A Retrospective Review on Dysregulated Autophagy in Polycystic Ovary Syndrome: From Pathogenesis to Therapeutic Strategies

Abstract

The main purpose of this article is to explore the relationship between autophagy and the pathological mechanism of PCOS, and to find potential therapeutic methods that can alleviate the pathological mechanism of PCOS by targeting autophagy. Relevant literatures were searched in the following databases, including: PubMed, MEDLINE, Web of Science, Scopus. The search terms were “autophagy”, “PCOS”, “polycystic ovary syndrome”, “ovulation”, “hyperandrogenemia”, “insulin resistance”, “inflammatory state”, “circadian rhythm” and “treatment”, which were combined according to the retrieval methods of different databases. Through analysis, we uncovered that abnormal levels of autophagy were closely related to abnormal ovulation, insulin resistance, hyperandrogenemia, and low-grade inflammation in patients with PCOS. Lifestyle intervention, melatonin, vitamin D, and probiotics, etc. were able to improve the pathological mechanism of PCOS via targeting autophagy. In conclusion, autophagy disorder is a key pathological mechanism in PCOS and is also a potential target for drug development and design.

Keywords

polycystic ovary syndrome -  adrenal - autophagy - pathogenesis - therapeutic strategies - retrospective review

Introduction

Polycystic ovary syndrome (PCOS) is the most common reproductive endocrine disease among women of reproductive age today, with an incidence of about 10–15% [1]. According to the consensus of ESHRE/ASRM, the diagnosis of PCOS should meet at least two of the following three characteristics: 1) ovulation dysfunction (rare ovulation and/or anovulation); 2) hyperandrogenism biochemical or clinical characteristics of excessive androgen; and 3) the appearance of polycystic ovaries in ultrasound examination. In addition to its effects on reproduction and body shape, PCOS is also closely associated with a high risk of metabolic disorders, including insulin resistance (IR) and compensatory hyperinsulinemia, obesity, lipid metabolism disorders, and often accompanied by a low-grade inflammatory state, which interact with each other and collectively contribute to diabetes, cardiovascular diseases, and gynecologic tumor in the long term [1] [2] [3] [4].

The etiology and pathogenesis of PCOS is still confusing, which seriously hinders the advancement of clinical therapies. Nowadays, the impact of autophagy on the pathological mechanism of PCOS has deeply fascinated scholars. Autophagy is an evolution-conserved self-digestion pathway of cells, which realizes energy cycling and tissue remodeling by degrading non-functional cytoplasmic proteins and organelles [5] [6], and this process can be initiated by a variety of stressors and is a mechanism that kills stress cells and maintains environmental balance in the body [7]. The onset of apoptosis is related to changes in the mitochondrial membrane caused by cellular stimulation, allowing pro-apoptotic proteins to enter the cytoplasm through mitochondria [8]. Both apoptosis and autophagy are important ways to ensure the orderly renewal of cells and maintain the stability of the internal environment of the organism. Studies in recent years have found that some pathological changes in PCOS seem to be related to the disruption of this balance [9]. In the context of PCOS, studies have revealed that abnormal autophagy can lead to abnormal ovulation, disorder of glucose and lipid metabolism, and obesity, which are closely related to the pathological manifestations of PCOS [10] [11]. Therefore, we review in this manuscript relevant literatures in PubMed, MEDLINE, Web of Science and Scopus, with the search terms of "autophagy", "PCOS", "polycystic ovary syndrome", "ovulation", "hyperandrogenemia", "insulin resistance", "inflammatory state", "circadian rhythm" and "treatment". And then, we summarize the association between autophagy and abnormal ovulation, hyperandrogenemia and IR in PCOS. In addition, we also sort out the current therapeutic methods that can improve the pathological mechanism of PCOS by regulating autophagy, thus providing a new direction for the treatment of PCOS.

