).

4ROS have important roles in reproductive processes such as influencing follicular growth, oocyte maturation, ovulation, fertilization, embryo implantation, and embryo development. During ovulation, following a surge of luteinizing hormone (LH) that induces the release of ova, large amounts of ROS are generated by vascular endothelial cells and macrophages as neovascularization progresses within the follicles. The ROS generated within follicles provides the stimulation needed for oocyte maturation and follicular rupture. Excessive ROS, however, cause cytotoxicity. As a defense mechanism against ROS, there are antioxidative enzymes and antioxidants present in follicles and in the oocytes themselves, which protect oocytes and granulosa cells from oxidative stress. If an imbalance between ROS concentrations and antioxidant activities occurs, oocytes and granulosa cells are readily damaged due to oxidative stress, which results in poor oocyte quality. Studies have reported that in mice after the induction of ovulation with human chorionic gonadotropin (hCG) following stimulation of superovulation using pregnant mare serum gonadotropin (PMSG), increased concentrations of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a marker of DNA damage, and of hexanoyl-lysine (HEL), a marker of lipid peroxidation, prior to ovulation were measured. Also, in humans, it has been reported that patients undergoing in vitro fertilization (IVF) with a high rate of denatured oocytes (poor oocyte quality) exhibit a high levels of 8-OHdG in the follicular fluid; also, low fertilization rates were noted for oocytes retrieved from follicles with an elevated concentration of 8-OHdG in the follicular fluid. Thus, it appears that ROS generated during the ovulatory process is a source of oxidative stress to oocytes, in follicles, as well as granulosa cells, providing an explanation for reduced oocyte quality and infertility.

5It is becoming clear that melatonin acts directly on the ovaries. In a report that examined tissue penetration of melatonin in cats, it was found that, compared to other organs, the accumulation was highest in the ovaries. Melatonin also is found at a high concentrations in human follicular fluid, and it increases in proportion to follicular growth. Hence, there is a mechanism whereby melatonin is taken up into the follicles as they enlarge. The physiological significance as to why melatonin is taken up into follicles and is present at high concentrations in follicles has long been unclear. Since ovulation is perceived as a phenomenon that resembles inflammation, and as a defense mechanism against ROS generated in follicles, antioxidative enzymes and antioxidants, including melatonin, protect oocytes and granulosa cells from oxidative damage. It is highly likely that melatonin carries out major roles as an antioxidant in both follicles and oocytes. In a study of follicular fluid collected during oocyte retrieval from women who have undergone IVF-ET, there were no marked correlations observed between the concentration of 8-OHdG and antioxidative enzymes, including copper (Cu) and zinc (Zn) superoxide dismutase (Cu, Zn-SOD), as well as for glutathione; however, a significantly negative correlation was observed between the concentration of melatonin and 8-OHdG. Furthermore, in patients who received melatonin (3 mg/day, taken at 22:00), the levels of melatonin in follicular fluid significantly increased compared to the period when melatonin was not given as a supplement, and 8-OHdG concentrations were significantly decreased as a result of melatonin administration. These results suggest that melatonin reduces oxidative stress in ovarian follicles via its antioxidative action, thereby protecting oocytes and granulosa cells.

6Tanabe et al. reported that melatonin protects granulosa cells from ROS by reducing oxidative stress of cellular components including nucleus, mitochondria, and cell membranes. Mouse granulosa cells were incubated with H2O2 (0.1–10 mM) in the presence or absence of melatonin (100 μg/mL). DNA damage (8-OHdG and γH2AX), mitochondrial dysfunction, and lipid peroxidation of membranes (HEL) were elevated after H2O2 treatment, whereas these harmful effects of ROS on cellular components were alleviated by melatonin supplementation. Moreover, H2O2 treatment increased the number of apoptotic cells and caspase 3/7 (Csp3/7) activities, which were inhibited upon melatonin administration. These results document that while ROS damage DNA, mitochondria, and cell membranes of granulosa cells, melatonin prevents this mutilation, thereby protecting granulosa cells (Figure 1).

7Tamura et al. examined the effect of ROS and melatonin on the maturation process of oocytes from mice. Germinal vesicle (GV)-stage oocytes were cultured with H2O2. After 12 h incubation, oocytes with the first polar body (MII stage oocytes) were significantly decreased as a result of the addition of H2O2 in a dose-dependent manner (>200 μM). However, melatonin treatment dose-dependently blocked the inhibitory effect of H2O2 on oocyte maturation. To further investigate the intracellular role of melatonin, oocytes were incubated with dichlorofluorescein (DCF-DA), a fluorescent dye that identifies ROS. High fluorescence intensities were observed in the presence of H2O2 (300 μM), whereas the increased fluorescence intensity was significantly depressed by melatonin treatment. This is consistent with antioxidative action of melatonin, which reduces ROS in oocytes.

8Based on these findings, it appears that melatonin, both locally-synthesized and secreted into the blood by the pineal gland, is taken up by ovarian follicles, where it locally reduces ROS in the follicles and limits oxidative stress, thereby protecting oocytes, as well as granulosa cells, and contributes to oocyte maturation and the luteinization of granulosa cells (Figure 1).

9In recent years, remarkable advances have been made in assisted reproductive technology (ART) such as IVF-ET for infertility treatment; however, satisfactory conception rates have not been achieved. The major cause of this is attributed to problems in the quality of oocytes. In IVF-ET, the frequent unsuccessful cases in which fertilization, embryo development, and implantation do not progress optimally are a result of poor oocyte quality. While the processes underlying reduced oocyte quality have not been fully elucidated, it is thought that oxidative stress caused by ROS in the follicle is a critical factor. In IVF-ET program, in vitro incubation including oocyte incubation, insemination (co-incubation with spermatozoa), fertilization, embryo development, and embryo transfer are performed in a high oxygen environment compared with the in vivo physiological condition. As long-term in vitro incubation conditions have a major negative impact on the quality of the oocyte and embryo, extreme caution must be exercised to control oxidative stress caused by ROS during incubation.

10As an application of the antioxidative effects of melatonin for reproductive medicine, it is possible that in vivo administration may improve the quality of oocytes until ovulation (oocyte retrieval) and improve oocyte maturation, fertilization, and embryo development during in vitro incubation when melatonin is added to the incubation medium (Figure 2).

11Follicular melatonin protects granulosa cells and oocytes from ROS. If follicular concentrations of melatonin were increased by administering melatonin to female patients, it may well improve the quality of oocytes. Tamura et al. performed an examination of IVF-ET with infertile patients with poor oocyte quality (fertilization rate < 50%) who were divided into two groups, including a treatment group given melatonin tablets (3 mg/day) for one month up to the time of oocyte retrieval for the next IVF-ET (melatonin group) and a group without melatonin treatment (control group). In the melatonin treated subjects, the fertilization rate was approximately 50% (vs. approximately 20% for the control group) and the pregnancy rate was roughly 20% (vs. approximately 10% for the control group), indicating improved outcomes for IVF-ET. It is possible that the administration of melatonin to patients suffering from infertility increases follicular concentrations of melatonin, which thereby inhibits oxidative stress and improves the quality of oocytes, thus improving fertilization and pregnancy rates. After this initial study, other reports also examined whether melatonin therapy improves the clinical outcome of IVF-ET, with the findings likewise also showing that the concurrent use of melatonin increases the number of mature oocytes, the fertilization rate, and number of high-quality embryos (Table 1). It is suggested that the effects of melatonin include an increased concentration of melatonin in the follicular fluid and a reduced 8-OHdG concentration, which leads to the conclusion that melatonin’s multiple antioxidant actions lower oxidative stress in oocytes. In ART, melatonin would be expected to elevate the pregnancy rate by improving the quality of oocytes as well as promoting the fertilization rate and embryo development.

12In studies using oocytes from mice, cows, and pigs, it has been reported that oocyte maturation, fertilization, and embryo development are promoted upon in vitro incubation of oocytes in culture medium supplemented with melatonin (Table 2). As oxygen concentrations are higher in in vitro incubations than in vivo, oxidative stress caused by ROS generated during incubation often has an adverse effect on oocyte maturation and embryo development. The addition melatonin to the culture medium wound detoxifies ROS, thereby reducing oxidative damage so as to protect the oocytes and granulosa cells. Reducing oxidative stress and apoptosis of oocytes and promoting mitochondrial function via melatonin treatment have been shown to improve oocyte maturation, fertilization rate, and rate of blastocyst formation (blastocyst cell count). Furthermore, the effect of oxidative stress induced by substances that generate ROS, such as bisphenol A (BPA) and aflatoxin B1 (AFB1), is reduced when melatonin is added to the culture medium. This action of melatonin reduces ROS and oxidative stress due to its direct antioxidative actions.