Autophagy is involved in the pathological mechanism of PCOS

Autophagy is a highly conserved process of self-renewal in eukaryotic cells, characterized by the cytoplasmic material being swallowed into double membrane vesicles (autophagosomes) and then degraded in lysosomes [12]. In general, autophagy is necessary for the clearance of dysfunctional proteins and organelles [13], of which the components can be recycled to produce new cellular structures, or can be further processed and used as an energy source. Studies have corroborated that autophagy is involved in the physiological and pathological processes of reproduction by regulating the growth, atresia and differentiation of follicles [5] [13]. For example, active autophagy in ovaries induced by accumulated reactive oxygen is closely related to ovarian granulocyte death in obese women [14] [15], leading to an increased incidence of anovulation-induced infertility. In addition, autophagy has been verified to participate in PCOS-related metabolic disorders, such as lipid metabolism and insulin sensitivity [15]. All of these findings suggest that autophagy may play an influential role in the reproductive and metabolic problems of PCOS. Therefore, understanding the role of autophagy in the pathological mechanism of PCOS is expected to provide novel clues for the discovery of precise targeted treatment strategies for PCOS.

Autophagy and abnormal ovulation

Increasing evidence supports the essential role of autophagy in the atresia, development and maturation of follicles, and the cellular mechanisms of rare ovulation or anovulation in PCOS are closely related to abnormal autophagy. Data have shown that only about 0.1% of the follicles in mammals are expelled [16], and the rest are atretic. It has been suggested that autophagy in granulocyte contributes to digesting abnormal proteins and damaged organelles in the atretic follicles so as to achieve energy cycle and support the development and excretion of the dominant follicle [17] [18]. Studies verify that the LC3 protein staining is weak in the early/middle stage of unatresia follicles and in the granulosa layer of large sinus follicles, while LC3 immunoreactivity is strong in the atretic follicles [19]. LC3 is currently the most widely used autophagosome marker, which can conjugate to phosphatidylethanolamine to form LC3-II. And the amount of LC3-II reflects the number of autophagosomes and autophagy-related structures. Besides, the current study displays that follicular atresia depends on the degree of the granulosa cell apoptosis, which can be facilitated by autophagy, thus determining the fate of follicles and female fertility [20]. Cellular autophagy and apoptosis coexist and are dynamic at different follicular stages. Autophagy is commonly observed in medium-sized follicles, whereas apoptosis is more common in large follicles [21]. In young healthy women of normal weight, cells choose to self-repair by inducing autophagy rather than undergo apoptosis in response to low levels of reactive oxygen species. In contrast, in obese women granulosa autophagy is activated by excess oxidized low-density lipoprotein (oxLDL), which in turn leads to apoptosis, resulting in dysfunction of follicular development and ovulation [22]. As granulosa cells express FSH receptors at cell surface, excessive apoptosis of granulosa cells leads to a lack of binding receptors for FSH, which in turn affects its follicle-promoting biological effects [23]. Therefore, we speculate that the abnormal autophagy level is closely related to the occurrence of clinical ovulation disorders in PCOS. Yi et al. found that autophagy of granulosa cells in PCOS patients was increased, characterized by a high level of autophagosomes, Beclin-1 and LC3-II and a decreased level of the autophagy substrate p62 [24], which was consistent with the findings of Li et al. who uncovered that transcription profiles of 34 autophagy-related genes were upregulated and autophagy was active in ovarian tissue of PCOS model rats [25]. In addition, disturbances in the balance of autophagy and apoptosis were also observed in PCOS [26] [27]. In contrast, ovulatory function can be improved in PCOS when over-activated autophagy of granulosa cells was alleviated [28] [29]. From the above research, we project that the abnormal autophagy level of granulosa cells may be one of the pivotal mechanisms for the pathogenesis of PCOS. The involvement of autophagy in ovulation dysregulation in PCOS is shown in [Fig. 1]. However, due to the complex mechanisms regulating cellular autophagy, the molecular cascade activating autophagy in granulocytes of PCOS patients is currently poorly understood. Thus, an in-depth exploration of this area is imminent so as to provide potential targets for precision therapy of PCOS.

Fig. 1 Autophagy is involved in abnormal ovulation of PCOS by regulating the growth, atresia, and differentiation of follicles.