13Because melatonin reduces ROS and oxidative stress via its direct antioxidative functions, it improves cellular integrity (Figure 3). However, indirect effects of melatonin via membrane receptors (MT1, MT2) and/or nuclear receptors (RORα) are also important to understand the processes by which melatonin alters the level of oxidative stress. Figure 3 summarizes the mechanism of melatonin that are assumed to improve oocyte maturation and quality based on reports to date. Melatonin membrane receptors (MT1, MT2) are located in oocytes and granulosa cells (cumulus cells); it would be of great interest to determine the intracellular signaling pathway by which melatonin promotes oocyte maturation and embryo development. While some reports have examined these intracellular processes of melatonin in oocytes, the details remain unclear. It has been reported that melatonin controls the expression of genes related to oocyte maturation including mitochondrial function, antioxidative enzymes, apoptosis, cumulus cell expansion, and oocyte maturation factors. Furthermore, epigenetic mechanisms such as DNA methylation and histone acetylation have also been reported. Further study may identify how these epigenetic mechanisms contribute to oocyte maturation by regulating the expression of some specific genes in oocyte and granulosa cells. In addition to a direct antioxidative action, it is essential to elucidate the detailed mechanisms of melatonin on oocytes and granulosa cells (cumulus cells) that involve both the membrane and nuclear receptors.

14ROS play crucial roles in reproductive functions such as the ovulatory process. However, in excess, they may adversely affect oocytes in the form of oxidative stress which would cause infertility. Melatonin existing in follicles may possibly protect oocytes from ROS with its antioxidant activity and its involvement in oocyte maturation, fertilization, and embryo development. The trials of clinical application of melatonin for infertile women have reported improved outcomes of ART. It should be noted that melatonin supplementation could become a new treatment for improving oocyte quality and it may benefit women who suffer from infertility.

15Age is the critical factor that has a major effect on fertility. Infertility caused by ovarian aging is the most important challenge in reproductive medicine. ART success decreases with increased age, with a sudden decline in the pregnancy rate and elevation in the miscarriage rate observed from 35 years of age onwards. Ovarian aging has two problems and includes both reduced oocyte number and reduced oocyte quality. At birth, females are born with 1–2 million oocytes in their ovaries. However, they never produce any new oocytes thereafter and many are continuously being lost. It has been shown that at puberty, the number already is reduced to 100,000–300,000 oocytes, and that by the late 30s, particularly after 37 years of age, the number of oocytes rapidly decreases.

16As women grow older, the quality of each oocyte also is reduced. Oocytes are subjected to various types of damage in an age-dependent manner over the long period of several decades, with dysfunction noted in organelles such as mitochondria and nuclei. This is perceived as poor oocyte quality, and is a major cause of frequent unbalanced chromosome segregation in the first meiotic division accompanied by impaired fertilization. Although the mechanisms that cause a decline oocyte quality have not been fully elucidated, it is generally accepted that oxidative stress caused by ROS contributes to age-induced dysfunction of oocyte mitochondria and nuclei. It has also been frequently reported that reduced antioxidative function caused by aging are associated with lower oocyte quality. While attempts have been made to prevent the lower function and to improve the impaired quality of oocytes, at present no effective method has been established.