Autophagy and hyperandrogenemia

Hyperandrogenemia (HA) is a prominent feature of PCOS [30]. HA can cause clinical symptoms such as hirsutism, obesity, acne, and alopecia, which also tends to hinder the normal growth of follicles, resulting in anovulation or rare ovulation. Through literature, we found that HA and autophagy of granule cells can affect each other and jointly contribute to the complex reproductive endocrine disorder in PCOS [25] [31] [32]. As we mentioned above, active autophagy in granulosa cells of PCOS patients leads to increased cell apoptosis, which can then bring about disfunction of hormone transformation from testosterone and androstenedione to estradiol and estrone, thus facilitating HA. In addition, HA can directly promote the autophagy and apoptosis of granulosa cells and affect the synthesis of steroid hormones and the development of follicles. Li et al. found that compared with non-PCOS patients, autophagy of granulosa cells in PCOS was induced. In addition, the beclin1 mRNA abundance in human granulosa cells was positively correlated with serum total testosterone level, and the expression of beclin1 mRNA and LC3II/LC3I in granulosa cells induced by androgen was dose-dependent [31]. Beclin1 has been confirmed to be involved in membrane transport and recombination process in autophagy and can interact with multiple autophagy factors to promote autophagy pathway. Including Bcl-2 homology domain 3 (BH3), central coiled coil domain (CCD), and C-terminal evolutionarily conserved domain (ECD) [33]. Similarly, another study confirmed that the LC3II and beclin1 expression levels were increased and the p62 and p-mTOR levels were decreased in vivo in ovarian tissue from the PCOS mice [34]. p62 is a selective autophagy adaptor protein, which is involved in the degradation of ubiquitin-proteasome and autophagy lysosome. And elevated p62 implies that autophagic substrates are not efficiently degraded. p-mTOR can activate Unc-51 like autophagy activating kinase 1 (ULK1), block the activation of Adenosine 5′-monophosphate-activated protein kinase (AMPK), and thus inhibit autophagy process. Besides, the in vitro data were similarly with the in vivo by stimulation of mouse granulosa cells with dihydrotestosterone. These effects could be diminished by the autophagy inhibitor (MHY1485) or by androgen receptor antagonists (ARN509) [34]. To sum up, the active autophagy and increased apoptosis of granulosa cells can promote the increase of serum androgen levels, while hyperandrogenemia in turn can further lead to the increase of autophagy and apoptosis in granulosa cells, forming a recurrent pathological ring and promoting the development of PCOS. The involvement of autophagy in hyperandrogenemia in PCOS is shown in [Fig. 2]. Besides, future studies are needed to further focus on the effects of hyperandrogenemia on autophagy levels in different tissues and cells of PCOS, so as to fully understand the regulatory role of autophagy in the pathogenic mechanism of hyperandrogenemia in PCOS.

Fig. 2 Autophagy is involved in hyperandrogenemia, insulin resistance, and abnormal circadian rhythm of PCOS.

Autophagy and insulin resistance

Insulin resistance (IR) is present in 50–70% of women with PCOS [35] and is a high risk factor for metabolic syndrome. Compensatory hyperinsulinemia in PCOS can lead to increased value of free testosterone through synergy with luteinizing hormone (LH) and decreased hepatic synthetic sex hormone binding globulin (SHBG) [36] [37] [38], and jointly participate in the complex reproductive endocrine disorders in PCOS. Recently, studies have displayed that the significant decreased level of autophagy in fat, liver, skeletal muscle and other tissues is closely related to the occurrence of IR. Wang et al. established IR model in vitro, and observed decreased level of autophagy [39]. On the contrary, when autophagy level was increased, IR was mitigated. Besides, in order to further study the role of autophagy in regulating insulin sensitivity, the autophagy related gene (ATG)7 or ATG5 were knocked out, respectively, which significantly interfered with the establishment of IR model. Based on the above experiments, we can find that the abnormal level of autophagy is closely related to the occurrence of IR, and targeting the regulatory factors of autophagy is expected to become a potential therapeutic direction to improve IR in the future. Whether autophagy is also involved in the development of IR in PCOS is a question worth exploring. Scholars have confirmed that the regulation of autophagy contributes to improving IR in PCOS. Sumarac-Dumanovic et al. observed that mRNA profiling of autophagy-related molecules are significantly reduced in endometrium of PCOS. Moreover, metformin treatment (2 g/d, 3 months) can significantly increase the mRNA level of ATG14, beclin-1, and ATG3 in endometrium of patients with PCOS [40]. In addition, a study by Song et al. demonstrated that DHEA-exposed mice presented IR in whole-body, along with autophagy inhibition [41]. Besides, DHEA-induced IR could be alleviated by electroacupuncture, and this effect was reversed by autophagy inhibitor 3-MA [42]. Abuelezz et al. suggested that the mechanism of ameliorating IR by modulating autophagy may be related to its protection against oxidative stress and the effects of chronic inflammation on the function and fate of b cells [43]. Therefore, it can be concluded that autophagy in PCOS is closely related to IR, which may be a novel target for the treatment of IR in PCOS. The involvement of autophagy in IR in PCOS is shown in [Fig. 2].

Autophagy and abnormal circadian rhythm

The circadian rhythm is a physiological and behavioral pattern controlled by the circadian clock located in the suprachiasmatic nucleus (SCN) and mainly affected by light. Secondary oscillators are present in many brain regions and peripheral organs, which can affect function of local tissue [44] [45] [46] [47] [48]. At the molecular level, circadian rhythms are generated by a transcription-translation feedback loop regulated by molecular clock transcription factors, in which brain and muscle arant-like-1 (BMAL1), CLOCK, cryptochrome (CRY), and period circadian regulator (PER) are the core circadian rhythm genes [49]. In 2007, Nakao et al. found the role of circadian rhythm genes in Japanese quail ovaries, which broke the inherent research model around neuroendocrine control of ovarian steroidogenesis and ovulation [50]. Evidence of large-scale cohort epidemiological studies shows that the circadian rhythm disorder caused by shift work will cause women to face such reproductive physiological abnormalities as menstrual disorder, infertility or abortion [51]. Moreover, the disorder of circadian rhythm is closely related to the occurrence and development of reproductive diseases such as PCOS. It has been reported that pinealectomy or long-term light exposure can induce polycystic ovary and androgen excess in rodents [52] [53], while androgen excess can cause abnormal expression of PER2, one of the main control genes as one of the core elements of molecular oscillation [54] [55], thus leading to disorder of steroid hormone production in granulosa cells and promoting the development of PCOS [56]. In addition, scholars also confirmed that the expression of BMAL1 in granulosa cells of PCOS decreased significantly, which was closely related to the expression of FSHR, aromatase, abnormal follicular development and ovulation disorder [57] [58] [59]. Besides, the disorder of circadian rhythm is closely related to the abnormality metabolism of PCOS. Several studies have shown a significant increase in the incidence of IR and metabolic syndrome in women exposed to night-light changes [60] [61]. Simon et al. conducted a cross-sectional study comparing circadian rhythms in women with PCOS (n=559) and obese girls without PCOS (n=533). The result displayed that women with PCOS showed a later secretion of melatonin during puberty than controls, with a delayed circadian rhythm, which showed a significant correlation with serum free testosterone and insulin sensitivity [62]. In addition, Chu et al. also corroborated that circadian rhythm could promote the occurrence and development of PCOS through gut microbiota. In their studies, female Sprague Dawley (SD) rats exposed to continuous light showed changes in gut microbiota, estrous cycle, and ovarian morphology, which was similar to that of PCOS [63]. Based on the above experimental results, it can be found that abnormalities in the circadian system can affect many aspects in PCOS such as endocrine, reproduction and metabolism systems.

Circadian rhythm is also influenced by autophagy. Studies by Toledo et al. have revealed that autophagy controls the liver’s circadian clock by degrading a circadian protein, cryptochrome 1 (Cry1), which can reduce glucose production in the liver. While the degradation of CRY1 is aggravated by autophagy in obese individuals. Olsvik et al. confirmed that the circadian rhythm protein BMAL1, CLOCK, REV- ERB a and CRY1 were the targets of lysosome, and autophagy can selectively degrade CRY1 to influence circadian rhythm. In addition, they also documented CRY1 contained light chain 3 (LC3)-interacting region (LIR) motifs [64] that promoted the interaction of cargo proteins with LC3-labeled autophagosome, thereby regulating the autophagic degradation of CRY1 to achieve circadian glycemic control [65]. In recent years, active autophagy has been observed in the ovarian tissues of PCOS women and rat models, especially in ovarian granulosa cells [25]. Therefore, excessive autophagy of PCOS may be closely related to the degradation of circadian proteins, thus affecting glucose and lipid metabolism, steroid hormone synthesis and follicular development. In the future, the correlation between autophagy and changes of circadian genes in the pathological mechanism of PCOS should be further explored in vivo or in vitro, which is conducive for us to understanding the disease and discovering new direction for the treatment. The involvement of autophagy in abnormal circadian rhythm in PCOS is shown in [Fig. 2].