17Melatonin has drawn attention as an anti-aging molecule. The life expectancy of mice treated with melatonin is reportedly prolonged, and the life expectancy of mice with the pineal gland removed is shortened. Therefore, there is a possibility that melatonin has the ability to delay ovarian aging. Tamura et al. examined the protective effect of melatonin on ovarian aging using mice. Female mice were administered melatonin (drinking water containing melatonin) over a period from 10 to 43 weeks of age, after which oocytes were retrieved from follicles and IVF was performed. In 43-week-old control mice, there were fewer follicles of all developmental stages within the ovaries (primordial follicles, primary follicles, secondary follicles, and antral follicles). In the melatonin-treated animals, however, there were more follicles remaining compared to the number in the control animals. Also in the melatonin group, there were more ovulated oocytes, and the age-related decline in the number of oocytes was reduced. The results of IVF indicated that the number of fertilized oocytes and the number of blastocysts declined with age, but were maintained in the melatonin-treated animals. Thus, melatonin also appears to reduce the decline in oocyte quality. The authors also analyzed changes in ovarian gene expression using microarray. The expression of 77 genes decreased with age and were elevated as a result of melatonin treatment. Among these genes, approximately half (40 genes) were involved in ribosomal function. Furthermore, upon performing pathway analysis, the authors extracted eukaryotic initiation factor 2 (eIF2) signaling, which maintains the accuracy of protein synthesis (translation) (Figure 4). This was interpreted to mean that melatonin maintains ribosomal function, accuracies of gene translation, and protein synthesis, thereby slowing the processes of aging. On pathway analysis, the growth arrest and DNA-damage-inducible 45 (GADD45) signaling involved in DNA repair and checkpoint functions were predominantly noted, suggesting that melatonin enhances the mechanism underlying DNA damage repair (Figure 4). Melatonin also suppresses autophagy-related protein (light-chain 3a: LC3a; light-chain 3b: LC3b) by enhancing intracellular pathways, including eIF2, GADD45, and alternative reading frame (ARF) pathways. Furthermore, the network analysis found that melatonin had a stimulatory effect on antioxidative mechanisms. Moreover, the telomere length, which typically decreases during aging, and expressions of the sirtuin longevity genes (SIRT1, SIRT3) were significantly higher in the melatonin-treated animals compared to the control mice. Collectively, the results suggest that, through various mechanisms, melatonin reduces at least some aging processes in the ovaries and oocytes (Figure 4).

18Differences in the time of initiating melatonin treatment might result in different anti-aging effects on the ovaries. Therefore, melatonin treatment was started from 23 or 33 weeks of age in mice, and the results of IVF outcomes at 43 weeks of age were analyzed according to the previous report (Figure 5). In the group in which treatment was initiated at 23 weeks, the number of ovulated oocytes (8.5 ± 2.2, C23 (control group); 16.8 ± 3.0, M23 (melatonin group)), fertilization rate (32.3%, C23; 59.5%, M23), and blastocyst rate (17.6%, C23; 39.2%, M23) were all significantly higher in the melatonin-supplemented mice than in control animals (Figure 5). These results show that melatonin treatment that began at 23 weeks delayed aging of the ovaries. Conversely, in the group that started at 33 weeks, the number of ovulated oocytes (9.6 ± 1.8, C33; 9.4 ± 2.7, M33), fertilization rate (30.2%, C33; 37.6%, M33), and blastocyst rate (22.9%, C33; 36.4%, M33) showed no significant difference as a result of melatonin treatment compared to controls (Figure 5). Melatonin was not found to be beneficial if melatonin treatment began at 33 weeks. The implication is that, to reduce aging of the ovaries, melatonin supplementation should be initiated well in advance of reproductive deterioration.

19In another study where melatonin was given to mice for 6–12 months, it was reported that it inhibited the age-related reduction in the number of follicles, litter sizes, and blastocyst rates while improving mitochondrial function, lowering ROS production, reducing oxidative stress, increasing adenosine triphosphate (ATP) production, lowering apoptosis, and elevating antioxidative enzymes, thus demonstrating that melatonin can reduce ovarian aging. A recent study that investigated the effect of melatonin in delaying ovarian aging also reported on the importance of intracellular signaling (MT1/MAPK pathway) via melatonin receptors in oocytes (MT1) to modulate the age-related changes.

20The mechanisms related to the anti-ovarian aging effects of melatonin are clearly not yet well defined. The clinical application of melatonin for the treatment of humans to improve ovarian physiology should have high priority. Many women suffer from the infertility due to ovarian aging. If long-term melatonin treatment prevents ovarian aging as represented by a decline in the number and quality of oocytes, they would have more oocytes of better quality when they undergo IVF-ET program. Melatonin treatment may make a major contribution to reproductive medicine to improve ART outcomes. There are currently no effective methods or established medications that prevent ovarian aging; the administration of melatonin may be a promising candidate for this purpose.