Autophagy and state of chronic inflammation

Recently, PCOS has been identified as a chronic inflammatory disease [66], manifested as significantly increased levels of C-reactive protein (CRP), tumor necrosis factor-a (TNF-a), and interleukin-6 (IL-6) in the peripheral circulation. In addition to disrupting ovarian function, chronic low-grade inflammation in PCOS patients also affects glucose and lipid metabolism, exerting a vital part in PCOS [67] [68]. Studies documented that low-grade inflammatory response and autophagy were involved in the crosstalk of signaling pathways to promote the progression of pathological mechanisms [69]. Toll-like receptor (TLR) and NOD-like receptor (NLR) signaling pathways mainly contribute to chronic inflammation state, which can interact with autophagy and jointly promote the process of disease. Gu et al. confirmed that the expression of TLR-related proteins, TLR4 and TLR9, were upregulated in the cumulus cells of PCOS, and were associated with decreased oocyte quality and low embryo rate [70]. Besides, TLR is the main pathogen associated molecular pattern (PAMP) cell sensors, which can activate autophagy. Xu et al. has shown that lipopolysaccharide (LPS), acted as PAMP molecular, is able to induce autophagy flux in macrophages by stimulating TLR4 so as to promote the clearance to pathogen [71]. Later, Shi et al. further demonstrated that not only TLR4, but also other TLR family members could interact with beclin-1 (a key factor of autophagosome formation) through the cohesion protein MyD88 or TRIF, to reduce the binding of beclin-1 and bcl-2, thus removing the inhibition of beclin-1 by bcl-2 and promote the occurrence of autophagy. Furthermore, TLR4 and TLR9 on the surface of granulosa cells are controlled by LH and androgen. So, we suspect that the TLR signaling pathway of oocyte granulosa cells in PCOS patients is activated by high levels of LH and androgen, which lead to autophagic cell death in granulosa cells and ovulation disorders. Of course, this hypothesis needs to be further validated in vivo and in vitro. The involvement of autophagy in chronic inflammation state in PCOS is shown in [Fig. 3].

Fig. 3 Autophagy is involved in the state of chronic inflammation of PCOS.

In addition, autophagy can in turn regulate the TLR signaling pathway and affect inflammatory response. Lee et al. found that autophagosomes could capture the RNA of Sendai virus and deliver it to the endosomes. Then, TLR on the endosome membrane is activated, promoting the induction of interferon (IFN) [72]. Other studies have shown that human follicular granulosa cells exposed to resveratrol (RES) can induce protective autophagy to reduce the expression of TLR4, CD36, and LX1, and thus playing a protective role on granulosa cells [73]. In addition, NLRP3, as one of the important members of the NLR family, has also been proved to participate in the inflammation response of PCOS by promoting the release of IL-1 and IL-18 [74], while autophagy can negatively regulate the activation of NLRP3 and thereby inhibits the inflammation state of PCOS [75].

Besides, low-grade inflammatory response and autophagy are linked by intestinal microorganisms. Studies have shown that patients with PCOS show imbalance in gut microbiota, with an increased number of Gram-negative bacteria. Thus, level of LPS is increased and then enter the blood circulation, which can bind to lipopolysaccharide binding protein (LBP) synthesized by the liver and acts on TLR4 on the surface of immune cells to activate the downstream MyD88 signaling pathway, then promote the expression of TNF-α, IL-6, etc. Moreover, TNF-α can also upregulate the expression of autophagy genes (LC3) and beclin1 [76] [77]. In turn, hyperactivated autophagy may promote inflammation through gut microbiota. Mice lacking autophagy-related gene Atg7 showed abnormal fecal microbiota and significantly increased Gram-negative bacteria such as Bacteroides fragilis [78]. In addition, autophagy can also influence the intestinal barrier function by inducing lysosomal degradation of tight junction protein CLDN2, increasing its permeability, accelerating the entry of LPS into the circulation process, and causing systemic inflammation response [79]. Therefore, we can conclude that that autophagy can promote the contribution of gut microbiota to chronic inflammation in PCOS by influencing the number of bacteria and the permeability of host intestinal wall.

To sum up, autophagy and inflammatory signaling pathways can influence each other and jointly affect the pathological mechanism development in PCOS. However, the mechanisms are intricate and complicated, which remain to be further explored.

Drugs targeting autophagy may have potential to treat PCOS

Over the past decades, the treatment strategies of PCOS have remained controversial. Up to now, more than 160 recommendations and practical guidelines have been published around the world [80] [81], which generally include lifestyle intervention drug therapy and surgical procedures. Treatment targets include improving abnormal ovulation, hyperandrogen, and metabolic abnormalities. In addition to symptom management, treatment objectives also contain the prevention of long-term complications associated with PCOS, such as diabetes and cardiovascular disease [82]. But in general, current treatments are symptomatic rather than parenchymal, due to a superficial understanding of disease mechanisms. For example, oral contraceptives are the first-line treatment for menstrual androgen disorders, clomiphene and letrozole are used for ovulation induction, and metformin is recommended for metabolic therapy, etc. [83]. Current studies have confirmed that current therapeutic drugs can also play a therapeutic role in PCOS by targeting autophagy. Xu et al. demonstrated that metformin ameliorated PCOS in a rat model by downregulating autophagy in granulosa cells, and metformin could decrease the levels of oxidative stress and autophagy in H₂O₂-induced granulosa cells and affected the PI3K/AKT/mTOR signaling pathway [19]. Taken together, these results indicate that metformin ameliorates PCOS by decreasing excessive autophagy in granulosa cells. In addition, a study by Liu revealed that Guizhi Fuling pill, a traditional Chinese medicine used for PCOS, could inhibit granulosa cell autophagy and promote follicular development to attenuate ovulation disorder in PCOS-IR rats [84]. In order to broaden the decision-making range of PCOS and make up for the deficiencies of existing treatment plans, this study proposes several potential methods that can improve the pathological mechanism of PCOS by regulating autophagy ([Fig. 4]).

Fig. 4 The treatment strategies of PCOS.

Lifestyle intervention

Various lifestyle interventions for PCOS are suggested, including diet. So far, nearly 10 studies have been published involving dietary interventions, including low-calorie diets, vegan or soy diets. However, there are no guidelines or studies confirming the benefits of restrictive feeding (RF) for PCOS. Currently intermittent fasting (for example, limiting energy intake by 60% two days a week or every other day), periodic fasting (for example, a 5-day diet providing 750–1100 kcal), and time restrictive feeding (TRF, limiting daily food intake to 8 hours or less) in normal and overweight subjects have been shown to be effective in weight loss and has improved various health indicators and reduced risk factors for cardiovascular disease [85]. So far, nine trials of TRF on human have been shown to reduce weight, improve glucose metabolism, inflammation cytokines and extend life span, even when food intake was comparable to that of the control group [86] [87]. Later, scholars further explored the molecular mechanism of the metabolic improvement of RF, which revealed that the benefits of intermittent fasting are partly due to regulation on autophagy [88]. Jamshed et al. conducted a randomized, cross-sectional study in overweight adults who were required to take food between 8 AM and 2 PM (early TRF, eTRF) or between 8 AM and 8 PM (control schedule). The results showed that fasting glucose and insulin in the morning, fasting insulin in the evening, and 24-hour glucose fluctuations were reduced in TRF group. And the expression of LC3 was increased by 22% in eTRF group, which encoded an important structural component of the autophagosome membrane [89]. This study reveals that the improvement of glucose metabolism by RF may be related to the influence of autophagy. Martinez et al. confirmed the effect of trial-a-day (ITAD) feeding on autophagy and metabolism through animal experiments. They established an isocaloric twice-a-day (ITAD) bean model wherein ITAD-fed mice consume the same food amount as controls but were limited to take food at two short windows: 8–10 AM (feeding window 1) and 5–7 PM (feeding window 2). The results displayed that ITAD fasting activated autophagy in the liver, fat and muscle tissues, promotes diverse metabolic benefits in multiple systems, and prevents the age-and obesity-associated metabolic defects [90]. However, the efficacy of intermittent fasting in the treatment of PCOS is still lacking. According to the above experiments, we speculate that RF tends to improve the glucose and lipid metabolism disorders in PCOS, and whether it can improve the secretion of steroid hormones and restore ovulation remains to be confirmed by experiments.

Drug therapy

Melatonin

Melatonin (N-acetyl-5-methoxytryptamine), an indoleamine hormone secreted by the pineal gland, exerts a variety of functions in regulating circadian rhythm, immune response, inflammation, etc. [91]. Melatonin is widely distributed in the body, including follicular fluid, and its concentration is significantly higher than that in the blood (36.5, 4.8 pg/ml vs. 10.0, 1.4 pg/ml) [92]. Accordingly, the expression of melatonin receptors can be detected throughout the ovary [93] [94], especially in the granulosa of sinus follicles. Thus, we speculate that melatonin is essential for the reproductive and endocrine functions of the ovaries. Recent studies have confirmed the therapeutic effect of melatonin on PCOS. For instance, Pai et al. found that the use of melatonin (1–2 mg/kg, 35 days) in PCOS rats could improve its reproductive and metabolic disorders [95]. Prata Lima et al. discovered that the number of ovarian cysts and the ovarian weight were decreased after 4 months of melatonin treatment, suggesting that melatonin may improve chronic anovulatory symptoms [59]. Afterwards, some scholars further explored the molecular mechanism of melatonin in the treatment of PCOS.

In addition, melatonin is able to alleviate PCOS by targeting autophagy. Studies have shown that the inhibition of autophagy mediated by melatonin improves the resistance of cells to harmful stimuli [96] [97]. They found that melatonin protects granulosa cells from oxidative damage by weakening the autophagy signal activated by oxidative stress. Cell experiments showed that the loss of activity in ovarian granulosa cells induced by oxidative stress was significantly ameliorated by melatonin treatment. In addition, PI3K-AKT is the key downstream effector of melatonin. It not only improves the resistance of granulosa cells to oxidative stress, but also inhibits autophagic responses, from gene expression to the formation of autophagy vacuoles [98]. Besides, melatonin has been proven to protect against mitochondrial injury in granulosa cells of PCOS by enhancing SIRT1 expression and inhibiting excessive PINK1/Parkin-mediated mitophagy [99]. These findings suggest a novel mechanism that melatonin protects against oxidative damage to granulosa cells by regulating autophagy, which could be a potential therapeutic target for anovulatory disorders.

Vitamin D

It is well known that vitamin D helps promote calcium and bone mineralization. Accumulated evidence shows that deficiency of vitamin D is also a risk factor for IR, cardiovascular, autoimmune diseases, and low fertility [100] [101]. Interestingly, numerous studies authenticate that vitamin D is associated with the pathogenesis and symptoms of PCOS [102] [103]. Women with PCOS show a higher incidence of vitamin D deficiency (defined as a concentration of 25[OH]D<75 nmol/l) when compared with healthy women matched by age and body mass index (BMI). Current researches confirm that vitamin D can effectively improve the metabolism, endocrine and fertility of PCOS [104]. Wehr et al. [102] conducted a cross-sectional study and they found that 25(OH)D concentration is an independent predictor of homeostasis model assessment of insulin resistance (HOMA-IR) and BMI. And serum 25(OH)D concentration was negatively correlated with BMI and other obesity markers. Trummer summarized current cross-sectional studies and randomized controlled trials (RCTs) and concluded that vitamin D showed a regulatory role in PCOS-related symptoms such as ovulation disorders, IR, and HA [105]. Azadi et al. conducted a meta-analysis to evaluate the effect of vitamin D supplementation on PCOS patients with HA. This meta-analysis included 6 clinical trials involving 183 participants. The results showed that vitamin D supplementation significantly reduced total testosterone levels [106]. From the above studies, we can definitely conclude that vitamin D supplementation is of great benefit to the treatment of PCOS. However, the molecular mechanism remains to be explored.

Studies have confirmed that vitamin D can enhance autophagy so as to improve metabolic problems [107] [108]. The autophagy process can be influenced by the levels of Ca2⁺ [109]. And vitamin D is able to regulate Ca²⁺ levels by enhancing the effects of Ca^{2 +} pumps and Ca²⁺ buffers. Thus, vitamin D is capable of maintaining a moderate autophagic flux [110]. Besides, the effects of vitamin D in regulating autophagy in PCOS has been confirmed by Lajtai et al. [111]. They divided female Wistar rats into four groups. Two groups were PCOS model groups, one of which received a low vitamin D diet (D–T+), and the other received a normal vitamin D3 diet (D+T+). The other two groups without testosterone treatment served as controls, one of which received vitamin D3 (D+T–) orally, and the other of which were lack of vitamin D (D–T–). The results displayed that D–T+animals showed a decrease in LC3 II levels in the liver and increased insulin levels. This reflected that decreased level of autophagy in the liver of PCOS patients was closely related to IR. In addition, in (D–T+) group, the level of LC3 II was reduced and phosphorylated Akt was increased in ovary, and excessive activation of PI3K/Akt may lead to impaired follicular development and the appearance of a large number of immature follicles, which contributes to the formation of polycystic ovarian status. While in (D+T+) group, IR was alleviated and the level of autophagy was increased, with decreased level of phosphorylated Akt. Therefore, we can infer that vitamin D supplementation play a therapeutic role in the recovery of PCOS, which is closely related to its regulation on autophagy. In the future, it should be further observed whether vitamin D exerts an effect on PCOS via mediating autophagy in human, and further exploration should be conducted to explore the pathway by which vitamin D interferes with autophagy.

Probiotics

Probiotics can improve the balance of intestinal microbiota by regulating microbial composition and metabolites, enabling the host to obtain health benefits from living microorganisms [112]. Common probiotic strains include lactobacillus, bifidobacterium, and yeast, etc. Currently, probiotic supplementation is also used in the treatment of PCOS, as it is suggested that the gut microbiota of PCOS is abnormal [113] [114]. The addition of probiotics can restore the diversity of gut microbiota and improve the reproductive and metabolic capacity of PCOS rats [115]. Ahmadi et al. conducted a randomized double-blind placebo-controlled clinical trial on 60 patients with PCOS who were randomly divided into probiotic capsule group (n=30) and placebo group (n=30) for 12 weeks of treatment [116]. The results showed that probiotic supplementation could significantly reduce the BMI, fasting blood glucose, serum insulin concentration, and lipid levels in patients with PCOS. And another randomized double-blind controlled trial [117] found that treatment of probiotics combined with vitamin D for 12 weeks could significantly reduce total level of testosterone in patients with PCOS, improve the hairy symptoms, and alleviate chronic inflammation. In addition, the addition of probiotics can help to improve the long-term complications of PCOS. Tabrizi R et al. performed a meta-analysis involving 11 RCTs, which indicated that probiotics could not only improve HA and IR, but also reduce the level of total cholesterol and triglyceride, thereby reducing the risk of cardiovascular disease in patients with PCOS [118]. Giorgia et al. studied adult female zebrafish to explore the relationship between probiotics and autophagy [119]. The control group (n=10) was given a commercial diet and the treatment group (n=10) was fed with diets containing the lyophilized probiotic Lactobacillus rhamnosus IMC 501. After 10 days of treatment, the zebrafish were sacrificed, and their ovaries were removed. The results showed that autophagosomes in the preovulation follicles were increased in the probiotics treatment group, which were more obvious in stage IV follicles. The expression of autophagy related genes (i. e., Ambra1, beclin1, LC3 and Uvrag) was increased, and the expression profiling of apoptosis-related genes (i. e., P53, Bax, Apaf and Cas3) was decreased, leading to a reduced apoptosis rate and an increased survival rate of follicles. This study confirms the supportive role of probiotics in follicular development by regulating follicular autophagy and apoptosis. Moreover, probiotics can regulate inflammation through autophagy. Studies have shown that probiotics can activate autophagy in bone marrow dendritic cells in which autophagy were reduced or Atg16L1 was knocked down, thus reducing the release of inflammatory cytokines [120]. In addition, probiotics can also amplify the effect of beneficial signaling pathways by modulating autophagy and enhance the therapeutic effect of PCOS. For instance, probiotics has been confirmed to be able to exert anti-inflammation activity and induce autophagy by activating the Vitamin D receptor-autophagy signaling pathways [121] [122], which is closely related to the improvement of PCOS-related pathological mechanisms. However, the effect of probiotics on autophagy, especially the effects of different probiotics on the autophagy level of different tissue cells in patients with PCOS needs to be further clarified.

Conclusion

Up to now, the exact pathological mechanism of PCOS is still unclear and its clinical intervention is also controversial. This study innovatively and systematically analyzed the role of autophagy in the pathological mechanism of PCOS and provided sufficient literature evidence for autophagy-related exploration in the field of PCOS. Furthermore, we summarized the potential of innovative methods or drugs to improve the pathological mechanism of PCOS from the aspects of regulating autophagy, and provided inspiration for drug discovery, which is conducive to broadening clinical decision-making and filling gaps in today’s treatments.

Conflict of Interest

The authors declare that they have no conflict of interest.

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Publikationsverlauf

Eingereicht: 26. November 2023

Angenommen nach Revision: 26. Februar 2024

Artikel online veröffentlicht:
02. April 2024